CRISPR-based technologies represent a major breakthrough in biomedical science as they offer a powerful platform for unbiased screening and functional genomics in various fields, including immunology. Pooled and arrayed CRISPR screens have uncovered previously unknown intracellular drivers in innate and adaptive immune cells for immune regulation as well as intercellular regulators mediating cell–cell interactions. Recent single-cell CRISPR screening platforms expand the readouts to the transcriptome and enable the inference of gene regulatory networks for better mechanistic insights. CRISPR screens also allow for mapping of genetic interactions to identify genes that synergize or alleviate complex immune phenotypes. Here, we review the progress in and emerging adaptation of CRISPR technologies to advance our fundamental immunological knowledge and identify novel disease targets for immunotherapy of infection, inflammation and cancer.
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Cho, S. W., Kim, S., Kim, J. M. & Kim, J. S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).
Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Bock, C. et al. High-content CRISPR screening. Nat. Rev. Methods Prim. 2, 8 (2022).
Klompe, S. E., Vo, P. L. H., Halpin-Healy, T. S. & Sternberg, S. H. Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration. Nature 571, 219–225 (2019).
Schumann, K. et al. Functional CRISPR dissection of gene networks controlling human regulatory T cell identity. Nat. Immunol. 21, 1456–1466 (2020).
Chow, R. D. et al. AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma. Nat. Neurosci. 20, 1329–1341 (2017).
Ye, L. et al. In vivo CRISPR screening in CD8 T cells with AAV-Sleeping Beauty hybrid vectors identifies membrane targets for improving immunotherapy for glioblastoma. Nat. Biotechnol. 37, 1302–1313 (2019).
Shifrut, E. et al. Genome-wide CRISPR screens in primary human T cells reveal key regulators of immune function. Cell 175, 1958–1971 (2018). This study has developed the SLICE approach for CRISPR–Cas9 delivery (sgRNA-encoding lentiviral infection followed by Cas9 protein electroporation) and describes its use for a genome-wide pooled CRISPR screen in primary human T cells.
Doench, J. G. Am I ready for CRISPR? A user’s guide to genetic screens. Nat. Rev. Genet. 19, 67–80 (2018).
Joung, J. et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat. Protoc. 12, 828–863 (2017).
Platt, R. J. et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014).
Chu, V. T. et al. Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes. BMC Biotechnol. 16, 4 (2016).
Parnas, O. et al. A Genome-wide CRISPR screen in primary immune cells to dissect regulatory networks. Cell 162, 675–686 (2015). This study is the first to report a genome-wide pooled CRISPR screen in primary immune cells, namely bone marrow-derived DCs stimulated with LPS.
Pulendran, B. & Davis, M. M. The science and medicine of human immunology. Science 369, essay4014 (2020).
Jost, M. et al. CRISPR-based functional genomics in human dendritic cells. eLife 10, e65856 (2021).
Yeung, A. T. Y. et al. A genome-wide knockout screen in human macrophages identified host factors modulating Salmonella infection. mBio 10, e02169–19 (2019).
Lai, Y. et al. High-throughput CRISPR screens to dissect Macrophage-Shigella interactions. mBio 12, e0215821 (2021).
Sedlyarov, V. et al. The bicarbonate transporter SLC4A7 plays a key role in macrophage phagosome acidification. Cell Host Microbe 23, 766–774.e5 (2018).
Haney, M. S. et al. Identification of phagocytosis regulators using magnetic genome-wide CRISPR screens. Nat. Genet. 50, 1716–1727 (2018).
Shi, J. et al. A genome-wide CRISPR screen identifies WDFY3 as a novel regulator of macrophage efferocytosis. Preprint at bioRxiv https://doi.org/10.1101/2022.01.21.477299 (2022).
Swanson, K. V., Deng, M. & Ting, J. P. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat. Rev. Immunol. 19, 477–489 (2019).
Locati, M., Curtale, G. & Mantovani, A. Diversity, mechanisms, and significance of macrophage plasticity. Annu. Rev. Pathol. 15, 123–147 (2020).
Schmid-Burgk, J. L. et al. A Genome-wide CRISPR (clustered regularly interspaced short palindromic repeats) screen identifies NEK7 as an essential component of NLRP3 inflammasome activation. J. Biol. Chem. 291, 103–109 (2016).
Tong, J. et al. Pooled CRISPR screening identifies m(6)A as a positive regulator of macrophage activation. Sci. Adv. 7, eabd4742 (2021).
Covarrubias, S. et al. High-throughput CRISPR screening identifies genes involved in macrophage viability and inflammatory pathways. Cell Rep. 33, 108541 (2020).
Harding, C. V. & Boom, W. H. Regulation of antigen presentation by Mycobacterium tuberculosis: a role for toll-like receptors. Nat. Rev. Microbiol. 8, 296–307 (2010).
Kiritsy, M. C. et al. A genetic screen in macrophages identifies new regulators of IFNγ-inducible MHCII that contribute to T cell activation. eLife 10, e65110 (2021).
Jiang, C. et al. CRISPR/Cas9 screens reveal multiple layers of B cell CD40 regulation. Cell Rep. 28, 1307–1322 (2019).
Chu, V. T. et al. Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line. Proc. Natl Acad. Sci. USA 113, 12514–12519 (2016).
Carnevale, J. et al. RASA2 ablation in T cells boosts antigen sensitivity and long-term function. Nature 609, 174–182 (2022).
Shang, W. et al. Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation. Proc. Natl Acad. Sci. USA 115, E4051–E4060 (2018).
Johansen, K. H. et al. A CRISPR screen targeting PI3K effectors identifies RASA3 as a negative regulator of LFA-1-mediated adhesion in T cells. Sci. Signal. 15, eabl9169 (2022).
Rosenberg, S. A. & Restifo, N. P. Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348, 62–68 (2015).
Gurusamy, D. et al. Multi-phenotype CRISPR-Cas9 screen identifies p38 kinase as a target for adoptive immunotherapies. Cancer Cell 37, 818–833 (2020).
Dong, M. B. et al. Systematic immunotherapy target discovery using genome-scale in vivo CRISPR screens in CD8 T cells. Cell 178, 1189–1204 (2019).
Ye, L. et al. A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-T therapy. Cell Metab. 34, 595–614 (2022).
Chapman, N. M., Boothby, M. R. & Chi, H. Metabolic coordination of T cell quiescence and activation. Nat. Rev. Immunol. 20, 55–70 (2020).
Long, L. et al. CRISPR screens unveil signal hubs for nutrient licensing of T cell immunity. Nature 600, 308–313 (2021).
Saravia, J., Chapman, N. M. & Chi, H. Helper T cell differentiation. Cell. Mol. Immunol. 16, 634–643 (2019).
Chapman, N. M. & Chi, H. Metabolic adaptation of lymphocytes in immunity and disease. Immunity 55, 14–30 (2022).
Henriksson, J. et al. Genome-wide CRISPR screens in T helper cells reveal pervasive crosstalk between activation and differentiation. Cell 176, 882–896 (2019).
Cortez, J. T. et al. CRISPR screen in regulatory T cells reveals modulators of Foxp3. Nature 582, 416–420 (2020).
Loo, C. S. et al. A Genome-wide CRISPR screen reveals a role for the non-canonical nucleosome-remodeling BAF complex in Foxp3 expression and regulatory T cell function. Immunity 53, 143–157 (2020). Long et al.40, Henriksson et al.43 and Loo et al.45 conducted genome-wide CRISPR screens in primary mouse T cells in vitro to explore new regulators of mTORC1 signalling, TH2 cell differentiation programmes and FOXP3 expression, respectively.
Legut, M. et al. A genome-scale screen for synthetic drivers of T cell proliferation. Nature 603, 728–735 (2022).
Schmidt, R. et al. CRISPR activation and interference screens decode stimulation responses in primary human T cells. Science 375, eabj4008 (2022). This study conducted comprehensive bulk and single-cell CRISPR screens in primary human T cells for regulators of cytokine production.
Theisen, D. J. et al. WDFY4 is required for cross-presentation in response to viral and tumor antigens. Science 362, 694–699 (2018). This study reports an intercellular CRISPR screening platform through a DC–T cell co-culture that enables identification of novel regulators of antigen cross-presentation.
Scott, A. M., Wolchok, J. D. & Old, L. J. Antibody therapy of cancer. Nat. Rev. Cancer 12, 278–287 (2012).
Kamber, R. A. et al. Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis. Nature 597, 549–554 (2021). This study reports an intercellular CRISPR screening platform that facilitates identification of ligand–receptor pairs between tumour cells and macrophages that mediate phagocytosis of tumour cells.
Li, R. et al. Generation and validation of versatile inducible CRISPRi embryonic stem cell and mouse model. PLoS Biol. 18, e3000749 (2020).
Deng, Y. et al. Generation of a CRISPR activation mouse that enables modelling of aggressive lymphoma and interrogation of venetoclax resistance. Nat. Commun. 13, 4739 (2022).
Zhou, H. et al. In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR-dCas9-activator transgenic mice. Nat. Neurosci. 21, 440–446 (2018).
Wangensteen, K. J. et al. Combinatorial genetics in liver repopulation and carcinogenesis with a in vivo CRISPR activation platform. Hepatology 68, 663–676 (2018).
Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020).
Fu, G. et al. Metabolic control of TFH cells and humoral immunity by phosphatidylethanolamine. Nature 595, 724–729 (2021). This study conducted in vivo metabolic CRISPR screening in primary mouse CD4+ T cells for specific regulators of TFH cell versus TH1 cell differentiation.
Huang, B. et al. In vivo CRISPR screens reveal a HIF-1α-mTOR-network regulates T follicular helper versus Th1 cells. Nat. Commun. 13, 805 (2022).
Sugiura, A. et al. MTHFD2 is a metabolic checkpoint controlling effector and regulatory T cell fate and function. Immunity 55, 65–81 (2022).
Sutra Del Galy, A. et al. In vivo genome-wide CRISPR screens identify SOCS1 as intrinsic checkpoint of CD4+ TH1 cell response. Sci. Immunol. 6, eabe8219 (2021).
Crompton, J. G., Sukumar, M. & Restifo, N. P. Uncoupling T-cell expansion from effector differentiation in cell-based immunotherapy. Immunol. Rev. 257, 264–276 (2014).
Huang, H. et al. In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8+ T cell fate decisions. Cell 184, 1245–1261 (2021).
Chen, Z. et al. In vivo CD8+ T cell CRISPR screening reveals control by Fli1 in infection and cancer. Cell 184, 1262–1280 (2021). Huang et al.61 and Chen et al.62 conducted in vivo CRISPR screens in primary CD8+ T cells to explore new pathways affecting their clonal expansion and differentiation, with the goals of engineering more efficacious T cell responses against infections and tumours.
Ellis, G. I., Sheppard, N. C. & Riley, J. L. Genetic engineering of T cells for immunotherapy. Nat. Rev. Genet. 22, 427–447 (2021).
Wei, J. et al. Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy. Nature 576, 471–476 (2019). Dong et al.37 and Wei et al.64 conducted in vivo CRISPR screens in primary CD8+ T cells to identify targets in T cells that can be reprogrammed to promote the antitumour response.
Zhao, H. et al. Genome-wide fitness gene identification reveals Roquin as a potent suppressor of CD8 T cell expansion and anti-tumor immunity. Cell Rep. 37, 110083 (2021).
Kumar, S. et al. CARM1 inhibition enables immunotherapy of resistant tumors by dual action on tumor cells and T cells. Cancer Discov. 11, 2050–2071 (2021).
Wang, D. et al. CRISPR screening of CAR T cells and cancer stem cells reveals critical dependencies for cell-based therapies. Cancer Discov. 11, 1192–1211 (2021).
LaFleur, M. W. et al. A CRISPR-Cas9 delivery system for in vivo screening of genes in the immune system. Nat. Commun. 10, 1668 (2019). This study developed a novel tool for in vivo CRISPR screening (CHIME) using bone marrow cells from Cas9-expressing mice to identify regulators of immune cell homeostasis.
Liu, B. et al. Large-scale multiplexed mosaic CRISPR perturbation in the whole organism. Cell 185, 3008–3024 (2022). This study developed an inducible mosaic animal for perturbation, which enables in situ CRISPR targeting of at least 100 genes in parallel throughout the mouse body, and showed mapping of a miniature Perturb-Atlas by phenotyping across perturbations in multiple tissues.
Kalbasi, A. & Ribas, A. Tumour-intrinsic resistance to immune checkpoint blockade. Nat. Rev. Immunol. 20, 25–39 (2020).
Shah, N. N. & Fry, T. J. Mechanisms of resistance to CAR T cell therapy. Nat. Rev. Clin. Oncol. 16, 372–385 (2019).
Burr, M. L. et al. An evolutionarily conserved function of polycomb silences the MHC class I antigen presentation pathway and enables immune evasion in cancer. Cancer Cell 36, 385–401 (2019).
Burr, M. L. et al. CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity. Nature 549, 101–105 (2017).
Gu, S. S. et al. Therapeutically increasing MHC-I expression potentiates immune checkpoint blockade. Cancer Discov. 11, 1524–1541 (2021).
Barkal, A. A. et al. CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy. Nature 572, 392–396 (2019).
Wang, J. et al. Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy. Nat. Med. 25, 656–666 (2019).
Wisnovsky, S. et al. Genome-wide CRISPR screens reveal a specific ligand for the glycan-binding immune checkpoint receptor Siglec-7. Proc. Natl Acad. Sci. USA 118, e2015024118 (2021).
Patel, S. J. et al. Identification of essential genes for cancer immunotherapy. Nature 548, 537–542 (2017).
Pan, D. et al. A major chromatin regulator determines resistance of tumor cells to T cell-mediated killing. Science 359, 770–775 (2018).
Vredevoogd, D. W. et al. Augmenting immunotherapy impact by lowering tumor TNF cytotoxicity threshold. Cell 178, 585–599 (2019).
Han, P. et al. Genome-wide CRISPR screening identifies JAK1 deficiency as a mechanism of T-cell resistance. Front. Immunol. 10, 251 (2019).
Young, T. M. et al. Autophagy protects tumors from T cell-mediated cytotoxicity via inhibition of TNFα-induced apoptosis. Sci. Immunol. 5, eabb9561 (2020).
Singh, N. et al. Impaired death receptor signaling in leukemia causes antigen-independent resistance by inducing CAR T-cell dysfunction. Cancer Discov. 10, 552–567 (2020).
Dufva, O. et al. Integrated drug profiling and CRISPR screening identify essential pathways for CAR T-cell cytotoxicity. Blood 135, 597–609 (2020).
Hou, J. et al. Integrating genome-wide CRISPR immune screen with multi-omic clinical data reveals distinct classes of tumor intrinsic immune regulators. J. Immunother. Cancer 9, e001819 (2021).
Upadhyay, R. et al. A critical role for fas-mediated off-target tumor killing in T-cell immunotherapy. Cancer Discov. 11, 599–613 (2021).
Shen, Y. et al. Cancer cell-intrinsic resistance to BiTE therapy is mediated by loss of CD58 costimulation and modulation of the extrinsic apoptotic pathway. J. Immunother. Cancer 10, e004348 (2022).
Lawson, K. A. et al. Functional genomic landscape of cancer-intrinsic evasion of killing by T cells. Nature 586, 120–126 (2020). This study conducted genome-wide CRISPR screens across six genetically diverse mouse cancer cell lines co-cultured with CD8+ CTLs and identified 182 core cancer-intrinsic, CTL-evasion genes.
Zhuang, X., Veltri, D. P. & Long, E. O. Genome-wide CRISPR screen reveals cancer cell resistance to NK cells induced by NK-derived IFN-γ. Front. Immunol. 10, 2879 (2019).
Larson, R. C. et al. CAR T cell killing requires the IFNγR pathway in solid but not liquid tumours. Nature 604, 563–570 (2022).
Manguso, R. T. et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature 547, 413–418 (2017). This study is the first to report in vivo CRISPR screening in tumour cells under immune pressure to identify new targets to improve the response to immunotherapy.
Griffin, G. K. et al. Epigenetic silencing by SETDB1 suppresses tumour intrinsic immunogenicity. Nature 595, 309–314 (2021).
Li, F. et al. In vivo epigenetic CRISPR screen identifies Asf1a as an immunotherapeutic target in Kras-mutant lung adenocarcinoma. Cancer Discov. 10, 270–287 (2020).
Ishizuka, J. J. et al. Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade. Nature 565, 43–48 (2019).
Zhu, X. G. et al. Functional genomics in vivo reveal metabolic dependencies of pancreatic cancer cells. Cell Metab. 33, 211–221 (2021).
Martin, T. D. et al. The adaptive immune system is a major driver of selection for tumor suppressor gene inactivation. Science 373, 1327–1335 (2021). This study conducted in vivo CRISPR screening in multiple tissue-derived tumour cell lines with or without adaptive immune pressure, showing that cancer is largely driven by tumour immune evasion.
Wang, X. et al. In vivo CRISPR screens identify the E3 ligase Cop1 as a modulator of macrophage infiltration and cancer immunotherapy target. Cell 184, 5357–5374 (2021).
Dubrot, J. et al. In vivo CRISPR screens reveal the landscape of immune evasion pathways across cancer. Nat. Immunol. 23, 1495–1506 (2022).
Ramos, A. et al. Leukemia-intrinsic determinants of CAR-T response revealed by in vivo genome-wide CRISPR screening. Preprint at bioRxiv https://doi.org/10.1101/2022.02.15.480217 (2022).
