Bio


Dr. Lei (Stanley) Qi is Associate Professor of Bioengineering, Sarafan ChEM-H, and a Chan Zuckerberg Biohub Investigator. Dr. Qi is a principal contributor to the development of CRISPR technologies for genome engineering beyond gene editing. His lab created the first nuclease-deactivated Cas9 (dCas9) for targeted gene regulation in cells. His lab has invented a CRISPR toolbox for engineering the epigenome, including CRISPRi and CRISPRa for targeted gene repression and activation, epigenome editing, LiveFISH for real-time DNA/RNA imaging, CRISPR-GO for 3D genome manipulation, CasMINI as a compact CRISPR system for gene therapy, hyperCas12a for multi-gene engineering, and CRISPR antivirals aimed at treating broad RNA viruses.

Dr. Qi obtained B.S. in Physics and Math from Tsinghua University in 2005, and Ph.D. in Bioengineering from the University of California, Berkeley in 2012. He was a Systems Biology Faculty Fellow at UCSF between 2012-2014, and joined Stanford faculty in 2014. His research focuses on mammalian synthetic biology, epigenetic engineering, immune cell engineering, directed evolution, and novel approaches for gene therapy.

Administrative Appointments


  • Systems Biology Faculty Fellow, University of California San Francisco (2012 - 2014)
  • Assistant Professor, Stanford University (2014 - 2022)
  • Associate Professor, Stanford University (2022 - Present)
  • Chan Zuckerberg Biohub Investigator, Chan Zuckerberg Biohub (2022 - Present)

Honors & Awards


  • NIH Director's Independence Award, National Institutes of Health (2013)
  • Pew Biomedical Scholar, The Pew Charitable Trusts (2016)
  • Alfred P. Sloan Fellow, Alfred P. Sloan Foundation (2017)
  • Frontiers of Science Award, Society of Cosmetic Chemists (2017)
  • 35 Innovators Under 35, MIT Technology Review (2018)
  • Bioengineering Rising Star Lectureship, University of California Berkeley (2018)
  • SN 10: Scientists to Watch, Science News (2019)
  • ACS Synthetic Biology Young Innovator Award, American Chemical Society (2021)
  • Blavatnik Fellow in the Life Sciences, Blavatnik Family Foundation (2021)
  • NSF CAREER Award, National Science Foundation (2021)
  • Thomas and Salma Haider Biomedical Breakthrough Lectureship, University of California Riverside (2021)
  • Blavatnik Fellow in the Life Sciences, Blavatnik Family Foundation (2022)
  • Chan Zuckerberg Biohub Investigator, Chan Zuckerberg Biohub (2022)
  • Kenneth Fong Young Investigator Award, SCBA (2022)
  • AIMBE Fellow, American Institute for Medical and Biological Engineering (AIMBE) (2023)
  • Blavatnik Fellow in the Life Sciences, Blavatnik Family Foundation (2023)
  • NIH Director’s Pioneer Award, National Institutes of Health (2023)

Boards, Advisory Committees, Professional Organizations


  • Member, Phi Beta Kappa (2011 - Present)
  • Scientific Advisory Board, Caribou Biosciences (2015 - 2016)
  • Scientific founder, Refuge Biotechnologies (2016 - 2021)
  • Associate Editor, The CRISPR Journal (2017 - 2022)
  • Member, Sigma Xi (2017 - Present)
  • Scientific Advisory Board, NIH Center for Genome Editing and Recording (CGER) (2018 - Present)
  • Founder and Scientific Advisory Board, Epicrispr Biotechnologies (2019 - Present)
  • Board of Reviewing Editors (BoRE), Science (2020 - Present)
  • Advisory Board Editor, Cell Reports Methods (2021 - Present)
  • Scientific Advisory Board, Laboratory of Genomics Research (2022 - Present)
  • Editorial Board Member, The CRISPR Journal (2023 - Present)
  • Fellow, American Institute for Medical and Biological Engineering (AIMBE) (2023 - Present)
  • Scientific Advisory Board, Kytopen Corp (2023 - Present)

Professional Education


  • B.S., Tsinghua University, Math and Physics (2005)
  • M.A., University of California, Berkeley, Physics (2007)
  • Ph.D., University of California, Berkeley/UCSF, Bioengineering (2012)

Patents


  • Qi LS, Wang H. "United States Patent US provisional patent application No. 62/722,684 Systems and methods for polynucleotide spatial organization", Leland Stanford Junior University, Sep 1, 2018
  • Qi LS, Dingal DCPD. "United States Patent US Patent NO. 9,856,497 Recombinant chimeric receptors for antigen sensing and genome manipulation", Leland Stanford Junior University, Dec 20, 2017
  • Lei S Qi, Rachel E Haurwitz, Jennifer A Doudna, Adam P Arkin. "United States Patent US Patent application NO. 14/248,980 & WO 2011/143124; US Patent No. 9,745,610. Methods and compositions for controlling gene expression by RNA processing", University of California, Sep 29, 2017
  • Lei S Qi, Chang Liu, Adam P Arkin. "United States Patent US Patent NO. 9,593,338 Synthetic transcriptional control elements and methods of generating and using such elements", University of California, Mar 14, 2017
  • Qi LS, Liu Y. "United States Patent US provisional patent application NO. 27/998,407 Compositions and methods identifying and using stem cell differentiation markers", Leland Stanford Junior University, Jan 8, 2017
  • Zalatan J, Lim WA, Qi LS. "United States Patent US Patent application No. 15/514,892 Scaffold RNAs", University of California, Dec 1, 2015
  • Qi LS, Tanenbaum ME, Gilbert LA, Weissman JS, Vale RD. "United States Patent US provisional patent application NO. 62/024,241 A protein tagging system for in vivo single molecule imaging and control of gene transcription", University of California, Sep 1, 2014
  • Gilbert LA, Horlbeck MA, Kampmann M, Weissman JS, Qi LS. "United States Patent US provisional patent application NO. 62/024,373 Genome-scale CRISPR-mediated control of gene expression", University of California, Sep 1, 2014
  • Qi LS, Chen B, Huang B. "United States Patent International patent provisional application NO. PCT/US2014/058133 Optimized small guide RNAs and methods of use", University of California, San Francisco, Sep 1, 2013
  • Lei S Qi, Jennifer A Doudna, Martin Jinek, Emmanuelle Charpentier,Krzysztof Chylinski, James HD Cate, Wendell A Lim. "United States Patent US Patent App. 13/842,859 Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription", University of California, Mar 15, 2013

Stanford Advisees


Graduate and Fellowship Programs


All Publications


  • A versatile CRISPR-Cas13d platform for multiplexed transcriptomic regulation and metabolic engineering in primary human T cells. Cell Tieu, V., Sotillo, E., Bjelajac, J. R., Chen, C., Malipatlolla, M., Guerrero, J. A., Xu, P., Quinn, P. J., Fisher, C., Klysz, D., Mackall, C. L., Qi, L. S. 2024

    Abstract

    CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.

    View details for DOI 10.1016/j.cell.2024.01.035

    View details for PubMedID 38387457

  • Sonogenetic control of multiplexed genome regulation and base editing. Nature communications Liu, P., Foiret, J., Situ, Y., Zhang, N., Kare, A. J., Wu, B., Raie, M. N., Ferrara, K. W., Qi, L. S. 2023; 14 (1): 6575

    Abstract

    Manipulating gene expression in the host genome with high precision is crucial for controlling cellular function and behavior. Here, we present a precise, non-invasive, and tunable strategy for controlling the expression of multiple endogenous genes both in vitro and in vivo, utilizing ultrasound as the stimulus. By engineering a hyper-efficient dCas12a and effector under a heat shock promoter, we demonstrate a system that can be inducibly activated through thermal energy produced by ultrasound absorption. This system allows versatile thermal induction of gene activation or base editing across cell types, including primary T cells, and enables multiplexed gene activation using a single guide RNA array. In mouse models, localized temperature elevation guided by high-intensity focused ultrasound effectively triggers reporter gene expression in implanted cells. Our work underscores the potential of ultrasound as a clinically viable approach to enhance cell and gene-based therapies via precision genome and epigenome engineering.

    View details for DOI 10.1038/s41467-023-42249-8

    View details for PubMedID 37852951

    View details for PubMedCentralID PMC10584809

  • Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus. Nature biomedical engineering Chavez, M., Rane, D. A., Chen, X., Qi, L. S. 2023

    Abstract

    The targeted insertion and stable expression of a large genetic payload in primary human cells demands methods that are robust, efficient and easy to implement. Large payload insertion via retroviruses is typically semi-random and hindered by transgene silencing. Leveraging homology-directed repair to place payloads under the control of endogenous essential genes can overcome silencing but often results in low knock-in efficiencies and cytotoxicity. Here we report a method for the knock-in and stable expression of a large payload and for the simultaneous knock-in of two genes at two endogenous loci. The method, which we named CLIP (for 'CRISPR for long-fragment integration via pseudovirus'), leverages an integrase-deficient lentivirus encoding a payload flanked by homology arms and 'cut sites' to insert the payload upstream and in-frame of an endogenous essential gene, followed by the delivery of a CRISPR-associated ribonucleoprotein complex via electroporation. We show that CLIP enables the efficient insertion and stable expression of large payloads and of two difficult-to-express viral antigens in primary T cells at low cytotoxicity. CLIP offers a scalable and efficient method for manufacturing engineered primary cells.

    View details for DOI 10.1038/s41551-023-01037-x

    View details for PubMedID 37127707

  • Advances in CRISPR therapeutics. Nature reviews. Nephrology Chavez, M., Chen, X., Finn, P. B., Qi, L. S. 2022

    Abstract

    The clustered regularly interspaced short palindromic repeats (CRISPR) renaissance was catalysed by the discovery that RNA-guided prokaryotic CRISPR-associated (Cas) proteins can create targeted double-strand breaks in mammalian genomes. This finding led to the development of CRISPR systems that harness natural DNA repair mechanisms to repair deficient genes more easily and precisely than ever before. CRISPR has been used to knock out harmful mutant genes and to fix errors in coding sequences to rescue disease phenotypes in preclinical studies and in several clinical trials. However, most genetic disorders result from combinations of mutations, deletions and duplications in the coding and non-coding regions of the genome and therefore require sophisticated genome engineering strategies beyond simple gene knockout. To overcome this limitation, the toolbox of natural and engineered CRISPR-Cas systems has been dramatically expanded to include diverse tools that function in human cells for precise genome editing and epigenome engineering. The application of CRISPR technology to edit the non-coding genome, modulate gene regulation, make precise genetic changes and target infectious diseases has the potential to lead to curative therapies for many previously untreatable diseases.

    View details for DOI 10.1038/s41581-022-00636-2

    View details for PubMedID 36280707

  • Nested epistasis enhancer networks for robust genome regulation. Science (New York, N.Y.) Lin, X., Liu, Y., Liu, S., Zhu, X., Wu, L., Zhu, Y., Zhao, D., Xu, X., Chemparathy, A., Wang, H., Cao, Y., Nakamura, M., Noordermeer, J. N., La Russa, M., Wong, W. H., Zhao, K., Qi, L. S. 2022: eabk3512

    Abstract

    Mammalian genomes possess multiple enhancers spanning an ultralong distance (>megabases) to modulate important genes, yet it is unclear how these enhancers coordinate to achieve this task. Here, we combine multiplexed CRISPRi screening with machine learning to define quantitative enhancer-enhancer interactions. We find that the ultralong distance enhancer network possesses a nested multi-layer architecture that confers functional robustness of gene expression. Experimental characterization reveals that enhancer epistasis is maintained by three-dimensional chromosomal interactions and BRD4 condensation. Machine learning prediction of synergistic enhancers provides an effective strategy to identify non-coding variant pairs associated with pathogenic genes in diseases beyond Genome-Wide Association Studies (GWAS) analysis. Our work unveils nested epistasis enhancer networks, which can better explain enhancer functions within cells and in diseases.

    View details for DOI 10.1126/science.abk3512

    View details for PubMedID 35951677

  • Scalable biological signal recording in mammalian cells using Cas12a base editors. Nature chemical biology Kempton, H. R., Love, K. S., Guo, L. Y., Qi, L. S. 2022

    Abstract

    Biological signal recording enables the study of molecular inputs experienced throughout cellular history. However, current methods are limited in their ability to scale up beyond a single signal in mammalian contexts. Here, we develop an approach using a hyper-efficient dCas12a base editor for multi-signal parallel recording in human cells. We link signals of interest to expression of guide RNAs to catalyze specific nucleotide conversions as a permanent record, enabled by Cas12's guide-processing abilities. We show this approach is plug-and-play with diverse biologically relevant inputs and extend it for more sophisticated applications, including recording of time-delimited events and history of chimeric antigen receptor T cells' antigen exposure. We also demonstrate efficient recording of up to four signals in parallel on an endogenous safe-harbor locus. This work provides a versatile platform for scalable recording of signals of interest for a variety of biological applications.

    View details for DOI 10.1038/s41589-022-01034-2

    View details for PubMedID 35637351

  • Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro. Nature communications Zeng, L., Liu, Y., Nguyenla, X. H., Abbott, T. R., Han, M., Zhu, Y., Chemparathy, A., Lin, X., Chen, X., Wang, H., Rane, D. A., Spatz, J. M., Jain, S., Rustagi, A., Pinsky, B., Zepeda, A. E., Kadina, A. P., Walker, J. A., Holden, K., Temperton, N., Cochran, J. R., Barron, A. E., Connolly, M. D., Blish, C. A., Lewis, D. B., Stanley, S. A., La Russa, M. F., Qi, L. S. 2022; 13 (1): 2766

    Abstract

    A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.

    View details for DOI 10.1038/s41467-022-30546-7

    View details for PubMedID 35589813

  • Multiplexed genome regulation in vivo with hyper-efficient Cas12a. Nature cell biology Guo, L. Y., Bian, J., Davis, A. E., Liu, P., Kempton, H. R., Zhang, X., Chemparathy, A., Gu, B., Lin, X., Rane, D. A., Xu, X., Jamiolkowski, R. M., Hu, Y., Wang, S., Qi, L. S. 2022

    Abstract

    Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR-Cas12a systems enable versatile multiple-genomic-loci targeting by processing numerous CRISPR RNAs (crRNAs) from a single transcript; however, their low efficiency has hindered in vivo applications. Through structure-guided protein engineering, we developed a hyper-efficient Lachnospiraceae bacterium Cas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low concentrations of crRNA. We demonstrate that hyperdCas12a has comparable off-target effects compared with the wild-type system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a activator and a single crRNA array simultaneously activating the endogenous Oct4, Sox2 and Klf4 genes in the retina of post-natal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene-modulation and gene-therapy applications.

    View details for DOI 10.1038/s41556-022-00870-7

    View details for PubMedID 35414015

  • Engineering 3D genome organization. Nature reviews. Genetics Wang, H., Han, M., Qi, L. S. 2021

    Abstract

    Cancers and developmental disorders are associated with alterations in the 3D genome architecture in space and time (the fourth dimension). Mammalian 3D genome organization is complex and dynamic and plays an essential role in regulating gene expression and cellular function. To study the causal relationship between genome function and its spatio-temporal organization in the nucleus, new technologies for engineering and manipulating the 3D organization of the genome have been developed. In particular, CRISPR-Cas technologies allow programmable manipulation at specific genomic loci, enabling unparalleled opportunities in this emerging field of 3D genome engineering. We review advances in mammalian 3D genome engineering with a focus on recent manipulative technologies using CRISPR-Cas and related technologies.

    View details for DOI 10.1038/s41576-020-00325-5

    View details for PubMedID 33558716

  • Interrogation of the dynamic properties of higher-order heterochromatin using CRISPR-dCas9. Molecular cell Gao, Y., Han, M., Shang, S., Wang, H., Qi, L. S. 2021

    Abstract

    Eukaryotic chromosomes feature large regions of compact, repressed heterochromatin hallmarked by Heterochromatin Protein 1 (HP1). HP1 proteins play multi-faceted roles in shaping heterochromatin, and in cells, HP1 tethering to individual gene promoters leads to epigenetic modifications and silencing. However, emergent properties of HP1 at supranucleosomal scales remain difficult to study in cells because of a lack of appropriate tools. Here, we develop CRISPR-engineered chromatin organization (EChO), combining live-cell CRISPR imaging with inducible large-scale recruitment of chromatin proteins to native genomic targets. We demonstrate that human HP1α tiled across kilobase-scale genomic DNA form novel contacts with natural heterochromatin, integrates two distantly targeted regions, and reversibly changes chromatin from a diffuse to compact state. The compact state exhibits delayed disassembly kinetics and represses transcription across over 600 kb. These findings support a polymer model of HP1α-mediated chromatin regulation and highlight the utility of CRISPR-EChO in studying supranucleosomal chromatin organization in living cells.

