title: Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.
abstract: 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).
  
  
  
  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.
  
  
  
  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.
authors.affiliation: State Key Laboratory of Experimental Hematology, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
authors.forename: Jian-Ping
authors.initials: JP
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Experimental Hematology, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
authors.forename: Xiao-Lan
authors.initials: XL
authors.lastname: Li
authors.affiliation: State Key Laboratory of Experimental Hematology, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
authors.forename: Guo-Hua
authors.initials: GH
authors.lastname: Li
authors.affiliation: Division of Regenerative Medicine MC1528B, Department of Medicine, Loma Linda University, 11234 Anderson Street, Loma Linda, CA, 92354, USA.
authors.forename: Wanqiu
authors.initials: W
authors.lastname: Chen
authors.affiliation: Division of Regenerative Medicine MC1528B, Department of Medicine, Loma Linda University, 11234 Anderson Street, Loma Linda, CA, 92354, USA.
authors.forename: Cameron
authors.initials: C
authors.lastname: Arakaki
authors.affiliation: Department of Orthopaedic Surgery, Loma Linda University, Loma Linda, CA, USA.
authors.forename: Gary D
authors.initials: GD
authors.lastname: Botimer
authors.affiliation: Division of Regenerative Medicine MC1528B, Department of Medicine, Loma Linda University, 11234 Anderson Street, Loma Linda, CA, 92354, USA.
authors.forename: David
authors.initials: D
authors.lastname: Baylink
authors.affiliation: State Key Laboratory of Experimental Hematology, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Experimental Hematology, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wen
authors.affiliation: State Key Laboratory of Experimental Hematology, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
authors.forename: Ya-Wen
authors.initials: YW
authors.lastname: Fu
authors.affiliation: State Key Laboratory of Experimental Hematology, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.affiliation: Division of Regenerative Medicine MC1528B, Department of Medicine, Loma Linda University, 11234 Anderson Street, Loma Linda, CA, 92354, USA.
authors.forename: Noah
authors.initials: N
authors.lastname: Chun
authors.affiliation: State Key Laboratory of Experimental Hematology, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
authors.forename: Weiping
authors.initials: W
authors.lastname: Yuan
authors.affiliation: State Key Laboratory of Experimental Hematology, Tianjin, China. chengtao@ihcams.ac.cn.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China. chengtao@ihcams.ac.cn.|Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Tianjin, China. chengtao@ihcams.ac.cn.|Department of Stem Cell & Regenerative Medicine, Peking Union Medical College, Tianjin, China. chengtao@ihcams.ac.cn.|Collaborative Innovation Center for Cancer Medicine, Tianjin, China. chengtao@ihcams.ac.cn.|Tianjin Key Laboratory of Blood Cell Therapy and Technology, Tianjin, China. chengtao@ihcams.ac.cn.
authors.forename: Tao
authors.initials: T
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Experimental Hematology, Tianjin, China. xzhang@llu.edu.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China. xzhang@llu.edu.|Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Tianjin, China. xzhang@llu.edu.|Department of Stem Cell & Regenerative Medicine, Peking Union Medical College, Tianjin, China. xzhang@llu.edu.|Division of Regenerative Medicine MC1528B, Department of Medicine, Loma Linda University, 11234 Anderson Street, Loma Linda, CA, 92354, USA. xzhang@llu.edu.
authors.forename: Xiao-Bing
authors.initials: XB
authors.lastname: Zhang
mesh_terms: D064113:CRISPR-Cas Systems; D002453:Cell Cycle / Q000235:genetics; D004247:DNA; D053837:DNA Cleavage; D004268:DNA-Binding Proteins; D018390:Gene Targeting / Q000379:methods; D056426:Genetic Loci; D057809:HEK293 Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism; D016601:RNA-Binding Proteins; D059767:Recombinational DNA Repair; D012097:Repressor Proteins / Q000235:genetics; D014157:Transcription Factors; D051176:beta Catenin / Q000235:genetics
chemical_list: C495270:CTNNB1 protein, human; D004268:DNA-Binding Proteins; C524724:PRDM14 protein, human; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D051176:beta Catenin; D004247:DNA
keywords: 293 T; CRISPR; Donor design; Genome editing; Homology-directed repair (HDR); Human induced pluripotent stem cells; Knockin; Non-homologous end joining (NHEJ)