exoCasMINI: A T5 exonuclease fused CRISPR-Cas12f system with enhanced gene editing efficiency.

CRISPR-Cas9 and Cas12a are widely used for genome editing, but their large size limits delivery efficiency. Compact Cas12f proteins offer delivery advantages but suffer from low activity. To address this limitation, we engineered an enhanced Cas12f system (exoCasMINI) by fusing T5 exonuclease to CasMINI, achieving 1.1- 21.1-fold higher editing efficiency while maintaining specificity. exoCasMINI matched the activity of SpCas9 and LbCas12a, induced longer deletions, and exhibited superior specificity to SpCas9. In addition, exoCasMINI was more efficient than CasMINI to induce tumorigenesis in adult mouse liver by integrating the oncogenic Kras (G12D) into the Trp53 locus and disrupting the tumor suppressor genes Trp53 and Pten. We extended this approach to another Cas12f subtype (RhCas12f1), generating exoRhCas12f1 with 1.2-3.6-fold enhanced activity. Overall, our work establishes the engineered exoCasMINI and exoRhCas12f1 systems as highly efficient tools for genome editing in mammalian cells, holding great potential for gene therapy in the future.