Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.
Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.
复制标题
CRISPR/Cas9 介导的双链 DNA 切割后，使用双切割 HDR 供体进行高效精确的敲入。
DOI：
10.1186/s13059-017-1164-8
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发表时间：
2017-02-20
期刊：
影响因子：
12.3
通讯作者：
Zhang XB
中科院分区：
文献类型：
--
作者：
Zhang JP;Li XL;Li GH;Chen W;Arakaki C;Botimer GD;Baylink D;Zhang L;Wen W;Fu YW;Xu J;Chun N;Yuan W;Cheng T;Zhang XB
BackgroundPrecise 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).ResultsHere, 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.ConclusionsTaken 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.
