Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes

Dev Biol. 2014 Sep 1;393(1):3-9. doi: 10.1016/j.ydbio.2014.06.017. Epub 2014 Jun 28.

Abstract

Tyrosinase is the rate-limiting enzyme for the production of melanin pigmentation. In the mouse and other animals, homozygous null mutations in the Tyrosinase gene (Tyr) result in the absence of pigmentation, i.e. albinism. Here we used the CRISPR/Cas9 system to generate mono- and bi-allelic null mutations in the Tyr locus by zygote injection of two single-guide and Cas9 RNAs. Injection into C57BL/6N wild-type embryos resulted in one completely albino founder carrying two different Tyr mutations. In addition, three pigmentation mosaics and fully pigmented littermates were obtained that transmitted new mutant Tyr alleles to progeny in test crosses with albinos. Injection into Tyr heterozygous (B6CBAF1/J×FVB/NJ) zygotes resulted in the generation of numerous albinos and also mice with a graded range of albino mosaicism. Deep sequencing revealed that the majority of the albinos and the mosaics had more than two new mutant alleles. These visual phenotypes and molecular genotypes highlight the somatic mosaicism and allele complexity in founders that occurs for targeted genes during CRISPR/Cas9-mediated mutagenesis by zygote injection in mice.

Keywords: Gene targeting; Genome editing; Knockout; Melanocyte; Mosaic; Pigmentation.

Publication types

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

MeSH terms

  • Albinism / genetics*
  • Alleles
  • Animals
  • Bacterial Proteins / genetics*
  • Base Sequence
  • CRISPR-Associated Protein 9
  • CRISPR-Associated Proteins*
  • CRISPR-Cas Systems*
  • Endonucleases / genetics*
  • Female
  • Gene Editing / methods*
  • High-Throughput Nucleotide Sequencing
  • Male
  • Melanins / genetics
  • Mice
  • Mice, Inbred C57BL
  • Monophenol Monooxygenase / genetics*
  • Mosaicism / embryology*
  • Mutagenesis
  • Mutation
  • Pigmentation / genetics*
  • RNA, Guide / genetics
  • RNA, Messenger / genetics
  • Sequence Analysis, DNA
  • Zygote / cytology

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • Melanins
  • RNA, Guide
  • RNA, Messenger
  • Monophenol Monooxygenase
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases