Theodore Roth

All Publications

• Latent human herpesvirus 6 is reactivated in CAR T cells. Nature Lareau, C. A., Yin, Y., Maurer, K., Sandor, K. D., Daniel, B., Yagnik, G., Peña, J., Crawford, J. C., Spanjaart, A. M., Gutierrez, J. C., Haradhvala, N. J., Riberdy, J. M., Abay, T., Stickels, R. R., Verboon, J. M., Liu, V., Buquicchio, F. A., Wang, F., Southard, J., Song, R., Li, W., Shrestha, A., Parida, L., Getz, G., Maus, M. V., Li, S., Moore, A., Roberts, Z. J., Ludwig, L. S., Talleur, A. C., Thomas, P. G., Dehghani, H., Pertel, T., Kundaje, A., Gottschalk, S., Roth, T. L., Kersten, M. J., Wu, C. J., Majzner, R. G., Satpathy, A. T. 2023

  Abstract

  Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are understudied. For example, we lack a comprehensive understanding of the mechanisms of toxicities observed in patients receiving T cell therapies, including recent reports of encephalitis caused by reactivation of human herpesvirus 6 (HHV-6)1. Here, through petabase-scale viral genomics mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in cultures of human CD4+ T cells. Using single-cell sequencing, we identify a rare population of HHV-6 'super-expressors' (about 1 in 300-10,000 cells) that possess high viral transcriptional activity, among research-grade allogeneic chimeric antigen receptor (CAR) T cells. By analysing single-cell sequencing data from patients receiving cell therapy products that are approved by the US Food and Drug Administration2 or are in clinical studies3-5, we identify the presence of HHV-6-super-expressor CAR T cells in patients in vivo. Together, the findings of our study demonstrate the utility of comprehensive genomics analyses in implicating cell therapy products as a potential source contributing to the lytic HHV-6 infection that has been reported in clinical trials1,6-8 and may influence the design and production of autologous and allogeneic cell therapies.

  View details for DOI 10.1038/s41586-023-06704-2

  View details for PubMedID 37938768

  View details for PubMedCentralID 9827115

• Modular pooled discovery of synthetic knockin sequences to program durable cell therapies. Cell Blaeschke, F., Chen, Y. Y., Apathy, R., Daniel, B., Chen, A. Y., Chen, P. A., Sandor, K., Zhang, W., Li, Z., Mowery, C. T., Yamamoto, T. N., Nyberg, W. A., To, A., Yu, R., Bueno, R., Kim, M. C., Schmidt, R., Goodman, D. B., Feuchtinger, T., Eyquem, J., Jimmie Ye, C., Carnevale, J., Satpathy, A. T., Shifrut, E., Roth, T. L., Marson, A. 2023; 186 (19): 4216-4234.e33

  Abstract

  Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

  View details for DOI 10.1016/j.cell.2023.08.013

  View details for PubMedID 37714135

• High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails NATURE BIOTECHNOLOGY Shy, B. R., Vykunta, V. S., Ha, A., Talbot, A., Roth, T. L., Nguyen, D. N., Pfeifer, W. G., Chen, Y., Blaeschke, F., Shifrut, E., Vedova, S., Mamedov, M. R., Chung, J., Li, H., Yu, R., Wu, D., Wolf, J., Martin, T. G., Castro, C. E., Ye, L., Esensten, J. H., Eyquem, J., Marson, A. 2023; 41 (4): 521-+

  Abstract

  Enhancing CRISPR-mediated site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of around two- to threefold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional roughly two- to threefold on average. Our method works across a variety of target loci, knock-in constructs and primary human cell types, reaching HDR efficiencies of >80-90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for nonviral chimeric antigen receptor-T cell manufacturing, with knock-in efficiencies (46-62%) and yields (>1.5 × 109 modified cells) exceeding those of conventional approaches.

  View details for DOI 10.1038/s41587-022-01418-8

  View details for Web of Science ID 000844596500003

  View details for PubMedID 36008610

  View details for PubMedCentralID PMC10065198

• RASA2 ablation in T cells boosts antigen sensitivity and long-term function. Nature Carnevale, J., Shifrut, E., Kale, N., Nyberg, W. A., Blaeschke, F., Chen, Y. Y., Li, Z., Bapat, S. P., Diolaiti, M. E., O'Leary, P., Vedova, S., Belk, J., Daniel, B., Roth, T. L., Bachl, S., Anido, A. A., Prinzing, B., Ibanez-Vega, J., Lange, S., Haydar, D., Luetke-Eversloh, M., Born-Bony, M., Hegde, B., Kogan, S., Feuchtinger, T., Okada, H., Satpathy, A. T., Shannon, K., Gottschalk, S., Eyquem, J., Krenciute, G., Ashworth, A., Marson, A. 2022

  Abstract

  The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulationandcan increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in thebone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

  View details for DOI 10.1038/s41586-022-05126-w

  View details for PubMedID 36002574

• A functional map of HIV-host interactions in primary human T cells NATURE COMMUNICATIONS Hiatt, J., Hultquist, J. F., McGregor, M. J., Bouhaddou, M., Leenay, R. T., Simons, L. M., Young, J. M., Haas, P., Roth, T. L., Tobin, V., Wojcechowskyj, J. A., Woo, J. M., Rathore, U., Cavero, D. A., Shifrut, E., Nguyen, T. T., Haas, K. M., Malik, H. S., Doudna, J. A., May, A. P., Marson, A., Krogan, N. J. 2022; 13 (1): 1752

  Abstract

  Human Immunodeficiency Virus (HIV) relies on host molecular machinery for replication. Systematic attempts to genetically or biochemically define these host factors have yielded hundreds of candidates, but few have been functionally validated in primary cells. Here, we target 426 genes previously implicated in the HIV lifecycle through protein interaction studies for CRISPR-Cas9-mediated knock-out in primary human CD4+ T cells in order to systematically assess their functional roles in HIV replication. We achieve efficient knockout (>50% of alleles) in 364 of the targeted genes and identify 86 candidate host factors that alter HIV infection. 47 of these factors validate by multiplex gene editing in independent donors, including 23 factors with restrictive activity. Both gene editing efficiencies and HIV-1 phenotypes are highly concordant among independent donors. Importantly, over half of these factors have not been previously described to play a functional role in HIV replication, providing numerous novel avenues for understanding HIV biology. These data further suggest that host-pathogen protein-protein interaction datasets offer an enriched source of candidates for functional host factor discovery and provide an improved understanding of the mechanics of HIV replication in primary T cells.

