嵌合抗原受体
生物
清脆的
基因组编辑
Cas9
遗传增强
电穿孔
同源定向修复
逆转录病毒
计算生物学
基因
免疫系统
T细胞
遗传学
DNA修复
DNA错配修复
作者
Renpeng Ding,Cheng‐Chi Chao,Qianqian Gao
标识
DOI:10.1016/bs.mcb.2021.08.001
摘要
Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated promising efficacy in several kinds of blood cancers, including diffuse large B-cell lymphoma and acute and chronic lymphoblastic leukemia, etc. It is essential to effectively generate more potent and safer CAR-T cells through gene editing technologies for immune cell therapy. Conventional methods based on lentivirus, retrovirus and transposon, randomly integrate CAR sequence into T cell genome, which could lead to safety issues. Therefore, precise knock-in of CAR cassette into specific gene locus like TRAC and PDCD1 can lower the risks caused by random integration, as well as enhance the stability and function of the modified CAR-T cells. Current approaches of CRISPR/Cas9-based gene-editing have limitations in knock-in efficiency of the chimeric antigen receptor, while Cpf1, a CRISPR-Cas/RNA-guided nuclease, shows higher homology-directed repair (HDR) rate compared to Cas9 due to its unique biochemical characteristics. Here, we introduce a method combining electroporation and adeno-associated virus (AAV) infection to deliver CRISPR/Cpf1 components and a HDR template into T cells, thus precisely integrate CAR sequence at a specific gene locus with high efficiency.
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