Li, J. et al. Epigenetic and transcriptional control of the epidermal growth factor receptor regulates the tumor immune microenvironment in pancreatic cancer. Cancer Discov. 11, 736–753 (2021).
Wagner, D. L. et al. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat. Med. 25, 242–248 (2019).
Chew, W. L. et al. A multifunctional AAV-CRISPR-Cas9 and its host response. Nat. Methods 13, 868–874 (2016).
Dubrot, J. et al. In vivo screens using a selective CRISPR antigen removal lentiviral vector system reveal immune dependencies in renal cell carcinoma. Immunity 54, 571–585 (2021).
Chen, R. et al. In vivo RNA interference screens identify regulators of antiviral CD4+ and CD8+ T cell differentiation. Immunity 41, 325–338 (2014).
Stripecke, R. et al. Immune response to green fluorescent protein: implications for gene therapy. Gene Ther. 6, 1305–1312 (1999).
Wang, G. et al. CRISPR-GEMM pooled mutagenic screening identifies KMT2D as a major modulator of immune checkpoint blockade. Cancer Discov. 10, 1912–1933 (2020).
Yim, S., Hwang, W., Han, N. & Lee, D. Computational discovery of cancer immunotherapy targets by intercellular CRISPR screens. Front. Immunol. 13, 884561 (2022).
Dixit, A. et al. Perturb-Seq: dissecting molecular circuits with scalable single-cell RNA profiling of pooled genetic screens. Cell 167, 1853–1866 (2016).
Jaitin, D. A. et al. Dissecting immune circuits by linking CRISPR-pooled screens with single-cell RNA-seq. Cell 167, 1883–1896 (2016). Dixit et al.108 and Jaitin et al.109 are among the first studies to report a single-cell CRISPR screen in primary immune cells.
Drager, N. M. et al. A CRISPRi/a platform in human iPSC-derived microglia uncovers regulators of disease states. Nat. Neurosci. 25, 1149–1162 (2022).
Zhou, W., Gao, F., Romero-Wolf, M., Jo, S. & Rothenberg, E. V. Single-cell deletion analyses show control of pro-T cell developmental speed and pathways by Tcf7, Spi1, Gata3, Bcl11a, Erg, and Bcl11b. Sci. Immunol. 7, eabm1920 (2022).
Wagner, D. E. & Klein, A. M. Lineage tracing meets single-cell omics: opportunities and challenges. Nat. Rev. Genet. 21, 410–427 (2020).
Havel, J. J., Chowell, D. & Chan, T. A. The evolving landscape of biomarkers for checkpoint inhibitor immunotherapy. Nat. Rev. Cancer 19, 133–150 (2019).
Costanzo, M. et al. Global genetic networks and the genotype-to-phenotype relationship. Cell 177, 85–100 (2019).
Perez-Perez, J. M., Candela, H. & Micol, J. L. Understanding synergy in genetic interactions. Trends Genet. 25, 368–376 (2009).
Guo, A. et al. cBAF complex components and MYC cooperate early in CD8+ T cell fate. Nature 607, 135–141 (2022).
Belk, J. A. et al. Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence. Cancer Cell 40, 768–786 (2022). Guo et al.116 and Belk et al.117 conducted in vivo CRISPR screens in primary CD8+ T cells to elucidate the inhibitory role of the SWI/SNF complex in T cell persistence in infection and tumours.
Wang, T. et al. Gene essentiality profiling reveals gene networks and synthetic lethal interactions with oncogenic Ras. Cell 168, 890–903 (2017).
Bayraktar, E. C. et al. Metabolic coessentiality mapping identifies C12orf49 as a regulator of SREBP processing and cholesterol metabolism. Nat. Metab. 2, 487–498 (2020).
Aregger, M. et al. Systematic mapping of genetic interactions for de novo fatty acid synthesis identifies C12orf49 as a regulator of lipid metabolism. Nat. Metab. 2, 499–513 (2020).
Chow, R. D. et al. In vivo profiling of metastatic double knockouts through CRISPR-Cpf1 screens. Nat. Methods 16, 405–408 (2019).
Gonatopoulos-Pournatzis, T. et al. Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform. Nat. Biotechnol. 38, 638–648 (2020).
Gier, R. A. et al. High-performance CRISPR-Cas12a genome editing for combinatorial genetic screening. Nat. Commun. 11, 3455 (2020).
DeWeirdt, P. C. et al. Optimization of AsCas12a for combinatorial genetic screens in human cells. Nat. Biotechnol. 39, 94–104 (2021).