  View details for DOI 10.1038/s41467-022-29346-w

  View details for Web of Science ID 000777408600045

  View details for PubMedID 35365639

• Robust T cell activation requires an eIF3-driven burst in T cell receptor translation ELIFE De Silva, D., Ferguson, L., Chin, G. H., Smith, B. E., Apathy, R. A., Roth, T. L., Blaeschke, F., Kudla, M., Marson, A., Ingolia, N. T., Cate, J. D. 2021; 10

  Abstract

  Activation of T cells requires a rapid surge in cellular protein synthesis. However, the role of translation initiation in the early induction of specific genes remains unclear. Here, we show human translation initiation factor eIF3 interacts with select immune system related mRNAs including those encoding the T cell receptor (TCR) subunits TCRA and TCRB. Binding of eIF3 to the TCRA and TCRB mRNA 3'-untranslated regions (3'-UTRs) depends on CD28 coreceptor signaling and regulates a burst in TCR translation required for robust T cell activation. Use of the TCRA or TCRB 3'-UTRs to control expression of an anti-CD19 chimeric antigen receptor (CAR) improves the ability of CAR-T cells to kill tumor cells in vitro. These results identify a new mechanism of eIF3-mediated translation control that can aid T cell engineering for immunotherapy applications.

  View details for DOI 10.7554/eLife.74272.sa2

  View details for Web of Science ID 000746406200001

  View details for PubMedID 34970966

  View details for PubMedCentralID PMC8758144

• Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins CELL REPORTS Hiatt, J., Cavero, D. A., McGregor, M. J., Zheng, W., Budzik, J. M., Roth, T. L., Haas, K. M., Wu, D., Rathore, U., Meyer-Franke, A., Bouzidi, M. S., Shifrut, E., Lee, Y., Kumar, V., Dang, E., Gordon, D. E., Wojcechowskyj, J. A., Hultquist, J. F., Fontaine, K. A., Pillai, S. K., Cox, J. S., Ernst, J. D., Krogan, N. J., Marson, A. 2021; 35 (6): 109105

  Abstract

  Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically edited cells that retain transcript and protein markers of myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection >50-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible, and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate the development of myeloid cellular therapies.

  View details for DOI 10.1016/j.celrep.2021.109105

  View details for Web of Science ID 000649197800014

  View details for PubMedID 33979618

  View details for PubMedCentralID PMC8188731

• The CD28-Transmembrane Domain Mediates Chimeric Antigen Receptor Heterodimerization With CD28 FRONTIERS IN IMMUNOLOGY Muller, Y. D., Nguyen, D. P., Ferreira, L. R., Ho, P., Raffin, C., Valencia, R., Congrave-Wilson, Z., Roth, T. L., Eyquem, J., Van Gool, F., Marson, A., Perez, L., Wells, J. A., Bluestone, J. A., Tang, Q. 2021; 12: 639818

  Abstract

  Anti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge domain (HD) and the transmembrane domain (TMD) between the extracellular antigen-targeting CARs and the intracellular signaling modalities of CARs has not been systemically studied. In this study, a series of 19-CARs differing only by their HD (CD8, CD28, or IgG4) and TMD (CD8 or CD28) was generated. CARs containing a CD28-TMD, but not a CD8-TMD, formed heterodimers with the endogenous CD28 in human T cells, as shown by co-immunoprecipitation and CAR-dependent proliferation of anti-CD28 stimulation. This dimerization was dependent on polar amino acids in the CD28-TMD and was more efficient with CARs containing CD28 or CD8 HD than IgG4-HD. The CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but had a significantly reduced CD28 cell-surface expression. These data unveiled a fundamental difference between CD28-TMD and CD8-TMD and indicated that CD28-TMD can modulate CAR T-cell activities by engaging endogenous partners.

  View details for DOI 10.3389/fimmu.2021.639818

  View details for Web of Science ID 000637003000001

  View details for PubMedID 33833759

  View details for PubMedCentralID PMC8021955

• TCF-1 regulates HIV-specific CD8+ T cell expansion capacity. JCI insight Rutishauser, R. L., Deguit, C. D., Hiatt, J., Blaeschke, F., Roth, T. L., Wang, L., Raymond, K. A., Starke, C. E., Mudd, J. C., Chen, W., Smullin, C., Matus-Nicodemos, R., Hoh, R., Krone, M., Hecht, F. M., Pilcher, C. D., Martin, J. N., Koup, R. A., Douek, D. C., Brenchley, J. M., Sékaly, R. P., Pillai, S. K., Marson, A., Deeks, S. G., McCune, J. M., Hunt, P. W. 2021; 6 (3)

  Abstract

  Although many HIV cure strategies seek to expand HIV-specific CD8+ T cells to control the virus, all are likely to fail if cellular exhaustion is not prevented. A loss in stem-like memory properties (i.e., the ability to proliferate and generate secondary effector cells) is a key feature of exhaustion; little is known, however, about how these properties are regulated in human virus-specific CD8+ T cells. We found that virus-specific CD8+ T cells from humans and nonhuman primates naturally controlling HIV/SIV infection express more of the transcription factor TCF-1 than noncontrollers. HIV-specific CD8+ T cell TCF-1 expression correlated with memory marker expression and expansion capacity and declined with antigenic stimulation. CRISPR-Cas9 editing of TCF-1 in human primary T cells demonstrated a direct role in regulating expansion capacity. Collectively, these data suggest that TCF-1 contributes to the regulation of the stem-like memory property of secondary expansion capacity of HIV-specific CD8+ T cells, and they provide a rationale for exploring the enhancement of this pathway in T cell-based therapeutic strategies for HIV.

  View details for DOI 10.1172/jci.insight.136648

  View details for PubMedID 33351785

  View details for PubMedCentralID PMC7934879

• XYZeq: Spatially resolved single-cell RNA sequencing reveals expression heterogeneity in the tumor microenvironment. Science advances Lee, Y., Bogdanoff, D., Wang, Y., Hartoularos, G. C., Woo, J. M., Mowery, C. T., Nisonoff, H. M., Lee, D. S., Sun, Y., Lee, J., Mehdizadeh, S., Cantlon, J., Shifrut, E., Ngyuen, D. N., Roth, T. L., Song, Y. S., Marson, A., Chow, E. D., Ye, C. J. 2021; 7 (17)

  Abstract

  Single-cell RNA sequencing (scRNA-seq) of tissues has revealed remarkable heterogeneity of cell types and states but does not provide information on the spatial organization of cells. To better understand how individual cells function within an anatomical space, we developed XYZeq, a workflow that encodes spatial metadata into scRNA-seq libraries. We used XYZeq to profile mouse tumor models to capture spatially barcoded transcriptomes from tens of thousands of cells. Analyses of these data revealed the spatial distribution of distinct cell types and a cell migration-associated transcriptomic program in tumor-associated mesenchymal stem cells (MSCs). Furthermore, we identify localized expression of tumor suppressor genes by MSCs that vary with proximity to the tumor core. We demonstrate that XYZeq can be used to map the transcriptome and spatial localization of individual cells in situ to reveal how cell composition and cell states can be affected by location within complex pathological tissue.

