The development of potent, off-the-shelf T cell therapies often necessitates multiple genetic alterations. Traditional CRISPR-Cas nucleases, which induce DNA double-strand breaks (DSBs), can lead to genomic rearrangements, posing safety concerns for edited cells. We integrate a non-viral CRISPR-Cas nuclease-assisted method for gene knock-in with a SpCas9-derived base editing technique for DSB-free knock-outs. This combination allows for simultaneous and efficient gene modification within a single intervention.

Co-delivery of SpCas9 nuclease for knock-in and SpCas9 base editors enabled the insertion of a chimeric antigen receptor (CAR) into the T cell receptor alpha constant (TRAC) gene and the concurrent knock-out of major histocompatibility complexes (MHC) class I and II expression. While translocations between the targeted sites were reduced over conventional SpCas9 multiplexing, insertions and deletions at the base editing sites suggest significant frequency of DSB, presumably due to guide RNA interchange. By combining AsCas12a nuclease for CAR knock-in with a SpCas9-derived base editor, we efficiently produce triple-edited CAR T cells with significantly reduced translocation frequency, akin to that of unedited cells. These cells demonstrate resistance to allogeneic T cell targeting in vitro. The single-step method was also adapted to improve large-scale manufacturing of allogeneic MHC-I/II-silenced regulatory T cells overexpressing HLA-E-B2M fusion gene to evade NK cell lysis. 

Our findings suggest a viable strategy for non-viral gene transfer and effective gene silencing or epitope masking through the use of distinct CRISPR enzymes for knock-in and base editing. This approach could pave the way for safer multiplex-edited cell products.