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

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

Author information

1
Program in Developmental Biology, Baylor College of Medicine, USA; Department of Genetics, University of Texas M.D. Anderson Cancer Center, USA.
2
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, USA.
3
Department of Genetics, University of Texas M.D. Anderson Cancer Center, USA.
4
Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, USA.
5
Texas Heart Institute, Houston, TX 77030, USA.
6
Program in Developmental Biology, Baylor College of Medicine, USA; Department of Molecular Physiology and Biophysics, Baylor College of Medicine, USA; Texas Heart Institute, Houston, TX 77030, USA.
7
Program in Developmental Biology, Baylor College of Medicine, USA; Department of Genetics, University of Texas M.D. Anderson Cancer Center, USA. Electronic address: rrb@mdanderson.org.

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

PMID:
24984260
PMCID:
PMC4166609
DOI:
10.1016/j.ydbio.2014.06.017
[Indexed for MEDLINE]
Free PMC Article

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