1. Academic Validation
  2. Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression

Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression

  • Nucleic Acids Res. 2018 Nov 2;46(19):10195-10215. doi: 10.1093/nar/gky804.
Xiao-Lan Li 1 2 Guo-Hua Li 1 2 Juan Fu 3 4 Ya-Wen Fu 1 2 Lu Zhang 1 2 Wanqiu Chen 5 Cameron Arakaki 5 Jian-Ping Zhang 1 2 6 Wei Wen 1 2 Mei Zhao 1 2 Weisheng V Chen 7 Gary D Botimer 8 David Baylink 5 Leslie Aranda 5 Hannah Choi 5 Rachel Bechar 9 Prue Talbot 9 Chang-Kai Sun 3 10 11 Tao Cheng 1 2 10 12 13 14 Xiao-Bing Zhang 1 2 5 12 13
Affiliations

Affiliations

  • 1 State Key Laboratory of Experimental Hematology, Tianjin 300020, China.
  • 2 Institute of Hematology and Blood Disease Hospital, Tianjin 300020, China.
  • 3 Liaoning Provincial Key Laboratory of Cerebral Diseases, Institute for Brain Disorders, Dalian Medical University, Dalian 116044, China.
  • 4 Department of Obstetrics and Gynecology, the First Affiliated Hospital of Dalian Medical University, Dalian 116044, China.
  • 5 Department of Medicine, Loma Linda University, Loma Linda, CA 92350, USA.
  • 6 CAMS Key Laboratory of Gene Therapy for Blood Diseases, Tianjin 300020, China.
  • 7 Leveragen, Inc., Cambridge, MA 01605, USA.
  • 8 Department of Orthopaedic Surgery, Loma Linda University, Loma Linda, CA 92350, USA.
  • 9 UCR Stem Cell Center and Core, University of California at Riverside, Riverside, CA 92521, USA.
  • 10 Research & Educational Center for the Control Engineering of Translational Precision Medicine (R-ECCE-TPM), School of Biomedical Engineering, Faculty of Electronic Information and Electrical Engineering, Dalian University of Technology, Dalian 116024, China.
  • 11 State Key Laboratory of Fine Chemicals, Dalian R&D Center for Stem Cell and Tissue Engineering, Dalian University of Technology, Dalian 116024, China.
  • 12 Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Tianjin 300020, China.
  • 13 Department of Stem Cell & Regenerative Medicine, Peking Union Medical College, Tianjin 300020, China.
  • 14 Collaborative Innovation Center for Cancer Medicine, Tianjin 300020, China.
Abstract

Genome editing of human induced pluripotent stem cells (iPSCs) is instrumental for functional genomics, disease modeling, and regenerative medicine. However, low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockin (KI) or knockout (KO) iPSC lines, which is largely due to massive cell death after electroporation with editing plasmids. Here, we report that the transient delivery of Bcl-xL increases iPSC survival by ∼10-fold after plasmid transfection, leading to a 20- to 100-fold increase in homology-directed repair (HDR) KI efficiency and a 5-fold increase in non-homologous end joining (NHEJ) KO efficiency. Treatment with a BCL inhibitor ABT-263 further improves HDR efficiency by 70% and KO efficiency by 40%. The increased genome editing efficiency is attributed to higher expressions of Cas9 and sgRNA in surviving cells after electroporation. HDR or NHEJ efficiency reaches 95% with dual editing followed by selection of cells with HDR insertion of a selective gene. Moreover, KO efficiency of 100% can be achieved in a bulk population of cells with biallelic HDR KO followed by double selection, abrogating the necessity for single cell cloning. Taken together, these simple yet highly efficient editing strategies provide useful tools for applications ranging from manipulating human iPSC genomes to creating gene-modified animal models.

Figures
Products
  • Cat. No.
    Product Name
    Description
    Target
    Research Area
  • HY-12202
    98.35%, MEK抑制剂
    MEK