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

Shuo Ting Yen, Min Zhang, Jian Min Deng, Shireen J. Usman, Chad N. Smith, Jan Parker-Thornburg, Paul G. Swinton, James F. Martin, Richard R. Behringer

Research output: Contribution to journalArticlepeer-review

236 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)3-9
Number of pages7
JournalDevelopmental Biology
Volume393
Issue number1
DOIs
StatePublished - Sep 1 2014

Keywords

  • Gene targeting
  • Genome editing
  • Knockout
  • Melanocyte
  • Mosaic
  • Pigmentation

ASJC Scopus subject areas

  • Molecular Biology
  • Developmental Biology
  • Cell Biology

MD Anderson CCSG core facilities

  • Advanced Technology Genomics Core
  • Genetically Engineered Mouse Facility
  • Research Animal Support Facility

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