  View details for DOI 10.1126/sciadv.abg4755

  View details for PubMedID 33883145

  View details for PubMedCentralID PMC8059935

• Epithelial miR-141 regulates IL-13-induced airway mucus production. JCI insight Siddiqui, S., Johansson, K., Joo, A., Bonser, L. R., Koh, K. D., Le Tonqueze, O., Bolourchi, S., Bautista, R. A., Zlock, L., Roth, T. L., Marson, A., Bhakta, N. R., Ansel, K. M., Finkbeiner, W. E., Erle, D. J., Woodruff, P. G. 2021; 6 (5)

  Abstract

  IL-13-induced goblet cell metaplasia contributes to airway remodeling and pathological mucus hypersecretion in asthma. miRNAs are potent modulators of cellular responses, but their role in mucus regulation is largely unexplored. We hypothesized that airway epithelial miRNAs play roles in IL-13-induced mucus regulation. miR-141 is highly expressed in human and mouse airway epithelium, is altered in bronchial brushings from asthmatic subjects at baseline, and is induced shortly after airway allergen exposure. We established a CRISPR/Cas9-based protocol to target miR-141 in primary human bronchial epithelial cells that were differentiated at air-liquid-interface, and goblet cell hyperplasia was induced by IL-13 stimulation. miR-141 disruption resulted in decreased goblet cell frequency, intracellular MUC5AC, and total secreted mucus. These effects correlated with a reduction in a goblet cell gene expression signature and enrichment of a basal cell gene expression signature defined by single cell RNA sequencing. Furthermore, intranasal administration of a sequence-specific mmu-miR-141-3p inhibitor in mice decreased Aspergillus-induced secreted mucus and mucus-producing cells in the lung and reduced airway hyperresponsiveness without affecting cellular inflammation. In conclusion, we have identified a miRNA that regulates pathological airway mucus production and is amenable to therapeutic manipulation through an inhaled route.

  View details for DOI 10.1172/jci.insight.139019

  View details for PubMedID 33682796

  View details for PubMedCentralID PMC8021117

• Genetic Disease and Therapy. Annual review of pathology Roth, T. L., Marson, A. 2021; 16: 145-166

  Abstract

  Genetic diseases cause numerous complex and intractable pathologies. DNA sequences encoding each human's complexity and many disease risks are contained in the mitochondrial genome, nuclear genome, and microbial metagenome. Diagnosis of these diseases has unified around applications of next-generation DNA sequencing. However, translating specific genetic diagnoses into targeted genetic therapies remains a central goal. To date, genetic therapies have fallen into three broad categories: bulk replacement of affected genetic compartments with a new exogenous genome, nontargeted addition of exogenous genetic material to compensate for genetic errors, and most recently, direct correction of causative genetic alterations using gene editing. Generalized methods of diagnosis, therapy, and reagent delivery into each genetic compartment will accelerate the next generations of curative genetic therapies. We discuss the structure and variability of the mitochondrial, nuclear, and microbial metagenomic compartments, as well as the historical development and current practice of genetic diagnostics and gene therapies targeting each compartment.

  View details for DOI 10.1146/annurev-pathmechdis-012419-032626

  View details for PubMedID 33497260

• Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms SCIENCE Gordon, D. E., Hiatt, J., Bouhaddou, M., Rezelj, V. V., Ulferts, S., Braberg, H., Jureka, A. S., Obernier, K., Guo, J. Z., Batra, J., Kaake, R. M., Weckstein, A. R., Owens, T. W., Gupta, M., Pourmal, S., Titus, E. W., Cakir, M., Soucheray, M., McGregor, M., Cakir, Z., Jang, G., O'Meara, M. J., Tummino, T. A., Zhang, Z., Foussard, H., Rojc, A., Zhou, Y., Kuchenov, D., Huttenhain, R., Xu, J., Eckhardt, M., Swaney, D. L., Fabius, J. M., Ummadi, M., Tutuncuoglu, B., Rathore, U., Modak, M., Haas, P., Haas, K. M., Naing, Z., Pulido, E. H., Shi, Y., Barrio-Hernandez, I., Memon, D., Petsalaki, E., Dunham, A., Marrero, M., Burke, D., Koh, C., Vallet, T., Silvas, J. A., Azumaya, C. M., Billesbolle, C., Brilot, A. F., Campbell, M. G., Diallo, A., Dickinson, M., Diwanji, D., Herrera, N., Hoppe, N., Kratochvil, H. T., Liu, Y., Merz, G. E., Moritz, M., Nguyen, H. C., Nowotny, C., Puchades, C., Rizo, A. N., Schulze-Gahmen, U., Smith, A. M., Sun, M., Young, I. D., Zhao, J., Asarnow, D., Biel, J., Bowen, A., Braxton, J. R., Chen, J., Chio, C. M., Chio, U., Deshpande, I., Doan, L., Faust, B., Flores, S., Jin, M., Kim, K., Lam, V. L., Li, F., Li, J., Li, Y., Li, Y., Liu, X., Lo, M., Lopez, K. E., Melo, A. A., Moss, F. R., Phuong Nguyen, Paulino, J., Pawar, K., Peters, J. K., Pospiech, T. H., Safari, M., Sangwan, S., Schaefer, K., Thomas, P., Thwin, A. C., Trenker, R., Tse, E., Tsui, T., Wang, F., Whitis, N., Yu, Z., Zhang, K., Zhang, Y., Zhou, F., Saltzberg, D., Hodder, A. J., Shun-Shion, A. S., Williams, D. M., White, K. M., Rosales, R., Kehrer, T., Miorin, L., Moreno, E., Patel, A. H., Rihn, S., Khalid, M. M., Vallejo-Gracia, A., Fozouni, P., Simoneau, C. R., Roth, T. L., Wu, D., Karim, M., Ghoussaini, M., Dunham, I., Berardi, F., Weigang, S., Chazal, M., Park, J., Logue, J., McGrath, M., Weston, S., Haupt, R., Hastie, C., Elliott, M., Brown, F., Burness, K. A., Reid, E., Dorward, M., Johnson, C., Wilkinson, S. G., Geyer, A., Giesel, D. M., Baillie, C., Raggett, S., Leech, H., Toth, R., Goodman, N., Keough, K. C., Lind, A. L., Klesh, R. J., Hemphill, K. R., Carlson-Stevermer, J., Oki, J., Holden, K., Maures, T., Pollard, K. S., Sali, A., Agard, D. A., Cheng, Y., Fraser, J. S., Frost, A., Jura, N., Kortemme, T., Manglik, A., Southworth, D. R., Stroud, R. M., Alessi, D. R., Davies, P., Frieman, M. B., Ideker, T., Abate, C., Jouvenet, N., Kochs, G., Shoichet, B., Ott, M., Palmarini, M., Shokat, K. M., Garcia-Sastre, A., Rassen, J. A., Grosse, R., Rosenberg, O. S., Verba, K. A., Basler, C. F., Vignuzzi, M., Peden, A. A., Beltrao, P., Krogan, N. J. 2020; 370 (6521): 1181-+