    View details for DOI 10.1016/j.molcel.2021.07.034

    View details for PubMedID 34428454

  • Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Molecular cell Xu, X., Chemparathy, A., Zeng, L., Kempton, H. R., Shang, S., Nakamura, M., Qi, L. S. 2021

    Abstract

    Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here, we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.

    View details for DOI 10.1016/j.molcel.2021.08.008

    View details for PubMedID 34480847

  • CRISPR technologies for precise epigenome editing. Nature cell biology Nakamura, M., Gao, Y., Dominguez, A. A., Qi, L. S. 2021; 23 (1): 11–22

    Abstract

    The epigenome involves a complex set of cellular processes governing genomic activity. Dissecting this complexity necessitates the development of tools capable of specifically manipulating these processes. The repurposing of prokaryotic CRISPR systems has allowed for the development of diverse technologies for epigenome engineering. Here, we review the state of currently achievable epigenetic manipulations along with corresponding applications. With future optimization, CRISPR-based epigenomic editing stands as a set of powerful tools for understanding and controlling biological function.

    View details for DOI 10.1038/s41556-020-00620-7

    View details for PubMedID 33420494

  • Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza. Cell Abbott, T. R., Dhamdhere, G. n., Liu, Y. n., Lin, X. n., Goudy, L. n., Zeng, L. n., Chemparathy, A. n., Chmura, S. n., Heaton, N. S., Debs, R. n., Pande, T. n., Endy, D. n., La Russa, M. F., Lewis, D. B., Qi, L. S. 2020

    Abstract

    The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.

    View details for DOI 10.1016/j.cell.2020.04.020

    View details for PubMedID 32353252

  • Multiple Input Sensing and Signal Integration Using a Split Cas12a System. Molecular cell Kempton, H. R., Goudy, L. E., Love, K. S., Qi, L. S. 2020

    Abstract

    The ability to integrate biological signals and execute a functional response when appropriate is critical for sophisticated cell engineering using synthetic biology. Although the CRISPR-Cas system has been harnessed for synthetic manipulation of the genome, it has not been fully utilized for complex environmental signal sensing, integration, and actuation. Here, we develop a split dCas12a platform and show that it allows for the construction of multi-input, multi-output logic circuits in mammalian cells. The system is highly programmable and can generate expandable AND gates with two, three, and four inputs. It can also incorporate NOT logic by using anti-CRISPR proteins as an OFF switch. By coupling the split dCas12a design to multiple tumor-relevant promoters, we provide a proof of concept that the system can implement logic gating to specifically detect breast cancer cells and execute therapeutic immunomodulatory responses.

    View details for DOI 10.1016/j.molcel.2020.01.016

    View details for PubMedID 32027839

  • Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells. Nature communications Nakamura, M., Srinivasan, P., Chavez, M., Carter, M. A., Dominguez, A. A., La Russa, M., Lau, M. B., Abbott, T. R., Xu, X., Zhao, D., Gao, Y., Kipniss, N. H., Smolke, C. D., Bondy-Denomy, J., Qi, L. S. 2019; 10 (1): 194

    Abstract

    Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate "write-protected" cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells.

    View details for PubMedID 30643127

  • CRISPR-mediated live imaging of genome editing and transcription. Science (New York, N.Y.) Wang, H. n., Nakamura, M. n., Abbott, T. R., Zhao, D. n., Luo, K. n., Yu, C. n., Nguyen, C. M., Lo, A. n., Daley, T. P., La Russa, M. n., Liu, Y. n., Qi, L. S. 2019

    Abstract

    We report a robust, versatile approach named CRISPR Live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligos for genome tracking in broad cell types including primary cells. An intrinsic stability switch of CRISPR guide RNAs enables LiveFISH to accurately detect chromosomal disorders such as Patau Syndrome in prenatal amniotic fluid cells and track multiple loci in human T lymphocytes. In addition, LiveFISH tracks the real-time movement of DNA double-strand breaks induced by CRISPR-Cas9-mediated editing and consequent chromosome translocations. Finally, combining Cas9 and Cas13 systems, LiveFISH allows for simultaneous visualization of genomic DNA and RNA transcripts in living cells. The LiveFISH approach enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.

    View details for DOI 10.1126/science.aax7852

    View details for PubMedID 31488703

  • CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization. Cell Wang, H., Xu, X., Nguyen, C. M., Liu, Y., Gao, Y., Lin, X., Daley, T., Kipniss, N. H., La Russa, M., Qi, L. S. 2018

    Abstract

    Programmable control of spatial genome organization is a powerful approach for studying how nuclear structure affects gene regulation and cellular function. Here, we develop a versatile CRISPR-genome organization (CRISPR-GO) system that can efficiently control the spatial positioning of genomic loci relative to specific nuclear compartments, including the nuclear periphery, Cajal bodies, and promyelocytic leukemia (PML) bodies. CRISPR-GO is chemically inducible and reversible, enabling interrogation of real-time dynamics of chromatin interactions with nuclear compartments in living cells. Inducible repositioning of genomic loci to the nuclear periphery allows for dissection of mitosis-dependent and -independent relocalization events and also for interrogation of the relationship between gene position and gene expression. CRISPR-GO mediates rapid de novo formation of Cajal bodies at desired chromatin loci and causes significant repression of endogenous gene expression over long distances (30-600 kb). The CRISPR-GO system offers a programmable platform to investigate large-scale spatial genome organization and function.

    View details for PubMedID 30318144

  • CRISPR Activation Screens Systematically Identify Factors that Drive Neuronal Fate and Reprogramming. Cell stem cell Liu, Y., Yu, C., Daley, T. P., Wang, F., Cao, W. S., Bhate, S., Lin, X., Still, C. 2., Liu, H., Zhao, D., Wang, H., Xie, X. S., Ding, S., Wong, W. H., Wernig, M., Qi, L. S. 2018

    Abstract

    Comprehensive identification of factors that can specify neuronal fate could provide valuable insights into lineage specification and reprogramming, but systematic interrogation of transcription factors, and their interactions with each other, has proven technically challenging. We developed a CRISPR activation (CRISPRa) approach to systematically identify regulators of neuronal-fate specification. We activated expression of all endogenous transcription factors and other regulators via a pooled CRISPRa screen in embryonic stem cells, revealing genes including epigenetic regulators such as Ezh2 that can induce neuronal fate. Systematic CRISPR-based activation of factor pairs allowed us to generate a genetic interaction map for neuronal differentiation, with confirmation of top individual and combinatorial hits as bona fide inducers of neuronal fate. Several factor pairs could directly reprogram fibroblasts into neurons, which shared similar transcriptional programs with endogenous neurons. This study provides an unbiased discovery approach for systematic identification of genes that drive cell-fate acquisition.

    View details for PubMedID 30318302

  • Engineering cell sensing and responses using a GPCR-coupled CRISPR-Cas system. Nature communications Kipniss, N. H., Dingal, P. C., Abbott, T. R., Gao, Y. n., Wang, H. n., Dominguez, A. A., Labanieh, L. n., Qi, L. S. 2017; 8 (1): 2212

    Abstract

    G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and detect a wide array of cues in the human body. Here we describe a molecular device that couples CRISPR-dCas9 genome regulation to diverse natural and synthetic extracellular signals via GPCRs. We generate alternative architectures for fusing CRISPR to GPCRs utilizing the previously reported design, Tango, and our design, ChaCha. Mathematical modeling suggests that for the CRISPR ChaCha design, multiple dCas9 molecules can be released across the lifetime of a GPCR. The CRISPR ChaCha is dose-dependent, reversible, and can activate multiple endogenous genes simultaneously in response to extracellular ligands. We adopt the design to diverse GPCRs that sense a broad spectrum of ligands, including synthetic compounds, chemokines, mitogens, fatty acids, and hormones. This toolkit of CRISPR-coupled GPCRs provides a modular platform for rewiring diverse ligand sensing to targeted genome regulation for engineering cellular functions.

    View details for PubMedID 29263378

  • Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Nature methods Gao, Y., Xiong, X., Wong, S., Charles, E. J., Lim, W. A., Qi, L. S. 2016

    Abstract

    The ability to dynamically manipulate the transcriptome is important for studying how gene networks direct cellular functions and how network perturbations cause disease. Nuclease-dead CRISPR-dCas9 transcriptional regulators, while offering an approach for controlling individual gene expression, remain incapable of dynamically coordinating complex transcriptional events. Here, we describe a flexible dCas9-based platform for chemical-inducible complex gene regulation. From a screen of chemical- and light-inducible dimerization systems, we identified two potent chemical inducers that mediate efficient gene activation and repression in mammalian cells. We combined these inducers with orthogonal dCas9 regulators to independently control expression of different genes within the same cell. Using this platform, we further devised AND, OR, NAND, and NOR dCas9 logic operators and a diametric regulator that activates gene expression with one inducer and represses with another. This work provides a robust CRISPR-dCas9-based platform for enacting complex transcription programs that is suitable for large-scale transcriptome engineering.

    View details for DOI 10.1038/nmeth.4042

    View details for PubMedID 27776111

  • Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds CELL Zalatan, J. G., Lee, M. E., Almeida, R., Gilbert, L. A., Whitehead, E. H., La Russa, M., Tsai, J. C., Weissman, J. S., Dueber, J. E., Qi, L. S., Lim, W. A. 2015; 160 (1-2): 339-350

    Abstract

    Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications, including rewiring cell fates or engineering metabolic pathways.

    View details for DOI 10.1016/j.cell.2014.11.052

    View details for Web of Science ID 000347923200029

    View details for PubMedID 25533786

  • Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System CELL Chen, B., Gilbert, L. A., Cimini, B. A., Schnitzbauer, J., Zhang, W., Li, G., Park, J., Blackburn, E. H., Weissman, J. S., Qi, L. S., Huang, B. 2013; 155 (7): 1479-1491

    Abstract

    The spatiotemporal organization and dynamics of chromatin play critical roles in regulating genome function. However, visualizing specific, endogenous genomic loci remains challenging in living cells. Here, we demonstrate such an imaging technique by repurposing the bacterial CRISPR/Cas system. Using an EGFP-tagged endonuclease-deficient Cas9 protein and a structurally optimized small guide (sg) RNA, we show robust imaging of repetitive elements in telomeres and coding genes in living cells. Furthermore, an array of sgRNAs tiling along the target locus enables the visualization of nonrepetitive genomic sequences. Using this method, we have studied telomere dynamics during elongation or disruption, the subnuclear localization of the MUC4 loci, the cohesion of replicated MUC4 loci on sister chromatids, and their dynamic behaviors during mitosis. This CRISPR imaging tool has potential to significantly improve the capacity to study the conformation and dynamics of native chromosomes in living human cells.

    View details for DOI 10.1016/j.cell.2013.12.001

    View details for Web of Science ID 000328693300006

    View details for PubMedID 24360272

  • CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes CELL Gilbert, L. A., Larson, M. H., Morsut, L., Liu, Z., Brar, G. A., Torres, S. E., Stern-Ginossar, N., Brandman, O., Whitehead, E. H., Doudna, J. A., Lim, W. A., Weissman, J. S., Qi, L. S. 2013; 154 (2): 442-451

    Abstract

    The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

    View details for DOI 10.1016/j.cell.2013.06.044

    View details for Web of Science ID 000321950700019

    View details for PubMedID 23849981

  • Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression CELL Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., Lim, W. A. 2013; 152 (5): 1173-1183

    Abstract

    Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.

    View details for DOI 10.1016/j.cell.2013.02.022

    View details for Web of Science ID 000315710300022

    View details for PubMedID 23452860

  • Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles. bioRxiv : the preprint server for biology Andronov, L., Han, M., Zhu, Y., Roy, A. R., Barentine, A. E., Garhyan, J., Qi, L. S., Moerner, W. E. 2023

    Abstract

    The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelle where the replication of viral genomic RNA (vgRNA) occurs. To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain vgRNA clusters along with viral double-stranded RNA (dsRNA) clusters and the replication enzyme, encapsulated by membranes derived from the host endoplasmic reticulum (ER). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of ER labels and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are enclosed by DMVs at early infection stages which then merge into vesicle packets as infection progresses. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

    View details for DOI 10.1101/2023.11.07.566110

    View details for PubMedID 37986994

  • Inactive S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in human stem cell model of Parkinson's disease. Scientific reports Sastre, D., Zafar, F., Torres, C. A., Piper, D., Kirik, D., Sanders, L. H., Qi, L. S., Schüle, B. 2023; 13 (1): 17796

    Abstract

    Parkinson's disease (PD) is one of the most common neurodegenerative diseases, but no disease modifying therapies have been successful in clinical translation presenting a major unmet medical need. A promising target is alpha-synuclein or its aggregated form, which accumulates in the brain of PD patients as Lewy bodies. While it is not entirely clear which alpha-synuclein protein species is disease relevant, mere overexpression of alpha-synuclein in hereditary forms leads to neurodegeneration. To specifically address gene regulation of alpha-synuclein, we developed a CRISPR interference (CRISPRi) system based on the nuclease dead S. aureus Cas9 (SadCas9) fused with the transcriptional repressor domain Krueppel-associated box to controllably repress alpha-synuclein expression at the transcriptional level. We screened single guide (sg)RNAs across the SNCA promoter and identified several sgRNAs that mediate downregulation of alpha-synuclein at varying levels. CRISPRi downregulation of alpha-synuclein in iPSC-derived neuronal cultures from a patient with an SNCA genomic triplication showed functional recovery by reduction of oxidative stress and mitochondrial DNA damage. Our results are proof-of-concept in vitro for precision medicine by targeting the SNCA gene promoter. The SNCA CRISPRi approach presents a new model to understand safe levels of alpha-synuclein downregulation and a novel therapeutic strategy for PD and related alpha-synucleinopathies.

    View details for DOI 10.1038/s41598-023-45078-3

    View details for PubMedID 37853101

    View details for PubMedCentralID PMC10584834

  • Building smart CAR T cell therapies: The path to overcome current challenges. Cancer cell Cai, Q., Warren, S., Pietrobon, V., Maeurer, M., Qi, L. S., Lu, T. K., Lajoie, M. J., Barrett, D., Stroncek, D. F., Marincola, F. M. 2023

    Abstract

    Successful implementation of adoptive cell therapy (ACT) of cancer requires comprehensively addressing biological and practical challenges. This approach has been largely overlooked, resulting in a gap between the potential of ACT and its actual effectiveness. We summarize the most promising technical strategies in creating an "ideal" ACT product, focusing on chimeric antigen receptor (CAR)-engineered cells. Since many requirements for effective ACT are common to most cancers, what we outline here might have a broader impact.

    View details for DOI 10.1016/j.ccell.2023.08.011

    View details for PubMedID 37714150

  • Cas9-mediated knockout of Ndrg2 enhances the regenerative potential of dendritic cells for wound healing. Nature communications Henn, D., Zhao, D., Sivaraj, D., Trotsyuk, A., Bonham, C. A., Fischer, K. S., Kehl, T., Fehlmann, T., Greco, A. H., Kussie, H. C., Moortgat Illouz, S. E., Padmanabhan, J., Barrera, J. A., Kneser, U., Lenhof, H., Januszyk, M., Levi, B., Keller, A., Longaker, M. T., Chen, K., Qi, L. S., Gurtner, G. C. 2023; 14 (1): 4729

    Abstract

    Chronic wounds impose a significant healthcare burden to a broad patient population. Cell-based therapies, while having shown benefits for the treatment of chronic wounds, have not yet achieved widespread adoption into clinical practice. We developed a CRISPR/Cas9 approach to precisely edit murine dendritic cells to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing of tolerogenic dendritic cells, we identified N-myc downregulated gene 2 (Ndrg2), which marks a specific population of dendritic cell progenitors, as a promising target for CRISPR knockout. Ndrg2-knockout alters the transcriptomic profile of dendritic cells and preserves an immature cell state with a strong pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a therapeutic hydrogel for in vivo cell delivery and developed an effective translational approach for dendritic cell-based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in dendritic cells for treating various types of chronic wounds.

