Gene editing in mouse zygotes using the CRISPR/Cas9 system

Methods. 2017 May 15:121-122:55-67. doi: 10.1016/j.ymeth.2017.02.008. Epub 2017 Mar 2.

Abstract

The generation of targeted mouse mutants is a key technology for biomedical research. Using the CRISPR/Cas9 system for induction of targeted double-strand breaks, gene editing can be performed in a single step directly in mouse zygotes. This article covers the design of knockout and knockin alleles, preparation of reagents, microinjection or electroporation of zygotes and the genotyping of pups derived from gene editing projects. In addition we include a section for the control of experimental settings by targeting the Rosa26 locus and PCR based genotyping of blastocysts.

Keywords: CRISPR; Cas9; Gene editing; Mouse; Zygotes.

MeSH terms

  • Animals
  • Animals, Newborn
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • DNA / genetics
  • DNA / metabolism
  • DNA Breaks, Double-Stranded
  • DNA End-Joining Repair
  • Electroporation / methods
  • Endonucleases / genetics*
  • Endonucleases / metabolism
  • Gene Editing / methods*
  • Gene Knock-In Techniques*
  • Gene Knockout Techniques*
  • Gene Targeting / methods
  • Gene Transfer Techniques*
  • Genome
  • Mice
  • Mice, Transgenic
  • Microinjections
  • RNA, Guide, CRISPR-Cas Systems / genetics*
  • RNA, Guide, CRISPR-Cas Systems / metabolism
  • RNA, Untranslated / genetics
  • RNA, Untranslated / metabolism
  • Recombinational DNA Repair
  • Zygote / cytology
  • Zygote / metabolism

Substances

  • Bacterial Proteins
  • Gt(ROSA)26Sor non-coding RNA, mouse
  • RNA, Guide, CRISPR-Cas Systems
  • RNA, Untranslated
  • DNA
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases