Table 2.
CRISPR screens in T cells to develop advanced T-cell-based products for cancer immunotherapy
| Species | Target cells | Loss or gain of function | CRISPR library | Transduction methods | Selection methods | Genes identified | Corresponding proteins | Gene/protein functions | Years and references |
|---|---|---|---|---|---|---|---|---|---|
| Mouse | CD4+ T cells | loss of function | library pMSCV-U6gRNA(lib)-PGKpuroT2ABFP (Addgene: #104861) | retrovirus transduction | expression of IRF4, XBP1, or GATA3 | Pparg and Bhlhe40 | PPARG (peroxisome proliferator activated receptor gamma) and BHLHE40 (basic-helix-loop-helix protein 40) | PPARG and BHLHE40 are crucial to TH2 gene regulation and differentiation. Genes regulating TH2 activation and genes regulating TH2 differentiation are highly overlapped. | Henriksson et al. (2019)93 |
| CD8+ T cells | loss of function | retroviral mouse genome-wide CRISPR knockout library (Addgene #104861), containing 90,230 sgRNAs with 4 guides per gene | retrovirus transduction | in vitro T cell exhaustion assay | Arid1a | ARID1A | ARID1A depletion limits the acquisition of exhaustion-associated chromatin accessibility and leads to improved anti-tumor immunity. | Belk et al. (2022)94 | |
| CD8+ T cells | loss of function | a self-designed sgRNA library targeting exonic regions of 25 kinases showing kinase activity in T cells after TCR stimulation, with three sgRNAs per gene | electroporation | cell expansion, differentiation, oxidative stress, and genomic stress | Mapk14 | MAPK14 (p38-α) | Low level of MAPK14 improves the efficacy of mouse anti-tumor T cells. | Gurusamy et al. (2020)95 | |
| CD8+ T cell | loss of function | a self-designed domain-focused sgRNA library against 120 TFs, including 675 sgRNAs in total, with 4–5 sgRNAs per DNA-binding domain, positive selection controls (sgPdcd1), and non-selection controls | retrovirus transduction | cell proliferation | Fli1 | FLI1 | FLI1 depletion enhances effector T cells’ responses without compromising memory or exhaustion precursors whereas high level of FLI1 restrains differentiation. CD8+ T cells lacking FLI1 provides substantially better protection against multiple infections and tumors. | Chen et al. (2021)96 | |
| CD8+ T cells | loss of function | sgRNA Brie library; two lentiviral sub-libraries of sgRNAs (six sgRNAs per gene) targeting 3,017 metabolic enzymes, small molecule transporters and metabolism-related transcriptional regulators | lentivirus transduction | cell proliferation in tumor-infiltrating lymphocytes | Zc3h12a (Regnase-1) | REGNASE-1 | REGNASE-1-deficient CD8+ T cells are reprogrammed in the TME to long-lived effector cells by enhancing BATF function and mitochondrial metabolism, thereby improving adoptive cell therapy for cancer. | Wei et al. (2019)97 | |
| CD8+ T cells | loss of function | a focused sgRNA library (mouse surface and membrane protein-encoding gene library, Surf) targeting 1,658 membrane-bound protein-coding genes (four sgRNAs were chosen per gene similar to the mBrie library design49), with 6,628 sgRNAs and 1,000 NTCs | AAV transduction | cell proliferation in brain | Mgat5 and Pdia3 | MGAT5 and PDIA3 | Adoptive transfer of CD8+ T cells deficient in PDIA3, MGAT5, EMP1, or LAG3 enhances the survival of glioblastoma-bearing mice in both syngeneic and T cell receptor transgenic models. | Ye et al. (2019)98 | |
| CD8+ T cells | loss of function | a mouse genome-scale sgRNA library (MKO) containing 128,209 gene-specific sgRNAs that target every gene in the genome and 1,000 NTCs | lentivirus transduction | cell number in tumor | Dhx37 | DHX37 | DHX37 modulates CD8 T cell activation, cytokine production, and cytotoxicity. Dhx37 knockout in CD8 T cells enhances adoptive transfer efficacy. | Dong et al. (2019)99 | |
| CD8+ T cell | loss of function | a self-designed sgRNA library of 110 sgRNAs targeting 21 genes relevant to T cell biology and 50 NTC sgRNAs. | lentivirus transduction | cell proliferation | Ptpn2 | PTPN2 | PTPN2 is a negative regulator of CD8+ T-cell-mediated responses to LCMV clone 13 viral infection. | Lafleur et al. (2019)100 | |
| CD8+ hEAR2 targeting CAR-T cells | loss of function | a self-designed sgRNA library with 5 sgRNAs per gene, targeting 1,316 genes that are expressed differentially in in vivo activated T cells and naive T cells | retrovirus transduction | cell number in circulation | St3gal1 | ST3GAL1 | ST3GAL1 is a negative regulator of the tumor-specific CAR-T cell migration. | Hong et al. (2023)101 | |