    View details for DOI 10.1038/s41467-023-40519-z

    View details for PubMedID 37550295

  • Targeted knock-ins with pseudovirus for the stable expression of large transgenes NATURE BIOMEDICAL ENGINEERING Chavez, M., Qi, L. 2023: 612-613

    Abstract

    We engineered integrase-deficient lentiviruses to act as vectors for the delivery of large gene knock-ins via homology-directed repair. This technology enables the non-cytotoxic, targeted insertion of difficult-to-express transgenes into genomic loci that are essential to cell survival, thereby overcoming the gene silencing that otherwise limits primary immune cell engineering.

    View details for DOI 10.1038/s41551-023-01044-y

    View details for Web of Science ID 000986322600001

    View details for PubMedID 37173433

    View details for PubMedCentralID PMC10177726

  • Nuclease-Dead Cas9-Mediated Downregulation of alpha-Synuclein as a Disease-Modulatory Therapeutic Strategy for Parkinson's Disease Schuele, B., Torres, C., Zafar, F., Hermesky, D., Vazquez, J., Piper, D., Sastre, D., Qi, L., Sanders, L., Kirik, D. CELL PRESS. 2023: 339
  • Statins improve endothelial function via suppression of epigenetic-driven EndMT. Nature cardiovascular research Liu, C., Shen, M., Tan, W. L., Chen, I. Y., Liu, Y., Yu, X., Yang, H., Zhang, A., Liu, Y., Zhao, M. T., Ameen, M., Zhang, M., Gross, E. R., Qi, L. S., Sayed, N., Wu, J. C. 2023; 2 (5): 467-485

    Abstract

    The pleiotropic benefits of statins in cardiovascular diseases that are independent of their lipid-lowering effects have been well documented, but the underlying mechanisms remain elusive. Here we show that simvastatin significantly improves human induced pluripotent stem cell-derived endothelial cell functions in both baseline and diabetic conditions by reducing chromatin accessibility at transcriptional enhanced associate domain elements and ultimately at endothelial-to-mesenchymal transition (EndMT)-regulating genes in a yes-associated protein (YAP)-dependent manner. Inhibition of geranylgeranyltransferase (GGTase) I, a mevalonate pathway intermediate, repressed YAP nuclear translocation and YAP activity via RhoA signaling antagonism. We further identified a previously undescribed SOX9 enhancer downstream of statin-YAP signaling that promotes the EndMT process. Thus, inhibition of any component of the GGTase-RhoA-YAP-SRY box transcription factor 9 (SOX9) signaling axis was shown to rescue EndMT-associated endothelial dysfunction both in vitro and in vivo, especially under diabetic conditions. Overall, our study reveals an epigenetic modulatory role for simvastatin in repressing EndMT to confer protection against endothelial dysfunction.

    View details for DOI 10.1038/s44161-023-00267-1

    View details for PubMedID 37693816

    View details for PubMedCentralID PMC10489108

  • Nanoscale, antigen encounter-dependent, IL-12 delivery by CAR T cells plus PD-L1 blockade for cancer treatment. Journal of translational medicine Yang, Z., Pietrobon, V., Bobbin, M., Stefanson, O., Yang, J., Goswami, A., Alphson, B., Choi, H., Magallanes, K., Cai, Q., Barrett, D., Wang, B., Qi, L. S., Marincola, F. M. 2023; 21 (1): 158

    Abstract

    Chimeric antigen receptor (CAR)-T cell therapies for the treatment of hematological malignancies experienced tremendous progress in the last decade. However, essential limitations need to be addressed to further improve efficacy and reduce toxicity to assure CAR-T cell persistence, trafficking to the tumor site, resistance to an hostile tumor microenvironment (TME), and containment of toxicity restricting production of powerful but potentially toxic bioproducts to the TME; the last could be achieved through contextual release upon tumor antigen encounter of factors capable of converting an immune suppressive TME into one conducive to immune rejection.We created an HER2-targeting CAR-T (RB-312) using a clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system, which induces the expression of the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. This circuit includes two lentiviral constructs. The first one (HER2-TEV) expresses an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3z co-stimulatory domains linked to the tobacco etch virus (TEV) protease and two single guide RNAs (sgRNA) targeting the interleukin (IL)-12A and IL12B transcription start site (TSS), respectively. The second construct (LdCV) encodes linker for activation of T cells (LAT) fused to nuclease-deactivated Streptococcus Pyogenes Cas9 (dCas9)-VP64-p65-Rta (VPR) via a TEV-cleavable sequence (TCS). Activation of the CAR brings HER2-TEV in close proximity to LdCV releasing dCas9 for nuclear localization. This conditional circuit leads to conditional and reversible induction of the IL-12/p70 heterodimer. RB-312 was compared in vitro to controls (cRB-312), lacking the IL-12 sgRNAs and conventional HER2 CAR (convCAR).The inducible CRISPRa system activated endogenous IL-12 expression resulting in enhanced secondary interferon (FN)-γ production, cytotoxicity, and CAR-T proliferation in vitro, prolonged in vivo persistence and greater suppression of HER2+ FaDu oropharyngeal cancer cell growth compared to the conventional CAR-T cell product. No systemic IL-12 was detected in the peripheral circulation. Moreover, the combination with programmed death ligand (PD-L1) blockade demonstrated robust synergistic effects.RB-312, the first clinically relevant product incorporating a CRISPRa system with non-gene editing and reversible upregulation of endogenous gene expression that promotes CAR-T cells persistence and effectiveness against HER2-expressing tumors. The autocrine effects of reversible, nanoscale IL-12 production limits the risk of off-tumor leakage and systemic toxicity.

    View details for DOI 10.1186/s12967-023-04014-9

    View details for PubMedID 36855120

    View details for PubMedCentralID 9820257

  • Multicolor super-resolution imaging to study human coronavirus RNA during cellular infection Roy, A. R., Wang, J., Han, M., Wang, H., Moeckl, L., Zeng, L., Moerner, W. E., Qi, L. S. CELL PRESS. 2023: 16A
  • Multicolor super-resolution imaging to study human coronavirus RNA during cellular infection. Biophysical journal Roy, A. R., Wang, J., Han, M., Wang, H., Mockl, L., Zeng, L., Moerner, W. E., Qi, L. S. 2023; 122 (3S1): 16a

    View details for DOI 10.1016/j.bpj.2022.11.313

    View details for PubMedID 36782799

  • Reversing the Central Dogma: RNA-guided control of DNA in epigenetics and genome editing. Molecular cell Chang, H. Y., Qi, L. S. 2023; 83 (3): 442-451

    Abstract

    The Central Dogma of the flow of genetic information is arguably the crowning achievement of 20th century molecular biology. Reversing the flow of information from RNA to DNA or chromatin has come to the fore in recent years, from the convergence of fundamental discoveries and synthetic biology. Inspired by the example of long noncoding RNAs (lncRNAs) in mammalian genomes that direct chromatin modifications and gene expression, synthetic biologists have repurposed prokaryotic RNA-guided genome defense systems such as CRISPR to edit eukaryotic genomes and epigenomes. Here we explore the parallels of these two fields and highlight opportunities for synergy and future breakthroughs.

    View details for DOI 10.1016/j.molcel.2023.01.010

    View details for PubMedID 36736311

  • Nuclease-dead S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in patient-derived stem cell model of Parkinson's disease. bioRxiv : the preprint server for biology Sastre, D., Zafar, F., Torres, C. A., Piper, D., Kirik, D., Sanders, L. H., Qi, S., Schule, B. 2023

    Abstract

    Parkinson's disease (PD) is one of the most common neurodegenerative diseases, but no disease modifying therapies have been successful in clinical translation presenting a major unmet medical need. A promising target is alpha-synuclein or its aggregated form, which accumulates in the brain of PD patients as Lewy bodies. While it is not entirely clear which alpha-synuclein protein species is disease relevant, mere overexpression of alpha-synuclein in hereditary forms leads to neurodegeneration. To specifically address gene regulation of alpha-synuclein, we developed a CRISPR interference (CRISPRi) system based on the nuclease dead S. aureus Cas9 (SadCas9) fused with the transcriptional repressor domain Krueppel-associated box to controllably repress alpha-synuclein expression at the transcriptional level. We screened single guide (sg)RNAs across the SNCA promoter and identified several sgRNAs that mediate downregulation of alpha-synuclein at varying levels. CRISPRi downregulation of alpha-synuclein in iPSC-derived neuronal cultures from a patient with an SNCA genomic triplication showed functional recovery by reduction of oxidative stress and mitochondrial DNA damage. Our results are proof-of-concept in vitro for precision medicine by targeting the SNCA gene promoter. The SNCA CRISPRi approach presents a new model to understand safe levels of alpha-synuclein downregulation and a novel therapeutic strategy for PD and related alpha-synucleinopathies.

    View details for DOI 10.1101/2023.01.24.525105

    View details for PubMedID 36747875

  • Programmable Drug Control of Receptor Valency Modulates the Potency of Cell Therapeutics. bioRxiv : the preprint server for biology Finn, P. B., Chavez, M., Chen, X., Wang, H., Rane, D. A., Gurjar, J., Qi, L. S. 2023

    Abstract

    An increasing number of preclinical and clinical studies are exploring the use of receptor-engineered cells that can respond to disease states for the treatment of cancer, infectious disease, autoimmunity, and regeneration. However, receptor-based cell therapies, including chimeric antigen receptor (CAR), face many critical issues including target recognition escape, adverse side effects, and lack of in vivo control. Drug-controllable receptors offer a promising solution to overcome these issues through precise in vivo tuning of cells via enhanced sensing and therapeutic efficacy. Here we develop a novel class of modular and tunable receptors, termed valency-controlled receptors (VCRs), which can leverage customized small molecules to mediate cell signaling strength via controlled spatial clustering. We first develop DNA origami activated VCRs to demonstrate that receptor valency is a core mechanism that modulates immune cell activation. We design a series of customized valency-control ligands (VCLs) by transforming small molecule drugs into a multivalency format and modularly fusing VCR onto the CAR architecture. We demonstrate that VCL induction allows enhanced target sensitivity of engineered cells. Using medicinal chemistry, we develop programmable bioavailable VCL drugs to demonstrate that the VCR system enables drug-induced highly potent responses towards low antigen cancers in vitro and in vivo . Valency controlled receptors and customizable drug ligands provide a new synthetic biology platform to precisely tune engineered cell therapeutic potency, which can address existing safety and efficacy barriers in cell therapy.

    View details for DOI 10.1101/2023.01.04.522664

    View details for PubMedID 36712002

  • SARS-CoV-2 escapes direct NK cell killing through Nsp1-mediated downregulation of ligands for NKG2D. Cell reports Lee, M. J., Leong, M. W., Rustagi, A., Beck, A., Zeng, L., Holmes, S., Qi, L. S., Blish, C. A. 2022: 111892

    Abstract

    Natural killer (NK) cells are cytotoxic effector cells that target and lyse virally infected cells; many viruses therefore encode mechanisms to escape such NK cell killing. Here, we interrogate the ability of SARS-CoV-2 to modulate NK cell recognition and lysis of infected cells. We find that NK cells exhibit poor cytotoxic responses against SARS-CoV-2-infected targets, preferentially killing uninfected bystander cells. We demonstrate that this escape is driven by downregulation of ligands for the activating receptor NKG2D (NKG2D-L). Indeed, early in viral infection, prior to NKG2D-L downregulation, NK cells are able to target and kill infected cells; however, this ability is lost as viral proteins are expressed. Finally, we find that SARS-CoV-2 non-structural protein 1 (Nsp1) mediates downregulation of NKG2D-L and that Nsp1 alone is sufficient to confer resistance to NK cell killing. Collectively, our work demonstrates that SARS-CoV-2 evades direct NK cell cytotoxicity and describes a mechanism by which this occurs.

    View details for DOI 10.1016/j.celrep.2022.111892

    View details for PubMedID 36543165

  • Computation empowers CRISPR discovery and technology NATURE COMPUTATIONAL SCIENCE Shang, S., Cai, X. S., Qi, L. S. 2022; 2 (9): 533-535
  • Computation empowers CRISPR discovery and technology. Nature computational science Shang, S., Cai, X. S., Qi, L. S. 2022; 2 (9): 533-535

    View details for DOI 10.1038/s43588-022-00321-1

    View details for PubMedID 38177471

  • What are the current bottlenecks in developing and applying CRISPR technologies? CELL SYSTEMS Kellogg, E. H., Gootenberg, J., Abudayyeh, O., Wong, A. L., Dahlman, J. E., Lapinaite, A., Myhrvoid, C., Liu, C. C., Hsu, P. D., Mali, P., Qi, L. 2022; 13 (8): 589-593

    View details for Web of Science ID 000944344800001

    View details for PubMedID 35981511

  • Single-cell transcriptome analysis of regenerating RGCs reveals potent glaucoma neural repair genes. Neuron Li, L., Fang, F., Feng, X., Zhuang, P., Huang, H., Liu, P., Liu, L., Xu, A. Z., Qi, L. S., Cong, L., Hu, Y. 2022

    Abstract

    Axon regeneration holds great promise for neural repair of CNS axonopathies, including glaucoma. Pten deletion in retinal ganglion cells (RGCs) promotes potent optic nerve regeneration, but only a small population of Pten-null RGCs are actually regenerating RGCs (regRGCs); most surviving RGCs (surRGCs) remain non-regenerative. Here, we developed a strategy to specifically label and purify regRGCs and surRGCs, respectively, from the same Pten-deletion mice after optic nerve crush, in which they differ only in their regeneration capability. Smart-Seq2 single-cell transcriptome analysis revealed novel regeneration-associated genes that significantly promote axon regeneration. The most potent of these, Anxa2, acts synergistically with its ligand tPA in Pten-deletion-induced axon regeneration. Anxa2, its downstream effector ILK, and Mpp1 dramatically protect RGC somata and axons and preserve visual function in a clinically relevant model of glaucoma, demonstrating the exciting potential of this innovative strategy to identify novel effective neural repair candidates.

    View details for DOI 10.1016/j.neuron.2022.06.022

    View details for PubMedID 35952672

  • Corrigendum to "Hormone sensitive lipase ablation promotes bone regeneration" [Biochim. Biophys. Acta Mol. Basis Dis. Volume 1868, Issue 9, 1 September 2022, 166449]. Biochimica et biophysica acta. Molecular basis of disease Shen, W. J., Still, C., Han, L., Yang, P., Chen, J., Wosczyna, M., Rando, T. A., Salmon, B. J., Perez, K. C., Li, J., Cuevas, P. L., Liu, B., Azhar, S., Helms, J., Qi, L. S., Kraemer, F. B. 2022; 1868 (11): 166506

    View details for DOI 10.1016/j.bbadis.2022.166506

    View details for PubMedID 35926435

  • Enhanced Myogenesis by Silencing Myostatin with Nonviral Delivery of dCas9 Ribonucleoprotein Complex. The CRISPR journal Chen, Y., Banie, L., Breyer, B. N., Tan, Y., Wang, Z., Zhou, F., Wang, G., Lin, G., Liu, J., Qi, L. S., Lue, T. F. 2022

    Abstract

    Stress urinary incontinence (SUI) and pelvic floor disorder (PFD) are common conditions with limited treatment options in women worldwide. Regenerative therapy to restore urethral striated and pelvic floor muscles represents a valuable therapeutic approach. We aim to determine the CRISPR interference-mediated gene silencing effect of the nonviral delivery of nuclease-deactivated dCas9 ribonucleoprotein (RNP) complex on muscle regeneration at the cellular and molecular level. We designed four myostatin (MSTN)-targeting sgRNAs and transfected them into rat myoblast L6 cells together with the dCas9 protein. Myogenesis assay and immunofluorescence staining were performed to evaluate muscle differentiation, while CCK8 assay, cell cycle assay, and 5-ethynyl-2'-deoxyuridine staining were used to measure muscle proliferation. Reverse transcription-polymerase chain reaction and Western blotting were also performed to examine cellular signaling. Myogenic factors (including myosin heavy chain, MSTN, myocardin, and serum response factor) increased significantly after day 5 during myogenesis. MSTN was efficiently silenced after transfecting the dCas9 RNP complex, which significantly promoted more myotube formation and a higher fusion index for L6 cells. In cellular signaling, MSTN repression enhanced the expression of MyoG and MyoD, phosphorylation of Smad2, and the activity of Wnt1/GSK-3β/β-catenin pathway. Moreover, MSTN repression accelerated L6 cell growth with a higher cell proliferation index as well as a higher expression of cyclin D1 and cyclin E. Nonviral delivery of the dCas9 RNP complex significantly promoted myoblast differentiation and proliferation, providing a promising approach to improve muscle regeneration for SUI and PFD. Further characterization and validation of this approach in vivo are needed.