  Abstract

  The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study.

  View details for DOI 10.1126/science.abe9403

  View details for Web of Science ID 000596071300050

  View details for PubMedID 33060197

  View details for PubMedCentralID PMC7808408

• Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency. Nature biotechnology Nguyen, D. N., Roth, T. L., Li, P. J., Chen, P. A., Apathy, R., Mamedov, M. R., Vo, L. T., Tobin, V. R., Goodman, D., Shifrut, E., Bluestone, J. A., Puck, J. M., Szoka, F. C., Marson, A. 2020; 38 (1): 44-49

  Abstract

  Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies1-5. Here we report two improvements that increase the efficiency of CRISPR-Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3+) T cells, CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived6 hematopoietic stem progenitor cells (HSPCs).

  View details for DOI 10.1038/s41587-019-0325-6

  View details for PubMedID 31819258

  View details for PubMedCentralID PMC6954310

• Editing of Endogenous Genes in Cellular Immunotherapies. Current hematologic malignancy reports Roth, T. L. 2020; 15 (4): 235-240

  Abstract

  T cell-based cellular and antibody immunotherapies have dramatically altered the landscape of cancer treatment over the past decade. Over the same time span, gene editing technologies have enabled unprecedented degrees of genetic control.Knock-outs of endogenous genes, especially based on electroporation of targetable nucleases such as CRISPR/Cas9, have rapidly proliferated. Simultaneous introduction of large DNA sequences can integrate new synthetic genetic instructions with specific endogenous loci to alter T cell function and specificity. Recently developed discovery technologies to perform genome-wide knock-out and large-scale knock-in screens in T cells can rapidly identify endogenous gene targets and therapeutic knock-in programs. Endogenous gene knock-outs and targeted knock-ins may offer the chance to expand beyond the current limitations of randomly integrating viral vector-based T cell therapies, and extend immunotherapies' therapeutic advances to wider hematologic and solid tumor indications.

  View details for DOI 10.1007/s11899-020-00587-0

  View details for PubMedID 32415407

  View details for PubMedCentralID PMC7415559

• Functional CRISPR dissection of gene networks controlling human regulatory T cell identity. Nature immunology Schumann, K., Raju, S. S., Lauber, M., Kolb, S., Shifrut, E., Cortez, J. T., Skartsis, N., Nguyen, V. Q., Woo, J. M., Roth, T. L., Yu, R., Nguyen, M. L., Simeonov, D. R., Nguyen, D. N., Targ, S., Gate, R. E., Tang, Q., Bluestone, J. A., Spitzer, M. H., Ye, C. J., Marson, A. 2020; 21 (11): 1456-1466

  Abstract

  Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Treg cell identity, yet the complete set of key transcription factors that control Treg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Treg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from Treg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in Treg cell function, coregulates another gene network with SATB1 and is important for Treg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human Treg cells that could be targeted for immunotherapies.

  View details for DOI 10.1038/s41590-020-0784-4

  View details for PubMedID 32989329

  View details for PubMedCentralID PMC7577958

• A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature Gordon, D. E., Jang, G. M., Bouhaddou, M., Xu, J., Obernier, K., White, K. M., O'Meara, M. J., Rezelj, V. V., Guo, J. Z., Swaney, D. L., Tummino, T. A., Hüttenhain, R., Kaake, R. M., Richards, A. L., Tutuncuoglu, B., Foussard, H., Batra, J., Haas, K., Modak, M., Kim, M., Haas, P., Polacco, B. J., Braberg, H., Fabius, J. M., Eckhardt, M., Soucheray, M., Bennett, M. J., Cakir, M., McGregor, M. J., Li, Q., Meyer, B., Roesch, F., Vallet, T., Mac Kain, A., Miorin, L., Moreno, E., Naing, Z. Z., Zhou, Y., Peng, S., Shi, Y., Zhang, Z., Shen, W., Kirby, I. T., Melnyk, J. E., Chorba, J. S., Lou, K., Dai, S. A., Barrio-Hernandez, I., Memon, D., Hernandez-Armenta, C., Lyu, J., Mathy, C. J., Perica, T., Pilla, K. B., Ganesan, S. J., Saltzberg, D. J., Rakesh, R., Liu, X., Rosenthal, S. B., Calviello, L., Venkataramanan, S., Liboy-Lugo, J., Lin, Y., Huang, X. P., Liu, Y., Wankowicz, S. A., Bohn, M., Safari, M., Ugur, F. S., Koh, C., Savar, N. S., Tran, Q. D., Shengjuler, D., Fletcher, S. J., O'Neal, M. C., Cai, Y., Chang, J. C., Broadhurst, D. J., Klippsten, S., Sharp, P. P., Wenzell, N. A., Kuzuoglu-Ozturk, D., Wang, H. Y., Trenker, R., Young, J. M., Cavero, D. A., Hiatt, J., Roth, T. L., Rathore, U., Subramanian, A., Noack, J., Hubert, M., Stroud, R. M., Frankel, A. D., Rosenberg, O. S., Verba, K. A., Agard, D. A., Ott, M., Emerman, M., Jura, N., von Zastrow, M., Verdin, E., Ashworth, A., Schwartz, O., d'Enfert, C., Mukherjee, S., Jacobson, M., Malik, H. S., Fujimori, D. G., Ideker, T., Craik, C. S., Floor, S. N., Fraser, J. S., Gross, J. D., Sali, A., Roth, B. L., Ruggero, D., Taunton, J., Kortemme, T., Beltrao, P., Vignuzzi, M., García-Sastre, A., Shokat, K. M., Shoichet, B. K., Krogan, N. J. 2020; 583 (7816): 459-468

  Abstract

  A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.

