Two type II-C Cas9 orthologs (Nm1Cas9 and Nm2Cas9) were recently identified from Neisseria meningitidis and have been extensively used in mammalian cells, but whether these NmCas9 orthologs or other type II-C Cas9 proteins can mediate genome editing in plants remains unclear. In this study, we developed and optimized targeted mutagenesis systems from NmCas9s for plants. Efficient genome editing at the target with N₄GATT and N₄CC protospacer adjacent motifs (PAMs) was achieved with Nm1Cas9 and Nm2Cas9 respectively. These results indicated that a highly active editing system could be developed from type II-C Cas9s with distinct PAM preferences, thus providing a reliable strategy to extend the scope of genome editing in plants. Base editors (BEs) were further developed from the NmCas9s. The editing efficiency of adenine BEs (ABEs) of TadA*-7.10 and cytosine BEs (CBEs) of rat APOBEC1 (rAPO1) or human APOBEC3a (hA3A) were extremely limited, whereas ABEs of TadA-8e and CBEs of Petromyzon marinus cytidine deaminase 1 (PmCDA1) exhibited markedly improved performance on the same targets. In addition, we found that fusion of a single-stranded DNA-binding domain from the human Rad51 protein enhanced the base editing capability of rAPO1-CBEs of NmCas9s. Together, our results suggest that the engineering of NmCas9s or other type II-C Cas9s can provide useful alternatives for crop genome editing.

中文翻译：

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水稻脑膜炎奈瑟菌 II-C CRISPR-Cas9 系统的基因组编辑

最近从脑膜炎奈瑟球菌中鉴定出两种 II-C Cas9 直系同源物（Nm1Cas9 和 Nm2Cas9），并已广泛用于哺乳动物细胞，但这些 NmCas9 直系同源物或其他 II-C Cas9 蛋白是否可以介导植物中的基因组编辑仍不清楚。在这项研究中，我们开发并优化了来自 NmCas9s 的植物靶向诱变系统。_{使用 N 4} GATT 和 N ₄在目标上进行高效的基因组编辑CC protospacer 相邻基序 (PAM) 分别用 Nm1Cas9 和 Nm2Cas9 实现。这些结果表明，可以从具有不同 PAM 偏好的 II-C Cas9s 开发出一种高度活跃的编辑系统，从而为扩展植物基因组编辑的范围提供了可靠的策略。碱基编辑器 (BE) 是从 NmCas9s 进一步发展而来的。TadA*-7.10 的腺嘌呤 BEs (ABEs) 和大鼠 APOBEC1 (rAPO1) 或人 APOBEC3a (hA3A) 的胞嘧啶 BEs (CBEs) 的编辑效率极其有限，而 TadA-8e 的 ABEs 和Petromyzon marinus的 CBEs胞苷脱氨酶 1 (PmCDA1) 在相同靶标上表现出显着提高的性能。此外，我们发现来自人类 Rad51 蛋白的单链 DNA 结合结构域的融合增强了 NmCas9s 的 rAPO1-CBE 的碱基编辑能力。总之，我们的结果表明 NmCas9s 或其他 II-C Cas9s 的工程可以为作物基因组编辑提供有用的替代方案。