    View details for DOI 10.1089/crispr.2022.0009

    View details for PubMedID 35758824

  • Multiplex CRISPR genome regulation in the retina Guo, L., Bian, J., Davis, A. E., Liu, P., Kempton, H., Zhang, X., Chemparathy, A., Gu, B., Lin, X., Rane, D., Jamiolkowski, R. M., Hu, Y., Wang, S., Qi, L. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2022
  • Hormone sensitive lipase ablation promotes bone regeneration. Biochimica et biophysica acta. Molecular basis of disease Shen, W. J., Chris Still, I. I., Han, L., Yang, P., Chen, J., Wosczyna, M., Salmon, B. J., Perez, K. C., Li, J., Cuevas, P. L., Liu, B., Azhar, S., Helms, J., Qi, L. S., Kraemer, F. B. 2022: 166449

    Abstract

    There is an inverse relationship between the differentiation of mesenchymal stem cells (MSCs) along either an adipocyte or osteoblast lineage, with lineage differentiation known to be mediated by transcription factors PPARγ and Runx2, respectively. Endogenous ligands for PPARγ are generated during the hydrolysis of triacylglycerols to fatty acids through the actions of lipases such as hormone sensitive lipase (HSL). To examine whether reduced production of endogenous PPARγ ligands would influence bone regeneration, we examined the effects of HSL knockout on fracture repair in mice using a tibial mono-cortical defect as a model. We found an improved rate of fracture repair in HSL-ko mice documented by serial μCT and bone histomorphometry compared to wild-type (WT) mice. Similarly, accelerated rates of bone regeneration were observed with a calvarial model where implantation of bone grafts from HSL-ko mice accelerated bone regeneration at the injury site. Further analysis revealed improved MSC differentiation down osteoblast and chondrocyte lineage with inhibition of HSL. MSC recruitment to the injury site was greater in HSL-ko mice than WT. Finally, we used single cell RNAseq to understand the osteoimmunological differences between WT and HSL-ko mice and found changes in the pre-osteoclast population. Our study shows HSL-ko mice as an interesting model to study improvements to bone injury repair. Furthermore, our study highlights the potential importance of pre-osteoclasts and osteoclasts in bone repair.

    View details for DOI 10.1016/j.bbadis.2022.166449

    View details for PubMedID 35618183

  • Multiplexed Genome Regulation In Vivo with Hyper-Efficient Cas12a Guo, L., Bian, J., Davis, A. E., Liu, P., Kempton, H. R., Zhang, X., Chemparathy, A., Gu, B., Lin, X., Rane, D. A., Jamiolkowski, R. M., Hu, Y., Wang, S., Qi, L. S. CELL PRESS. 2022: 103
  • CRISPR-mediated Synergistic Epigenetic and Transcriptional Control. The CRISPR journal Dominguez, A. A., Chavez, M. G., Urke, A., Gao, Y., Wang, L., Qi, L. S. 2022

    Abstract

    Targeted activation of endogenous genes is an important approach for cell engineering. Here, we report that the nuclease-deactivated dCas9 fused to a transcriptional activator (VPR) and an epigenetic effector (the catalytic domain of histone acetyltransferase p300core) simultaneously, sequentially, or as a single quadripartite effector can lead to enhanced activation of target genes. The composite activator, VPRP, behaves more efficiently than individual activators across a set of genes in different cell types. We characterize off-target effects for host chromatin acetylation and transcriptome using the effectors. Our work demonstrates that transcriptional and epigenetic effectors can be used together to enhance gene activation and suggests the need for further optimization of epigenetic effectors to reduce off-targets.

    View details for DOI 10.1089/crispr.2021.0099

    View details for PubMedID 35271371

  • High-content CRISPR screening NATURE REVIEWS METHODS PRIMERS Bock, C., Datlinger, P., Chardon, F., Coelho, M. A., Dong, M. B., Lawson, K. A., Lu, T., Maroc, L., Norman, T. M., Song, B., Stanley, G., Chen, S., Garnett, M., Li, W., Moffat, J., Qi, L. S., Shapiro, R. S., Shendure, J., Weissman, J. S., Zhuang, X. 2022; 2 (1)
  • Dual CRISPR interference and activation for targeted reactivation of X-linked endogenous FOXP3 in human breast cancer cells. Molecular cancer Cui, X., Zhang, C., Xu, Z., Wang, S., Li, X., Stringer-Reasor, E., Bae, S., Zeng, L., Zhao, D., Liu, R., Qi, L. S., Wang, L. 2022; 21 (1): 38

    Abstract

    BACKGROUND: Unlike autosomal tumor suppressors, X-linked tumor suppressors can be inactivated by a single hit due to X-chromosome inactivation (XCI). Here, we argue that targeted reactivation of the non-mutated allele from XCI offers a potential therapy for female breast cancers.METHODS: Towards this goal, we developed a dual CRISPR interference and activation (CRISPRi/a) approach for simultaneously silencing and reactivating multiple X-linked genes using two orthogonal, nuclease-deficient CRISPR/Cas9 (dCas9) proteins.RESULTS: Using Streptococcus pyogenes dCas9-KRAB for silencing XIST and Staphylococcus aureus dCas9-VPR for activating FOXP3, we achieved CRISPR activation of FOXP3 in various cell lines of human female breast cancers. In human breast cancer HCC202 cells, which express a synonymous heterozygous mutation in the coding region of FOXP3, simultaneous silencing of XIST from XCI led to enhanced and prolonged FOXP3 activation. Also, reactivation of endogenous FOXP3 in breast cancer cells by CRISPRi/a inhibited tumor growth in vitro and in vivo. We further optimized CRISPRa by fusing dCas9 to the demethylase TET1 and observed enhanced FOXP3 activation. Analysis of the conserved CpG-rich region of FOXP3 intron 1 confirmed that CRISPRi/a-mediated simultaneous FOXP3 activation and XIST silencing were accompanied by elevated H4 acetylation, including H4K5ac, H4K8ac, and H4K16ac, and H3K4me3 and lower DNA methylation. This indicates that CRISPRi/a targeting to XIST and FOXP3 loci alters their transcription and their nearby epigenetic modifications.CONCLUSIONS: The simultaneous activation and repression of the X-linked, endogenous FOXP3 and XIST from XCI offers a useful research tool and a potential therapeutic for female breast cancers.

    View details for DOI 10.1186/s12943-021-01472-x

    View details for PubMedID 35130925

  • Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection. bioRxiv : the preprint server for biology Wang, J., Han, M., Roy, A. R., Wang, H., Mockl, L., Zeng, L., Moerner, W. E., Qi, L. S. 2022

    Abstract

    The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus within 20 years that gave rise to a life-threatening disease and the first to reach pandemic spread. To make therapeutic headway against current and future coronaviruses, the biology of coronavirus RNA during infection must be precisely understood. Here, we present a robust and generalizable framework combining high-throughput confocal and super-resolution microscopy imaging to study coronavirus infection at the nanoscale. Employing the model human coronavirus HCoV-229E, we specifically labeled coronavirus genomic RNA (gRNA) and double-stranded RNA (dsRNA) via multicolor RNA-immunoFISH and visualized their localization patterns within the cell. The exquisite resolution of our approach uncovers a striking spatial organization of gRNA and dsRNA into three distinct structures and enables quantitative characterization of the status of the infection after antiviral drug treatment. Our approach provides a comprehensive framework that supports investigations of coronavirus fundamental biology and therapeutic effects.

    View details for DOI 10.1101/2021.06.09.447760

    View details for PubMedID 34127974

  • Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection. Cell reports methods Wang, J., Han, M., Roy, A. R., Wang, H., Möckl, L., Zeng, L., Moerner, W. E., Qi, L. S. 2022: 100170

    Abstract

    The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus within 20 years that gave rise to a life-threatening disease and the first to reach pandemic spread. To make therapeutic headway against current and future coronaviruses, the biology of coronavirus RNA during infection must be precisely understood. Here, we present a robust and generalizable framework combining high-throughput confocal and super-resolution microscopy imaging to study coronavirus infection at the nanoscale. Employing the model human coronavirus HCoV-229E, we specifically labeled coronavirus genomic RNA (gRNA) and double-stranded RNA (dsRNA) via multicolor RNA-immunoFISH and visualized their localization patterns within the cell. The 20nm resolution achieved by of our approach uncovers a striking spatial organization of gRNA and dsRNA into three distinct structures and enables quantitative characterization of the status of the infection after antiviral drug treatment. Our approach provides a comprehensive imaging framework that will enable future investigations of coronavirus fundamental biology and therapeutic effects.

    View details for DOI 10.1016/j.crmeth.2022.100170

    View details for PubMedID 35128513

    View details for PubMedCentralID PMC8806145

  • High-content CRISPR screening. Nature reviews. Methods primers Bock, C., Datlinger, P., Chardon, F., Coelho, M. A., Dong, M. B., Lawson, K. A., Lu, T., Maroc, L., Norman, T. M., Song, B., Stanley, G., Chen, S., Garnett, M., Li, W., Moffat, J., Qi, L. S., Shapiro, R. S., Shendure, J., Weissman, J. S., Zhuang, X. 2022; 2 (1)

    Abstract

    CRISPR screens are a powerful source of biological discovery, enabling the unbiased interrogation of gene function in a wide range of applications and species. In pooled CRISPR screens, various genetically encoded perturbations are introduced into pools of cells. The targeted cells proliferate under a biological challenge such as cell competition, drug treatment or viral infection. Subsequently, the perturbation-induced effects are evaluated by sequencing-based counting of the guide RNAs that specify each perturbation. The typical results of such screens are ranked lists of genes that confer sensitivity or resistance to the biological challenge of interest. Contributing to the broad utility of CRISPR screens, adaptations of the core CRISPR technology make it possible to activate, silence or otherwise manipulate the target genes. Moreover, high-content read-outs such as single-cell RNA sequencing and spatial imaging help characterize screened cells with unprecedented detail. Dedicated software tools facilitate bioinformatic analysis and enhance reproducibility. CRISPR screening has unravelled various molecular mechanisms in basic biology, medical genetics, cancer research, immunology, infectious diseases, microbiology and other fields. This Primer describes the basic and advanced concepts of CRISPR screening and its application as a flexible and reliable method for biological discovery, biomedical research and drug development - with a special emphasis on high-content methods that make it possible to obtain detailed biological insights directly as part of the screen.

    View details for DOI 10.1038/s43586-022-00098-7

    View details for PubMedID 37214176

  • The use of new CRISPR tools in cardiovascular research and medicine. Nature reviews. Cardiology Nishiga, M., Liu, C., Qi, L. S., Wu, J. C. 2022

    Abstract

    Many novel CRISPR-based genome-editing tools, with a wide variety of applications, have been developed in the past few years. The original CRISPR-Cas9 system was developed as a tool to alter genomic sequences in living organisms in a simple way. However, the functions of new CRISPR tools are not limited to conventional genome editing mediated by non-homologous end-joining or homology-directed repair but expand into gene-expression control, epigenome editing, single-nucleotide editing, RNA editing and live-cell imaging. Furthermore, genetic perturbation screening by multiplexing guide RNAs is gaining popularity as a method to identify causative genes and pathways in an unbiased manner. New CRISPR tools can also be applied to ex vivo or in vivo therapeutic genome editing for the treatment of conditions such as hyperlipidaemia. In this Review, we first provide an overview of the diverse new CRISPR tools that have been developed to date. Second, we summarize how these new CRISPR tools are being used to study biological processes and disease mechanisms in cardiovascular research and medicine. Finally, we discuss the prospect of therapeutic genome editing by CRISPR tools to cure genetic cardiovascular diseases.

    View details for DOI 10.1038/s41569-021-00669-3

    View details for PubMedID 35145236

  • Contextual reprogramming of CAR-T cells for treatment of HER2+ cancers. Journal of translational medicine Yang, Z., Li, L., Turkoz, A., Chen, P., Harari-Steinfeld, R., Bobbin, M., Stefanson, O., Choi, H., Pietrobon, V., Alphson, B., Goswami, A., Balan, V., Kearney, A., Patel, D., Yang, J., Inel, D., Vinod, V., Cesano, A., Wang, B., Roh, K., Qi, L. S., Marincola, F. M. 2021; 19 (1): 459

    Abstract

    BACKGROUND: Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells combined with checkpoint inhibition may prevent T cell exhaustion and improve clinical outcomes. However, the approach is limited by cumulative costs and toxicities.METHODS: To overcome this drawback, we created a CAR-T (RB-340-1) that unites in one product the two modalities: a CRISPR interference-(CRISPRi) circuit prevents programmed cell death protein 1 (PD-1) expression upon antigen-encounter. RB-340-1 is engineered to express an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3zeta co-stimulatory domains linked to the tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the PD-1 transcription start site (TSS). A second constructs includes linker for activation of T cells (LAT) fused to nuclease-deactivated spCas9 (dCas9)-Kruppel-associated box (KRAB) via a TEV-cleavable sequence (TCS). Upon antigen encounter, the LAT-dCas9-KRAB (LdCK) complex is cleaved by TEV allowing targeting of dCas9-KRAB to the PD-1 gene TSS.RESULTS: Here, we show that RB-340-1 consistently demonstrated higher production of homeostatic cytokines, enhanced expansion of CAR-T cells in vitro, prolonged in vivo persistence and more efficient suppression of HER2+ FaDu oropharyngeal cancer growth compared to the respective conventional CAR-T cell product.CONCLUSIONS: As the first application of CRISPRi toward a clinically relevant product, RB-340-1 with the conditional, non-gene editing and reversible suppression promotes CAR-T cells resilience to checkpoint inhibition, and their persistence and effectiveness against HER2-expressing cancer xenografts.

    View details for DOI 10.1186/s12967-021-03132-6

    View details for PubMedID 34743703

  • Enhanced Cas12a multi-gene regulation using a CRISPR array separator. eLife Magnusson, J. P., Rios, A. R., Wu, L., Qi, L. S. 2021; 10

    Abstract

    The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple guide RNA (gRNAs). Here, we report that Cas12a array performance is hypersensitive to the GC content of gRNA spacers, as high-GC spacers can impair activity of the downstream gRNA. We analyze naturally occurring CRISPR arrays and observe that natural repeats always contain an AT-rich fragment that separates gRNAs, which we term a CRISPR separator. Inspired by this observation, we design short, AT-rich synthetic separators (synSeparators) that successfully remove the disruptive effects between gRNAs. We further demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously underexplored feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.

    View details for DOI 10.7554/eLife.66406

    View details for PubMedID 34499031

  • Enhanced Cas12a multi-gene regulation using a CRISPR array separator ELIFE Magnusson, J. P., Rios, A., Wu, L., Qi, L. S. 2021; 10
  • Single-cell transcriptomic profiling reveals distinct mechanical responses between normal and diseased tendon progenitor cells. Cell reports. Medicine Still, C. 2., Chang, W., Sherman, S. L., Sochacki, K. R., Dragoo, J. L., Qi, L. S. 2021; 2 (7): 100343

    Abstract

    Regenerative medicine approaches utilizing stem cells offer a promising strategy to address tendinopathy, a class of common tendon disorders associated with pain and impaired function. Tendon progenitor cells (TPCs) are important in healing and maintaining tendon tissues. Here we provide a comprehensive single cell transcriptomic profiling of TPCs from three normal and three clinically classified tendinopathy samples in response to mechanical stimuli. Analysis reveals seven distinct TPC subpopulations including subsets that are responsive to the mechanical stress, highly clonogenic, and specialized in cytokine or growth factor expression. The single cell transcriptomic profiling of TPCs and their subsets serves as a foundation for further investigation into the pathology and molecular hallmarks of tendinopathy in mechanical stimulation conditions.

