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

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).

SUBMITTER: Ling X

PROVIDER: S-EPMC9038777 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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

Ling Xinyu X Chang Liying L Chen Heqi H Liu Tao T

STAR protocols 20220414 2

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, hi ...[more]

PMID: 35496795

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