Dr. Lei (Stanley) Qi is an assistant professor in the Department of Bioengineering and Department of Chemical and Systems Biology at Stanford University. He is a faculty fellow in ChEM-H. He is one of the developers and pioneers in the CRISPR technology for sequence-specfic genome engineering. He has developed a series of CRISPR-based gene regulation and imaging technologies, including CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), CRISPR imaging, and high-throughput CRISPRi/a screening. He works in the field of Synthetic Biology, and developed synthetic noncoding RNA approaches for control of the Central Dogma including ranscription and translation. He obtained his Ph.D. in Bioengineering from the University of California Berkeley in 2012, and performed independent research as a Systems Biology Fellow at UCSF from 2012 to 2014. His lab is currently applying genome engineering approaches for the interrogation of genomics related to cell differentiation, proliferation, epigenetic regulation and diseases.
Honors & Awards
Phi Beta Kappa, Phi Beta Kappa (2011)
Chinese Government Award For Outstanding Students Abroad, Chinese government (2012)
NIH Director's Independence Award, National Institutes of Health (2013)
Pew Biomedical Scholar, The Pew Charitable Trusts (2016)
Alfred P. Sloan Fellowship, Alfred P. Sloan Foundation (2017)
Next Power Honorary Chair Professor, National Tsing Hua University (2017)
B.S., Tsinghua University, Math and Physics (2005)
M.A., University of California, Berkeley, Physics (2007)
Ph.D., University of California, Berkeley, Bioengineering (2012)
Systems Biology Fellow, University of California, San Francisco, Systems and Synthetic Biology (2014)
Qi LS; Ding S; Chen Y. "United StatesSystems and methods for modulating CRISPR/Cas9 genome editing.", Leland Stanford Junior University
Jennifer A Doudna, Lei S Qi, Rachel E Haurwitz, Adam P Arkin. "United States Patent 14/248,980 Methods and compositions of controlling gene expression by RNA processing", University of California, Berkeley, Oct 9, 2014
Qi LS, Chen B, Huang B. "United States Patent US provisional patent application number 61/883,929. Optimized small guide RNAs and methods of use", University of California, San Francisco, Sep 1, 2013
Lei S Qi, Chang Liu, Adam P Arkin. "United States Patent WO Patent 2,013,049,330 Synthetic transcriptional control elements and methods of generating and using such elements", University of California, Berkeley, Apr 5, 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, Berkeley & University of California, San Francisco, Mar 15, 2013
- Fundamentals for Engineering Biology Lab
BIOE 44 (Aut, Spr)
Independent Studies (15)
- Bioengineering Problems and Experimental Investigation
BIOE 191 (Aut, Win, Spr, Sum)
- Directed Investigation
BIOE 392 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
CBIO 299 (Win, Spr)
- Directed Reading in Chemical and Systems Biology
CSB 299 (Win, Spr)
- Directed Reading in Stem Cell Biology and Regenerative Medicine
STEMREM 299 (Aut, Win, Spr)
- Directed Study
BIOE 391 (Aut)
- Graduate Research
CBIO 399 (Aut, Win, Spr)
- Graduate Research
CSB 399 (Win, Spr)
- Graduate Research
STEMREM 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
CSB 370 (Win, Spr)
- Medical Scholars Research
MED 370 (Sum)
- Medical Scholars Research
STEMREM 370 (Aut, Win, Spr)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Undergraduate Research
CSB 199 (Win, Spr)
- Undergraduate Research
STEMREM 199 (Aut, Win, Spr)
- Bioengineering Problems and Experimental Investigation
- Prior Year Courses
Doctoral Dissertation Reader (AC)
Zahra Bahrami nejad
Postdoctoral Faculty Sponsor
Antonia Dominguez, Marie La Russa, Xueqiu Lin, Yanxia Liu, Albert Lo, Ingrid Vogelaar, Haifeng Wang, Xiaoshu Xu
Doctoral Dissertation Advisor (AC)
Salil Bhate, Hannah Kempton
Salil Bhate, Akshay Maheshwari
Postdoctoral Research Mentor
Muneaki Nakamura, Ingrid Vogelaar
Complex transcriptional modulation with orthogonal and inducible dCas9 regulators.
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
View details for PubMedCentralID PMC5436902
A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria
2016; 165 (6): 1493-1506
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
Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells.
Cell stem cell
2015; 16 (2): 142-147
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
Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds
2015; 160 (1-2): 339-350
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
2013; 155 (7): 1479-1491
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
2013; 154 (2): 442-451
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
2013; 152 (5): 1173-1183
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
RNA processing enables predictable programming of gene expression
2012; 30 (10): 1002-?
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
Applications of CRISPR Genome Engineering in Cell Biology.
Trends in cell biology
2016; 26 (11): 875-888
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
CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs
CELL STEM CELL
2016; 18 (4): 541-553
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
YAP Induces Human Naive Pluripotency.
2016; 14 (10): 2301-2312
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
2016; 2016 (1): pdb prot090175-?
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
2016; 2016 (1): pdb top086835-?
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
2016; 85: 227-264
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 Web of Science ID 000379324700011
View details for PubMedID 27145843
Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation
NATURE REVIEWS MOLECULAR CELL BIOLOGY
2016; 17 (1)
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
2016; 17: 131-154
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 2015; 31 (22): 3676-3678
The New State of the Art: Cas9 for Gene Activation and Repression
MOLECULAR AND CELLULAR BIOLOGY
2015; 35 (22): 3800-3809
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 Web of Science ID 000365714400001
View details for PubMedID 26370509
View details for PubMedCentralID PMC4609748
- Bacterial CRISPR: accomplishments and prospects CURRENT OPINION IN MICROBIOLOGY 2015; 27: 121-126
Specific Gene Repression by CRISPRi System Transferred through Bacterial Conjugation
ACS SYNTHETIC BIOLOGY
2014; 3 (12): 929-931
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
A versatile framework for microbial engineering using synthetic non-coding RNAs
NATURE REVIEWS MICROBIOLOGY
2014; 12 (5): 341-354
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 2014; 156 (1-2): 373-373
CRISPR interference (CRISPRi) for sequence-specific control of gene expression
2013; 8 (11): 2180-2196
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
2012; 9 (11): 1088-?
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
Engineering naturally occurring trans-acting non-coding RNAs to sense molecular signals
NUCLEIC ACIDS RESEARCH
2012; 40 (12): 5775-5786
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
2012; 8 (5): 447-454
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
2011; 108 (21): 8617-8622
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
2011; 29 (2): 164-U111
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
2008; 11 (6): 567-573
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