| regulatory T cells | loss of function | a self-designed sgRNA library against 489 targets with 4 guides per gene on the basis of the Brie library to identify gene regulatory programs that promote or disrupt Foxp3 expression | retrovirus transduction | Foxp3 expression | Usp22 and Rnf20 | USP22 and RNF20 | Usp22 is revealed to be a positive regulator that stabilized Foxp3 expression. Rnf20 can serve as a negative regulator of Foxp3. | Cortex et al. (2020)102 | |
| Human | CD4+ and CD8+ T cells | gain of function | a lentiviral library of barcoded human ORFs; nearly 12,000 full-length genes with around 6 barcodes per gene | lentivirus transduction | cell proliferation | LTBR | LTBR | When overexpressed in T cells, LTBR induces profound transcriptional and epigenomic remodeling, leading to increased T cell effector functions and resistance to exhaustion in chronic stimulation settings through constitutive activation of the canonical NF-κB pathway. | Legut et al. (2022)103 |
| CD8+ T cells | loss of function | pMD2.G (Addgene, catalog #12259) and psPAX2 (Addgene, catalog #12260) containing 77,441 sgRNAs (19,114 genes) | SLICE | cell division tested by CFSE | DGKA, DGKZ, TCEB2, SOCS1, UBASH3A, CBLB, CD5, RNF7, CUL5, TNFAIP3, TNIP1, and RASA2 | DGKA, DGKZ, TCEB2, SOCS1, UBASH3A, CBLB, CD5, RNF7, CUL5, TNFAIP3, TNIP1, and RASA2 | Cells deficient in identified proteins show a marked increase in number of divisions post stimulation compared with controls. | Shifrut et al. (2018)35 | |
| CD8+ NY-ESO-1 TCR-specific T cells | loss of function | CRISPR-Cas9 pooled library (rank candidate genes → single gene validation experiment for high ranking genes) | lentivirus transduction | immunofluorescence staining (CD107a+) after an exhaustion assay | SNX9 | SNX9 | Depletion of SNX9 enhances memory differentiation, prevents T cell exhaustion, and improves anti-tumor efficacy. | Trifny et al. (2023)104 | |
| HA-28z targeting CAR-T cells | loss of function | a self-designed sgRNA library containing 19,885 genes targeted with at least four sgRNAs per gene | SLICE | replicate expansion screen; cytokine production screen | MED12 and CCNC | MED12 and CCNC | Deletion of MED12 or CCNV in human CAR-T cells results in increased proliferation, cytokine production, and increases tumor clearance by reducing steric hindrance between core Mediator and RNAPII. | Freitas et al. (2022)105 | |
| CD8+ T cells for screening; CD19 targeting CAR-T cells (for validation) | loss of function | the genome-wide Brunello sgRNA library | SLICE | cell proliferation tested by CFSE | RASA2 | RASA2 | RASA2-deficient T cells show increased activation, cytokine production, and metabolic activity in repeated tumor antigen stimulations, and demonstrate an advantage in persistent cancer cell killing. | Carnevale et al. (2022)106 | |
| CD8+ CAR-T cells | gain of function | a self-designed lentiviral mouse genome-scale dead-guide RNA library (mm10dgLib) using the promoter sequences of all annotated protein-coding transcripts from the mm10 genome assembly. The final mm10dgLib consists of 84,601 dead-guide RNAs that target 22,391 coding transcripts and 1,000 NTCs. | lentivirus transduction | intracellular flow cytometry (CD107+) after a kill assay | PRODH2 | PRODH2 | High level of PRODH2 enhances CD8+ T cell effector function. | Ye et al. (2022)107 |
AAV, adeno-associated virus; ARID1A, AT-rich interactive domain-containing protein 1A; CCNC, cyclin C; DHX37, DEAH-box helicase 37; FLI1, friend leukemia integration 1 transcription factor; LCMV, lymphocytic choriomeningitis virus; LTBR, lymphotoxin beta receptor; MAPK14, mitogen-activated protein kinase 14; MED12, mediator complex subunit 2; MGAT5, alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase A; NTC, nontargeting control; ORF, open reading frame; PDIA3, protein disulfide isomerase associated 3; PRODH2, proline dehydrogenase 2; PTPN2, protein tyrosine phosphatase nonreceptor type 2; RASA2, RAS p21 protein activator 2; REGNASE-1, regulatory RNase 1; RNF20, ring finger protein 20; SLICE, sgRNA lentiviral infection with Cas 9 electroporation; SNX9, sorting nexin 9; ST3GAL1, ST3 β-galactoside α-2,3-sialyltransferase 1; USP22, ubiquitin-specific peptidase 22.