Highly Efficient Genome Editing via CRISPR/Cas9 to Create Clock Gene Knockout Cells

J Biol Rhythms. 2015 Oct;30(5):389-95. doi: 10.1177/0748730415597519. Epub 2015 Aug 4.

Abstract

Targeted genome editing using CRISPR/Cas9 is a relatively new, revolutionary technology allowing for efficient and directed alterations of the genome. It has been widely used for loss-of-function studies in animals and cell lines but has not yet been used to study circadian rhythms. Here, we describe the application of CRISPR/Cas9 genome editing for the generation of an F-box and leucine-rich repeat protein 3 (Fbxl3) knockout in a human cell line. Genomic alterations at the Fbxl3 locus occurred with very high efficiency (70%-100%) and specificity at both alleles, resulting in insertions and deletions that led to premature stop codons and hence FBXL3 knockout. Fbxl3 knockout cells displayed low amplitude and long period oscillations of Bmal1-luciferase reporter activity as well as increased CRY1 protein stability in line with previously published phenotypes for Fbxl3 knockout in mice. Thus, CRISPR/Cas9 genome editing should be highly valuable for studying circadian rhythms not only in human cells but also in classic model systems as well as nonmodel organisms.

Keywords: CRISPR/Cas9; Fbxl3; circadian rhythms; clock genes; genome editing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ARNTL Transcription Factors / genetics
  • ARNTL Transcription Factors / metabolism
  • Base Sequence
  • Blotting, Western
  • CRISPR-Cas Systems / genetics*
  • Cell Line, Tumor
  • Circadian Clocks / genetics*
  • Circadian Clocks / physiology
  • Cryptochromes / genetics
  • Cryptochromes / metabolism
  • F-Box Proteins / genetics*
  • F-Box Proteins / metabolism
  • Gene Knockout Techniques / methods*
  • Genome, Human / genetics*
  • HEK293 Cells
  • Humans
  • Luciferases / genetics
  • Luciferases / metabolism
  • Models, Genetic
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • ARNTL Transcription Factors
  • ARNTL protein, human
  • CRY1 protein, human
  • Cryptochromes
  • F-Box Proteins
  • FBXL3 protein, human
  • Luciferases