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.

微型 CRISPR-Cas12f 核酸酶能够通过货物大小有限的载体进行有效递送，从而显示出体内治疗应用的前景。氧化硫酸性杆菌Cas12f1（AsCas12f1，422 个氨基酸）是最紧凑的 Cas12f 核酸酶之一，与 Cas9 和 Cas12a 相比，在人类细胞中表现出中等的基因组编辑活性。了解如此紧凑的核酸酶为何对基因组编辑具有活性的机制将有助于其合理的工程设计。在这里，我们展示了 AsCas12f1-sgRNA-dsDNA 三元复合物的冷冻电子显微镜结构，并揭示了 AsCas12f1 作为 sgRNA 结合和 DNA 靶向的不对称二聚体。阐明了二聚体形成、原型间隔子相邻基序识别和 sgRNA 调节的机制。基于这些发现，我们对该系统进行了广泛的设计，并产生了一种进化的 AsCas12f1-sgRNA 组合，其在人类细胞中的基因组编辑活性显着增强。