Interrogating immune cells and cancer with CRISPR-Cas9

Recent methods have enabled primary human and mouse T cell CRISPR-Cas9 screens, identifying novel regulators of T cell differentiation and function. In vivo T cell screens revealed regulators of T cell differentiation in the context of infection and cancer, including FLI1-mediated restriction of effector CD8+ T cell differentiation. Regulatory T cell (Treg) CRISPR-Cas9 screens unveiled new mechanisms of FOXP3 regulation, including deubiquitination of the Foxp3 gene locus by USP22 in mice. CRISPR-Cas9 screens in cancer cells identified novel mechanisms of tumor cell-intrinsic immune evasion, such as negative regulation of IFNγ signaling by the SWI/SNF chromatin remodeling complex. Preclinical studies have shown that CRISPR-Cas9 can be used to enhance CD8+ T cell function, leading to the first-in-human clinical trial using CRISPR-Cas9 edited T cells. CRISPR-Cas9 technologies have transformed the study of genetic pathways governing cellular differentiation and function. Recent advances have adapted these methods to immune cells, which has accelerated the pace of functional genomics in immunology and enabled new avenues for the design of cellular immunotherapies for cancer. In this review, we summarize recent developments in CRISPR-Cas9 technology and discuss how they have been leveraged to discover and manipulate novel genetic regulators of the immune system. We envision that these results will provide a valuable resource to aid in the design, implementation, and interpretation of CRISPR-Cas9-based screens in immunology and immuno-oncology. CRISPR-Cas9 technologies have transformed the study of genetic pathways governing cellular differentiation and function. Recent advances have adapted these methods to immune cells, which has accelerated the pace of functional genomics in immunology and enabled new avenues for the design of cellular immunotherapies for cancer. In this review, we summarize recent developments in CRISPR-Cas9 technology and discuss how they have been leveraged to discover and manipulate novel genetic regulators of the immune system. We envision that these results will provide a valuable resource to aid in the design, implementation, and interpretation of CRISPR-Cas9-based screens in immunology and immuno-oncology. assay for transposase-accessible chromatin using sequencing; assays genome-wide chromatin accessibility by transposition with Tn5 transposase. Cas9-mediated conversion of single base pairs within a specified window, mediated by fusion of Cas9 to a deaminase enzyme. C57BL/6J mouse genetically engineered to constitutively express Cas9; Gt(ROSA)26Sortm1.1(CAG-cas9⁎,-EGFP)Fezh/Gt(ROSA)26Sortm1.1(CAG-cas9⁎,-EGFP)Fezh. Cas9, engineered to have one or both of its nuclease domains; retains genome homing but loses DSB potential. blocking antibodies used to inhibit the action of immune checkpoint receptors such as PD-1 or CTLA-4. T cells that are genetically modified to express a chimeric antigen receptor, typically specific for a tumor-associated antigen and used for adoptive cell transfer immunotherapy. assays for 3D DNA interactions in the nucleus. immune cell type required for antigen presentation and T cell priming. methods for CRISPR screening paired with a single-cell RNA sequencing readout. CRISPR activation or inhibition; leverages dCas9 fusions to activate or inhibit transcription of sgRNA target genes. presentation of cell exogenous antigen on MHC class I by professional antigen presenting cells; MHC class I required for activation of antigen specific CD8+ T cells. cleavage under targets and release using nuclease; chromatin profiling method in which antibody-targeted regions of DNA are cleaved by nucleases and sequenced. homologous DNA strands that flank gene insertion cassette, used to activate cell-intrinsic homology directed repair pathways. Cas9 with a single nuclease domain inactivation; creates single-stranded nicks on target DNA. DNA damage response pathway that prioritizes end-joining. CD8+ T cells engineered to express the OT-1 TCR, specific for the SIINFEKL antigen on ovalbumin (Ova). short DNA motif required for Cas9 binding to target site; different Cas9 orthologs have different PAM sequence requirements. RNA engineered to contain both the crRNA (targeting RNA) and trRNA (Cas9 binding RNA).