BGI-Shenzhen, Shenzhen, China; BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, China; Guangdong Provincial Key Laboratory of Human Disease Genomics, Shenzhen Key Laboratory of Genomics, Guangdong, China.
BGI-Shenzhen, Shenzhen, China.
Methods Cell Biol. 2022;167:1-14. doi: 10.1016/bs.mcb.2021.08.001. Epub 2021 Sep 15.
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.
嵌合抗原受体 T（CAR-T）细胞疗法在多种血液癌症中显示出了有前景的疗效，包括弥漫性大 B 细胞淋巴瘤、急性和慢性淋巴细胞白血病等。通过基因编辑技术有效地生成更有效和更安全的 CAR-T 细胞对于免疫细胞治疗至关重要。基于慢病毒、逆转录病毒和转座子的传统方法，随机地将 CAR 序列整合到 T 细胞基因组中，这可能会导致安全性问题。因此，将 CAR 盒精确地敲入 TRAC 和 PDCD1 等特定基因座，可以降低随机整合引起的风险，同时增强修饰的 CAR-T 细胞的稳定性和功能。基于 CRISPR/Cas9 的基因编辑的当前方法在嵌合抗原受体的敲入效率方面存在限制，而 Cpf1（一种 CRISPR-Cas/RNA 指导的核酸内切酶）由于其独特的生化特性，与 Cas9 相比具有更高的同源定向修复（HDR）率。在这里，我们介绍了一种结合电穿孔和腺相关病毒（AAV）感染的方法，将 CRISPR/Cpf1 组件和 HDR 模板递送到 T 细胞中，从而以高效率将 CAR 序列精确地整合到特定基因座。
