Methods to Increase the Efficiency of Knock-in of a Construct Encoding the HIV-1 Fusion Inhibitor, MT-C34 Peptide, into the CXCR4 Locus in the CEM/R5 T Cell Line

IF 1.5 4区生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Molecular Biology Pub Date : 2024-08-07 DOI:10.1134/s0026893324700249

D. S. Golubev, D. S. Komkov, M. V. Shepelev, D. V. Mazurov, N. A. Kruglova

{"title":"Methods to Increase the Efficiency of Knock-in of a Construct Encoding the HIV-1 Fusion Inhibitor, MT-C34 Peptide, into the CXCR4 Locus in the CEM/R5 T Cell Line","authors":"D. S. Golubev, D. S. Komkov, M. V. Shepelev, D. V. Mazurov, N. A. Kruglova","doi":"10.1134/s0026893324700249","DOIUrl":null,"url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Abstract</h3><p>The low knock-in efficiency, especially in primary human cells, limits the use of the genome editing technology for therapeutic purposes, rendering it important to develop approaches for increasing the knock-in levels. In this work, the efficiencies of several approaches were studied using a model of knock-in of a construct coding for the peptide HIV fusion inhibitor MT-C34 into the human <i>CXCR4</i> locus in the CEM/R5 T cell line. First, donor DNA modification was evaluated as a means to improve the efficiency of plasmid transport into the nucleus. The donor plasmid was modified to include the simian virus 40 (SV40) DNA nuclear targeting sequence (DTS) or binding sites for the transcription factor NF-κB, whose effects on the knock-in levels have not been described. The modification was ineffective in the model of MT-C34 knock-in into the <i>CXCR4</i> locus. A second approach consisted in modification of Cas9 nuclease by introducing two additional nuclear localization signals (NLSs) and increased the knock-in level by 30%. Finally, blocking DNA repair via the nonhomologous end joining (NHEJ) pathway with DNA-dependent protein kinase inhibitors caused a 1.8-fold increase in knock-in. A combination of the last two approaches caused an additive effect. Thus, increasing the number of NLSs in the Cas9 protein and inhibiting DNA repair via the NHEJ pathway significantly increased the level of knock-in of the HIV-1 fusion inhibitory peptide into the clinically relevant locus <i>CXCR4.</i> The finding can be used to develop effective gene therapy approaches for treating HIV infection.</p>","PeriodicalId":18734,"journal":{"name":"Molecular Biology","volume":"92 1","pages":""},"PeriodicalIF":1.5000,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1134/s0026893324700249","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}

引用次数: 0

Abstract

The low knock-in efficiency, especially in primary human cells, limits the use of the genome editing technology for therapeutic purposes, rendering it important to develop approaches for increasing the knock-in levels. In this work, the efficiencies of several approaches were studied using a model of knock-in of a construct coding for the peptide HIV fusion inhibitor MT-C34 into the human CXCR4 locus in the CEM/R5 T cell line. First, donor DNA modification was evaluated as a means to improve the efficiency of plasmid transport into the nucleus. The donor plasmid was modified to include the simian virus 40 (SV40) DNA nuclear targeting sequence (DTS) or binding sites for the transcription factor NF-κB, whose effects on the knock-in levels have not been described. The modification was ineffective in the model of MT-C34 knock-in into the CXCR4 locus. A second approach consisted in modification of Cas9 nuclease by introducing two additional nuclear localization signals (NLSs) and increased the knock-in level by 30%. Finally, blocking DNA repair via the nonhomologous end joining (NHEJ) pathway with DNA-dependent protein kinase inhibitors caused a 1.8-fold increase in knock-in. A combination of the last two approaches caused an additive effect. Thus, increasing the number of NLSs in the Cas9 protein and inhibiting DNA repair via the NHEJ pathway significantly increased the level of knock-in of the HIV-1 fusion inhibitory peptide into the clinically relevant locus CXCR4. The finding can be used to develop effective gene therapy approaches for treating HIV infection.

Abstract Image

查看原文

微信好友朋友圈 QQ好友复制链接

本刊更多论文

提高将编码 HIV-1 融合抑制剂 MT-C34 肽的构建基因敲入 CEM/R5 T 细胞系 CXCR4 基因座的效率的方法

摘要基因敲入效率低，尤其是在原代人类细胞中，限制了基因组编辑技术在治疗方面的应用，因此开发提高基因敲入水平的方法非常重要。在这项工作中，研究人员利用在 CEM/R5 T 细胞系中将编码多肽 HIV 融合抑制剂 MT-C34 的构建体敲入人类 CXCR4 基因座的模型，研究了几种方法的效率。首先，对供体 DNA 修饰进行了评估，以提高质粒转运到细胞核的效率。对供体质粒进行了修饰，使其包含猿猴病毒 40（SV40）DNA 核靶向序列（DTS）或转录因子 NF-κB 的结合位点。在将 MT-C34 基因敲入 CXCR4 基因座的模型中，这种修饰没有效果。第二种方法是通过引入两个额外的核定位信号（NLSs）对 Cas9 核酸酶进行修饰，从而将基因敲入水平提高了 30%。最后，用DNA依赖性蛋白激酶抑制剂阻断通过非同源末端连接（NHEJ）途径进行的DNA修复，使基因敲入增加了1.8倍。后两种方法的组合产生了叠加效应。因此，增加 Cas9 蛋白中 NLS 的数量并通过 NHEJ 途径抑制 DNA 修复，可显著提高将 HIV-1 融合抑制肽敲入临床相关基因座 CXCR4 的水平。这一发现可用于开发治疗艾滋病毒感染的有效基因治疗方法。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文去求助

来源期刊

Molecular Biology 生物-生化与分子生物学

CiteScore

1.30

自引率

8.30%

发文量

审稿时长

3 months

期刊介绍： Molecular Biology is an international peer reviewed journal that covers a wide scope of problems in molecular, cell and computational biology including genomics, proteomics, bioinformatics, molecular virology and immunology, molecular development biology, molecular evolution and related areals. Molecular Biology publishes reviews, experimental and theoretical works. Every year, the journal publishes special issues devoted to most rapidly developing branches of physical-chemical biology and to the most outstanding scientists.