One-Step Generation of Seamless Luciferase Gene Knockin Using CRISPR/Cas9 Genome Editing in Human Pluripotent Stem Cells

Methods Mol Biol. 2019:1942:61-69. doi: 10.1007/978-1-4939-9080-1_5.

Abstract

Human pluripotent stem cells (hPSCs) offer powerful platforms for studying mechanisms of human diseases and for evaluating potential treatments. Genome editing, particularly the CRISPR/Cas9-based method, is highly effective for generating cell and animal models to study genetic human diseases. However, the procedure for generating gene-edited hPSCs is laborious, time consuming and unintentional genetic changes may confound the consequent experiments and conclusions. Here we describe one-step knockin of the NanoLuc luciferase gene (Nluc) to the fragile X syndrome gene, FMR1, in a human embryonic stem cell line (hESC), H1, and a fragile X disease model human induced pluripotent stem cell line (hiPSC), FX-iPSC. The luciferase reporter cell lines provide new platforms for exploring potential treatments for fragile X syndrome. The shortened and scarless targeting method described here can be effectively applied to other genes.

Keywords: CRISPR/Cas9; FMR1; Fragile X; Genome editing; Human pluripotent stem cells; Knockin; Luciferase reporter.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Cells, Cultured
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / metabolism
  • Fragile X Mental Retardation Protein / genetics*
  • Fragile X Syndrome / genetics
  • Gene Editing / methods*
  • Gene Knock-In Techniques / methods*
  • Genome, Human*
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / metabolism*
  • Luciferases / metabolism*

Substances

  • FMR1 protein, human
  • Fragile X Mental Retardation Protein
  • Luciferases