Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells

STAR Protoc. 2021 Nov 16;2(4):100965. doi: 10.1016/j.xpro.2021.100965. eCollection 2021 Dec 17.

Abstract

Selection-free, scarless genome editing in human pluripotent stem cells (PSCs) by utilizing ribonucleoprotein (RNP) of CRISPR-Cas9 is a useful tool for a variety of applications. However, the process can be hampered by time-consuming subcloning steps and inefficient delivery of the RNP complex and ssDNA template. Here, we describe the optimized protocol to introduce a single nucleotide change or a loxP site insertion in feeder-free, xeno-free iPSCs by utilizing MaxCyte and 4D-Nucleofector electroporators. For complete details on the use and execution of this protocol, please refer to Kagita et al. (2021) and Xu et al. (2019).

Keywords: CRISPR; Cell isolation; Genetics; Molecular Biology; Stem Cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • CRISPR-Cas Systems / genetics*
  • DNA, Single-Stranded / genetics
  • Electroporation / methods*
  • Female
  • Gene Editing / methods*
  • Homologous Recombination / genetics
  • Humans
  • Male
  • Pluripotent Stem Cells* / cytology
  • Pluripotent Stem Cells* / metabolism
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • RNA, Guide, CRISPR-Cas Systems / metabolism
  • Ribonucleoproteins* / genetics
  • Ribonucleoproteins* / metabolism

Substances

  • DNA, Single-Stranded
  • RNA, Guide, CRISPR-Cas Systems
  • Ribonucleoproteins