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></div

CRISPR targeting and mutation detection by heteroduplex mobility assay in the <i>Tyr</i> gene.

<p>(A) Schematic showing CRISPR targeting regions (blue bold arrows), PCR primer binding sites, and amplicon sizes. (B) Breeding scheme with genotype of the zygotes used for CRISPR-Cas9 injections, and the CRISPR target sequences on both chromosomes. (C, D, E) Images of ethidium bromide stained polyacrylamide gels (6%) showing separation of homoduplex and heteroduplex PCR amplicons from CRISPR-Cas9 RNA injected, single cultured blastocysts (C, arrowheads) and tail DNA of potential founder mice (D, E). Gels on the left correspond to the 5’CRISPR target site, and those on the right correspond to the 3’CRISPR target site. Small and large square brackets indicate homoduplex and heteroduplex bands, respectively. L = 100 bp ladder; C = uninjected wildtype control.</p

Details of the F0 animals obtained from CRISPR-Cas9 injections.

<p>Details of the F0 animals obtained from CRISPR-Cas9 injections.</p

Schematic of possible sequence of events producing related <i>dhoosara</i> and <i>chandana</i> alleles.

<p>CRISPR-Cas9 nuclease activity in the zygote at the 5’ target site on the wildtype chromosome results in a 15 bp deletion. After the cell division, nuclease activity persists and creates a DSB at the 3’ target site on the previously modified chromosome. NHEJ repair in this daughter cell results in a second, 3 bp deletion.</p

Comparison of <i>dhoosara</i> and <i>chandana</i> with the albino and black/wild type mice.

<p>(A) Dorsal view showing the coat color of albino, <i>chandana</i>, <i>dhoosara</i>, and black animals. Increasing levels of pigmentation can be seen in the hind limbs and tips of tails as well. (B) Frontal view of the face with increasing pigmentation in the nose region. Differences in the eye color can be noted. (C) Brightfield images of RPE wholemounts from Albino, <i>chandana</i>, <i>dhoosara</i> and Black mice. (D) Brightfield images of retinal cryosections from Albino, <i>chandana</i>, <i>dhoosara</i> and black mice. The RPE layer is the only layer within the eyeball that contains pigment. Scale bar = 50 μm. (E) Mean gray values (quantified in ImageJ) obtained from the RPE wholemounts reflecting relative intensity of RPE cell pigmentation. Results are presented in a bar chart with standard error of mean used for error bars. All groups are significantly different from each other (p<0.05, t-test). (F) Western Blot of tyrosinase protein isolated from skin of albino (alb), <i>chandana (cha)</i>, <i>dhoosara (dho)</i>, and black animals (arrow points to the 60 kDa tyrosinase band); 50 kDa tubulin protein (lower panel) is used as a control.</p

Multiples mutant alleles resulting from NHEJ at 5’ and 3’ CRISPR target sites.

<p>Multiples mutant alleles resulting from NHEJ at 5’ and 3’ CRISPR target sites.</p

A391V is a potential pathogenic allele.

<p>(A) Sanger Sequence confirmation of the 1172 C>T point mutation in Gas8^AV mice reproducing the A391V missense mutation found in the human patient. (B) Ciliary beat frequency analysis on tracheal cilia of Gas8^GT/WT and Gas8^GT/AV mice shows no difference between controls and compound heterozygotes (n = 86 points from 3 trachea for Gas8^GT/WT (13.04 Hz), n = 76 points from 3 tracheas for Gas8^GT/AV (13.34 Hz)). (C) Tracking of red fluorescent latex beads added to lateral ventricles shows a trending but not significant decrease in ability of Gas8^GT/AV cilia to move fluid. (n = 3 for Gas8^GT/WT (163.7μm/sec), n = 2 for Gas8^GT/AV (135.9μm/sec)). (D) Nissl stained brains of 10 week old Gas8^GT/WT, Gas8^WT/AV, and Gas8^GT/AV mice. Mild to moderate hydrocephalus is present in the Gas8^GT/AV brains. Scale is 1mm (Arrowhead indicates mild, arrow indicates moderate) (n = 4). (E) Swim speed quantification of rescue of DRC4-D198K construct in <i>pf2</i> deficient <i>Chlamydomonas</i>. “<i>pf2</i>” denotes <i>pf2</i> deficient <i>Chlamydomonas</i>, “DK-GFP” denotes <i>pf2</i> deficient <i>Chlamydomonas</i> expressing the DRC4-D198K-GFP construct, “DRC4-GFP” denotes <i>pf2</i> deficient <i>Chlamydomonas</i> expressing the DRC4-GFP wild-type construct. * = significant difference from WT (p<0.05), # = significant difference from <i>pf2</i> (p<0.05), § = significant difference between DK-GFP and DRC4-GFP (p<0.05) (n = 390 for WT (123.9μm/sec), n = 271 for <i>pf2</i> (40.1μm/sec), n = 180 for DK-GFP (108.3μm/sec), n = 299 for DRC4-GFP (120.9μm/sec)).</p

PCD patient missense mutations in highly conserved regions of Gas8.

<p>(A) Sanger Sequence trace and amino acid sequence of human mutations. (B) ClustalOmega amino acid alignment indicates the high conservation of the missense mutant residues found in patients which are indicated by black boxes.</p

Gas8 localizes to cilia.

<p>Gas8 immunofluorescence staining on Gas8^WT and Gas8^GT p21 trachea (Scale Bar = 10μm). Gas8 is present in wild-type axonemes but absent from mutant axonemes. DIC images are shown to visualize the cilia on the tracheal epithelium.</p

Generation of mutant Gas8^GT mice and phenotype description.

<p>(A) Schematic of the wildtype Gas8 allele (Gas8^WT) and the Gas8 genetrap allele (Gas8^GT). The relative position of the β-geo cassette is indicated by the blue box. Arrows indicate the primers used for RT-PCR analysis. (B) RT-PCR expression analysis of Gas8 transcript in Gas8^WT, Gas8^GT/WT, Gas8^GT whole embryos shows the presence of the 5’ end of Gas8 transcript (left panels) and absence of the 3’ end (right panels). Actin served as a positive template control in all samples. Reactions with reverse transcriptase are indicated (+) and negative RT controls (-). (C) Western blot for Gas8 protein on Gas8^WT, Gas8^GT/WT, and Gas8^GT trachea. Wildtype Gas8 is located at 57 kDa (Gas8^WT) while the genetrap allele is at approximately 230kDa (Gas8^GT). * denotes a spurious band recognized by polyclonal antibody. GAPDH was used as a loading control. (D) Nissl stained coronal section of P21 Gas8^WT and Gas8^GT brain. (E) Gas8^GT mice display <i>situs inversus</i> as noted by the reversed direction of the heart apex (white lines indicate heart axis) and (F) the stomach location in P2 pups (arrow).</p