Wang Kangkang, Wang Jiuyu, Yang Xiaoqi, Sun Wei, Sheng Gang, Wang Yanli
State Key Laboratory of RNA Innovation, Science and Engineering, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
State Key Laboratory of Epigenetic Regulation and Intervention, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Nat Commun. 2025 Aug 11;16(1):7396. doi: 10.1038/s41467-025-62128-8.
Type II-D Cas9 proteins (Cas9d) are more compact than typical Type II-A/B/C Cas9s. Here, we demonstrate that NsCas9d from Nitrospirae bacterium RBG_13_39_12 derived from a metagenomic assembly exhibits robust dsDNA cleavage activity comparable to SpCas9 in vitro. Unlike typical Cas9 enzymes that generate blunt ends, NsCas9d produces 3-nucleotide staggered overhangs. Our high-resolution cryo-EM structure of the NsCas9d-sgRNA-dsDNA complex in its catalytic state reveals the target and non-target DNA strands positioned within the HNH and RuvC catalytic pockets, respectively. NsCas9d recognizes the 5'-NRG-3' protospacer adjacent motif (PAM), with 5'-NGG-3' showing the highest cleavage efficiency. Its sgRNA structure, resembling the 5' end of IscB ωRNA, along with structural features shared with other Cas9 variants, suggests that Cas9d are hypothesized to resemble evolutionary intermediates between other Cas9 sub-types and IscB. These findings deepen our understanding of Cas9 evolution and mechanisms, highlighting NsCas9d as a promising genome-editing tool due to its compact size, DNA cleavage pattern, and efficient PAM recognition.
II-D型Cas9蛋白（Cas9d）比典型的II-A/B/C型Cas9蛋白更为紧凑。在此，我们证明，从宏基因组组装获得的硝化螺旋菌RBG_13_39_12中的NsCas9d在体外表现出与SpCas9相当的强大双链DNA切割活性。与产生平端的典型Cas9酶不同，NsCas9d产生3个核苷酸的交错突出端。我们对处于催化状态的NsCas9d-sgRNA-dsDNA复合物的高分辨率冷冻电镜结构揭示了靶DNA链和非靶DNA链分别位于HNH和RuvC催化口袋内。NsCas9d识别5'-NRG-3'原间隔相邻基序（PAM），其中5'-NGG-3'的切割效率最高。其sgRNA结构类似于IscB ωRNA的5'端，以及与其他Cas9变体共有的结构特征，表明推测Cas9d类似于其他Cas9亚型和IscB之间的进化中间体。这些发现加深了我们对Cas9进化和机制的理解，突出了NsCas9d因其紧凑的尺寸、DNA切割模式和高效的PAM识别而成为一种有前途的基因组编辑工具。
