title: Novel Hypomorphic Alleles of the Mouse Tyrosinase Gene Induced by CRISPR-Cas9 Nucleases Cause Non-Albino Pigmentation Phenotypes.
abstract: Tyrosinase is a key enzyme in melanin biosynthesis. Mutations in the gene encoding tyrosinase (Tyr) cause oculocutaneous albinism (OCA1) in humans. Alleles of the Tyr gene have been useful in studying pigment biology and coat color formation. Over 100 different Tyr alleles have been reported in mice, of which ≈24% are spontaneous mutations, ≈60% are radiation-induced, and the remaining alleles were obtained by chemical mutagenesis and gene targeting. Therefore, most mutations were random and could not be predicted a priori. Using the CRISPR-Cas9 system, we targeted two distinct regions of exon 1 to induce pigmentation changes and used an in vivo visual phenotype along with heteroduplex mobility assays (HMA) as readouts of CRISPR-Cas9 activity. Most of the mutant alleles result in complete loss of tyrosinase activity leading to an albino phenotype. In this study, we describe two novel in-frame deletion alleles of Tyr, dhoosara (Sanskrit for gray) and chandana (Sanskrit for sandalwood). These alleles are hypomorphic and show lighter pigmentation phenotypes of the body and eyes. This study demonstrates the utility of CRISPR-Cas9 system in generating domain-specific in-frame deletions and helps gain further insights into structure-function of Tyr gene.
authors.affiliation: Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Anil K
authors.identifier: 0000-0002-3633-3304
authors.initials: AK
authors.lastname: Challa
authors.affiliation: Department of Optometry and Vision Sciences, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Evan R
authors.initials: ER
authors.lastname: Boitet
authors.affiliation: Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Ashley N
authors.initials: AN
authors.lastname: Turner
authors.affiliation: Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Larry W
authors.initials: LW
authors.lastname: Johnson
authors.affiliation: Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kennedy
authors.affiliation: Science and Technology Honors Program, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Ethan R
authors.initials: ER
authors.lastname: Downs
authors.affiliation: Science and Technology Honors Program, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Hymel
authors.affiliation: Department of Optometry and Vision Sciences, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Alecia K
authors.initials: AK
authors.lastname: Gross
authors.affiliation: Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Kesterson
mesh_terms: D000483:Alleles; D000818:Animals; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D005260:Female; D006200:Hair Color / Q000235:genetics*; D008297:Male; D051379:Mice; D014442:Monophenol Monooxygenase / Q000235:genetics*; D017384:Sequence Deletion
chemical_list: D014442:Monophenol Monooxygenase