The CRISPR/Cas9 system, originally derived from the prokaryotic adaptive immune system, has been developed as efficient genome editing tools. It enables precise gene manipulation on chromosomal DNA through the specific binding of programmable sgRNA to target DNA, and the Cas9 protein, which has endonuclease activity, will cut a double strand break at specific locus. However, Cas9 is a foreign protein in mammalian cells, and the potential risks associated with its introduction into mammalian cells are not fully understood. In this study, we performed pull-down and mass spectrometry (MS) analysis of Streptococcus pyogenes Cas9 (SpyCas9) interacting proteins in HEK293T cells and showed that the majority of Cas9-associated proteins identified by MS were localized in the nucleolus. Interestingly, we further discovered that the Cas9 protein contains a sequence encoding a nucleolus detention signal (NoDS). Compared with wild-type (WT) Cas9, NoDS-mutated variants of Cas9 (mCas9) are less stable, although their gene editing activity is minimally affected. Overexpression of WT Cas9, but not mCas9, causes general effects on transcription and protein translation in the host cell. Overall, identification of NoDS in Cas9 will improve the understanding of Cas9's biological function in vivo, and the removal of NoDS in Cas9 may enhance its safety for future clinical use.

最初源自原核适应性免疫系统的 CRISPR/Cas9 系统已被开发为高效的基因组编辑工具。它通过可编程 sgRNA 与靶 DNA 的特异性结合，实现对染色体 DNA 的精确基因操作，具有内切酶活性的 Cas9 蛋白将在特定位点切割双链断裂。然而，Cas9 是哺乳动物细胞中的一种外源蛋白，其引入哺乳动物细胞的潜在风险尚不完全清楚。在这项研究中，我们对化脓性链球菌进行了下拉和质谱 (MS) 分析HEK293T 细胞中的 Cas9 (SpyCas9) 相互作用蛋白，并表明 MS 鉴定的大多数 Cas9 相关蛋白都位于核仁中。有趣的是，我们进一步发现 Cas9 蛋白包含一个编码核仁滞留信号 (NoDS) 的序列。与野生型 (WT) Cas9 相比，Cas9 (mCas9) 的 NoDS 突变变体不太稳定，尽管它们的基因编辑活性受到的影响很小。WT Cas9 而不是 mCas9 的过表达对宿主细胞中的转录和蛋白质翻译造成一般影响。总体而言，鉴定 Cas9 中的 NoDS 将提高对 Cas9在体内的生物学功能的认识，去除 Cas9 中的 NoDS 可能会增强其未来临床使用的安全性。