  View details for DOI 10.1038/s41586-020-2286-9

  View details for PubMedID 32353859

  View details for PubMedCentralID PMC7431030

• CRISPR screen in regulatory T cells reveals modulators of Foxp3. Nature Cortez, J. T., Montauti, E., Shifrut, E., Gatchalian, J., Zhang, Y., Shaked, O., Xu, Y., Roth, T. L., Simeonov, D. R., Zhang, Y., Chen, S., Li, Z., Woo, J. M., Ho, J., Vogel, I. A., Prator, G. Y., Zhang, B., Lee, Y., Sun, Z., Ifergan, I., Van Gool, F., Hargreaves, D. C., Bluestone, J. A., Marson, A., Fang, D. 2020; 582 (7812): 416-420

  Abstract

  Regulatory T (Treg) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties2, can promote autoimmunity and/or facilitate more effective tumour immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Treg cells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Treg cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Treg cells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.

  View details for DOI 10.1038/s41586-020-2246-4

  View details for PubMedID 32499641

  View details for PubMedCentralID PMC7305989

• Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies. Cell Roth, T. L., Li, P. J., Blaeschke, F., Nies, J. F., Apathy, R., Mowery, C., Yu, R., Nguyen, M. L., Lee, Y., Truong, A., Hiatt, J., Wu, D., Nguyen, D. N., Goodman, D., Bluestone, J. A., Ye, C. J., Roybal, K., Shifrut, E., Marson, A. 2020; 181 (3): 728-744.e21

  Abstract

  Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor β (TGF-β) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.

  View details for DOI 10.1016/j.cell.2020.03.039

  View details for PubMedID 32302591

  View details for PubMedCentralID PMC7219528

• Large dataset enables prediction of repair after CRISPR-Cas9 editing in primary T cells. Nature biotechnology Leenay, R. T., Aghazadeh, A., Hiatt, J., Tse, D., Roth, T. L., Apathy, R., Shifrut, E., Hultquist, J. F., Krogan, N., Wu, Z., Cirolia, G., Canaj, H., Leonetti, M. D., Marson, A., May, A. P., Zou, J. 2019

  Abstract

  Understanding of repair outcomes after Cas9-induced DNA cleavage is still limited, especially in primary human cells. We sequence repair outcomes at 1,656 on-target genomic sites in primary human T cells and use these data to train a machine learning model, which we have called CRISPR Repair Outcome (SPROUT). SPROUT accurately predicts the length, probability and sequence of nucleotide insertions and deletions, and will facilitate design of SpCas9 guide RNAs in therapeutically important primary human cells.

  View details for DOI 10.1038/s41587-019-0203-2

  View details for PubMedID 31359007

• A large CRISPR-induced bystander mutation causes immune dysregulation. Communications biology Simeonov, D. R., Brandt, A. J., Chan, A. Y., Cortez, J. T., Li, Z., Woo, J. M., Lee, Y., Carvalho, C. M., Indart, A. C., Roth, T. L., Zou, J., May, A. P., Lupski, J. R., Anderson, M. S., Buaas, F. W., Rokhsar, D. S., Marson, A. 2019; 2: 70

  Abstract

  A persistent concern with CRISPR-Cas9 gene editing has been the potential to generate mutations at off-target genomic sites. While CRISPR-engineering mice to delete a ~360bp intronic enhancer, here we discovered a founder line that had marked immune dysregulation caused by a 24kb tandem duplication of the sequence adjacent to the on-target deletion. Our results suggest unintended repair of on-target genomic cuts can cause pathogenic "bystander" mutations that escape detection by routine targeted genotyping assays.

  View details for PubMedID 30793048

• Helios enhances the preferential differentiation of human fetal CD4+ naïve T cells into regulatory T cells. Science immunology Ng, M. S., Roth, T. L., Mendoza, V. F., Marson, A., Burt, T. D. 2019; 4 (41)

  Abstract

  T cell receptor (TCR) stimulation and cytokine cues drive the differentiation of CD4+ naïve T cells into effector T cell populations with distinct proinflammatory or regulatory functions. Unlike adult naïve T cells, human fetal naïve CD4+ T cells preferentially differentiate into FOXP3+ regulatory T (Treg) cells upon TCR activation independent of exogenous cytokine signaling. This cell-intrinsic predisposition for Treg differentiation is implicated in the generation of tolerance in utero; however, the underlying mechanisms remain largely unknown. Here, we identify epigenetic and transcriptional programs shared between fetal naïve T and committed Treg cells that are inactive in adult naïve T cells and show that fetal-derived induced Treg (iTreg) cells retain this transcriptional program. We show that a subset of Treg-specific enhancers is accessible in fetal naïve T cells, including two active superenhancers at Helios Helios is expressed in fetal naïve T cells but not in adult naïve T cells, and fetal iTreg cells maintain Helios expression. CRISPR-Cas9 ablation of Helios in fetal naïve T cells impaired their differentiation into iTreg cells upon TCR stimulation, reduced expression of immunosuppressive genes in fetal iTreg cells such as IL10, and increased expression of proinflammatory genes including IFNG Consequently, Helios knockout fetal iTreg cells had reduced IL-10 and increased IFN-γ cytokine production. Together, our results reveal important roles for Helios in enhancing preferential fetal Treg differentiation and fine-tuning eventual Treg function. The Treg-biased programs identified within fetal naïve T cells could potentially be used to engineer enhanced iTreg populations for adoptive cellular therapies.