    View details for DOI 10.1016/j.xcrm.2021.100343

    View details for PubMedID 34337559

  • Nanoscale, antigen-dependent, reversible IL-12 secretion by CAR T cells for cancer treatment. Yang, Z., Bobbin, M., Choi, H., Stefanson, O., Magallanes, K., Yang, J., Wang, B., Cesano, A., Qi, L., Marincola, F. M. AMER ASSOC CANCER RESEARCH. 2021
  • CRISPR/Cas9 Editing Of Autologous Dendritic Cells To Enhance Angiogenesis And Wound Healing Henn, D., Zhao, D., Bonham, C. A., Chen, K., Greco, A. H., Padmanabhan, J., Sivaraj, D., Trotsyuk, A., Barrera, J. A., Januszyk, M., Qi, L., Gurtner, G. C. WILEY. 2021: A31-A32
  • CRISPR-based genome editing in primary human pancreatic islet cells. Nature communications Bevacqua, R. J., Dai, X., Lam, J. Y., Gu, X., Friedlander, M. S., Tellez, K., Miguel-Escalada, I., Bonas-Guarch, S., Atla, G., Zhao, W., Kim, S. H., Dominguez, A. A., Qi, L. S., Ferrer, J., MacDonald, P. E., Kim, S. K. 2021; 12 (1): 2397

    Abstract

    Gene targeting studies in primary human islets could advance our understanding of mechanisms driving diabetes pathogenesis. Here, we demonstrate successful genome editing in primary human islets using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). CRISPR-based targeting efficiently mutated protein-coding exons, resulting in acute loss of islet beta-cell regulators, like the transcription factor PDX1 and the KATP channel subunit KIR6.2, accompanied by impaired beta-cell regulation and function. CRISPR targeting of non-coding DNA harboring type 2 diabetes (T2D) risk variants revealed changes in ABCC8, SIX2 and SIX3 expression, and impaired beta-cell function, thereby linking regulatory elements in these target genes to T2D genetic susceptibility. Advances here establish a paradigm for genetic studies in human islet cells, and reveal regulatory and genetic mechanisms linking non-coding variants to human diabetes risk.

    View details for DOI 10.1038/s41467-021-22651-w

    View details for PubMedID 33893274

  • A comprehensive analysis and resource to use CRISPR-Cas13 for broad-spectrum targeting of RNA viruses. Cell reports. Medicine Lin, X., Liu, Y., Chemparathy, A., Pande, T., La Russa, M., Qi, L. S. 2021: 100245

    Abstract

    The COVID-19 pandemic caused by SARS-CoV-2 and variants has led to significant mortality. We recently reported that an RNA-targeting CRISPR-Cas13 system, termed prophylactic antiviral CRISPR in human (PAC-MAN), offered an antiviral strategy against SARS-CoV-2 and influenza A virus. Here, we expand in silico analysis to use PAC-MAN to target a broad spectrum of human- or livestock-infectious RNA viruses with high specificity, coverage, and predicted efficiency. Our analysis reveals that a minimal set of 14 crRNAs is able to target >90% of human-infectious viruses across 10 RNA virus families. We predict that a set of 5 experimentally validated crRNAs can target new SARS-CoV-2 variant sequences with zero mismatches. We also build an online resource (crispr-pacman.stanford.edu) to support community use of CRISPR-Cas13 for broad-spectrum RNA virus targeting. Our work provides a new bioinformatic resource for using CRISPR-Cas13 to target diverse RNA viruses in order to facilitate development of CRISPR-based antivirals.

    View details for DOI 10.1016/j.xcrm.2021.100245

    View details for PubMedID 33778788

  • CRISPRi/a Screening with Human iPSCs. Methods in molecular biology (Clifton, N.J.) Nishiga, M., Qi, L. S., Wu, J. C. 2021; 2320: 261-281

    Abstract

    Identifying causative genes in a given phenotype or disease model is important for biological discovery and drug development. The recent development of the CRISPR/Cas9 system has enabled unbiased and large-scale genetic perturbation screens to identify causative genes by knocking out many genes in parallel and selecting cells with desired phenotype of interest. However, compared to cancer cell lines, human somatic cells including cardiomyocytes (CMs), neuron cells, and endothelial cells are not easy targets of CRISPR screens because CRISPR screens require a large number of isogenic cells to be cultured and thus primary cells from patients are not ideal. The combination of CRISPR screens with induced pluripotent stem cell (iPSC) technology would be a powerful tool to identify causative genes and pathways because iPSCs can be expanded easily and differentiated to any cell type in principle. Here we describe a robust protocol for CRISPR screening using human iPSCs. Because each screening is different and needs to be customized depending on the cell types and phenotypes of interest, we show an example of CRISPR knockdown screening using CRISPRi system to identify essential genes to differentiate iPSCs to CMs.

    View details for DOI 10.1007/978-1-0716-1484-6_23

    View details for PubMedID 34302664

  • Regenerating Urethral Striated Muscle by CRISPRi/dCas9-KRAB-Mediated Myostatin Silencing for Obesity-Associated Stress Urinary Incontinence. The CRISPR journal Yuan, H., Ruan, Y., Tan, Y., Reed-Maldonado, A. B., Chen, Y., Zhao, D., Wang, Z., Zhou, F., Peng, D., Banie, L., Wang, G., Liu, J., Lin, G., Qi, L. S., Lue, T. F. 2020; 3 (6): 562–72

    Abstract

    Overweight females are prone to obesity-associated stress urinary incontinence (OA-SUI), and there are no definitive medical therapies for this common urologic condition. This study was designed to test the hypothesis that regenerative therapy to restore urethral striated muscle (stM) and pelvic floor muscles might represent a valuable therapeutic approach. For the in vitro experiment, single-guide RNAs targeting myostatin (MSTN) were used for CRISPRi/dCas9-Kruppel associated box (KRAB)-mediated gene silencing. For the in vivo experiment, a total of 14 female lean ZUC-Leprfa 186 and 14 fatty ZUC-Leprfa 185 rats were used as control and CRISPRi-MSTN treated groups, respectively. The results indicated that lentivirus-mediated expression of MSTN CRISPRi/dCas9-KRAB caused sustained downregulation of MSTN in rat L6 myoblast cells and significantly enhanced myogenesis in vitro. In vivo, the urethral sphincter injection of lentiviral-MSTN sgRNA and lentiviral-dCas9-KRAB significantly increased the leak point pressure, the thickness of the stM layer, the ratio of stM to smooth muscle, and the number of neuromuscular junctions. Downregulation of MSTN with CRISPRi/dCas9-KRAB-mediated gene silencing significantly enhanced myogenesis in vitro and in vivo. It also improved urethral continence in the OA-SUI rat model.

    View details for DOI 10.1089/crispr.2020.0077

    View details for PubMedID 33346712

  • IMMUNOGENIC POTENTIAL OF CHIMERIC ANTIGEN RECEPTOR (CAR)-ENGINEERED T CELLS EXPRESSING INDUCIBLE NUCLEASE-DEACTIVATED SPCAS9 (DCAS9) Patel, D., Patel, D., Goswami, A., Balan, V., Yang, Z., Li, L., Rajavel, S., Kearney, A., Harari-Steinfeld, R., Bobbin, M., Wang, B., Cesano, A., Qi, S., Marincola, F. BMJ PUBLISHING GROUP. 2020: A80
  • CONTEXTUAL REPROGRAMMING OF CAR T CELLS FOR THE TREATMENT OF HER2-EXPRESSING CANCERS Yang, Z., Li, L., Turkoz, A., Chen, P., Choi, H., Inel, D., Qi, S., Marincola, F. BMJ PUBLISHING GROUP. 2020: A86–A87
  • CONTEXTUAL SECRETION OF NANOSCALE INTERLEUKIN (IL)-12 BY CAR T CELLS FOR THE TREATMENT OF CANCER Yang, Z., Bobbins, M., Choi, H., Stefanson, O., Yang, J., Magallanes, K., Wang, B., Qi, L., Marincola, F. BMJ PUBLISHING GROUP. 2020: A458
  • Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS. Analytical chemistry Brower, K. K., Khariton, M., Suzuki, P. H., Still, C. 2., Kim, G., Calhoun, S. G., Qi, L. S., Wang, B., Fordyce, P. M. 2020

    Abstract

    In the past five years, droplet microfluidic techniques have unlocked new opportunities for the high-throughput genome-wide analysis of single cells, transforming our understanding of cellular diversity and function. However, the field lacks an accessible method to screen and sort droplets based on cellular phenotype upstream of genetic analysis, particularly for large and complex cells. To meet this need, we developed Dropception, a robust, easy-to-use workflow for precise single-cell encapsulation into picoliter-scale double emulsion droplets compatible with high-throughput screening via fluorescence-activated cell sorting (FACS). We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying sizes and morphologies as well as a heterogeneous cell mixture of a whole dissociated flatworm (5-25 mum in diameter) within highly monodisperse double emulsions (35 mum in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events (<2% of cell-containing droplets), thereby allowing direct linkage of cellular phenotype to genotype. Across all cell lines, cell loading efficiency approaches the theoretical limit with no observable bias by cell size. FACS measurements reveal the ability to discriminate empty droplets from those containing cells with good agreement to single-cell occupancies quantified via microscopy, establishing robust droplet screening at single-cell resolution. High-throughput FACS screening of cellular picoreactors has the potential to shift the landscape of single-cell droplet microfluidics by expanding the repertoire of current nucleic acid droplet assays to include functional phenotyping.

    View details for DOI 10.1021/acs.analchem.0c02499

    View details for PubMedID 32900183

  • Lentiviral delivery of combinatorial CAR/CRISPRi circuit into human primary T cells is enhanced by TBK1/IKKɛ complex inhibitor BX795. Journal of translational medicine Li, L., Gao, Y., Srivastava, R., Wang, W., Xiong, Q., Fang, Z., Pelayo, A., Denson, C., Goswami, A., Harari-Steinfeld, R., Yang, Z., Weng, L., Qi, L. S., Marincola, F. M. 2020; 18 (1): 363

    Abstract

    BACKGROUND: Adoptive transfer of engineered immune cells is a promising strategy for cancer treatment. However, low transduction efficiency particularly when large payload lentiviral vectors are used on primary T cells is a limitation for the development of cell therapy platforms that include multiple constructs bearing long DNA sequences. RB-340-1 is a new CAR T cell that combines two strategies in one product through a CRISPR interference (CRISPRi) circuit. Because multiple regulatory components are included in the circuit, RB-340-1 production needs delivery of two lentiviral vectors into human primary T cells, both containing long DNA sequences. To improve lentiviral transduction efficiency, we looked for inhibitors of receptors involved in antiviral response. BX795 is a pharmacological inhibitor of the TBK1/IKKɛ complex, which has been reported to augment lentiviral transduction of human NK cells and some cell lines, but it has not been tested with human primary T cells. The purpose of this study was to test if BX795 treatment promotes large payload RB-340-1 lentiviral transduction of human primary T cells.METHODS: To make the detection of gene delivery more convenient, we constructed another set of RB-340-1 constructs containing fluorescent labels named RB-340-1F. We incorporated BX795 treatment into the human primary T cell transduction procedure that was optimized for RB-340-1F. We tested BX795 with T cells collected from multiple donors, and detected the effect of BX795 on T cell transduction, phenotype, cell growth and cell function.RESULTS: We found that BX795 promotes RB-340-1F lentiviral transduction of human primary T cells, without dramatic change in cell growth and T cell functions. Meanwhile, BX795 treatment increased CD8+ T cell ratios in transduced T cells.CONCLUSIONS: These results indicate that BX795 treatment is effective, and might be a safe approach to promote RB-340-1F lentiviral transduction of human primary T cells. This approach might also be helpful for other T cell therapy products that need delivery of complicated platform via large payload lentiviral vectors.

    View details for DOI 10.1186/s12967-020-02526-2

    View details for PubMedID 32967676

  • Special Issue: Expanding the CRISPR Toolbox. The CRISPR journal Qi, S. 2020; 3 (3): 135

    View details for DOI 10.1089/crispr.2020.29086.cfp3

    View details for PubMedID 33560911

  • Fibrinogen Alpha Chain Knockout Promotes Tumor Growth and Metastasis through Integrin-AKT Signaling Pathway in Lung Cancer. Molecular cancer research : MCR Wang, M., Zhang, G., Zhang, Y., Cui, X., Wang, S., Gao, S., Wang, Y., Liu, Y., Bae, J. H., Yang, W., Qi, L. S., Wang, L., Liu, R. 2020

    Abstract

    Fibrinogen is an extracellular matrix protein composed of three polypeptide chains with fibrinogen alpha (FGA), beta (FGB) and gamma (FGG). While fibrinogen and its related fragments are involved in tumor angiogenesis and metastasis, their functional roles are incompatible. A recent genome-scale screening reveals that loss of FGA affects the acceleration of tumor growth and metastasis of lung cancer, but the mechanism remains elusive. We used CRISPR/Cas9 genome editing to knockout (KO) FGA in human lung adenocarcinoma (LUAD) cell lines A549 and H1299. By colony formation, transwell migration and matrix invasion assays, FGA KO increased cell proliferation, migration, and invasion but decreased the expressions of epithelial-mesenchymal transition marker E-cadherin and cytokeratin 5/8 in A549 and H1299 cells. However, administration of FGA inhibited cell proliferation and migration but induced apoptosis in A549 cells. Of note, FGA KO cells indirectly co-cultured by transwells with FGA wild-type cells increased FGA in the culture medium, leading to decreased migration of FGA KO cells. Furthermore, our functional analysis identified a direct interaction of FGA with integrin alpha5 as well as FGA-integrin signaling that regulated the AKT-mTOR signaling pathway in A549 cells. In addition, we validated that FGA KO increased tumor growth and metastasis through activation of AKT signaling in an A549 xenograft model. Implications: These findings demonstrate that that loss of FGA facilities tumor growth and metastasis through integrin-AKT signaling pathway in lung cancer.

    View details for DOI 10.1158/1541-7786.MCR-19-1033

    View details for PubMedID 32205365

  • A benchmark of algorithms for the analysis of pooled CRISPR screens. Genome biology Bodapati, S., Daley, T. P., Lin, X., Zou, J., Qi, L. S. 2020; 21 (1): 62

    Abstract

    Genome-wide pooled CRISPR-Cas-mediated knockout, activation, and repression screens are powerful tools for functional genomic investigations. Despite their increasing importance, there is currently little guidance on how to design and analyze CRISPR-pooled screens. Here, we provide a review of the commonly used algorithms in the computational analysis of pooled CRISPR screens. We develop a comprehensive simulation framework to benchmark and compare the performance of these algorithms using both synthetic and real datasets. Our findings inform parameter choices of CRISPR screens and provide guidance to researchers on the design and analysis of pooled CRISPR screens.

    View details for DOI 10.1186/s13059-020-01972-x

    View details for PubMedID 32151271

  • Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells. Nature communications Sarkar, T. J., Quarta, M. n., Mukherjee, S. n., Colville, A. n., Paine, P. n., Doan, L. n., Tran, C. M., Chu, C. R., Horvath, S. n., Qi, L. S., Bhutani, N. n., Rando, T. A., Sebastiano, V. n. 2020; 11 (1): 1545

    Abstract

    Aging is characterized by a gradual loss of function occurring at the molecular, cellular, tissue and organismal levels. At the chromatin level, aging associates with progressive accumulation of epigenetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell/tissue homeostasis. Nuclear reprogramming to pluripotency can revert both the age and the identity of any cell to that of an embryonic cell. Recent evidence shows that transient reprogramming can ameliorate age-associated hallmarks and extend lifespan in progeroid mice. However, it is unknown how this form of rejuvenation would apply to naturally aged human cells. Here we show that transient expression of nuclear reprogramming factors, mediated by expression of mRNAs, promotes a rapid and broad amelioration of cellular aging, including resetting of epigenetic clock, reduction of the inflammatory profile in chondrocytes, and restoration of youthful regenerative response to aged, human muscle stem cells, in each case without abolishing cellular identity.

    View details for DOI 10.1038/s41467-020-15174-3

    View details for PubMedID 32210226

  • Therapeutic genome editing in cardiovascular diseases. Advanced drug delivery reviews Nishiga, M. n., Qi, L. S., Wu, J. C. 2020

    Abstract

    During the past decade, developments in genome editing technology have fundamentally transformed biomedical research. In particular, the CRISPR/Cas9 system has been extensively applied because of its simplicity and ability to alter genomic sequences within living organisms, and an ever increasing number of CRISPR/Cas9-based molecular tools are being developed for a wide variety of applications. While genome editing tools have been used for many aspects of biological research, they also have enormous potential to be used for genome editing therapy to treat a broad range of diseases. For some hematopoietic diseases, clinical trials of therapeutic genome editing with CRISPR/Cas9 are already starting phase I. In the cardiovascular field, genome editing tools have been utilized to understand the mechanisms of diseases such as cardiomyopathy, arrythmia, and lipid metabolism, which now open the door to therapeutic genome editing. Currently, therapeutic genome editing in the cardiovascular field is centered on liver-targeting strategies to reduce cardiovascular risks. Targeting the heart is more challenging. In this review, we discuss the potential applications, recent advances, and current limitations of therapeutic genome editing in the cardiovascular field.

