Novel Hypomorphic Alleles of the Mouse Tyrosinase Gene Induced by CRISPR-Cas9 Nucleases Cause Non-Albino Pigmentation Phenotypes

<div><p>Tyrosinase is a key enzyme in melanin biosynthesis. Mutations in the gene encoding tyrosinase (<i>Tyr</i>) cause oculocutaneous albinism (OCA1) in humans. Alleles of the <i>Tyr</i> gene have been useful in studying pigment biology and coat color formation. Over 100 different <i>Tyr</i> 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 <i>a priori</i>. Using the CRISPR-Cas9 system, we targeted two distinct regions of exon 1 to induce pigmentation changes and used an <i>in vivo</i> 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 <i>Tyr</i>, <i>dhoosara</i> (Sanskrit for gray) and <i>chandana</i> (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 <i>Tyr</i> gene.</p>