Fu Wenhan, Ma Jiacheng, Wang Zhipeng, Tang Na, Pan Deng, Su Mengjiao, Wu Zhaowei, Gan Jianhua, Ji Quanjiang
School of Physical Science and Technology & State Key Laboratory of Advanced Medical Materials and Devices, ShanghaiTech University, Shanghai, China.
School of Life Sciences, Fudan University, Shanghai, China.
Nat Commun. 2025 Jul 1;16(1):5667. doi: 10.1038/s41467-025-61290-3.
Type V CRISPR-Cas12 systems are highly diverse in their functionality and molecular compositions, including miniature Cas12f1 and Cas12n genome editors that provide advantages for efficient in vivo therapeutic delivery due to their small size. In contrast to Cas12f1 nucleases that utilize a homodimer structure for DNA targeting and cleavage with a preference for T- or C-rich PAMs, Cas12n nucleases are likely monomeric proteins and uniquely recognize rare A-rich PAMs. However, the molecular mechanisms behind RNA-guided genome targeting and cleavage by Cas12n remain unclear. Here, we present the cryo-electron microscopy (cryo-EM) structure of Rothia dentocariosa Cas12n (RdCas12n) bound to a single guide RNA (sgRNA) and target DNA, illuminating the intricate molecular architecture of Cas12n and its sgRNA, as well as PAM recognition and nucleic-acid binding mechanisms. Through structural comparisons with other Cas12 nucleases and the ancestral precursor TnpB, we provide insights into the evolutionary significance of Cas12n in the progression from TnpB to various Cas12 nucleases. Additionally, we extensively modify the sgRNA and convert RdCas12n into an effective genome editor in human cells. Our findings enhance the understanding of the evolutionary mechanisms of type V CRISPR-Cas12 systems and offer a molecular foundation for engineering Cas12n genome editors.
V型CRISPR-Cas12系统在功能和分子组成上高度多样，包括微型Cas12f1和Cas12n基因组编辑器，由于其尺寸小，在体内治疗递送方面具有优势。与利用同型二聚体结构进行DNA靶向和切割且偏好富含T或C的原间隔序列临近基序（PAM）的Cas12f1核酸酶不同，Cas12n核酸酶可能是单体蛋白，且独特地识别罕见的富含A的PAM。然而，Cas12n介导的RNA引导的基因组靶向和切割背后的分子机制仍不清楚。在这里，我们展示了龋齿罗氏菌Cas12n（RdCas12n）与单导向RNA（sgRNA）和靶DNA结合的冷冻电镜（cryo-EM）结构，阐明了Cas12n及其sgRNA复杂的分子结构，以及PAM识别和核酸结合机制。通过与其他Cas12核酸酶和祖先前体TnpB的结构比较，我们深入了解了Cas12n在从TnpB进化到各种Cas12核酸酶过程中的进化意义。此外，我们对sgRNA进行了广泛修饰，并将RdCas12n转化为人类细胞中有效的基因组编辑器。我们的研究结果加深了对V型CRISPR-Cas12系统进化机制的理解，并为工程化Cas12n基因组编辑器提供了分子基础。