    View details for DOI 10.1016/j.addr.2020.02.003

    View details for PubMedID 32092381

  • Low-frequency ultrasound-mediated cytokine transfection enhances T cell recruitment at local and distant tumor sites. Proceedings of the National Academy of Sciences of the United States of America Ilovitsh, T. n., Feng, Y. n., Foiret, J. n., Kheirolomoom, A. n., Zhang, H. n., Ingham, E. S., Ilovitsh, A. n., Tumbale, S. K., Fite, B. Z., Wu, B. n., Raie, M. N., Zhang, N. n., Kare, A. J., Chavez, M. n., Qi, L. S., Pelled, G. n., Gazit, D. n., Vermesh, O. n., Steinberg, I. n., Gambhir, S. S., Ferrara, K. W. 2020

    Abstract

    Robust cytotoxic T cell infiltration has proven to be difficult to achieve in solid tumors. We set out to develop a flexible protocol to efficiently transfect tumor and stromal cells to produce immune-activating cytokines, and thus enhance T cell infiltration while debulking tumor mass. By combining ultrasound with tumor-targeted microbubbles, membrane pores are created and facilitate a controllable and local transfection. Here, we applied a substantially lower transmission frequency (250 kHz) than applied previously. The resulting microbubble oscillation was significantly enhanced, reaching an effective expansion ratio of 35 for a peak negative pressure of 500 kPa in vitro. Combining low-frequency ultrasound with tumor-targeted microbubbles and a DNA plasmid construct, 20% of tumor cells remained viable, and ∼20% of these remaining cells were transfected with a reporter gene both in vitro and in vivo. The majority of cells transfected in vivo were mucin 1+/CD45- tumor cells. Tumor and stromal cells were then transfected with plasmid DNA encoding IFN-β, producing 150 pg/106 cells in vitro, a 150-fold increase compared to no-ultrasound or no-plasmid controls and a 50-fold increase compared to treatment with targeted microbubbles and ultrasound (without IFN-β). This enhancement in secretion exceeds previously reported fourfold to fivefold increases with other in vitro treatments. Combined with intraperitoneal administration of checkpoint inhibition, a single application of IFN-β plasmid transfection reduced tumor growth in vivo and recruited efficacious immune cells at both the local and distant tumor sites.

    View details for DOI 10.1073/pnas.1914906117

    View details for PubMedID 32430322

  • Computational Methods for Analysis of Large-Scale CRISPR Screens ANNUAL REVIEW OF BIOMEDICAL DATA SCIENCE, VOL 3, 2020 Lin, X., Chemparathy, A., La Russa, M., Daley, T., Qi, L. S., Altman, R. B. 2020; 3: 137–62
  • Biodesign Research to Advance the Principles and Applications of Biosystems Design. Biodesign research Yang, X., Qi, L. S., Jaramillo, A., Cheng, Z. M. 2019; 2019: 9680853

    View details for DOI 10.34133/2019/9680853

    View details for PubMedID 37850090

    View details for PubMedCentralID PMC10530645

  • Identification of cell context-dependent YAP-associated proteins reveals beta1 and beta4 integrin mediate YAP translocation independently of cell spreading. Scientific reports Lee, J. Y., Dominguez, A. A., Nam, S., Stowers, R. S., Qi, L. S., Chaudhuri, O. 2019; 9 (1): 17188

    Abstract

    Yes-associated protein (YAP) is a transcriptional regulator and mechanotransducer, relaying extracellular matrix (ECM) stiffness into proliferative gene expression in 2D culture. Previous studies show that YAP activation is dependent on F-actin stress fiber mediated nuclear pore opening, however the protein mediators of YAP translocation remain unclear. Here, we show that YAP co-localizes with F-actin during activating conditions, such as sparse plating and culturing on stiff 2D substrates. To identify proteins mediating YAP translocation, we performed co-immunoprecipitation followed by mass spectrometry (co-IP/MS) for proteins that differentially associated with YAP under activating conditions. Interestingly, YAP preferentially associates with beta1 integrin under activating conditions, and beta4 integrin under inactivating conditions. In activating conditions, CRISPR/Cas9 knockout (KO) of beta1 integrin (DeltaITGB1) resulted in decreased cell area, which correlated with decreased YAP nuclear localization. DeltaITGB1 did not significantly affect the slope of the correlation between YAP nuclear localization with area, but did decrease overall nuclear YAP independently of cell spreading. In contrast, beta4 integrin KO (DeltaITGB4) cells showed no change in cell area and similarly decreased nuclear YAP. These results reveal proteins that differentially associate with YAP during activation, which may aid in regulating YAP nuclear translocation.

    View details for DOI 10.1038/s41598-019-53659-4

    View details for PubMedID 31748579

  • Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9. Molecular cell Shariati, S. A., Dominguez, A., Xie, S., Wernig, M., Qi, L. S., Skotheim, J. M. 2019; 74 (3): 622

    Abstract

    The control of gene expression by transcription factor binding sites frequently determines phenotype. However, it is difficult to determine the function of single transcription factor binding sites within larger transcription networks. Here, we use deactivated Cas9 (dCas9) to disrupt binding to specific sites, a method we term CRISPRd. Since CRISPR guide RNAs are longer than transcription factor binding sites, flanking sequence can be used to target specific sites. Targeting dCas9 to an Oct4 site in the Nanog promoter displaced Oct4 from this site, reduced Nanog expression, and slowed division. In contrast, disrupting the Oct4 binding site adjacent to Pax6 upregulated Pax6 transcription and disrupting Nanog binding its own promoter upregulated its transcription. Thus, we can easily distinguish between activating and repressing binding sites and examine autoregulation. Finally, multiple guide RNA expression allows simultaneous inhibition of multiple binding sites, and conditionally destabilized dCas9 allows rapid reversibility.

    View details for PubMedID 31051141

  • Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9 MOLECULAR CELL Shariati, S., Dominguez, A., Xie, S., Wernig, M., Qi, L. S., Skotheim, J. M. 2019; 74 (3): 622-+
  • YAP-independent mechanotransduction drives breast cancer progression NATURE COMMUNICATIONS Lee, J. Y., Chang, J. K., Dominguez, A. A., Lee, H., Nam, S., Chang, J., Varma, S., Qi, L. S., West, R. B., Chaudhuri, O. 2019; 10
  • When genome editing goes off-target. Science (New York, N.Y.) Kempton, H. R., Qi, L. S. 2019; 364 (6437): 234–36

    View details for PubMedID 31000651

  • When genome editing goes off-target SCIENCE Kempton, H. R., Qi, L. S. 2019; 364 (6437): 234–36
  • Identification of Novel Regulatory Genes in APAP Induced Hepatocyte Toxicity by a Genome-Wide CRISPR-Cas9 Screen. Scientific reports Shortt, K., Heruth, D. P., Zhang, N., Wu, W., Singh, S., Li, D., Zhang, L. Q., Wyckoff, G. J., Qi, L. S., Friesen, C. A., Ye, S. Q. 2019; 9 (1): 1396

    Abstract

    Acetaminophen (APAP) is a commonly used analgesic responsible for more than half of acute liver failure cases. Identification of previously unknown genetic risk factors would provide mechanistic insights and novel therapeutic targets for APAP-induced liver injury. This study used a genome-wide CRISPR-Cas9 screen to evaluate genes that are protective against, or cause susceptibility to, APAP-induced liver injury. HuH7 human hepatocellular carcinoma cells containing CRISPR-Cas9 gene knockouts were treated with 15mM APAP for 30minutes to 4 days. A gene expression profile was developed based on the 1) top screening hits, 2) overlap of expression data from APAP overdose studies, and 3) predicted affected biological pathways. We further demonstrated the implementation of intermediate time points for the identification of early and late response genes. This study illustrated the power of a genome-wide CRISPR-Cas9 screen to systematically identify novel genes involved in APAP-induced hepatotoxicity and to provide potential targets to develop novel therapeutic modalities.

    View details for PubMedID 30718897

  • A CRISPR-dCas Toolbox for Genetic Engineering and Synthetic Biology JOURNAL OF MOLECULAR BIOLOGY Xu, X., Oi, L. S. 2019; 431 (1): 34–47
  • YAP-independent mechanotransduction drives breast cancer progression. Nature communications Lee, J. Y., Chang, J. K., Dominguez, A. A., Lee, H. P., Nam, S. n., Chang, J. n., Varma, S. n., Qi, L. S., West, R. B., Chaudhuri, O. n. 2019; 10 (1): 1848

    Abstract

    Increased tissue stiffness is a driver of breast cancer progression. The transcriptional regulator YAP is considered a universal mechanotransducer, based largely on 2D culture studies. However, the role of YAP during in vivo breast cancer remains unclear. Here, we find that mechanotransduction occurs independently of YAP in breast cancer patient samples and mechanically tunable 3D cultures. Mechanistically, the lack of YAP activity in 3D culture and in vivo is associated with the absence of stress fibers and an order of magnitude decrease in nuclear cross-sectional area relative to 2D culture. This work highlights the context-dependent role of YAP in mechanotransduction, and establishes that YAP does not mediate mechanotransduction in breast cancer.

    View details for PubMedID 31015465

  • Site-Programmable Transposition: Shifting the Paradigm for CRISPR-Cas Systems. Molecular cell Chavez, M. n., Qi, L. S. 2019; 75 (2): 206–8

    Abstract

    Discoveries by Klompe et al. (2019) and Strecker et al. (2019) elucidate distinct CRISPR-Cas mechanisms for site-specific programmable transposition in prokaryotic organisms.

    View details for DOI 10.1016/j.molcel.2019.07.004

    View details for PubMedID 31348878

  • Evolution at the Cutting Edge: CRISPR-Mediated Directed Evolution. Molecular cell Abbott, T. R., Qi, L. S. 2018; 72 (3): 402–3

    Abstract

    In a recent issue of Nature, Halperin etal. (2018) develop a new technology to continuously diversify specific genomic loci by combining CRISPR-Cas9 with error-prone DNA polymerases.

    View details for PubMedID 30388408

  • CRISPhieRmix: a hierarchical mixture model for CRISPR pooled screens. Genome biology Daley, T. P., Lin, Z., Lin, X., Liu, Y., Wong, W. H., Qi, L. S. 2018; 19 (1): 159

    Abstract

    Pooled CRISPR screens allow researchers to interrogate genetic causes of complex phenotypes at the genome-wide scale and promise higher specificity and sensitivity compared to competing technologies. Unfortunately, two problems exist, particularly for CRISPRi/a screens: variability in guide efficiency and large rare off-target effects. We present a method, CRISPhieRmix, that resolves these issues by using a hierarchical mixture model with a broad-tailed null distribution. We show that CRISPhieRmix allows for more accurate and powerful inferences in large-scale pooled CRISPRi/a screens. We discuss key issues in the analysis and design of screens, particularly the number of guides needed for faithful full discovery.

    View details for PubMedID 30296940

  • DNMT3A and TET1 cooperate to regulate promoter epigenetic landscapes in mouse embryonic stem cells GENOME BIOLOGY Gu, T., Lin, X., Cullen, S. M., Luo, M., Jeong, M., Estecio, M., Shen, J., Hardikar, S., Sun, D., Su, J., Rux, D., Guzman, A., Lee, M., Qi, L., Chen, J., Kyba, M., Huang, Y., Chen, T., Li, W., Goodell, M. A. 2018; 19: 88

    Abstract

    DNA methylation is a heritable epigenetic mark, enabling stable but reversible gene repression. In mammalian cells, DNA methyltransferases (DNMTs) are responsible for modifying cytosine to 5-methylcytosine (5mC), which can be further oxidized by the TET dioxygenases to ultimately cause DNA demethylation. However, the genome-wide cooperation and functions of these two families of proteins, especially at large under-methylated regions, called canyons, remain largely unknown.Here we demonstrate that DNMT3A and TET1 function in a complementary and competitive manner in mouse embryonic stem cells to mediate proper epigenetic landscapes and gene expression. The longer isoform of DNMT3A, DNMT3A1, exhibits significant enrichment at distal promoters and canyon edges, but is excluded from proximal promoters and canyons where TET1 shows prominent binding. Deletion of Tet1 increases DNMT3A1 binding capacity at and around genes with wild-type TET1 binding. However, deletion of Dnmt3a has a minor effect on TET1 binding on chromatin, indicating that TET1 may limit DNA methylation partially by protecting its targets from DNMT3A and establishing boundaries for DNA methylation. Local CpG density may determine their complementary binding patterns and therefore that the methylation landscape is encoded in the DNA sequence. Furthermore, DNMT3A and TET1 impact histone modifications which in turn regulate gene expression. In particular, they regulate Polycomb Repressive Complex 2 (PRC2)-mediated H3K27me3 enrichment to constrain gene expression from bivalent promoters.We conclude that DNMT3A and TET1 regulate the epigenome and gene expression at specific targets via their functional interplay.

    View details for PubMedID 30001199

  • p300 and STAT3 drive YAP-independent mechanotransduction during breast cancer invasion Lee, J. Y., Chang, J., Nam, S., Lee, H., Dominguez, A. A., Varma, S., Qi, L. S., West, R. B., Chaudhuri, O. AMER ASSOC CANCER RESEARCH. 2018
  • A CRISPR-dCas Toolbox for Genetic Engineering and Synthetic Biology. Journal of molecular biology Xu, X., Qi, L. S. 2018

    Abstract

    Programmable control of gene expression is essential to understanding gene function, engineering cellular behaviors, and developing therapeutics. Beyond the gene editing applications enabled by the nuclease CRISPR-Cas9 and CRISPR-Cas12a, the invention of the nuclease-dead Cas molecules (dCas9 and dCas12a) offers a platform for the precise control of genome function without gene editing. Diverse dCas tools have been developed, which constitute a comprehensive toolbox that allows interrogation of gene function and modulation of the cellular behaviors. This review summarizes current applications of the dCas tools for transcription regulation, epigenetic engineering, genome imaging, genetic screens, and chromatin immunoprecipitation. We also highlight the advantages and existing challenges of the current dCas tools in genetic engineering and synthetic biology, and provide perspectives on future directions and applications.

    View details for PubMedID 29958882

  • A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription. ACS chemical biology Zhou, X. X., Zou, X., Chung, H. K., Gao, Y., Liu, Y., Qi, L. S., Lin, M. Z. 2018; 13 (2): 443-448

    Abstract

    Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

    View details for DOI 10.1021/acschembio.7b00603

    View details for PubMedID 28938067

    View details for PubMedCentralID PMC5820652

  • CRISPR-Based Chromatin Remodeling of the Endogenous Oct4 or Sox2 Locus Enables Reprogramming to Pluripotency CELL STEM CELL Liu, P., Chen, M., Liu, Y., Qi, L. S., Ding, S. 2018; 22 (2): 252-+
  • CRISPR-Based Chromatin Remodeling of the Endogenous Oct4 or Sox2 Locus Enables Reprogramming to Pluripotency. Cell stem cell Liu, P., Chen, M., Liu, Y., Qi, L. S., Ding, S. 2018; 22 (2): 252-261.e4

    Abstract

    Generation of induced pluripotent stem cells typically requires the ectopic expression of transcription factors to reactivate the pluripotency network. However, it remains largely unclear what remodeling events on endogenous chromatin trigger reprogramming toward induced pluripotent stem cells (iPSCs). Toward this end, we employed CRISPR activation to precisely target and remodel endogenous gene loci of Oct4 and Sox2. Interestingly, we found that single-locus targeting of Sox2 was sufficient to remodel and activate Sox2, which was followed by the induction of other pluripotent genes and establishment of the pluripotency network. Simultaneous remodeling of the Oct4 promoter and enhancer also triggered reprogramming. Authentic pluripotent cell lines were established in both cases. Finally, we showed that targeted manipulation of histone acetylation at the Oct4 gene locus could also initiate reprogramming. Our study generated authentic iPSCs with CRISPR activation through precise epigenetic remodeling of endogenous loci and shed light on how targeted chromatin remodeling triggers pluripotency induction.

