Journal
STAR PROTOCOLS
Volume 3, Issue 2, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.xpro.2022.101321
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Funding
- National Key Research and Development Program of China [2021YFA0909900]
- National Natural Science Foundation of China [21922701, 91853111, 21778005]
- Beijing Natural Science Foundation [JQ20034]
- Peking University and Innovation Fund for Outstanding Doctoral Candidates of Peking University Health Science Center [71006Y2460]
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A system utilizing conjugated Cas12a and its CRISPR RNA is developed to efficiently edit human cells and precisely integrate CAR genes into the T cell genome.
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).
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