Nucleolus localization of SpyCas9 affects its stability and interferes with host protein translation in mammalian cells.
Nucleolus localization of SpyCas9 affects its stability and interferes with host protein translation in mammalian cells.
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SpyCas9 的核仁定位影响其稳定性并干扰哺乳动物细胞中的宿主蛋白翻译
DOI：
10.1016/j.gendis.2020.09.003
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发表时间：
2022-05
期刊：
影响因子：
6.8
通讯作者：
Dang, Yongjun
中科院分区：
文献类型：
--
作者：
Tan, Renke;Du, Wenhao;Liu, Yiyang;Cong, Xiaoji;Bai, Meirong;Jiang, Chenxiao;Li, Zengxia;Tan, Minjia;Ma, Dengke K.;Huang, Qiang;Jiang, Wei;Dang, Yongjun
CRISPR/Cas9系统最初来源于原核适应性免疫系统，已被开发为有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，实现了对染色体DNA的精确基因操作，并且具有内切酶活性的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外源蛋白，与其引入哺乳动物细胞相关的潜在风险尚未完全了解。在这项研究中，我们对HEK 293 T细胞中的化脓链球菌Cas9（SpyCas 9）相互作用蛋白进行了下拉和质谱（MS）分析，并显示通过MS鉴定的大多数Cas9相关蛋白位于核仁中。有趣的是，我们进一步发现Cas9蛋白含有编码核仁滞留信号（NoDS）的序列。与野生型（WT）Cas9相比，Cas9的NoDS突变变体（mCas 9）不太稳定，尽管它们的基因编辑活性受到的影响最小。WT Cas9而不是mCas 9的过表达对宿主细胞中的转录和蛋白质翻译产生一般影响。总的来说，Cas9中NoDS的鉴定将提高对Cas9体内生物学功能的理解，并且Cas9中NoDS的去除可以增强其未来临床使用的安全性。
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.
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影响因子：
64.5
作者：
Jaitin, Diego Adhemar;Weiner, Assaf;Amit, Ido
通讯作者：
Amit, Ido
解决与 CRISPR/Cas9 技术相关的临床应用挑战和伦理问题，以防止其滥用。
影响因子：
21.1
作者：
Kang XJ;Caparas CIN;Soh BS;Fan Y
通讯作者：
Fan Y
DOI：
10.1083/jcb.201105143
发表时间：
2011-09-05
期刊：
The Journal of cell biology
影响因子：
--
作者：
Boyd MT;Vlatkovic N;Rubbi CP
通讯作者：
Rubbi CP
影响因子：
64.8
作者：
Eyquem J;Mansilla-Soto J;Giavridis T;van der Stegen SJ;Hamieh M;Cunanan KM;Odak A;Gönen M;Sadelain M
通讯作者：
Sadelain M
影响因子：
82.9
作者：
Charlesworth, Carsten T.;Deshpande, Priyanka S.;Porteus, Matthew H.
通讯作者：
Porteus, Matthew H.