Methods in cell biologyFeb 14, 2022

Highly efficient genetic editing using CRISPR/Cpf1 for improved CAR-T cell therapy

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Mar 21, 2026

Abstract

A new method achieves high efficiency in integrating chimeric antigen receptor (CAR) sequences into specific gene loci in T cells.

Current CAR-T cell therapies show effectiveness against various blood cancers.
Conventional gene editing methods may pose safety risks due to random integration of CAR sequences.
Precise integration of CAR sequences into specific sites like TRAC and PDCD1 may improve stability and functionality of CAR-T cells.
Cpf1, a CRISPR technology, may offer higher rates of targeted gene integration compared to traditional Cas9 methods.
The combination of electroporation and adeno-associated virus (AAV) infection enhances the delivery of CRISPR/Cpf1 components and HDR templates into T cells.

Simplified

Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated promising efficacy in several kinds of blood cancers, including diffuse large B-cell lymphoma and acute and chronic lymphoblastic leukemia, etc. It is essential to effectively generate more potent and safer CAR-T cells through gene editing technologies for immune cell therapy. Conventional methods based on lentivirus, retrovirus and transposon, randomly integrate CAR sequence into T cell genome, which could lead to safety issues. Therefore, precise knock-in of CAR cassette into specific gene locus like TRAC and PDCD1 can lower the risks caused by random integration, as well as enhance the stability and function of the modified CAR-T cells. Current approaches of CRISPR/Cas9-based gene-editing have limitations in knock-in efficiency of the chimeric antigen receptor, while Cpf1, a CRISPR-Cas/RNA-guided nuclease, shows higher homology-directed repair (HDR) rate compared to Cas9 due to its unique biochemical characteristics. Here, we introduce a method combining electroporation and adeno-associated virus (AAV) infection to deliver CRISPR/Cpf1 components and a HDR template into T cells, thus precisely integrate CAR sequence at a specific gene locus with high efficiency.

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Highly efficient genetic editing using CRISPR/Cpf1 for improved CAR-T cell therapy

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