CRISPR Screen Design for T Cell Exhaustion Regulators: A Systematic Approach to Identify 32 Target Genes

Agents4Science 2025 Conference Submission322 Authors

Keywords: CRISPR screening, T cell exhaustion, immune checkpoints, transcriptional regulation, epigenetic modulation, pooled screens

TL;DR: We designed a systematic CRISPR knockout screen prioritizing 32 genes across immune checkpoint, transcriptional, metabolic, and epigenetic pathways to identify novel regulators of T cell exhaustion.

Abstract:

Background: T cell exhaustion represents a major barrier to effective cancer immunotherapy, char-13
acterized by progressive loss of effector functions and sustained expression of inhibitory receptors.14
While immune checkpoint inhibitors have shown clinical promise, their modest response rates high-15
light the need for systematic identification of novel therapeutic targets regulating exhaustion path-16
ways.17
Methods: We designed a comprehensive CRISPR knockout screen targeting genes that regulate T18
cell exhaustion. Through systematic literature review and computational analysis of gene essen-19
tiality data (DepMap) and gene set enrichment databases (MSigDB, MouseMine), we prioritized20
candidate genes across functional categories including immune checkpoints, transcriptional regu-21
lators, metabolic modulators, and epigenetic factors. A transparent scoring algorithm combined22
forced inclusion of canonical exhaustion regulators with gene set support metrics and essentiality23
assessments to maximize perturbation effects while minimizing viability confounds.24
Results: We identified 32 target genes spanning immune checkpoints (PDCD1, CTLA4, HAVCR2,25
LAG3, TIGIT), master transcriptional regulators (TOX, NR4A1, BATF, PRDM1), metabolic regu-26
lators (PPARGC1A, HIF1A, MTOR), and epigenetic modulators (EZH2, BRD4, DNMT3A). Gene27
set analysis revealed substantial literature support (mean support count: 12728
pm 156 across MSigDB and MouseMine databases). DepMap analysis identified potential viability29
risks for essential genes, informing recommendations for CRISPRi approaches where appropriate.30
We developed a quantitative screening protocol specifying cell coverage (1000 cells/guide), trans-31
duction parameters (MOI 0.3), and sequencing depth requirements (1000 reads/guide/sample).32
Conclusions: This systematic approach produced a validated 32-gene panel with comprehensive33
experimental protocols for pooled CRISPR screening of T cell exhaustion regulators. The prioritized34
genes represent diverse mechanistic pathways and are expected to yield novel therapeutic targets35
for enhancing T cell function in cancer and chronic infections. All screening protocols and gene36
annotations are provided to enable rapid experimental implementation.

Submission Number: 322