PcoCas12a: A novel CRISPR enzyme from Prevotella copri enhancing TCR-T-cell tumor suppression.

Genome editing technologies have been widely utilized in cell engineering, demonstrating immense potential in cell and gene therapy. However, an optimal gene-editing enzyme for immune cell editing remains unidentified. In this study, we identified a novel gene editing enzyme, termed CRISPR/PcoCas12a, derived from Prevotella copri, which recognizes a 5'-YYN PAM sequence. We demonstrated that CRISPR/PcoCas12a offers a broader range of editing sites and superior editing efficiency at specific loci compared to AsCas12a. Furthermore, we illustrated its capability to enhance tumor suppression by targeting DGKα in TCR-T cells. DGKα functions as a negative regulator of T cell function, and its knockout significantly boosts the antitumor efficacy of TCR-T cells. The knockout efficiency and tumor suppressor ability of PcoCas12a targeting DGKα were markedly higher than those achieved with AsCas12a. Single-cell sequencing data confirmed that PcoCas12a-mediated DGKα gene knockout improves the tumor suppressive capabilities of T cells by promoting T-cell activation and strengthening immune regulatory responses. These findings establish PcoCas12a as a highly efficient enzyme for T cell editing, indicating its potential application in T-cell therapy.