  View details for DOI 10.1126/sciimmunol.aav5947

  View details for PubMedID 31757834

  View details for PubMedCentralID PMC7340007

• Orthotopic replacement of T-cell receptor α- and β-chains with preservation of near-physiological T-cell function. Nature biomedical engineering Schober, K., Müller, T. R., Gökmen, F., Grassmann, S., Effenberger, M., Poltorak, M., Stemberger, C., Schumann, K., Roth, T. L., Marson, A., Busch, D. H. 2019; 3 (12): 974-984

  Abstract

  Therapeutic T cells with desired specificity can be engineered by introducing T-cell receptors (TCRs) specific for antigens of interest, such as those from pathogens or tumour cells. However, TCR engineering is challenging, owing to the complex heterodimeric structure of the receptor and to competition and mispairing between endogenous and transgenic receptors. Additionally, conventional TCR insertion disrupts the regulation of TCR dynamics, with consequences for T-cell function. Here, we report the outcomes and validation, using five different TCRs, of the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) with non-virally delivered template DNA for the elimination of endogenous TCR chains and for the orthotopic placement of TCRs in human T cells. We show that, whereas the editing of a single receptor chain results in chain mispairing, simultaneous editing of α- and β-chains combined with orthotopic TCR placement leads to accurate αβ-pairing and results in TCR regulation similar to that of physiological T cells.

  View details for DOI 10.1038/s41551-019-0409-0

  View details for PubMedID 31182835

• CRISPR-Cas9 genome engineering of primary CD4+ T cells for the interrogation of HIV-host factor interactions. Nature protocols Hultquist, J. F., Hiatt, J., Schumann, K., McGregor, M. J., Roth, T. L., Haas, P., Doudna, J. A., Marson, A., Krogan, N. J. 2019; 14 (1): 1-27

  Abstract

  CRISPR-Cas9 gene-editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology for use in primary human CD4+ T cells to create a high-throughput platform for analyzing the role of host factors in HIV infection and pathogenesis. Briefly, CRISPR-Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated CD4+ T cells by nucleofection. These cells are then assayed for editing efficiency and expanded for use in downstream cellular, genetic, or protein-based assays. This platform supports the rapid, arrayed generation of multiple gene manipulations and is widely adaptable across culture conditions, infection protocols, and downstream applications. Here, we present detailed protocols for crRNP synthesis, primary T-cell culture, 96-well nucleofection, molecular validation, and HIV infection, and discuss additional considerations for guide and screen design, as well as crRNP multiplexing. Taken together, this procedure allows high-throughput identification and mechanistic interrogation of HIV host factors in primary CD4+ T cells by gene knockout, validation, and HIV spreading infection in as little as 2-3 weeks.

  View details for DOI 10.1038/s41596-018-0069-7

  View details for PubMedID 30559373

  View details for PubMedCentralID PMC6637941

• Reprogramming human T cell function and specificity with non-viral genome targeting NATURE Roth, T. L., Puig-Saus, C., Yu, R., Shifrut, E., Carnevale, J., Li, P., Hiatt, J., Saco, J., Krystofinski, P., Li, H., Tobin, V., Nguyen, D. N., Lee, M. R., Putnam, A. L., Ferris, A. L., Chen, J. W., Schickel, J., Pellerin, L., Carmody, D., Alkorta-Aranburu, G., del Gaudio, D., Matsumoto, H., Morell, M., Mao, Y., Cho, M., Quadros, R. M., Gurumurthy, C. B., Smith, B., Haugwitz, M., Hughes, S. H., Weissman, J. S., Schumann, K., Esensten, J. H., May, A. P., Ashworth, A., Kupfer, G. M., Greeley, S. W., Bacchetta, R., Meffre, E., Roncarolo, M., Romberg, N., Herold, K. C., Ribas, A., Leonetti, M. D., Marson, A. 2018; 559 (7714): 405-+

  Abstract

  Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

  View details for PubMedID 29995861

• Genetic engineering in primary human B cells with CRISPR-Cas9 ribonucleoproteins. Journal of immunological methods Wu, C. M., Roth, T. L., Baglaenko, Y., Ferri, D. M., Brauer, P., Zuniga-Pflucker, J. C., Rosbe, K. W., Wither, J. E., Marson, A., Allen, C. D. 2018; 457: 33-40

  Abstract

  Genome editing in human cells with targeted nucleases now enables diverse experimental and therapeutic genome engineering applications, but extension to primary human B cells remains limited. Here we report a method for targeted genetic engineering in primary human B cells, utilizing electroporation of CRISPR-Cas9 ribonucleoproteins (RNPs) to introduce gene knockout mutations at protein-coding loci with high efficiencies that in some cases exceeded 80%. Further, we demonstrate knock-in editing of targeted nucleotides with efficiency exceeding 10% through co-delivery of oligonucleotide templates for homology directed repair. We delivered Cas9 RNPs in two distinct in vitro culture systems to achieve editing in both undifferentiated B cells and activated B cells undergoing differentiation, reflecting utility in diverse experimental conditions. In summary, we demonstrate a powerful and scalable research tool for functional genetic studies of human B cell biology that may have further applications in engineered B cell therapeutics.

  View details for DOI 10.1016/j.jim.2018.03.009

  View details for PubMedID 29614266

  View details for PubMedCentralID PMC6124898

• Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function. Cell Shifrut, E., Carnevale, J., Tobin, V., Roth, T. L., Woo, J. M., Bui, C. T., Li, P. J., Diolaiti, M. E., Ashworth, A., Marson, A. 2018; 175 (7): 1958-1971.e15

  Abstract

  Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, single guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA sequencing (RNA-seq) revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.

  View details for DOI 10.1016/j.cell.2018.10.024

  View details for PubMedID 30449619

  View details for PubMedCentralID PMC6689405

• Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms. Journal of visualized experiments : JoVE Farboud, B., Jarvis, E., Roth, T. L., Shin, J., Corn, J. E., Marson, A., Meyer, B. J., Patel, N. H., Hochstrasser, M. L. 2018

  Abstract

  Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break. The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

  View details for DOI 10.3791/57350

  View details for PubMedID 29889198

  View details for PubMedCentralID PMC6101420

• Light-activated cell identification and sorting (LACIS) for selection of edited clones on a nanofluidic device. Communications biology Mocciaro, A., Roth, T. L., Bennett, H. M., Soumillon, M., Shah, A., Hiatt, J., Chapman, K., Marson, A., Lavieu, G. 2018; 1: 41

  Abstract

  Despite improvements in the CRISPR molecular toolbox, identifying and purifying properly edited clones remains slow, laborious, and low-yield. Here, we establish a method to enable clonal isolation, selection, and expansion of properly edited cells, using OptoElectroPositioning technology for single-cell manipulation on a nanofluidic device. Briefly, after electroporation of primary T cells with CXCR4-targeting Cas9 ribonucleoproteins, single T cells are isolated on a chip and expanded into colonies. Phenotypic consequences of editing are rapidly assessed on-chip with cell-surface staining for CXCR4. Furthermore, individual colonies are identified based on their specific genotype. Each colony is split and sequentially exported for on-target sequencing and further off-chip clonal expansion of the validated clones. Using this method, single-clone editing efficiencies, including the rate of mono- and bi-allelic indels or precise nucleotide replacements, can be assessed within 10 days from Cas9 ribonucleoprotein introduction in cells.

