Engineering single-cycle MeV vector for CRISPR-Cas9 gene editing

IF 4.6 2区医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-06-24 DOI:10.1016/j.omtm.2024.101290

Ramya Rallabandi, Brenna Sharp, Spencer Majerus, Austin Royster, Sarrianna Hoffer, Mia Ikeda, Patricia Devaux

引用次数: 0

Abstract

CRISPR-Cas9-mediated gene editing has vast applications in basic and clinical research and is a promising tool for several disorders. Our lab previously developed a non-integrating RNA virus, measles virus (MeV), as a single-cycle reprogramming vector by replacing the viral attachment protein with the reprogramming factors for induced pluripotent stem cell generation. Encouraged by the MeV reprogramming vector efficiency, in this study, we develop a single-cycle MeV vector to deliver the gRNA(s) and Cas9 nuclease to human cells for efficient gene editing. We show that the MeV vector achieved on-target gene editing of the reporter () and endogenous genes ( and ) in human cells. Additionally, the MeV vector achieved precise knock-in via homology-directed repair using a single-stranded oligonucleotide donor. The MeV vector is a new and flexible platform for gene knock-out and knock-in modifications in human cells, capable of incorporating new technologies as they are developed.

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为 CRISPR-Cas9 基因编辑设计单循环 MeV 载体

CRISPR-Cas9介导的基因编辑技术在基础和临床研究中有着广泛的应用，是治疗多种疾病的理想工具。我们实验室之前开发了一种非整合 RNA 病毒--麻疹病毒（MeV），通过将病毒附着蛋白替换为诱导多能干细胞生成的重编程因子，将其作为单周期重编程载体。受MeV重编程载体效率的鼓舞，在本研究中，我们开发了一种单循环MeV载体，将gRNA和Cas9核酸酶输送到人体细胞，进行高效的基因编辑。结果表明，MeV载体实现了对人体细胞中报告基因（）和内源基因（和）的靶向基因编辑。此外，MeV载体还利用单链寡核苷酸供体通过同源定向修复实现了精确的基因敲入。MeV载体是在人体细胞中进行基因敲除和基因敲入修饰的一个灵活的新平台，能够随着新技术的发展而不断融入新技术。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Molecular Therapy-Methods & Clinical Development Biochemistry, Genetics and Molecular Biology-Molecular Biology

CiteScore

9.90

自引率

4.30%

发文量

163

审稿时长

12 weeks

期刊介绍： The aim of Molecular Therapy—Methods & Clinical Development is to build upon the success of Molecular Therapy in publishing important peer-reviewed methods and procedures, as well as translational advances in the broad array of fields under the molecular therapy umbrella. Topics of particular interest within the journal''s scope include: Gene vector engineering and production, Methods for targeted genome editing and engineering, Methods and technology development for cell reprogramming and directed differentiation of pluripotent cells, Methods for gene and cell vector delivery, Development of biomaterials and nanoparticles for applications in gene and cell therapy and regenerative medicine, Analysis of gene and cell vector biodistribution and tracking, Pharmacology/toxicology studies of new and next-generation vectors, Methods for cell isolation, engineering, culture, expansion, and transplantation, Cell processing, storage, and banking for therapeutic application, Preclinical and QC/QA assay development, Translational and clinical scale-up and Good Manufacturing procedures and process development, Clinical protocol development, Computational and bioinformatic methods for analysis, modeling, or visualization of biological data, Negotiating the regulatory approval process and obtaining such approval for clinical trials.

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