High-Level Precise Knockin of iPSCs by Simultaneous Reprogramming and Genome Editing of Human Peripheral Blood Mononuclear Cells

Stem Cell Reports. 2018 Jun 5;10(6):1821-1834. doi: 10.1016/j.stemcr.2018.04.013. Epub 2018 May 10.

Abstract

We have developed an improved episomal vector system for efficient generation of integration-free induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells. More recently, we reported that the use of an optimized CRISPR-Cas9 system together with a double-cut donor increases homology-directed repair-mediated precise gene knockin efficiency by 5- to 10-fold. Here, we report the integration of blood cell reprogramming and genome editing in a single step. We found that expression of Cas9 and KLF4 using a single vector significantly increases genome editing efficiency, and addition of SV40LT further enhances knockin efficiency. After these optimizations, genome editing efficiency of up to 40% in the bulk iPSC population can be achieved without any selection. Most of the edited cells show characteristics of iPSCs and genome integrity. Our improved approach, which integrates reprogramming and genome editing, should expedite both basic research and clinical applications of precision and regenerative medicine.

Keywords: CRISPR-Cas9; HDR; episomal vector; genome editing; induced pluripotent stem cell; peripheral blood mononuclear cell; reprogramming.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems
  • Cellular Reprogramming Techniques
  • Cellular Reprogramming* / genetics
  • Gene Editing*
  • Gene Knock-In Techniques*
  • Gene Order
  • Gene Targeting
  • Genetic Vectors / genetics
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / metabolism*
  • Kruppel-Like Factor 4
  • Leukocytes, Mononuclear / cytology*
  • Leukocytes, Mononuclear / metabolism*
  • Plasmids / genetics