Structure and engineering of miniature Acidibacillus sulfuroxidans Cas12f1

The miniature CRISPR-Cas12f nucleases allow for efficient delivery via cargo-size-limited vehicles, thereby showing promising potential for in vivo therapeutic applications. Acidibacillus sulfuroxidans Cas12f1 (AsCas12f1, 422 amino acids) is one of the most compact Cas12f nucleases, showing a moderate genome-editing activity in human cells compared to Cas9 and Cas12a. Understanding the mechanisms of why such a compact nuclease is active for genome editing would facilitate its rational engineering. Here we show the cryo-EM structure of the AsCas12f1-sgRNA-dsDNA ternary complex, and reveal that AsCas12f1 functions as an asymmetric dimer for sgRNA binding and DNA targeting. The mechanisms of dimer formation, PAM recognition, and sgRNA accommodation are elucidated. Thereby, we extensively engineer this system and result in an evolved AsCas12f1-sgRNA combination with drastically enhanced genome editing activity in human cells. These results provide further understanding of compact CRISPR systems and expand the mini CRISPR toolbox for therapeutic applications. ### Competing Interest Statement Q.J., Zhaowei Wu, and D.P. have filed a patent application (PCT/CN2022/113357) related to this work through ShanghaiTech University. The remaining authors declare no competing interest.