  View details for DOI 10.1038/s42003-018-0034-6

  View details for PubMedID 30271925

  View details for PubMedCentralID PMC6123811

• Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature Simeonov, D. R., Gowen, B. G., Boontanrart, M. n., Roth, T. L., Gagnon, J. D., Mumbach, M. R., Satpathy, A. T., Lee, Y. n., Bray, N. L., Chan, A. Y., Lituiev, D. S., Nguyen, M. L., Gate, R. E., Subramaniam, M. n., Li, Z. n., Woo, J. M., Mitros, T. n., Ray, G. J., Curie, G. L., Naddaf, N. n., Chu, J. S., Ma, H. n., Boyer, E. n., Van Gool, F. n., Huang, H. n., Liu, R. n., Tobin, V. R., Schumann, K. n., Daly, M. J., Farh, K. K., Ansel, K. M., Ye, C. J., Greenleaf, W. J., Anderson, M. S., Bluestone, J. A., Chang, H. Y., Corn, J. E., Marson, A. n. 2017

  Abstract

  The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

  View details for PubMedID 28854172

• Migratory and adhesive cues controlling innate-like lymphocyte surveillance of the pathogen-exposed surface of the lymph node. eLife Zhang, Y., Roth, T. L., Gray, E. E., Chen, H., Rodda, L. B., Liang, Y., Ventura, P., Villeda, S., Crocker, P. R., Cyster, J. G. 2016; 5

  Abstract

  Lymph nodes (LNs) contain innate-like lymphocytes that survey the subcapsular sinus (SCS) and associated macrophages for pathogen entry. The factors promoting this surveillance behavior have not been defined. Here, we report that IL7R(hi)Ccr6(+) lymphocytes in mouse LNs rapidly produce IL17 upon bacterial and fungal challenge. We show that these innate-like lymphocytes are mostly LN resident. Ccr6 is required for their accumulation near the SCS and for efficient IL17 induction. Migration into the SCS intrinsically requires S1pr1, whereas movement from the sinus into the parenchyma involves the integrin LFA1 and its ligand ICAM1. CD169, a sialic acid-binding lectin, helps retain the cells within the sinus, preventing their loss in lymph flow. These findings establish a role for Ccr6 in augmenting innate-like lymphocyte responses to lymph-borne pathogens, and they define requirements for cell movement between parenchyma and SCS in what we speculate is a program of immune surveillance that helps achieve LN barrier immunity.

  View details for DOI 10.7554/eLife.18156

  View details for PubMedID 27487469

  View details for PubMedCentralID PMC5017864

• Single-molecule imaging of Hedgehog pathway protein Smoothened in primary cilia reveals binding events regulated by Patched1. Proceedings of the National Academy of Sciences of the United States of America Milenkovic, L., Weiss, L. E., Yoon, J., Roth, T. L., Su, Y. S., Sahl, S. J., Scott, M. P., Moerner, W. E. 2015; 112 (27): 8320-8325

  Abstract

  Accumulation of the signaling protein Smoothened (Smo) in the membrane of primary cilia is an essential step in Hedgehog (Hh) signal transduction, yet the molecular mechanisms of Smo movement and localization are poorly understood. Using ultrasensitive single-molecule tracking with high spatial/temporal precision (30 nm/10 ms), we discovered that binding events disrupt the primarily diffusive movement of Smo in cilia at an array of sites near the base. The affinity of Smo for these binding sites was modulated by the Hh pathway activation state. Activation, by either a ligand or genetic loss of the negatively acting Hh receptor Patched-1 (Ptch), reduced the affinity and frequency of Smo binding at the base. Our findings quantify activation-dependent changes in Smo dynamics in cilia and highlight a previously unknown step in Hh pathway activation.

  View details for DOI 10.1073/pnas.1510094112

  View details for PubMedID 26100903

  View details for PubMedCentralID PMC4500289

• Inflammation and neuroprotection in traumatic brain injury. JAMA neurology Corps, K. N., Roth, T. L., McGavern, D. B. 2015; 72 (3): 355-62

  Abstract

  Traumatic brain injury (TBI) is a significant public health concern that affects individuals in all demographics. With increasing interest in the medical and public communities, understanding the inflammatory mechanisms that drive the pathologic and consequent cognitive outcomes can inform future research and clinical decisions for patients with TBI.To review known inflammatory mechanisms in TBI and to highlight clinical trials and neuroprotective therapeutic manipulations of pathologic and inflammatory mechanisms of TBI.We searched articles in PubMed published between 1960 and August 1, 2014, using the following keywords: traumatic brain injury, sterile injury, inflammation, astrocytes, microglia, monocytes, macrophages, neutrophils, T cells, reactive oxygen species, alarmins, danger-associated molecular patterns, purinergic receptors, neuroprotection, and clinical trials. Previous clinical trials or therapeutic studies that involved manipulation of the discussed mechanisms were considered for inclusion. The final list of selected studies was assembled based on novelty and direct relevance to the primary focus of this review.Traumatic brain injury is a diverse group of sterile injuries induced by primary and secondary mechanisms that give rise to cell death, inflammation, and neurologic dysfunction in patients of all demographics. Pathogenesis is driven by complex, interacting mechanisms that include reactive oxygen species, ion channel and gap junction signaling, purinergic receptor signaling, excitotoxic neurotransmitter signaling, perturbations in calcium homeostasis, and damage-associated molecular pattern molecules, among others. Central nervous system resident and peripherally derived inflammatory cells respond to TBI and can provide neuroprotection or participate in maladaptive secondary injury reactions. The exact contribution of inflammatory cells to a TBI lesion is dictated by their anatomical positioning as well as the local cues to which they are exposed.The mechanisms that drive TBI lesion development as well as those that promote repair are exceedingly complex and often superimposed. Because pathogenic mechanisms can diversify over time or even differ based on the injury type, it is important that neuroprotective therapeutics be developed and administered with these variables in mind. Due to its complexity, TBI has proven particularly challenging to treat; however, a number of promising therapeutic approaches are now under pre-clinical development, and recent clinical trials have even yielded a few successes. Given the worldwide impact of TBI on the human population, it is imperative that research remains active in this area and that we continue to develop therapeutics to improve outcome in afflicted patients.

