Suppr超能文献

带有核定位蛋白的 Cas9 搭便车会损害其可控性,并导致无核定位信号的 Cas9 的高效基因组编辑。

Hitchhiking of Cas9 with nucleus-localized proteins impairs its controllability and leads to efficient genome editing of NLS-free Cas9.

机构信息

School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, P.R. China; Biopharmaceutical Institute, Guangdong Pharmaceutical University, Guangzhou, P.R. China.

School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, P.R. China; Biopharmaceutical Institute, Guangdong Pharmaceutical University, Guangzhou, P.R. China.

出版信息

Mol Ther. 2024 Apr 3;32(4):920-934. doi: 10.1016/j.ymthe.2024.02.008. Epub 2024 Feb 9.

Abstract

CRISPR-Cas9 is the most commonly used genome-editing tool in eukaryotic cells. To modulate Cas9 entry into the nucleus to enable control of genome editing, we constructed a light-controlled CRISPR-Cas9 system to control exposure of the Cas9 protein nuclear localization signal (NLS). Although blue-light irradiation was found to effectively control the entry of Cas9 protein into the nucleus with confocal microscopy observation, effective gene editing occurred in controls with next-generation sequencing analysis. To further clarify this phenomenon, a CRISPR-Cas9 editing system without the NLS and a CRISPR-Cas9 editing system containing a nuclear export signal were also constructed. Interestingly, both Cas9 proteins could achieve effective editing of target sites with significantly reduced off-target effects. Thus, we speculated that other factors might mediate Cas9 entry into the nucleus. However, NLS-free Cas9 was found to produce effective target gene editing even following inhibition of cell mitosis to prevent nuclear import caused by nuclear membrane disassembly. Furthermore, multiple nucleus-localized proteins were found to interact with Cas9, which could mediate the "hitchhiking" of NLS-free Cas9 into the nucleus. These findings will inform future attempts to construct controllable gene-editing systems and provide new insights into the evolution of the nucleus and compatible protein functions.

摘要

CRISPR-Cas9 是真核细胞中最常用的基因组编辑工具。为了调节 Cas9 进入细胞核以实现对基因组编辑的控制,我们构建了一个光控 CRISPR-Cas9 系统来控制 Cas9 蛋白核定位信号(NLS)的暴露。虽然通过共聚焦显微镜观察发现蓝光照射可以有效地控制 Cas9 蛋白进入细胞核,但通过下一代测序分析,对照组中也发生了有效的基因编辑。为了进一步阐明这一现象,我们还构建了一个没有 NLS 的 CRISPR-Cas9 编辑系统和一个含有核输出信号的 CRISPR-Cas9 编辑系统。有趣的是,两种 Cas9 蛋白都可以实现有效的靶位点编辑,同时靶基因脱靶效应显著降低。因此,我们推测可能有其他因素介导 Cas9 进入细胞核。然而,即使抑制了细胞有丝分裂以阻止核膜解体引起的核内输入,无 NLS 的 Cas9 也能产生有效的靶基因编辑。此外,还发现多个核定位蛋白与 Cas9 相互作用,这可能介导无 NLS 的 Cas9 搭便车进入细胞核。这些发现将为未来构建可控基因编辑系统提供信息,并为细胞核的进化和兼容蛋白功能提供新的见解。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4633/11163216/2e50e31f1c34/fx1.jpg

文献AI研究员

20分钟写一篇综述,助力文献阅读效率提升50倍。

立即体验

用中文搜PubMed

大模型驱动的PubMed中文搜索引擎

马上搜索

文档翻译

学术文献翻译模型,支持多种主流文档格式。

立即体验