Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907, USA.
Purdue University Center for Cancer Research, Purdue University, 915 W. State Street, West Lafayette, IN 47907, USA.
Nucleic Acids Res. 2021 Apr 19;49(7):4120-4128. doi: 10.1093/nar/gkab179.
Cas12f, also known as Cas14, is an exceptionally small type V-F CRISPR-Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR-Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.
Cas12f，也称为 Cas14，是一种非常小的 V-F CRISPR-Cas 核酸酶，大约只有同类核酸酶的一半大小。为了揭示其底物识别和切割的机制，我们分别以 3.1 Å 和 3.9 Å 的分辨率确定了 Cas12f-sgRNA-靶 DNA 和 Cas12f-sgRNA 复合物的冷冻电镜结构。不对称的 Cas12f 二聚体结合到一个 sgRNA 上，用于识别和切割具有富含 T 的 PAM 序列的 dsDNA 底物。尽管发生二聚化，Cas12f 仍采用了 V 型核酸酶中保守的激活机制，该机制需要由 crRNA-靶 DNA 异源双链体形成诱导的协调构象变化，包括 RuvC 结构域的盖结构域的近乎打开的转变。只有 Cas12f 二聚体中的一个 RuvC 结构域被底物识别激活，并且与激活的 RuvC 结构域结合的底物被捕获在结构中。结构辅助的截断 sgRNA，其长度不到原始 sgRNA 的一半，仍然能够有效切割靶 DNA。我们的结果扩展了对不同的 V 型 CRISPR-Cas 核酸酶的理解，并促进了使用微型 Cas12f 的潜在基因组编辑应用。
