Precision genome editing using combinatorial viral vector delivery of CRISPR-Cas9 nucleases and donor DNA constructs

IF 16.6 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Zhen Li, Xiaoling Wang, Josephine M Janssen, Jin Liu, Francesca Tasca, Rob C Hoeben, Manuel A F V Gonçalves
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引用次数: 0

Abstract

Genome editing based on programmable nucleases and donor DNA constructs permits introducing specific base-pair changes and complete transgenes or live-cell reporter tags at predefined chromosomal positions. A crucial requirement for such versatile genome editing approaches is, however, the need to co-deliver in an effective, coordinated and non-cytotoxic manner all the required components into target cells. Here, adenoviral (AdV) and adeno-associated viral (AAV) vectors are investigated as delivery agents for, respectively, engineered CRISPR-Cas9 nucleases and donor DNA constructs prone to homologous recombination (HR) or homology-mediated end joining (HMEJ) processes. Specifically, canonical single-stranded and self-complementary double-stranded AAVs served as sources of ectopic HR and HMEJ substrates, whilst second- and third-generation AdVs provided for matched CRISPR-Cas9 nucleases. We report that combining single-stranded AAV delivery of HR donors with third-generation AdV transfer of CRISPR-Cas9 nucleases results in selection-free and precise whole transgene insertion in large fractions of target-cell populations (i.e. up to 93%) and disclose that programmable nuclease-induced chromosomal breaks promote AAV transduction. Finally, besides investigating relationships between distinct AAV structures and genome-editing performance endpoints, we further report that high-fidelity CRISPR-Cas9 nucleases are critical for mitigating off-target chromosomal insertion of defective AAV genomes known to be packaged in vector particles.
使用CRISPR-Cas9核酸酶和供体DNA构建的组合病毒载体进行精确基因组编辑
基于可编程核酸酶和供体DNA构建的基因组编辑允许在预定义的染色体位置引入特定的碱基对变化和完整的转基因或活细胞报告标签。然而,这种通用基因组编辑方法的一个关键要求是,需要以有效、协调和无细胞毒性的方式将所有所需成分共同递送到靶细胞中。本文研究了腺病毒(AdV)和腺相关病毒(AAV)载体分别作为工程化CRISPR-Cas9核酸酶和易于同源重组(HR)或同源介导末端连接(HMEJ)过程的供体DNA构建的递送剂。具体来说,典型的单链和自互补双链aav作为异位HR和HMEJ底物的来源,而第二代和第三代adv提供匹配的CRISPR-Cas9核酸酶。我们报道,将HR供体的单链AAV传递与CRISPR-Cas9核酸酶的第三代AdV转移相结合,可以在大部分靶细胞群(即高达93%)中实现无选择和精确的全转基因插入,并揭示可编程核酸酶诱导的染色体断裂促进AAV转导。最后,除了研究不同AAV结构与基因组编辑性能端点之间的关系外,我们进一步报道了高保真CRISPR-Cas9核酸酶对于减轻已知包装在载体颗粒中的缺陷AAV基因组的脱靶染色体插入至关重要。
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
Nucleic Acids Research
Nucleic Acids Research 生物-生化与分子生物学
CiteScore
27.10
自引率
4.70%
发文量
1057
审稿时长
2 months
期刊介绍: Nucleic Acids Research (NAR) is a scientific journal that publishes research on various aspects of nucleic acids and proteins involved in nucleic acid metabolism and interactions. It covers areas such as chemistry and synthetic biology, computational biology, gene regulation, chromatin and epigenetics, genome integrity, repair and replication, genomics, molecular biology, nucleic acid enzymes, RNA, and structural biology. The journal also includes a Survey and Summary section for brief reviews. Additionally, each year, the first issue is dedicated to biological databases, and an issue in July focuses on web-based software resources for the biological community. Nucleic Acids Research is indexed by several services including Abstracts on Hygiene and Communicable Diseases, Animal Breeding Abstracts, Agricultural Engineering Abstracts, Agbiotech News and Information, BIOSIS Previews, CAB Abstracts, and EMBASE.
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