Molecular therapy : the journal of the American Society of Gene TherapyAug 28, 2025

Using separate CRISPR tools for precise gene insertion and multiple base changes to create off-the-shelf CAR-T cells without viruses

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jun 27, 2026

Abstract

Essence

An orthogonal CRISPR strategy multiplexed CAR-T cell engineering while reducing translocations from knockout editing.

Evidence

Preclinical primary human T-cell experiments combined SaCas9 base editing of B2M and REGNASE-1 with SpCas9 anti-CD19 CAR integration, reaching 66% and 84% knockout editing and 36% nonviral or 71% AAV6 integration.

Caveat

The results are platform and preclinical function data, not clinical evidence that the engineered allogeneic CAR-T cells are safer or more effective.

Simplified

Multiple genomic modifications, including targeted transgene integrations and knockouts, may be required to develop potent, allogeneic chimeric antigen receptor (CAR)-T cell therapies. Conventional CRISPR-Cas systems generate double-strand breaks (DSBs) associated with genomic rearrangements and genotoxicities. DSB-free base editing reduces these risks. Here, we facilitate multiplex editing by combining Staphylococcus aureus Cas9 (SaCas9) mRNA base editors for DSB-free knockout of B2M and REGNASE-1 with Streptococcus pyogenes Cas9 nucleases for targeted integration of an anti-CD19 CAR transgene at the T cell receptor α constant locus. Combined, these edits have been reported to generate safer allogeneic CAR-T cells with enhanced activity and persistence. We demonstrate multiplex gene editing in primary human T cells with B2M and REGNASE-1 base editing frequencies reaching 66% and 84%, respectively, while integrating the anti-CD19 CAR transgene in up to 36% or 71% of cells using nonviral single-stranded DNA repair templates or viral vector templates (AAV6), respectively. Importantly, no detrimental effects on CAR-T cell function were observed in vitro or in vivo, and knockout by base editing reduced rates of balanced chromosomal translocations by 210-fold. This orthogonal CRISPR-Cas engineering approach represents a novel and safer strategy for nonviral, multiplexed genetic engineering of CAR-T cells.