Abstract

CRISPR-Cas nucleases such as Cas9 and Cas12a have revolutionized genome editing, but their large size hinders delivery via adeno-associated viruses (AAV). The miniature AsCas12f1 from Acidibacillus sulfuroxidans (422 amino acids) offers therapeutic potential but is limited by its stringent T-rich PAM requirements in human cells. Here, we report an engineered AsCas12f1 variant, AsCas12f1-M5 (G57R/M114Y/M157V/S188R/D311K, M5), which efficiently recognizes both canonical 5′-TTR PAMs and a set of six non-canonical 5′-TYN PAMs, substantially expanding the accessible PAM landscape relative to the wild type AsCas12f1. In human cells, M5 exhibits up to an 11.6-fold enhancement in nuclease activity relative to the wild type. In vivo , we achieved AAV-mediated delivery of M5 to murine livers, and the intended loss-of-function edit reduced serum transthyretin levels by 30%. Moreover, zygote injection enabled efficient Tyr gene editing with visible pigmentation loss in offspring. These results collectively demonstrate AsCas12f-M5 as a compact nuclease with expanded PAM recognition and enhanced nuclease activity and supports efficient AAV-mediated genome editing.