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Massively parallel knock-in engineering of human T cells.
- Source :
-
Nature Biotechnology . Sep2023, Vol. 41 Issue 9, p1239-1255. 17p. - Publication Year :
- 2023
-
Abstract
- The efficiency of targeted knock-in for cell therapeutic applications is generally low, and the scale is limited. In this study, we developed CLASH, a system that enables high-efficiency, high-throughput knock-in engineering. In CLASH, Cas12a/Cpf1 mRNA combined with pooled adeno-associated viruses mediate simultaneous gene editing and precise transgene knock-in using massively parallel homology-directed repair, thereby producing a pool of stably integrated mutant variants each with targeted gene editing. We applied this technology in primary human T cells and performed time-coursed CLASH experiments in blood cancer and solid tumor models using CD3, CD8 and CD4 T cells, enabling pooled generation and unbiased selection of favorable CAR-T variants. Emerging from CLASH experiments, a unique CRISPR RNA (crRNA) generates an exon3 skip mutant of PRDM1 in CAR-Ts, which leads to increased proliferation, stem-like properties, central memory and longevity in these cells, resulting in higher efficacy in vivo across multiple cancer models, including a solid tumor model. The versatility of CLASH makes it broadly applicable to diverse cellular and therapeutic engineering applications. Unbiased selection of CAR-T cells is achieved by massively parallel knock-in engineering. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 10870156
- Volume :
- 41
- Issue :
- 9
- Database :
- Academic Search Index
- Journal :
- Nature Biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 171898008
- Full Text :
- https://doi.org/10.1038/s41587-022-01639-x