期刊
GENOME BIOLOGY
卷 18, 期 -, 页码 -出版社
BIOMED CENTRAL LTD
DOI: 10.1186/s13059-017-1164-8
关键词
CRISPR; Genome editing; Knockin; Homology-directed repair (HDR); Non-homologous end joining (NHEJ); Donor design; 293 T; Human induced pluripotent stem cells
资金
- Ministry of Science and Technology of China [2015CB964902, 2013CB966902, 2012CB966601]
- National Natural Science Foundation of China [81500148, 81570164, 81421002]
- Loma Linda University School of Medicine GCAT grant
- Telemedicine and Advanced Technology Research Center [W81XWH-08-1-0697]
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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据