  View details for DOI 10.1001/jamaneurol.2014.3558

  View details for PubMedID 25599342

  View details for PubMedCentralID PMC5001842

• A Rapid and Simple Method for DNA Engineering Using Cycled Ligation Assembly PLOS ONE Roth, T. L., Milenkovic, L., Scott, M. P. 2014; 9 (9)

  Abstract

  DNA assembly techniques have developed rapidly, enabling efficient construction of complex constructs that would be prohibitively difficult using traditional restriction-digest based methods. Most of the recent methods for assembling multiple DNA fragments in vitro suffer from high costs, complex set-ups, and diminishing efficiency when used for more than a few DNA segments. Here we present a cycled ligation-based DNA assembly protocol that is simple, cheap, efficient, and powerful. The method employs a thermostable ligase and short Scaffold Oligonucleotide Connectors (SOCs) that are homologous to the ends and beginnings of two adjacent DNA sequences. These SOCs direct an exponential increase in the amount of correctly assembled product during a reaction that cycles between denaturing and annealing/ligating temperatures. Products of early cycles serve as templates for later cycles, allowing the assembly of many sequences in a single reaction. To demonstrate the method's utility, we directed the assembly of twelve inserts, in one reaction, into a transformable plasmid. All the joints were precise, and assembly was scarless in the sense that no nucleotides were added or missing at junctions. Simple, efficient, and low-cost cycled ligation assemblies will facilitate wider use of complex genetic constructs in biomedical research.

  View details for DOI 10.1371/journal.pone.0107329

  View details for Web of Science ID 000344317700045

  View details for PubMedID 25226397

  View details for PubMedCentralID PMC4167330

• Microglia development and function. Annual review of immunology Nayak, D., Roth, T. L., McGavern, D. B. 2014; 32: 367-402

  Abstract

  Proper development and function of the mammalian central nervous system (CNS) depend critically on the activity of parenchymal sentinels referred to as microglia. Although microglia were first described as ramified brain-resident phagocytes, research conducted over the past century has expanded considerably upon this narrow view and ascribed many functions to these dynamic CNS inhabitants. Microglia are now considered among the most versatile cells in the body, possessing the capacity to morphologically and functionally adapt to their ever-changing surroundings. Even in a resting state, the processes of microglia are highly dynamic and perpetually scan the CNS. Microglia are in fact vital participants in CNS homeostasis, and dysregulation of these sentinels can give rise to neurological disease. In this review, we discuss the exciting developments in our understanding of microglial biology, from their developmental origin to their participation in CNS homeostasis and pathophysiological states such as neuropsychiatric disorders, neurodegeneration, sterile injury responses, and infectious diseases. We also delve into the world of microglial dynamics recently uncovered using real-time imaging techniques.

  View details for DOI 10.1146/annurev-immunol-032713-120240

  View details for PubMedID 24471431

  View details for PubMedCentralID PMC5001846

• Transcranial amelioration of inflammation and cell death after brain injury. Nature Roth, T. L., Nayak, D., Atanasijevic, T., Koretsky, A. P., Latour, L. L., McGavern, D. B. 2014; 505 (7482): 223-8

  Abstract

  Traumatic brain injury (TBI) is increasingly appreciated to be highly prevalent and deleterious to neurological function. At present, no effective treatment options are available, and little is known about the complex cellular response to TBI during its acute phase. To gain insights into TBI pathogenesis, we developed a novel murine closed-skull brain injury model that mirrors some pathological features associated with mild TBI in humans and used long-term intravital microscopy to study the dynamics of the injury response from its inception. Here we demonstrate that acute brain injury induces vascular damage, meningeal cell death, and the generation of reactive oxygen species (ROS) that ultimately breach the glial limitans and promote spread of the injury into the parenchyma. In response, the brain elicits a neuroprotective, purinergic-receptor-dependent inflammatory response characterized by meningeal neutrophil swarming and microglial reconstitution of the damaged glial limitans. We also show that the skull bone is permeable to small-molecular-weight compounds, and use this delivery route to modulate inflammation and therapeutically ameliorate brain injury through transcranial administration of the ROS scavenger, glutathione. Our results shed light on the acute cellular response to TBI and provide a means to locally deliver therapeutic compounds to the site of injury.

  View details for DOI 10.1038/nature12808

  View details for PubMedID 24317693

  View details for PubMedCentralID PMC3930079

• Type I interferon programs innate myeloid dynamics and gene expression in the virally infected nervous system. PLoS pathogens Nayak, D., Johnson, K. R., Heydari, S., Roth, T. L., Zinselmeyer, B. H., McGavern, D. B. 2013; 9 (5): e1003395

  Abstract

  Viral infections of central nervous system (CNS) often trigger inflammatory responses that give rise to a wide range of pathological outcomes. The CNS is equipped with an elaborate network of innate immune sentinels (e.g. microglia, macrophages, dendritic cells) that routinely serve as first responders to these infections. The mechanisms that underlie the dynamic programming of these cells following CNS viral infection remain undefined. To gain insights into this programming, we utilized a combination of genomic and two-photon imaging approaches to study a pure innate immune response to a noncytopathic virus (lymphocytic choriomeningitis virus) as it established persistence in the brain. This enabled us to evaluate how global gene expression patterns were translated into myeloid cell dynamics following infection. Two-photon imaging studies revealed that innate myeloid cells mounted a vigorous early response to viral infection characterized by enhanced vascular patrolling and a complete morphological transformation. Interestingly, innate immune activity subsided over time and returned to a quasi-normal state as the virus established widespread persistence in the brain. At the genomic level, early myeloid cell dynamics were associated with massive changes in CNS gene expression, most of which declined over time and were linked to type I interferon signaling (IFN-I). Surprisingly, in the absence of IFN-I signaling, almost no differential gene expression was observed in the nervous system despite increased viral loads. In addition, two-photon imaging studies revealed that IFN-I receptor deficient myeloid cells were unresponsive to viral infection and remained in a naïve state. These data demonstrate that IFN-I engages non-redundant programming responsible for nearly all innate immune activity in the brain following a noncytopathic viral infection. This Achilles' heel could explain why so many neurotropic viruses have acquired strategies to suppress IFN-I.

  View details for DOI 10.1371/journal.ppat.1003395

  View details for PubMedID 23737750

  View details for PubMedCentralID PMC3667771