    View details for DOI 10.1016/j.stem.2017.12.001

    View details for PubMedID 29358044

  • Low-intensity extracorporeal shock wave therapy promotes myogenesis through PERK/ATF4 pathway NEUROUROLOGY AND URODYNAMICS Wang, B., Zhou, J., Banie, L., Reed-Maldonado, A. B., Ning, H., Lu, Z., Ruan, Y., Zhou, T., Wang, H., Oh, B., Wang, G., Qi, S., Lin, G., Lue, T. F. 2018; 37 (2): 699–707

    Abstract

    Stress urinary incontinence (SUI) is a significant health problem for women. Treatments employing muscle derived stem cells (MDSCs) may be a promising approach to this prevalent, bothersome condition, but these treatments are invasive and require collection of cells from one site for injection into another. It is also unknown whether or not these cells establish themselves and function as muscle cells in the target tissues. Alternatively, low-intensity extracorporeal shock wave therapy (Li-ESWT) is non-invasive and has shown positive outcomes in the treatment of multiple musculoskeletal disorders, but the biological effects responsible for clinical success are not yet well understood. The aim of this study is to explore the possibility of employing Li-ESWT for activation of MDSCs in situ and to further elucidate the underlying biological effects and mechanisms of action in urethral muscle.Urethral muscle derived stem cells (uMDSCs) were harvest from Zucker Lean (ZUC-LEAN) (ZUC-Leprfa 186) rats and characterized with flow cytometry. Li-ESWT (0.02 mJ/mm2 , 3 Hz, 200 pulses) and GSK2656157, an inhibitor of PERK pathway, were applied to L6 rat myoblast cells. To assess for myotube formation, we used immunofluorescence staining and western blot analysis in uMDSCs and L6 cells.The results indicate that uMDSCs could form myotubes. Myotube formation was significantly increased by the Li-ESWT as was the expression of muscle heavy chain (MHC) and myogenic factor 5 (Myf5) in L6 cells in vitro. Li-ESWT activated protein kinase RNA-like ER kinase (PERK) pathway by increasing the phosphorylation levels of PERK and eukaryotic initiation factor 2a (eIF2α) and by increasing activating transcription factor 4 (ATF4). In addition, GSK2656157, an inhibitor of PERK, effectively inhibited the myotube formation in L6 rat myoblast cells. Furthermore, GSK2656157 also attenuated myotube formation induced by Li-ESWT.In conclusion, this experiment reveals that rat uMDSCs can be isolated successfully and can form myotubes in vitro. PERK/ATF4 pathway was involved in myotube formation, and L6 rat myoblast cells were activated by Li-ESWT to form myotubes. These findings suggest that PERK/ATF4 pathway is activated by Li-ESWT. This study elucidates one of the biochemical pathways responsible for the clinical improvements seen after Li-ESWT. It is possible that this information will help to establish Li-ESWT as an acceptable treatment modality and may help to further refine the use of Li-ESWT in the clinical practice of medicine.

    View details for PubMedID 28763567

    View details for PubMedCentralID PMC5794657

  • Using CRISPR-ERA Webserver for sgRNA Design. Bio-protocol Liu, H., Wang, X., Qi, L. S. 2017; 7 (17): e2522

    Abstract

    The CRISPR-Cas9 system is emerging as a powerful technology for gene editing (modifying the genome sequence) and gene regulation (without modifying the genome sequence). Designing sgRNAs for specific genes or regions of interest is indispensable to CRISPR-based applications. CRISPR-ERA (http://crispr-era.stanford.edu/) is one of the state-of-the-art designer webserver tools, which has been developed both for gene editing and gene regulation sgRNA design. This protocol discusses how to design sgRNA sequences and genome-wide sgRNA library using CRISPR-ERA.

    View details for DOI 10.21769/BioProtoc.2522

    View details for PubMedID 34541182

    View details for PubMedCentralID PMC8413616

  • Genetic interaction mapping in mammalian cells using CRISPR interference. Nature methods Du, D., Roguev, A., Gordon, D. E., Chen, M., Chen, S., Shales, M., Shen, J. P., Ideker, T., Mali, P., Qi, L. S., Krogan, N. J. 2017; 14 (6): 577-580

    Abstract

    We describe a combinatorial CRISPR interference (CRISPRi) screening platform for mapping genetic interactions in mammalian cells. We targeted 107 chromatin-regulation factors in human cells with pools of either single or double single guide RNAs (sgRNAs) to downregulate individual genes or gene pairs, respectively. Relative enrichment analysis of individual sgRNAs or sgRNA pairs allowed for quantitative characterization of genetic interactions, and comparison with protein-protein-interaction data revealed a functional map of chromatin regulation.

    View details for DOI 10.1038/nmeth.4286

    View details for PubMedID 28481362

  • Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions. Nature methods Shen, J. P., Zhao, D., Sasik, R., Luebeck, J., Birmingham, A., Bojorquez-Gomez, A., Licon, K., Klepper, K., Pekin, D., Beckett, A. N., Sanchez, K. S., Thomas, A., Kuo, C. C., Du, D., Roguev, A., Lewis, N. E., Chang, A. N., Kreisberg, J. F., Krogan, N., Qi, L., Ideker, T., Mali, P. 2017; 14 (6): 573-576

    Abstract

    We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision. From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies.

    View details for DOI 10.1038/nmeth.4225

    View details for PubMedID 28319113

    View details for PubMedCentralID PMC5449203

  • Multiplexed Dynamic Imaging of Genomic Loci by Combined CRISPR Imaging and DNA Sequential FISH BIOPHYSICAL JOURNAL Takei, Y., Shah, S., Harvey, S., Qi, L. S., Cai, L. 2017; 112 (9): 1773-1776

    Abstract

    Visualization of chromosome dynamics allows the investigation of spatiotemporal chromatin organization and its role in gene regulation and other cellular processes. However, current approaches to label multiple genomic loci in live cells have a fundamental limitation in the number of loci that can be labeled and uniquely identified. Here we describe an approach we call "track first and identify later" for multiplexed visualization of chromosome dynamics by combining two techniques: CRISPR imaging and DNA sequential fluorescence in situ hybridization. Our approach first labels and tracks chromosomal loci in live cells with the CRISPR-Cas9 system, then barcodes those loci by DNA sequential fluorescence in situ hybridization in fixed cells and resolves their identities. We demonstrate our approach by tracking telomere dynamics, identifying 12 unique subtelomeric regions with variable detection efficiencies, and tracking back the telomere dynamics of respective chromosomes in mouse embryonic stem cells.

    View details for DOI 10.1016/j.bpj.2017.03.024

    View details for Web of Science ID 000401301600006

    View details for PubMedID 28427715

  • A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription Zhou, X. X., Zou, X., Chung, H. K., Gao, Y., Liu, Y., QI, L. S., Lin, M. Z. 2017: 443–48

    Abstract

    Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

    View details for DOI 10.1021/acschembio.7b00603

    View details for PubMedCentralID PMC5820652

  • Genetic and epigenetic control of gene expression by CRISPR-Cas systems. F1000Research Lo, A. n., Qi, L. n. 2017; 6

    Abstract

    The discovery and adaption of bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems has revolutionized the way researchers edit genomes. Engineering of catalytically inactivated Cas variants (nuclease-deficient or nuclease-deactivated [dCas]) combined with transcriptional repressors, activators, or epigenetic modifiers enable sequence-specific regulation of gene expression and chromatin state. These CRISPR-Cas-based technologies have contributed to the rapid development of disease models and functional genomics screening approaches, which can facilitate genetic target identification and drug discovery. In this short review, we will cover recent advances of CRISPR-dCas9 systems and their use for transcriptional repression and activation, epigenome editing, and engineered synthetic circuits for complex control of the mammalian genome.

    View details for PubMedID 28649363

    View details for PubMedCentralID PMC5464239

  • Repurposing CRISPR System for Transcriptional Activation. Advances in experimental medicine and biology Chen, M., Qi, L. S. 2017; 983: 147–57

    Abstract

    In recent years, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become the most popular one for genome editing. When the nuclease domains of Cas9 protein are mutated into deactivated form (dCas9), CRISPR/dCas9 still retains the ability to bind the targeted DNA sequence, but loses the endonuclease cleavage activity. Taking advantage of the characteristics of this engineered nuclease inactive Cas9, the CRISPR/dCas system has been repurposed into versatile RNA-guided, DNA-targeting platforms, such as genome imaging, gene regulation, and epigenetic modification. Specifically, fusion of dCas9 with activation domains allows specific and efficient transcriptional activation on a genome-wide scale among diverse organisms. The purpose of this chapter is to review most important the recently published literature on CRISPR/dCas9-based transcriptional activation systems. Compared with the conventional approaches for enhancement of the expression of specific genes of interest, CRISPR/Cas9-based system has emerged as a promising technology for genome regulation, allowing specificity, convenience, robustness, and scalability for endogenous gene activation.

    View details for PubMedID 28639197

  • Applications of CRISPR Genome Engineering in Cell Biology. Trends in cell biology Wang, F., Qi, L. S. 2016; 26 (11): 875-888

    Abstract

    Recent advances in genome engineering are starting a revolution in biological research and translational applications. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided endonuclease CRISPR associated protein 9 (Cas9) and its variants enable diverse manipulations of genome function. In this review, we describe the development of Cas9 tools for a variety of applications in cell biology research, including the study of functional genomics, the creation of transgenic animal models, and genomic imaging. Novel genome engineering methods offer a new avenue to understand the causality between the genome and phenotype, thus promising a fuller understanding of cell biology.

    View details for DOI 10.1016/j.tcb.2016.08.004

    View details for PubMedID 27599850

    View details for PubMedCentralID PMC5077632

  • A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria CELL Peters, J. M., Colavin, A., Shi, H., Czarny, T. L., Larson, M. H., Wong, S., Hawkins, J. S., Lu, C. H., Koo, B., Marta, E., Shiver, A. L., Whitehead, E. H., Weissman, J. S., Brown, E. D., Qi, L. S., Huang, K. C., Gross, C. A. 2016; 165 (6): 1493-1506

    Abstract

    Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.

    View details for DOI 10.1016/j.cell.2016.05.003

    View details for Web of Science ID 000377045400021

    View details for PubMedID 27238023

    View details for PubMedCentralID PMC4894308

  • CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs CELL STEM CELL Mandegar, M. A., Huebsch, N., Frolov, E. B., Shin, E., Truong, A., Olvera, M. P., Chan, A. H., Miyaoka, Y., Holmes, K., Spencer, C. I., Judge, L. M., Gordon, D. E., Eskildsen, T. V., Villalta, J. E., Horlbeck, M. A., Gilbert, L. A., Krogan, N. J., Sheikh, S. P., Weissman, J. S., Qi, L. S., So, P., Conklin, B. R. 2016; 18 (4): 541-553

    Abstract

    Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease.

    View details for DOI 10.1016/j.stem.2016.01.022

    View details for Web of Science ID 000373722100017

    View details for PubMedID 26971820

    View details for PubMedCentralID PMC4830697

  • YAP Induces Human Naive Pluripotency. Cell reports Qin, H., Hejna, M., Liu, Y., Percharde, M., Wossidlo, M., Blouin, L., Durruthy-Durruthy, J., Wong, P., Qi, Z., Yu, J., Qi, L. S., Sebastiano, V., Song, J. S., Ramalho-Santos, M. 2016; 14 (10): 2301-2312

    Abstract

    The human naive pluripotent stem cell (PSC) state, corresponding to a pre-implantation stage of development, has been difficult to capture and sustain in vitro. We report that the Hippo pathway effector YAP is nuclearly localized in the inner cell mass of human blastocysts. Overexpression of YAP in human embryonic stem cells (ESCs) and induced PSCs (iPSCs) promotes the generation of naive PSCs. Lysophosphatidic acid (LPA) can partially substitute for YAP to generate transgene-free human naive PSCs. YAP- or LPA-induced naive PSCs have a rapid clonal growth rate, a normal karyotype, the ability to form teratomas, transcriptional similarities to human pre-implantation embryos, reduced heterochromatin levels, and other hallmarks of the naive state. YAP/LPA act in part by suppressing differentiation-inducing effects of GSK3 inhibition. CRISPR/Cas9-generated YAP(-/-) cells have an impaired ability to form colonies in naive but not primed conditions. These results uncover an unexpected role for YAP in the human naive state, with implications for early human embryology.

    View details for DOI 10.1016/j.celrep.2016.02.036

    View details for PubMedID 26947063

    View details for PubMedCentralID PMC4807727

  • CRISPR Technology for Genome Activation and Repression in Mammalian Cells. Cold Spring Harbor protocols Du, D., Qi, L. S. 2016; 2016 (1): pdb prot090175-?

    Abstract

    Targeted modulation of transcription is necessary for understanding complex gene networks and has great potential for medical and industrial applications. CRISPR is emerging as a powerful system for targeted genome activation and repression, in addition to its use in genome editing. This protocol describes how to design, construct, and experimentally validate the function of sequence-specific single guide RNAs (sgRNAs) for sequence-specific repression (CRISPRi) or activation (CRISPRa) of transcription in mammalian cells. In this technology, the CRISPR-associated protein Cas9 is catalytically deactivated (dCas9) to provide a general platform for RNA-guided DNA targeting of any locus in the genome. Fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in mammalian cells. Delivery of multiple sgRNAs further enables activation or repression of multiple genes. By using scaffold RNAs (scRNAs), different effectors can be recruited to different genes for simultaneous activation of some and repression of others. The CRISPRi and CRISPRa methods provide powerful tools for sequence-specific control of gene expression on a genome-wide scale to aid understanding gene functions and for engineering genetic regulatory systems.

    View details for DOI 10.1101/pdb.prot090175

    View details for PubMedID 26729910

  • An Introduction to CRISPR Technology for Genome Activation and Repression in Mammalian Cells. Cold Spring Harbor protocols Du, D., Qi, L. S. 2016; 2016 (1): pdb top086835-?

    Abstract

    CRISPR interference/activation (CRISPRi/a) technology provides a simple and efficient approach for targeted repression or activation of gene expression in the mammalian genome. It is highly flexible and programmable, using an RNA-guided nuclease-deficient Cas9 (dCas9) protein fused with transcriptional regulators for targeting specific genes to effect their regulation. Multiple studies have shown how this method is an effective way to achieve efficient and specific transcriptional repression or activation of single or multiple genes. Sustained transcriptional modulation can be obtained by stable expression of CRISPR components, which enables directed reprogramming of cell fate. Here, we introduce the basics of CRISPRi/a technology for genome repression or activation.

    View details for DOI 10.1101/pdb.top086835

    View details for PubMedID 26729914

  • CRISPR/Cas9 in Genome Editing and Beyond ANNUAL REVIEW OF BIOCHEMISTRY, VOL 85 Wang, H., La Russa, M., Qi, L. S. 2016; 85: 227-264

    Abstract

    The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.

    View details for DOI 10.1146/annurev-biochem-060815-014607

    View details for PubMedID 27145843

  • Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation NATURE REVIEWS MOLECULAR CELL BIOLOGY Dominguez, A. A., Lim, W. A., Qi, L. S. 2016; 17 (1)

    Abstract

    The bacterial CRISPR-Cas9 system has emerged as a multifunctional platform for sequence-specific regulation of gene expression. This Review describes the development of technologies based on nuclease-deactivated Cas9, termed dCas9, for RNA-guided genomic transcription regulation, both by repression through CRISPR interference (CRISPRi) and by activation through CRISPR activation (CRISPRa). We highlight different uses in diverse organisms, including bacterial and eukaryotic cells, and summarize current applications of harnessing CRISPR-dCas9 for multiplexed, inducible gene regulation, genome-wide screens and cell fate engineering. We also provide a perspective on future developments of the technology and its applications in biomedical research and clinical studies.

    View details for DOI 10.1038/nrm.2015.2

    View details for Web of Science ID 000366920600007

    View details for PubMedID 26670017

  • CRISPR/Cas9 for Human Genome Engineering and Disease Research ANNUAL REVIEW OF GENOMICS AND HUMAN GENETICS, VOL 17 Xiong, X., Chen, M., Lim, W. A., Zhao, D., Qi, L. S. 2016; 17: 131-154

    Abstract

    The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, a versatile RNA-guided DNA targeting platform, has been revolutionizing our ability to modify, manipulate, and visualize the human genome, which greatly advances both biological research and therapeutics development. Here, we review the current development of CRISPR/Cas9 technologies for gene editing, transcription regulation, genome imaging, and epigenetic modification. We discuss the broad application of this system to the study of functional genomics, especially genome-wide genetic screening, and to therapeutics development, including establishing disease models, correcting defective genetic mutations, and treating diseases.

