改进 CAR-T 细胞中的 CRISPR-Cas9 筛选：一种优化的文库制备方法

Chimeric antigen receptor (CAR) T cell therapies have demonstrated remarkable efficacy in several hematological malignancies, yet their success has not been fully replicated in solid tumors.

Moreover, even in hematological cancers, relapse after CAR T cell infusion continues to compromise long-term outcomes. These challenges highlight the urgent need to develop strategies that enhance CAR T cell efficacy, persistence, overcoming tumor and microenvironment-mediated resistance. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based screening platforms provide a powerful approach to systematically identify genes that regulate CAR T cell function.

By linking genetic perturbations to phenotypic outcomes, these assays enable the discovery of pathways controlling activation, proliferation, memory formation, and cytotoxicity. Standard workflows involve transduction of substantial numbers of cells with a single guide RNA (sgRNA) library, Cas9-mediated editing, selection of edited cells, and PCR amplification of sgRNA cassettes from genomic DNA (gDNA) prior to sequencing.

However, PCR amplification using large amounts of gDNA poses significant challenges and often fails to selectively amplify and retrieve sgRNAs.

Here, we describe an optimized CRISPR-Cas9 knockout screening protocol, which we have tested on primary human CAR T cells. The method here incorporates an intermediate step during sgRNA library preparation that reduces gDNA carryover through enzymatic digestion and selective pulldown of the sgRNA cassette, thereby increasing the efficiency of the first PCR amplification.

This modification allowed us to retrieve sgRNA information across our CAR T cell screens, which had remained elusive in our previous attempts using traditional 1 and 2-step PCR amplification protocols.

In conclusion, this optimized workflow facilitates CRISPR screening library preparation in challenging samples and enables the identification of key genetic determinants that can be targeted to improve therapeutic efficacy.