针对细胞治疗应用的靶向敲入效率一般较低，规模也有限。在本研究中，我们开发了CLASH系统，能够实现高效率、高通量的敲入工程。在CLASH中，Cas12a/Cpf1 mRNA与混合的腺相关病毒共同介导大规模同源重组，同时进行基因编辑和精确的转基因敲入，从而产生一批稳定整合的突变变异体，每个变异体都有针对性的基因编辑。我们将这项技术应用于人类原代T细胞，并在血液癌和实体瘤模型中进行了时间序列的CLASH实验，利用CD3、CD8和CD4 T细胞进行池化生成和无偏选择有利的CAR-T变异体。从CLASH实验中，一种独特的CRISPR RNA（crRNA）在CAR-T中产生了PRDM1的外显子3跳变型，导致这些细胞在增殖、干细胞样特性、中央记忆和寿命方面增强，在多个癌症模型中，包括实体瘤模型，表现出更高的体内效价。CLASH的通用性使其在各种细胞和治疗工程应用中广泛适用。 ©2023年。作者（们）独家许可Springer Nature America，Inc.

The efficiency of targeted knock-in for cell therapeutic applications is generally low, and the scale is limited. In this study, we developed CLASH, a system that enables high-efficiency, high-throughput knock-in engineering. In CLASH, Cas12a/Cpf1 mRNA combined with pooled adeno-associated viruses mediate simultaneous gene editing and precise transgene knock-in using massively parallel homology-directed repair, thereby producing a pool of stably integrated mutant variants each with targeted gene editing. We applied this technology in primary human T cells and performed time-coursed CLASH experiments in blood cancer and solid tumor models using CD3, CD8 and CD4 T cells, enabling pooled generation and unbiased selection of favorable CAR-T variants. Emerging from CLASH experiments, a unique CRISPR RNA (crRNA) generates an exon3 skip mutant of PRDM1 in CAR-Ts, which leads to increased proliferation, stem-like properties, central memory and longevity in these cells, resulting in higher efficacy in vivo across multiple cancer models, including a solid tumor model. The versatility of CLASH makes it broadly applicable to diverse cellular and therapeutic engineering applications.© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.