Park, J. J. et al. Double knockout CRISPR screen in cancer resistance to T cell cytotoxicity. Preprint at bioRxiv https://doi.org/10.1101/2022.03.01.482556 (2022).
Norman, T. M. et al. Exploring genetic interaction manifolds constructed from rich single-cell phenotypes. Science 365, 786–793 (2019).
Hiatt, J. et al. Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins. Cell Rep. 35, 109105 (2021).
Dhainaut, M. et al. Spatial CRISPR genomics identifies regulators of the tumor microenvironment. Cell 185, 1223–1239 (2022). This study integrates spatial transcriptomics with CRISPR screening in tumour cells for regulators of the TME in vivo.
Schoenfeld, A. J. & Hellmann, M. D. Acquired resistance to immune checkpoint inhibitors. Cancer Cell 37, 443–455 (2020).
Hanna, R. E. et al. Massively parallel assessment of human variants with base editor screens. Cell 184, 1064–1080 (2021).
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
Simeonov, D. R. et al. Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature 549, 111–115 (2017).
Rosenblum, D. et al. CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy. Sci. Adv. 6, eabc9450 (2020).
Gao, J., Luo, T., Lin, N., Zhang, S. & Wang, J. A new tool for CRISPR-Cas13a-based cancer gene therapy. Mol. Ther. Oncolytics 19, 79–92 (2020).
Waldman, A. D., Fritz, J. M. & Lenardo, M. J. A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat. Rev. Immunol. 20, 651–668 (2020).
Beltra, J. C. et al. Developmental relationships of four exhausted CD8+ T cell subsets reveals underlying transcriptional and epigenetic landscape control mechanisms. Immunity 52, 825–841.e8 (2020).
Hudson, W. H. et al. Proliferating transitory T cells with an effector-like transcriptional signature emerge from PD-1+ stem-like CD8+ T cells during chronic infection. Immunity 51, 1043–1058 (2019).
Zander, R. et al. CD4+ T cell help is required for the formation of a cytolytic CD8+ T cell subset that protects against chronic infection and cancer. Immunity 51, 1028–1042 (2019).
Santomasso, B., Bachier, C., Westin, J., Rezvani, K. & Shpall, E. J. The other side of CAR T-cell therapy: cytokine release syndrome, neurologic toxicity, and financial burden. Am. Soc. Clin. Oncol. Educ. Book 39, 433–444 (2019).
Nahmad, A. D. et al. Frequent aneuploidy in primary human T cells after CRISPR-Cas9 cleavage. Nat. Biotechnol. https://doi.org/10.1038/s41587-022-01377-0 (2022).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Doench, J. G. et al. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat. Biotechnol. 32, 1262–1267 (2014).
Adli, M. The CRISPR tool kit for genome editing and beyond. Nat. Commun. 9, 1911 (2018).
Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).
Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).
Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).
Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).
Maeder, M. L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).
Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).
Han, K. et al. Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions. Nat. Biotechnol. 35, 463–474 (2017).
Shen, J. P. et al. Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions. Nat. Methods 14, 573–576 (2017).
Najm, F. J. et al. Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens. Nat. Biotechnol. 36, 179–189 (2018).
Nissim, L., Perli, S. D., Fridkin, A., Perez-Pinera, P. & Lu, T. K. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol. Cell 54, 698–710 (2014).
Xie, K., Minkenberg, B. & Yang, Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc. Natl Acad. Sci. USA 112, 3570–3575 (2015).
Adamson, B. et al. A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response. Cell 167, 1867–1882 (2016).
Hegde, M., Strand, C., Hanna, R. E. & Doench, J. G. Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens. PLoS ONE 13, e0197547 (2018).
Hanna, R. E. & Doench, J. G. A case of mistaken identity. Nat. Biotechnol. 36, 802–804 (2018).
Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).
Zetsche, B. et al. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat. Biotechnol. 35, 31–34 (2017).
Mandal, P. K. et al. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell 15, 643–652 (2014).
Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015).
Seki, A. & Rutz, S. Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells. J. Exp. Med. 215, 985–997 (2018).
Stadtmauer, E. A. et al. CRISPR-engineered T cells in patients with refractory cancer. Science 367, eaba7365 (2020).
Choi, B. D. et al. CRISPR-Cas9 disruption of PD-1 enhances activity of universal EGFRvIII CAR T cells in a preclinical model of human glioblastoma. J. Immunother. Cancer 7, 304 (2019).
Lynn, R. C. et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 576, 293–300 (2019).
Su, S. et al. CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer. Oncoimmunology 6, e1249558 (2017).
Ren, J. et al. Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition. Clin. Cancer Res. 23, 2255–2266 (2017).
Rupp, L. J. et al. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Sci. Rep. 7, 737 (2017).
Guo, X. et al. Disruption of PD-1 enhanced the anti-tumor activity of chimeric antigen receptor T cells against hepatocellular carcinoma. Front. Pharmacol. 9, 1118 (2018).
Hu, W. et al. CRISPR/Cas9-mediated PD-1 disruption enhances human mesothelin-targeted CAR T cell effector functions. Cancer Immunol. Immunother. 68, 365–377 (2019).
Hu, B. et al. Nucleofection with plasmid DNA for CRISPR/Cas9-mediated inactivation of programmed cell death protein 1 in CD133-specific CAR T cells. Hum. Gene Ther. 30, 446–458 (2019).
Lu, Y. et al. Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat. Med. 26, 732–740 (2020).
Morton, L. T. et al. Simultaneous deletion of endogenous TCRalphabeta for TCR gene therapy creates an improved and safe cellular therapeutic. Mol. Ther. 28, 64–74 (2020).
Legut, M., Dolton, G., Mian, A. A., Ottmann, O. G. & Sewell, A. K. CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells. Blood 131, 311–322 (2018).
Torikai, H. et al. Toward eliminating HLA class I expression to generate universal cells from allogeneic donors. Blood 122, 1341–1349 (2013).
Cooper, M. L. et al. An “off-the-shelf” fratricide-resistant CAR-T for the treatment of T cell hematologic malignancies. Leukemia 32, 1970–1983 (2018).
Gomes-Silva, D. et al. CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies. Blood 130, 285–296 (2017).
Xie, S., Duan, J., Li, B., Zhou, P. & Hon, G. C. Multiplexed engineering and analysis of combinatorial enhancer activity in single cells. Mol. Cell 66, 285–299 (2017).
Datlinger, P. et al. Pooled CRISPR screening with single-cell transcriptome readout. Nat. Methods 14, 297–301 (2017).
Mimitou, E. P. et al. Multiplexed detection of proteins, transcriptomes, clonotypes and CRISPR perturbations in single cells. Nat. Methods 16, 409–412 (2019).
Frangieh, C. J. et al. Multimodal pooled Perturb-CITE-seq screens in patient models define mechanisms of cancer immune evasion. Nat. Genet. 53, 332–341 (2021).
Replogle, J. M. et al. Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing. Nat. Biotechnol. 38, 954–961 (2020).
Xie, S., Cooley, A., Armendariz, D., Zhou, P. & Hon, G. C. Frequent sgRNA-barcode recombination in single-cell perturbation assays. PLoS ONE 13, e0198635 (2018).
Hill, A. J. et al. On the design of CRISPR-based single-cell molecular screens. Nat. Methods 15, 271–274 (2018).
Datlinger, P. et al. Ultra-high-throughput single-cell RNA sequencing and perturbation screening with combinatorial fluidic indexing. Nat. Methods 18, 635–642 (2021).
Replogle, J. M. et al. Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq. Cell 185, 2559–2575 (2022).
Papalexi, E. et al. Characterizing the molecular regulation of inhibitory immune checkpoints with multimodal single-cell screens. Nat. Genet. 53, 322–331 (2021).
Rubin, A. J. et al. Coupled single-cell CRISPR screening and epigenomic profiling reveals causal gene regulatory networks. Cell 176, 361–376 (2019).
Liscovitch-Brauer, N. et al. Profiling the genetic determinants of chromatin accessibility with scalable single-cell CRISPR screens. Nat. Biotechnol. 39, 1270–1277 (2021).
Pierce, S. E., Granja, J. M. & Greenleaf, W. J. High-throughput single-cell chromatin accessibility CRISPR screens enable unbiased identification of regulatory networks in cancer. Nat. Commun. 12, 2969 (2021).
Feldman, D. et al. Optical pooled screens in human cells. Cell 179, 787–799 (2019).
Wroblewska, A. et al. Protein barcodes enable high-dimensional single-cell CRISPR screens. Cell 175, 1141–1155 (2018).
The authors acknowledge all investigators whose contributions could not be discussed owing to space limitations, and N. Chapman for critical reading and editing of the manuscript. Research in the Chi laboratory was supported by ALSAC, US National Institutes of Health AI105887, AI131703, AI140761, AI150241, AI150514 and CA253188.
J.G.D. consults for Microsoft Research, Abata Therapeutics, Servier, Maze Therapeutics, BioNTech, Sangamo and Pfizer; consults for and has equity in Tango Therapeutics; serves as a paid scientific adviser to the Laboratory for Genomics Research, funded in part by GlaxoSmithKline; and receives funding support from the Functional Genomics Consortium: Abbvie, Bristol Myers Squibb, Janssen, Merck and Vir Biotechnology. Interests of J.G.D. were reviewed and are managed by the Broad Institute in accordance with its conflict of interest policies. H.C. consults for Kumquat Biosciences. H.S. declares no competing interests.
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- Base editing
A CRISPR–Cas9-based genome editing technology that introduces point mutations in DNA without generating double-strand breaks by tethering a nuclease-defective Cas9-D10A nickase variant (Cas9n) to a deaminase.
The average number of cells perturbed by each sgRNA in a pooled CRISPR screen, with a coverage of 500 cells generally recommended for screens to achieve optimal signal to noise ratio, although a lower coverage (such as 200 cells) may be used for enrichment screens.
- CRISPR-mediated genetically engineered mouse models
(CRISPR-GEMMs). Mouse models enabled by CRISPR technology for pooled targeting of multiple genes through somatic genome editing and large-scale direct in vivo screening.
A method that simultaneously captures transcripts encoding an sgRNA and carries out single-cell combinatorial indexing ATAC-seq based on a unique combination of barcodes, which tag both the sgRNA and ATAC fragments from each cell.
A platform that integrates the resolution of massively parallel scRNA-seq with the genome editing scale of pooled CRISPR screens, with sgRNA identification inferred by a transcribed poly-adenylated unique guide index on the same vector.
This platform makes use of an sgRNA delivery vector system that duplicates the sequence of a single-encoded sgRNA during lentiviral transduction to produce two expression cassettes on the same construct. One cassette expresses a functional sgRNA and the other expresses a polyadenylated transcript carrying the sgRNA sequence at the 3′ end for detection of individual sgRNAs in droplet-based single-cell RNA sequencing.
Expanded CRISPR-compatible CITE-seq, which enables 5′ capture-based scRNA-seq, T cell receptor or B cell receptor V(D)J reconstruction, and surface protein marker detection as readouts, together with single-cell sgRNA sequence capture.
Mosaic single-cell analysis by indexed CRISPR sequencing, a method that jointly measures the transcriptome of a cell and its sgRNA modulators inferred from the barcode sequences on the transduced sgRNA lentiviral vector backbone.
A method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling (assay for transposase accessible chromatin (ATAC)) in single cells captured by the Integrated Fluidics Circuit (Fluidigm) chambers.
A combined platform of 3′ droplet-based scRNA-seq with extracellular protein detection (by CITE-seq, which uses DNA-barcoded antibodies to convert the detection of proteins into a quantitative readout) and single-cell sgRNA detection. The method expresses an sgRNA on a polyadenylated transcript using a modified CROP-seq vector and performs a targeted ‘dial-out’ PCR amplification, which robustly links sgRNA identities to single-cell transcriptional and protein profiles.
A platform that combines a pooled CRISPR screen with single-cell RNA sequencing (scRNA-seq) by encoding the identity of the perturbation on an expressed guide barcode.
- Prime editing
A CRISPR–Cas9-based genome editing technology that introduces new sequence information into the genome by fusing Cas9-H840A nickase to a reverse transcriptase enzyme that promotes genome modification via a sequence template encoded within an extended prime editing guide RNA.
A protein barcoding vector system with combinatorial arrangements of linear epitopes, each paired with a different CRISPR sgRNA, for the analysis of multiple proteins to identify cells expressing different CRISPR sgRNAs at single-cell resolution.
The ordering of each cell along a developmental lineage based on gene expression as profiled by scRNA-seq.
- Single-guide RNA
(sgRNA). A single RNA molecule used to direct Cas9 protein to bind and cleave a particular DNA sequence for genome editing.
A platform that enables simultaneous readouts of chromatin accessibility profiles and integrated sgRNA from thousands of individual cells by reading out sgRNA spacer sequences directly from genomic DNA rather than from RNA transcripts.
- Tiling sgRNA library
An sgRNA library that is designed to incorporate many editing sites across the length of a gene and its regulatory elements to comprehensively evaluate their associated phenotypes.
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Shi, H., Doench, J.G. & Chi, H. CRISPR screens for functional interrogation of immunity. Nat Rev Immunol (2022). https://doi.org/10.1038/s41577-022-00802-4