Assessment of Miniature AsCas12f1 Variants for Gene Editing and Activation

ABSTRACT Miniature CRISPR/Cas systems possess delivery advantages for gene therapy. The type V‐F Cas12f1 from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been engineered by several studies as compact genome editing tools through protein and single guide RNA (sgRNA) engineering. However, a comparative evaluation of gene editing and activation efficiencies mediated by different AsCas12f1 variants and sgRNA scaffolds is lacking. This study tested combinations of four AsCas12f1 protein variants and six sgRNA scaffolds for their gene editing and transcription activation efficiencies. The protein variant AsCas12f1‐HKRA performed the best in gene editing and activation when paired with sgRNA‐en_v2.1 scaffold. Furthermore, we validated a super miniature gene activator by fusing a small activation domain to AsCas12f1‐HKRA. Our findings recommend using AsCas12f1‐HKRA and sgRNA‐en_v2.1 for gene editing and activation applications.