Efficient generation of locus-specific human CAR-T cells with CRISPR/cCas12a

STAR Protocols

Tao Liu¹  Liying Chang¹  Xinyu Ling¹  Heqi Chen¹ 

[1] 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;

关键词: Cell Biology;    Cell culture;    Cell isolation;    Flow Cytometry/Mass Cytometry;    Immunology;    Molecular Biology;   

DOI  :  

来源: DOAJ

【 摘 要 】

Summary: 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).

【 授权许可】

Unknown