Double knockout CRISPR screen for cancer resistance to T cell cytotoxicity

Abstract

Immunotherapy has transformed cancer treatments; however, a large fraction of patients encounter resistance. Such resistance is mediated by complex factors, often involving interactions between multiple genes. Thus, it is crucially important to identify genetic interactions between genes that are significantly mutated in cancer patients and those involved in immune responses, ideally the ones that currently have chemical compounds for direct targeting. To systematically interrogate such genetic interactions that mediate cancer cells’ response to T cell killing, we designed an asymmetric dual perturbation library targeting the matched combinations between significantly mutated tumor suppressors and immune resistance genes. We performed a combinatorial double knockout screen on 1159 gene pairs and identified those where joint loss-of-function renders altered cellular response to T cell cytotoxicity. We also performed comparative transcriptomics-based analyses on tumor and normal samples from TCGA and GTEx cohorts, mutational profiling analyses, and survival analyses to further characterize the significance of identified hits in clinical patients. Interactions between significantly mutated tumor suppressors and potentially druggable immune resistance genes may offer insights on potential new concepts of how to target clinically relevant cancer mutations with currently available agents. This study also provides a technology platform and an asymmetric double knockout library for interrogating genetic interactions between cancer mutations and immune resistance pathways under various settings.

Combinatorialin vivogenome editing identifies widespread epistasis during lung tumorigenesis

“…Cellular barcoding has facilitated the quantification of clonal heterogeneity, and the integration of clonal barcoding into CRISPR screens increased resolution both in vitro and in vivo 34,36 . Viral vectors with pairs of sgRNAs have been used for combinatorial genome editing of cells in culture, but these have lacked the ability to provide simultaneous clonal information 37,38 . Conversely, while barcoded dual-sgRNA vectors have led to key insights into the tumorigenic potential of complex tumor genotypes in vivo , they have required laborious individual cloning 39–42 and/or have suffered from lentiviral template switching that decouples the barcode from the sgRNA 43 .…”

“…Cellular barcoding has facilitated the quantification of clonal heterogeneity, and the integration of clonal barcoding into CRISPR screens increased resolution both in vitro and in vivo 34,36 . Viral vectors with pairs of sgRNAs have been used for combinatorial genome editing of cells in culture, but these have lacked the ability to provide simultaneous clonal information 37,38 .…”

Applications of CRISPR technology in cellular immunotherapy

“…For example, Cas12a/Cpf1 can effectively use multiple guide sequences in the same cell, allowing for combinatorial screens. These can be used to identify pairs of co‐mutational drivers of metastasis 128 or immune escape 129 . The use of a double‐knockout screen also presents opportunities to explore epistatic interactions between genes, including synergistic and antagonistic interactions 128–130 .…”

“…These can be used to identify pairs of co‐mutational drivers of metastasis 128 or immune escape 129 . The use of a double‐knockout screen also presents opportunities to explore epistatic interactions between genes, including synergistic and antagonistic interactions 128–130 . Such studies can also be aided by the use of mice with conditional/constitutive Cpf1 expression 131 .…”

“…129 The use of a double-knockout screen also presents opportunities to explore epistatic interactions between genes, including synergistic and antagonistic interactions. [128][129][130] Such studies can also be aided by the use of mice with conditional/constitutive Cpf1 expression. 131 Furthermore, Cpf1 double-knockout strategies can be combined with floxed gRNAs and an inducible-CRE system to enable sequential genetic editing.…”