探索真核细胞中CRISPR-Cas9的细胞质保留:核定位信号和核糖体相互作用的作用。

IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY
CRISPR Journal Pub Date : 2025-04-01 Epub Date: 2025-02-28 DOI:10.1089/crispr.2024.0074
Rami M Major, Christine A Mills, Lei Xing, James L Krantz, Justin M Wolter, Mark J Zylka
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引用次数: 0

摘要

Cas9必须定位于细胞核才能进入哺乳动物细胞的基因组。对于大多数蛋白质来说,加入单个核定位信号(NLS)就足以促进核进入。然而,Cas9的核进入似乎是低效的,因为多个NLSs通常被添加到Cas9中。在这里,我们发现三种不同的Cas9变体与HEK293T细胞中的核糖体相互作用,并且这种相互作用是RNA介导的。通过细胞质定位的Cas9- 0nls和核定位的Cas9- 4nls构建物的免疫沉淀-质谱分析,我们在有丝分裂后神经元中发现了新的Cas9相互作用物,包括KEAP1和其他核糖体亚基,后者在Cas9- 0nls样本中富集。总的来说,我们的研究结果表明,Cas9部分通过与核糖体的相互作用被隔离在哺乳动物细胞的细胞质中。增加Cas9上NLSs的数量和/或增加细胞质引导RNA的数量有可能胜过核糖体RNA的结合,并促进CRISPR-Cas9变体的有效核定位。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Exploring the Cytoplasmic Retention of CRISPR-Cas9 in Eukaryotic Cells: The Role of Nuclear Localization Signals and Ribosomal Interactions.

Cas9 must be localized to the nucleus to access the genome of mammalian cells. For most proteins, adding a single nuclear localization signal (NLS) is sufficient to promote nuclear entry. However, Cas9 nuclear entry appears to be inefficient as multiple NLSs are typically added to Cas9. Here, we found that three different Cas9 variants interact with the ribosome in HEK293T cells, and that this interaction is RNA mediated. Following immunoprecipitation-mass spectrometry of cytoplasmic-localized Cas9-0NLS and nuclear-localized Cas9-4NLS constructs, we identified novel Cas9 interactors in postmitotic neurons, including KEAP1 and additional ribosomal subunits, the latter were enriched in Cas9-0NLS samples. Collectively, our results suggest that Cas9 is sequestered in the cytoplasm of mammalian cells, in part, via interaction with the ribosome. Increasing the number of NLSs on Cas9 and/or increasing the amount of cytoplasmic guide RNA has the potential to outcompete ribosomal RNA binding and promote efficient nuclear localization of CRISPR-Cas9 variants.

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来源期刊
CRISPR Journal
CRISPR Journal Biochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
6.30
自引率
2.70%
发文量
76
期刊介绍: In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR. Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.
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