    View details for DOI 10.1146/annurev-genom-083115-022258

    View details for Web of Science ID 000382615800007

    View details for PubMedID 27216776

  • CRISPR-ERA: a comprehensive design tool for CRISPR-mediated gene editing, repression and activation BIOINFORMATICS Liu, H., Wei, Z., Dominguez, A., Li, Y., Wang, X., Qi, L. S. 2015; 31 (22): 3676-3678

    Abstract

    The CRISPR/Cas9 system was recently developed as a powerful and flexible technology for targeted genome engineering, including genome editing (altering the genetic sequence) and gene regulation (without altering the genetic sequence). These applications require the design of single guide RNAs (sgRNAs) that are efficient and specific. However, this remains challenging, as it requires the consideration of many criteria. Several sgRNA design tools have been developed for gene editing, but currently there is no tool for the design of sgRNAs for gene regulation. With accumulating experimental data on the use of CRISPR/Cas9 for gene editing and regulation, we implement a comprehensive computational tool based on a set of sgRNA design rules summarized from these published reports. We report a genome-wide sgRNA design tool and provide an online website for predicting sgRNAs that are efficient and specific. We name the tool CRISPR-ERA, for clustered regularly interspaced short palindromic repeat-mediated editing, repression, and activation (ERA).http://CRISPR-ERA.stanford.edu.stanley.qi@stanford.edu or xwwang@tsinghua.edu.cnSupplementary data are available at Bioinformatics online.

    View details for DOI 10.1093/bioinformatics/btv423

    View details for PubMedID 26209430

  • The New State of the Art: Cas9 for Gene Activation and Repression MOLECULAR AND CELLULAR BIOLOGY La Russa, M. F., Qi, L. S. 2015; 35 (22): 3800-3809

    Abstract

    CRISPR-Cas9 technology has rapidly changed the landscape for how biologists and bioengineers study and manipulate the genome. Derived from the bacterial adaptive immune system, CRISPR-Cas9 has been coopted and repurposed for a variety of new functions, including the activation or repression of gene expression (termed CRISPRa or CRISPRi, respectively). This represents an exciting alternative to previously used repression or activation technologies such as RNA interference (RNAi) or the use of gene overexpression vectors. We have only just begun exploring the possibilities that CRISPR technology offers for gene regulation and the control of cell identity and behavior. In this review, we describe the recent advances of CRISPR-Cas9 technology for gene regulation and outline advantages and disadvantages of CRISPRa and CRISPRi (CRISPRa/i) relative to alternative technologies.

    View details for DOI 10.1128/MCB.00512-15

    View details for PubMedID 26370509

    View details for PubMedCentralID PMC4609748

  • Bacterial CRISPR: accomplishments and prospects CURRENT OPINION IN MICROBIOLOGY Peters, J. M., Silvis, M. R., Zhao, D., Hawkins, J. S., Gross, C. A., Qi, L. S. 2015; 27: 121-126

    View details for DOI 10.1016/j.mib.2015.08.007

    View details for PubMedID 26363124

  • Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells. Cell stem cell Yu, C., Liu, Y., Ma, T., Liu, K., Xu, S., Zhang, Y., Liu, H., La Russa, M., Xie, M., Ding, S., Qi, L. S. 2015; 16 (2): 142-147

    Abstract

    The bacterial CRISPR-Cas9 system has emerged as an effective tool for sequence-specific gene knockout through non-homologous end joining (NHEJ), but it remains inefficient for precise editing of genome sequences. Here we develop a reporter-based screening approach for high-throughput identification of chemical compounds that can modulate precise genome editing through homology-directed repair (HDR). Using our screening method, we have identified small molecules that can enhance CRISPR-mediated HDR efficiency, 3-fold for large fragment insertions and 9-fold for point mutations. Interestingly, we have also observed that a small molecule that inhibits HDR can enhance frame shift insertion and deletion (indel) mutations mediated by NHEJ. The identified small molecules function robustly in diverse cell types with minimal toxicity. The use of small molecules provides a simple and effective strategy to enhance precise genome engineering applications and facilitates the study of DNA repair mechanisms in mammalian cells.

    View details for DOI 10.1016/j.stem.2015.01.003

    View details for PubMedID 25658371

    View details for PubMedCentralID PMC4461869

  • Specific Gene Repression by CRISPRi System Transferred through Bacterial Conjugation ACS SYNTHETIC BIOLOGY Ji, W., Lee, D., Wong, E., Dadlani, P., Dinh, D., Huang, V., Kearns, K., Teng, S., Chen, S., Haliburton, J., Heimberg, G., Heineike, B., Ramasubramanian, A., Stevens, T., Helmke, K. J., Zepeda, V., Qi, L. S., Lim, W. A. 2014; 3 (12): 929-931

    Abstract

    In microbial communities, bacterial populations are commonly controlled using indiscriminate, broad range antibiotics. There are few ways to target specific strains effectively without disrupting the entire microbiome and local environment. Here, we use conjugation, a natural DNA horizontal transfer process among bacterial species, to deliver an engineered CRISPR interference (CRISPRi) system for targeting specific genes in recipient Escherichia coli cells. We show that delivery of the CRISPRi system is successful and can specifically repress a reporter gene in recipient cells, thereby establishing a new tool for gene regulation across bacterial cells and potentially for bacterial population control.

    View details for DOI 10.1021/sb500036q

    View details for Web of Science ID 000347140300010

    View details for PubMedID 25409531

    View details for PubMedCentralID PMC4277763

  • A versatile framework for microbial engineering using synthetic non-coding RNAs NATURE REVIEWS MICROBIOLOGY Qi, L. S., Arkin, A. P. 2014; 12 (5): 341-354

    Abstract

    Synthetic non-coding RNAs have emerged as a versatile class of molecular devices that have a diverse range of programmable functions, including signal sensing, gene regulation and the modulation of molecular interactions. Owing to their small size and the central role of Watson-Crick base pairing in determining their structure, function and interactions, several distinct types of synthetic non-coding RNA regulators that are functional at the DNA, mRNA and protein levels have been experimentally characterized and computationally modelled. These engineered devices can be incorporated into genetic circuits, enabling the more efficient creation of complex synthetic biological systems. In this Review, we summarize recent progress in engineering synthetic non-coding RNA devices and their application to genetic and cellular engineering in a broad range of microorganisms.

    View details for DOI 10.1038/nrmicro3244

    View details for Web of Science ID 000334846500011

    View details for PubMedID 24736794

  • Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System (vol 155, pg 1479, 2013) CELL Chen, B., Gilbert, L. A., Cimini, B. A., Schnitzbauer, J., Zhang, W., Li, G., Park, J., Blackburn, E. H., Weissman, J. S., Qi, L. S., Huang, B. 2014; 156 (1-2): 373-373
  • CRISPR interference (CRISPRi) for sequence-specific control of gene expression NATURE PROTOCOLS Larson, M. H., Gilbert, L. A., Wang, X., Lim, W. A., Weissman, J. S., Qi, L. S. 2013; 8 (11): 2180-2196

    Abstract

    Sequence-specific control of gene expression on a genome-wide scale is an important approach for understanding gene functions and for engineering genetic regulatory systems. We have recently described an RNA-based method, CRISPR interference (CRISPRi), for targeted silencing of transcription in bacteria and human cells. The CRISPRi system is derived from the Streptococcus pyogenes CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring only the coexpression of a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). The Cas9-sgRNA complex binds to DNA elements complementary to the sgRNA and causes a steric block that halts transcript elongation by RNA polymerase, resulting in the repression of the target gene. Here we provide a protocol for the design, construction and expression of customized sgRNAs for transcriptional repression of any gene of interest. We also provide details for testing the repression activity of CRISPRi using quantitative fluorescence assays and native elongating transcript sequencing. CRISPRi provides a simplified approach for rapid gene repression within 1-2 weeks. The method can also be adapted for high-throughput interrogation of genome-wide gene functions and genetic interactions, thus providing a complementary approach to RNA interference, which can be used in a wider variety of organisms.

    View details for DOI 10.1038/nprot.2013.132

    View details for Web of Science ID 000326164100008

    View details for PubMedID 24136345

  • An adaptor from translational to transcriptional control enables predictable assembly of complex regulation NATURE METHODS Liu, C. C., Qi, L., Lucks, J. B., Segall-Shapiro, T. H., Wang, D., Mutalik, V. K., Arkin, A. P. 2012; 9 (11): 1088-?

    Abstract

    Bacterial regulators of transcriptional elongation are versatile units for building custom genetic switches, as they control the expression of both coding and noncoding RNAs, act on multigene operons and can be predictably tethered into higher-order regulatory functions (a property called composability). Yet the less versatile bacterial regulators of translational initiation are substantially easier to engineer. To bypass this tradeoff, we have developed an adaptor that converts regulators of translational initiation into regulators of transcriptional elongation in Escherichia coli. We applied this adaptor to the construction of several transcriptional attenuators and activators, including a small molecule-triggered attenuator and a group of five mutually orthogonal riboregulators that we assembled into NOR gates of two, three or four RNA inputs. Continued application of our adaptor should produce large collections of transcriptional regulators whose inherent composability can facilitate the predictable engineering of complex synthetic circuits.

    View details for DOI 10.1038/NMETH.2184

    View details for Web of Science ID 000310848700022

    View details for PubMedID 23023598

  • RNA processing enables predictable programming of gene expression NATURE BIOTECHNOLOGY Qi, L., Haurwitz, R. E., Shao, W., Doudna, J. A., Arkin, A. P. 2012; 30 (10): 1002-?

    Abstract

    Complex interactions among genetic components often result in variable systemic performance in designed multigene systems. Using the bacterial clustered regularly interspaced short palindromic repeat (CRISPR) pathway we develop a synthetic RNA-processing platform, and show that efficient and specific cleavage of precursor mRNA enables reliable and predictable regulation of multigene operons. Physical separation of linked genetic elements by CRISPR-mediated cleavage is an effective strategy to achieve assembly of promoters, ribosome binding sites, cis-regulatory elements, and riboregulators into single- and multigene operons with predictable functions in bacteria. We also demonstrate that CRISPR-based RNA cleavage is effective for regulation in bacteria, archaea and eukaryotes. Programmable RNA processing using CRISPR offers a general approach for creating context-free genetic elements and can be readily used in the bottom-up construction of increasingly complex biological systems in a plug-and-play manner.

    View details for DOI 10.1038/nbt.2355

    View details for Web of Science ID 000309965500028

    View details for PubMedID 22983090

  • Engineering naturally occurring trans-acting non-coding RNAs to sense molecular signals NUCLEIC ACIDS RESEARCH Qi, L., Lucks, J. B., Liu, C. C., Mutalik, V. K., Arkin, A. P. 2012; 40 (12): 5775-5786

    Abstract

    Non-coding RNAs (ncRNAs) are versatile regulators in cellular networks. While most trans-acting ncRNAs possess well-defined mechanisms that can regulate transcription or translation, they generally lack the ability to directly sense cellular signals. In this work, we describe a set of design principles for fusing ncRNAs to RNA aptamers to engineer allosteric RNA fusion molecules that modulate the activity of ncRNAs in a ligand-inducible way in Escherichia coli. We apply these principles to ncRNA regulators that can regulate translation (IS10 ncRNA) and transcription (pT181 ncRNA), and demonstrate that our design strategy exhibits high modularity between the aptamer ligand-sensing motif and the ncRNA target-recognition motif, which allows us to reconfigure these two motifs to engineer orthogonally acting fusion molecules that respond to different ligands and regulate different targets in the same cell. Finally, we show that the same ncRNA fused with different sensing domains results in a sensory-level NOR gate that integrates multiple input signals to perform genetic logic. These ligand-sensing ncRNA regulators provide useful tools to modulate the activity of structurally related families of ncRNAs, and building upon the growing body of RNA synthetic biology, our ability to design aptamer-ncRNA fusion molecules offers new ways to engineer ligand-sensing regulatory circuits.

    View details for DOI 10.1093/nar/gks168

    View details for Web of Science ID 000305829000057

    View details for PubMedID 22383579

  • Rationally designed families of orthogonal RNA regulators of translation NATURE CHEMICAL BIOLOGY Mutalik, V. K., Qi, L., Guimaraes, J. C., Lucks, J. B., Arkin, A. P. 2012; 8 (5): 447-454

    Abstract

    Our ability to routinely engineer genetic networks for applications is limited by the scarcity of highly specific and non-cross-reacting (orthogonal) gene regulators with predictable behavior. Though antisense RNAs are attractive contenders for this purpose, quantitative understanding of their specificity and sequence-function relationship sufficient for their design has been limited. Here, we use rationally designed variants of the RNA-IN-RNA-OUT antisense RNA-mediated translation system from the insertion sequence IS10 to quantify >500 RNA-RNA interactions in Escherichia coli and integrate the data set with sequence-activity modeling to identify the thermodynamic stability of the duplex and the seed region as the key determinants of specificity. Applying this model, we predict the performance of an additional ~2,600 antisense-regulator pairs, forecast the possibility of large families of orthogonal mutants, and forward engineer and experimentally validate two RNA pairs orthogonal to an existing group of five from the training data set. We discuss the potential use of these regulators in next-generation synthetic biology applications.

    View details for DOI 10.1038/NCHEMBIO.919

    View details for Web of Science ID 000302962500011

    View details for PubMedID 22446835

  • Versatile RNA-sensing transcriptional regulators for engineering genetic networks PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lucks, J. B., Qi, L., Mutalik, V. K., Wang, D., Arkin, A. P. 2011; 108 (21): 8617-8622

    Abstract

    The widespread natural ability of RNA to sense small molecules and regulate genes has become an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering. Previous work in RNA synthetic biology has engineered RNA mechanisms that independently regulate multiple targets and integrate regulatory signals. However, intracellular regulatory networks built with these systems have required proteins to propagate regulatory signals. In this work, we remove this requirement and expand the RNA synthetic biology toolkit by engineering three unique features of the plasmid pT181 antisense-RNA-mediated transcription attenuation mechanism. First, because the antisense RNA mechanism relies on RNA-RNA interactions, we show how the specificity of the natural system can be engineered to create variants that independently regulate multiple targets in the same cell. Second, because the pT181 mechanism controls transcription, we show how independently acting variants can be configured in tandem to integrate regulatory signals and perform genetic logic. Finally, because both the input and output of the attenuator is RNA, we show how these variants can be configured to directly propagate RNA regulatory signals by constructing an RNA-meditated transcriptional cascade. The combination of these three features within a single RNA-based regulatory mechanism has the potential to simplify the design and construction of genetic networks by directly propagating signals as RNA molecules.

    View details for DOI 10.1073/pnas.1015741108

    View details for Web of Science ID 000290908000025

    View details for PubMedID 21555549

  • Regulation of transcription by unnatural amino acids NATURE BIOTECHNOLOGY Liu, C. C., Qi, L., Yanofsky, C., Arkin, A. P. 2011; 29 (2): 164-U111

    Abstract

    Small-molecule regulation of gene expression is intrinsic to cellular function and indispensable to the construction of new biological sensing, control and expression systems. However, there are currently only a handful of strategies for engineering such regulatory components and fewer still that can give rise to an arbitrarily large set of inducible systems whose members respond to different small molecules, display uniformity and modularity in their mechanisms of regulation, and combine to actuate universal logics. Here we present an approach for small-molecule regulation of transcription based on the combination of cis-regulatory leader-peptide elements with genetically encoded unnatural amino acids (amino acids that have been artificially added to the genetic code). In our system, any genetically encoded unnatural amino acid (UAA) can be used as a small-molecule attenuator or activator of gene transcription, and the logics intrinsic to the network defined by expanded genetic codes can be actuated.

    View details for DOI 10.1038/nbt.1741

    View details for Web of Science ID 000287023000025

    View details for PubMedID 21240267

  • Toward scalable parts families for predictable design of biological circuits CURRENT OPINION IN MICROBIOLOGY Lucks, J. B., Qi, L., Whitaker, W. R., Arkin, A. P. 2008; 11 (6): 567-573

    Abstract

    Our current ability to engineer biological circuits is hindered by design cycles that are costly in terms of time and money, with constructs failing to operate as desired, or evolving away from the desired function once deployed. Synthetic biologists seek to understand biological design principles and use them to create technologies that increase the efficiency of the genetic engineering design cycle. Central to the approach is the creation of biological parts--encapsulated functions that can be composited together to create new pathways with predictable behaviors. We define five desirable characteristics of biological parts--independence, reliability, tunability, orthogonality and composability, and review studies of small natural and synthetic biological circuits that provide insights into each of these characteristics. We propose that the creation of appropriate sets of families of parts with these properties is a prerequisite for efficient, predictable engineering of new function in cells and will enable a large increase in the sophistication of genetic engineering applications.

    View details for DOI 10.1016/j.mib.2008.10.002

    View details for Web of Science ID 000261866200015

    View details for PubMedID 18983935