上海科技大学知识管理系统

The miniature CRISPR-Cas12f nucleases enable efficient delivery via cargo-size-limited vehicles, thereby showing promise for in vivo therapeutic applications. Acidibacillus sulfuroxidans Cas12f1 (AsCas12f1, 422 amino acids) is one of the most compact Cas12f nucleases and exhibits moderate genome-editing activity in human cells compared with 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-electron microscopy 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, protospacer adjacent motif recognition and sgRNA accommodation are elucidated. Based on these findings, we extensively engineer this system and have produced 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. [Figure not available: see fulltext.]. © 2023, The Author(s), under exclusive licence to Springer Nature Limited.