Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.

IF 12.3 1区生物学 Q1 Agricultural and Biological Sciences

Genome Biology Pub Date : 2017-02-20 DOI:10.1186/s13059-017-1164-8

Jian-Ping Zhang, Xiao-Lan Li, Guo-Hua Li, Wanqiu Chen, Cameron Arakaki, Gary D Botimer, David Baylink, Lu Zhang, Wei Wen, Ya-Wen Fu, Jing Xu, Noah Chun, Weiping Yuan, Tao Cheng, Xiao-Bing Zhang

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引用次数: 0

Abstract

Background: Precise genome editing via homology-directed repair (HDR) after double-stranded DNA (dsDNA) cleavage facilitates functional genomic research and holds promise for gene therapy. However, HDR efficiency remains low in some cell types, including some of great research and clinical interest, such as human induced pluripotent stem cells (iPSCs).

Results: Here, we show that a double cut HDR donor, which is flanked by single guide RNA (sgRNA)-PAM sequences and is released after CRISPR/Cas9 cleavage, increases HDR efficiency by twofold to fivefold relative to circular plasmid donors at one genomic locus in 293 T cells and two distinct genomic loci in iPSCs. We find that a 600 bp homology in both arms leads to high-level genome knockin, with 97-100% of the donor insertion events being mediated by HDR. The combined use of CCND1, a cyclin that functions in G1/S transition, and nocodazole, a G2/M phase synchronizer, doubles HDR efficiency to up to 30% in iPSCs.

Conclusions: Taken together, these findings provide guidance for the design of HDR donor vectors and the selection of HDR-enhancing factors for applications in genome research and precision medicine.

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CRISPR/ cas9介导的双链DNA切割后，高效精确敲入双切HDR供体。

背景：双链DNA （dsDNA）切割后通过同源定向修复（HDR）进行精确的基因组编辑，促进了功能基因组研究，并为基因治疗带来了希望。然而，在某些类型的细胞中，HDR效率仍然很低，包括一些具有重大研究和临床意义的细胞，如人类诱导多能干细胞（iPSCs）。结果：在这里，研究人员发现，在CRISPR/Cas9切割后释放的双切HDR供体，在293 T细胞的一个基因组位点和iPSCs的两个不同的基因组位点上，比环状质粒供体的HDR效率提高了2到5倍。我们发现，在两条手臂上600 bp的同源性导致高水平的基因组敲入，97-100%的供体插入事件是由HDR介导的。联合使用CCND1（一种在G1/S过渡中起作用的细胞周期蛋白）和nocodazole（一种G2/M相位同步器），可使iPSCs的HDR效率提高一倍，达到30%。结论：本研究结果可为HDR供体载体的设计和HDR增强因子的选择在基因组研究和精准医学中的应用提供指导。

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

Genome Biology BIOTECHNOLOGY & APPLIED MICROBIOLOGY-GENETICS & HEREDITY

CiteScore

25.50

自引率

3.30%

发文量

审稿时长

14 weeks

期刊介绍： Genome Biology is a leading research journal that focuses on the study of biology and biomedicine from a genomic and post-genomic standpoint. The journal consistently publishes outstanding research across various areas within these fields. With an impressive impact factor of 12.3 (2022), Genome Biology has earned its place as the 3rd highest-ranked research journal in the Genetics and Heredity category, according to Thomson Reuters. Additionally, it is ranked 2nd among research journals in the Biotechnology and Applied Microbiology category. It is important to note that Genome Biology is the top-ranking open access journal in this category. In summary, Genome Biology sets a high standard for scientific publications in the field, showcasing cutting-edge research and earning recognition among its peers.