Generation of Efficient Knock-in Mouse and Human Pluripotent Stem Cells Using CRISPR-Cas9

Methods Mol Biol. 2021:2320:247-259. doi: 10.1007/978-1-0716-1484-6_22.

Abstract

A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.

Keywords: CRISPR-Cas9; Genome editing; Homology-directed repair; Knock-in; Pluripotent stem cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Cells, Cultured
  • Clone Cells
  • Culture Media
  • DNA Primers
  • Drug Resistance / genetics
  • Electroporation
  • Embryonic Stem Cells / cytology
  • Gene Editing / methods
  • Gene Knock-In Techniques / methods*
  • Genes, Reporter
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Puromycin / pharmacology
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Recombinational DNA Repair / genetics

Substances

  • Culture Media
  • DNA Primers
  • RNA, Guide, CRISPR-Cas Systems
  • Puromycin