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

Xinyu Ling; Liying Chang; Heqi Chen; Tao Liu

doi:10.1016/j.xpro.2022.101321

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

STAR Protoc. 2022 Apr 14;3(2):101321. doi: 10.1016/j.xpro.2022.101321. eCollection 2022 Jun 17.

Authors

Xinyu Ling¹, Liying Chang¹, Heqi Chen¹, Tao Liu¹

Affiliation

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.

Abstract

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).

Keywords: CRISPR; Cell Biology; Cell culture; Cell isolation; Chemistry; Flow Cytometry/Mass Cytometry; Immunology; Molecular Biology; Protein Biochemistry; Protein expression and purification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems* / genetics
Gene Editing* / methods
Humans