Abstract
Rapid modular CAR-T generation with CRISPR/Cpf1 and AAV systems
Highlights
We describe a new method, the AAV-Cpf[1] KIKO system, using a combination of viral and nonviral approaches to generate a stable CAR-T with homology-directed repair \(HDR) knock-in and immune checkpoint knockout at high efficiency in one step
Modified T cells expressing anti-CD19 CARs have showcased their efficacy in various liquid cancers 1-5, and have been approved for clinical use in B-cell lymphomas and leukemias 6-8
The generally used method to modify human T cells is currently based on the CRISPR/Cas[9] system 10
Summary
Modified T cells expressing anti-CD19 CARs have showcased their efficacy in various liquid cancers 1-5, and have been approved for clinical use in B-cell lymphomas and leukemias 6-8. We describe a new method, the AAV-Cpf[1] KIKO system, using a combination of viral and nonviral approaches to generate a stable CAR-T with homology-directed repair \(HDR) knock-in and immune checkpoint knockout at high efficiency in one step. We describe a new method for generating CAR-T cells using a combination of mRNA electroporation for LbCpf[1] and AAV6 for crRNA and homology-directed repair \(HDR) template. 1. Amplify left and right homologous arms of the TRAC or PDCD1 locus from primary CD4+ T cells by PCR using locus-specific primer sets with multiple cloning site \(MCS).
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