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

• Published in: STAR protocols Vol. 3; no. 2; p. 101321
• Main Authors: Ling, Xinyu, Chang, Liying, Chen, Heqi, Liu, Tao
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.06.2022; Elsevier
• Subjects: Cell Biology; Cell culture; Cell isolation; Chemistry; CRISPR; CRISPR-Cas Systems - genetics; Flow Cytometry/Mass Cytometry; Gene Editing - methods; Humans; Immunology; Molecular Biology; Protein Biochemistry; Protein expression and purification; Protocol; Cell culture; CRISPR; Protein Biochemistry; Chemistry; Immunology; Molecular Biology; Flow Cytometry/Mass Cytometry; Protein expression and purification; Cell isolation; Cell Biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101321

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). [Display omitted] •Site-specific modification of Cas12a using noncanonical amino acid mutagenesis•A covalent Cas12a-crRNA complex can be prepared using bio-orthogonal chemistry•Cas12a-crRNA conjugates afford site-specific CAR-T preparation with high efficiency Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.