State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191, China.
STAR Protoc. 2022 Apr 14;3(2):101321. doi: 10.1016/j.xpro.2022.101321. eCollection 2022 Jun 17.
We recently developed a system to create human chimeric antigen receptor (CAR)-T cells using conjugated Cas12a (cCas12a) in which Cas12a is covalently linked to its CRISPR RNA (crRNA). This protocol describes site-specific modification of Cas12a and the preparation of Cas12a-crRNA complex using bio-orthogonal chemistry, followed by CAR-T cell generation through electroporation and AAV infection. This system shows robust editing efficiency in human cells and can be used for precisely targeted, highly efficient integration of CAR genes into T cell genome. For complete details on the use and execution of this protocol, please refer to Ling et al. (2021).
我们最近开发了一种使用连接 Cas12a（cCas12a）的方法来产生嵌合抗原受体（CAR）-T 细胞的系统，其中 Cas12a 通过共价键与其 CRISPR RNA（crRNA）相连。本方案描述了 Cas12a 的定点修饰以及 Cas12a-crRNA 复合物的制备，使用生物正交化学方法，然后通过电穿孔和 AAV 感染产生 CAR-T 细胞。该系统在人细胞中显示出强大的编辑效率，可用于将 CAR 基因精确靶向、高效地整合到 T 细胞基因组中。如需了解本方案的使用和执行的详细信息，请参考 Ling 等人（2021 年）。
