CRISPR-Cas9中的发夹内核定位信号增强了人原代淋巴细胞的编辑作用。

IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY
CRISPR Journal Pub Date : 2025-04-01 Epub Date: 2025-03-31 DOI:10.1089/crispr.2024.0080
Eric A Noel, Srishti U Sahu, Stacia K Wyman, Netravathi Krishnappa, Chris Jeans, Ross C Wilson
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引用次数: 0

摘要

在CRISPR酶的一端或两端加入核定位信号(NLS)序列是一种广泛采用的促进基因组编辑的策略。具有不同NLS序列的CRISPR酶的工程变体表现出优异的性能,促进了核定位和高效的DNA编辑。然而,将NLS融合限制在CRISPR蛋白的末端会对重组表达的蛋白产量产生负面影响。在这里,我们提出了一种独特的策略,包括在CRISPR-Cas9主干内合理选择的位点上安装发夹内部NLS序列(hiNLS)。我们通过电穿孔或与两亲肽共孵育的方式将核糖核蛋白酶传递到人原代T细胞中,通过编辑基因来评估这些hiNLS Cas9变体的性能。我们发现,与末端融合NLS序列的构建体相比,hiNLS Cas9变体可以提高T细胞中的编辑效率。此外,许多hiNLS Cas9构建体可以以高纯度和产率生产,即使这些构建体含有多达9个NLS。这些hiNLS Cas9构建体代表了优化CRISPR效应体设计的关键进展,可能有助于改善研究和治疗应用中的编辑结果。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Hairpin Internal Nuclear Localization Signals in CRISPR-Cas9 Enhance Editing in Primary Human Lymphocytes.

The incorporation of nuclear localization signal (NLS) sequences at one or both termini of CRISPR enzymes is a widely adopted strategy to facilitate genome editing. Engineered variants of CRISPR enzymes with diverse NLS sequences have demonstrated superior performance, promoting nuclear localization and efficient DNA editing. However, limiting NLS fusion to the CRISPR protein's termini can negatively impact protein yield via recombinant expression. Here we present a distinct strategy involving the installation of hairpin internal NLS sequences (hiNLS) at rationally selected sites within the backbone of CRISPR-Cas9. We evaluated the performance of these hiNLS Cas9 variants by editing genes in human primary T cells following the delivery of ribonucleoprotein enzymes via either electroporation or co-incubation with amphiphilic peptides. We show that hiNLS Cas9 variants can improve editing efficiency in T cells compared with constructs with terminally fused NLS sequences. Furthermore, many hiNLS Cas9 constructs can be produced with high purity and yield, even when these constructs contain as many as nine NLS. These hiNLS Cas9 constructs represent a key advance in optimizing CRISPR effector design and may contribute to improved editing outcomes in research and therapeutic applications.

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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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