Programmed genome editing by a miniature CRISPR-Cas12f nuclease

The RNA-guided CRISPR-associated (Cas) nucleases are versatile tools for genome editing in various organisms. The large sizes of the commonly used Cas9 and Cas12a nucleases restrict their flexibility in therapeutic applications that use the cargo-size-limited adeno-associated virus delivery vehicle. More compact systems would thus offer more therapeutic options and functionality for this field. Here, we report a miniature class 2 type V-F CRISPR-Cas genome-editing system from Acidibacillus sulfuroxidans (AsCas12f1, 422 amino acids). AsCas12f1 is an RNA-guided endonuclease that recognizes 5 ' T-rich protospacer adjacent motifs and creates staggered double-stranded breaks to target DNA. We show that AsCas12f1 functions as an effective genome-editing tool in both bacteria and human cells using various delivery methods, including plasmid, ribonucleoprotein and adeno-associated virus. The small size of AsCas12f1 offers advantages for cellular delivery, and characterizations of AsCas12f1 may facilitate engineering more compact genome-manipulation technologies. Miniature CRISPR-AsCas12f1 has been biochemically characterized and engineered as an effective genome-editing tool for bacteria and human cells.