Work

Engineered Un1Cas12f1 for multiplex genome editing with enhanced activity and targeting scope

Year: 2026

Type: article

Abstract: The compact CRISPR-Cas12f system is promising for AAV-delivered gene therapy, but its application has been constrained by restrictive PAM recognition (e.g., TTTR) and suboptimal editing efficiency. Through bacterial library screening and mammalian cell validation, we engineer evoCas12f, an optimized variant incorporating five key mutations, that dramatically expands PAM recognition to NTNR/NYTR. This advancement reduces median distance between two neighbouring PAM sites to 2 nucleotides in the human genome. It also demonstrates 1.4-fold enhanced activity at TTTR sites compared to wild-type Un1Cas12f1, achieving up to 91% editing efficiency. Remarkably, evoCas12f enables efficient generation of homozygous mutations in F0 generation mice, even at non-canonical PAM sites. We further adapt this system for robust transcriptional activation and precise base editing with a well-defined editing window. As a compact yet highly efficient platform, evoCas12f represents a significant advance in CRISPR technology, enabling multiplexed editing for high-resolution targeting applications and expanding possibilities for therapeutic genome engineering. Compact Un1Cas12f1 enzyme suits AAV delivery but is limited by narrow PAMs and modest activity. Here, authors engineer evoCas12f to recognize broader NTNR/NYTR PAMs and boost editing efficiency up to 91%, enabling efficient multiplex editing, base editing, and gene activation in cells and mice.

Source: Nature Communications

DOI: 10.1038/s41467-026-69678-5