Xiang Wenwen, Lin Xiaofeng, Yang Yunqian, Huang Linglong, Chen Ying, Chen Jiyun, Liu Liang
State Key Laboratory of Cellular Stress Biology, Xiang'an Hospital, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen 361102, China.
State Key Laboratory of Cellular Stress Biology, Xiang'an Hospital, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen 361102, China.
Cell Rep. 2025 May 27;44(5):115718. doi: 10.1016/j.celrep.2025.115718. Epub 2025 May 14.
Type V-H CRISPR-Cas system, an important subtype of type V CRISPR-Cas systems, has remained enigmatic in terms of its structure and function despite being discovered several years ago. Here, we comprehensively characterize the type V-H CRISPR-Cas system and elucidate its role as a DNA nicking system. The unique CRISPR RNA (crRNA) employed by Cas12h effector protein enables specific targeting of double-stranded DNA (dsDNA), while its RuvC domain is responsible for cleaving the non-target strand (NTS) of dsDNA. We present the structure of Cas12h bound to crRNA and target DNA. Our structural analysis reveals that the RuvC domain possesses a narrow active pocket that facilitates recognition of NTS but potentially hinders access to the target strand. Furthermore, we demonstrate that Cas12h confers adaptive immunity against invading mobile genetic elements through transcriptional gene inhibition. We have engineered an adenine base editor by fusing Cas12h with an adenine deaminase, achieving effective A-to-G substitution.
V-H型CRISPR-Cas系统是V型CRISPR-Cas系统的一个重要亚型，尽管在几年前就已被发现，但其结构和功能仍然成谜。在此，我们全面表征了V-H型CRISPR-Cas系统，并阐明了其作为DNA切口系统的作用。Cas12h效应蛋白所采用的独特CRISPR RNA（crRNA）能够特异性靶向双链DNA（dsDNA），而其RuvC结构域负责切割dsDNA的非靶向链（NTS）。我们展示了与crRNA和靶向DNA结合的Cas12h的结构。我们的结构分析表明，RuvC结构域具有一个狭窄的活性口袋，有助于识别NTS，但可能会阻碍对靶向链的接近。此外，我们证明Cas12h通过转录基因抑制赋予针对入侵的可移动遗传元件的适应性免疫。我们通过将Cas12h与腺嘌呤脱氨酶融合构建了一种腺嘌呤碱基编辑器，实现了有效的A到G替换。
