title: Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes.
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.
authors.affiliation: Program in Developmental Biology, Baylor College of Medicine, USA; Department of Genetics, University of Texas M.D. Anderson Cancer Center, USA.
authors.forename: Shuo-Ting
authors.initials: ST
authors.lastname: Yen
authors.affiliation: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Genetics, University of Texas M.D. Anderson Cancer Center, USA.
authors.forename: Jian Min
authors.initials: JM
authors.lastname: Deng
authors.affiliation: Department of Genetics, University of Texas M.D. Anderson Cancer Center, USA.
authors.forename: Shireen J
authors.initials: SJ
authors.lastname: Usman
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, USA.
authors.forename: Chad N
authors.initials: CN
authors.lastname: Smith
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, USA.
authors.forename: Jan
authors.initials: J
authors.lastname: Parker-Thornburg
authors.affiliation: Texas Heart Institute, Houston, TX 77030, USA.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Swinton
authors.affiliation: 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.
authors.forename: James F
authors.initials: JF
authors.lastname: Martin
authors.affiliation: 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.
authors.forename: Richard R
authors.initials: RR
authors.lastname: Behringer
mesh_terms: D000417:Albinism / Q000235:genetics*; D000483:Alleles; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D000076987:CRISPR-Associated Protein 9; D064130:CRISPR-Associated Proteins; D064113:CRISPR-Cas Systems; D004720:Endonucleases / Q000235:genetics*; D005260:Female; D000072669:Gene Editing / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D008297:Male; D008543:Melanins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D014442:Monophenol Monooxygenase / Q000235:genetics*; D009030:Mosaicism / Q000196:embryology*; D016296:Mutagenesis; D009154:Mutation; D010858:Pigmentation / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D017422:Sequence Analysis, DNA; D015053:Zygote / Q000166:cytology; D058727:RNA, Small Untranslated
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D008543:Melanins; D012333:RNA, Messenger; D014442:Monophenol Monooxygenase; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D058727:RNA, Small Untranslated
keywords: Gene targeting; Genome editing; Knockout; Melanocyte; Mosaic; Pigmentation