Table 2: Summary of CRISPR Genetic Screens with T Cells.

Reports are listed in chronological order of publication.

Citation	Cell type	In vitro/In vivo	Screening details	Screening assay	Technological advance and/or discovery
Okada et al. (99)	Immortalized mouse T-cell line (68–41)	In vitro	Genome-wide loss-of-function screen to identify genes involved in PD-1 expression induction/maintenance	Identified gRNA enriched in sorted PD-1low cells	Discovery: knocking out genes associated with core fucosylation decreases surface expression of PD-1 on T cells; treating antigen-specific murine T cells with a core fucosylation inhibitor decreases PD-1 expression and improves antigen-specific tumor control (B16-Ovalbumin) in vivo
Shang et al. (100)	Immortalized human T-cell line (Jurkat)	In vitro	Genome-wide loss-of-function screen to identify genes that regulate T-cell activation	gRNA abundance compared in CD69low and CD69high cells	Discovery: identified FAM49B as a negative regulator of TCR activation, which is involved in actin cytoskeleton remodeling
Ting et al. (102)	Primary human CD4+ T cells	In vitro	Proof-of-principle loss-of-function screen with ~13,000 gRNAs; developed Guide Swap method	Proof-of-principle report demonstrated feasibility of this screening methodology	Technological advance: development of Guide Swap to enable large-scale pooled screening with primary human cells (including T cells) that cannot be transduced with Cas9-encoding vectors
Shifrut et al. (103)	Primary human CD8+ T cells	In vitro	Genome-wide loss-of-function screens to identify genes that regulate T-cell stimulation and suppression; developed SLICE method	gRNA abundance compared in CFSEhigh and CFSElow T cells after TCR stimulation	Technological advance: development of SLICE screening method to enable large-scale pooled screening in primary human T cells Discovery: identified genes involved in T-cell suppression that, upon knockout, result in improved antigen-specific killing of tumor cells
Dong et al. (104)	Antigen-specific primary mouse CD8+ T cells	In vivo and in vitro	Genome-wide loss-of-function screen to identify genes that modulate CD8+ T cell effector function in vitro and in vivo	Identify gRNA associated with increased infiltration in tumors (in vivo); identify gRNA associated with increased T-cell degranulation (in vitro)	Discovery: complementary in vivo and in vitro screening reveals genes that can be targeted to improve T-cell function in the context of immunotherapy; authors validated that Dxh37 knockout in CD8+ T cells improves effector function in vitro and tumor control following adoptive transfer in vivo
Ye et al. (105)	Primary mouse CD8+ T cells	In vivo	Loss-of-function screen focused on genes encoding for membrane-bound proteins	Identify gRNA associated with enhanced T-cell infiltration in glioblastoma tumors following adoptive transfer	Discovery: identified multiple gene targets (novel and known) that, upon knockout, can increase the antitumor efficacy of adoptively transferred T cells; validated the potential of PDIA3 as an immunotherapy target using multiple mouse models with mechanistic characterization
Wei et al. (106)	Antigen-specific primary mouse CD8+ T cells	In vivo	Loss-of-function screen focused on metabolism-associated genes aimed at identifying factors to improve adoptive cell therapy for cancer	Identify gRNA associated with enhanced T-cell infiltration in B16 melanomas following adoptive transfer	Discovery: loss of REGNASE-1 improves CD8+ T-cell accumulation and persistence in the tumor microenvironment, improving antitumor efficacy. Follow-up genome-wide loss-of-function screen identified mechanistic targets of REGNASE-1 and candidates for combination therapy
Roth et al. (23)	Primary human T cells	In vitro and in vivo	Pooled knock-in screen to identify synthetic constructs that enhance T-cell fitness and antitumor activity; nonviral HDR template library delivered via Cas9 RNP electroporation	Identify constructs that enhance T-cell expansion in various contexts (in vitro) and that enhance T-cell infiltration and an anti-tumor phenotype in tumors (in vivo)	Major technologic advance: developed methodology to screen large numbers of knock-in constructs in vivo and in vitro using primary T cells Discovery: identified a novel switch receptor (TGFBR2–41BB) that promotes solid tumor clearance by tgTCR T cells in vivo