1. Academic Validation
  2. 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

  • Genome Biol. 2017 Feb 20;18(1):35. doi: 10.1186/s13059-017-1164-8.
Jian-Ping Zhang 1 2 Xiao-Lan Li 1 2 Guo-Hua Li 1 2 Wanqiu Chen 3 Cameron Arakaki 3 Gary D Botimer 4 David Baylink 3 Lu Zhang 1 2 Wei Wen 1 2 Ya-Wen Fu 1 2 Jing Xu 1 2 Noah Chun 3 Weiping Yuan 1 2 Tao Cheng 5 6 7 8 9 10 Xiao-Bing Zhang 11 12 13 14 15
Affiliations

Affiliations

  • 1 State Key Laboratory of Experimental Hematology, Tianjin, China.
  • 2 Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
  • 3 Division of Regenerative Medicine MC1528B, Department of Medicine, Loma Linda University, 11234 Anderson Street, Loma Linda, CA, 92354, USA.
  • 4 Department of Orthopaedic Surgery, Loma Linda University, Loma Linda, CA, USA.
  • 5 State Key Laboratory of Experimental Hematology, Tianjin, China. chengtao@ihcams.ac.cn.
  • 6 Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China. chengtao@ihcams.ac.cn.
  • 7 Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Tianjin, China. chengtao@ihcams.ac.cn.
  • 8 Department of Stem Cell & Regenerative Medicine, Peking Union Medical College, Tianjin, China. chengtao@ihcams.ac.cn.
  • 9 Collaborative Innovation Center for Cancer Medicine, Tianjin, China. chengtao@ihcams.ac.cn.
  • 10 Tianjin Key Laboratory of Blood Cell Therapy and Technology, Tianjin, China. chengtao@ihcams.ac.cn.
  • 11 State Key Laboratory of Experimental Hematology, Tianjin, China. xzhang@llu.edu.
  • 12 Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China. xzhang@llu.edu.
  • 13 Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Tianjin, China. xzhang@llu.edu.
  • 14 Department of Stem Cell & Regenerative Medicine, Peking Union Medical College, Tianjin, China. xzhang@llu.edu.
  • 15 Division of Regenerative Medicine MC1528B, Department of Medicine, Loma Linda University, 11234 Anderson Street, Loma Linda, CA, 92354, USA. xzhang@llu.edu.
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.

Keywords

293 T; CRISPR; Donor design; Genome editing; Homology-directed repair (HDR); Human induced pluripotent stem cells; Knockin; Non-homologous end joining (NHEJ).

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