Morphological analysis of the photoreceptor cell layer in wild type and Q344X-hRho-GFP mice.

<p>A. Light micrographs of Epon resin embedded retinal sections of 4-wk-old littermates. The organization of outer segments (OS), inner segments (IS), and the outer nuclear layer (ONL) are shown for wild type mice (mRho+/+), heterozygous Q344x-hRho-GFP mice (Q344X/+), and homozygous Q344X-hRho-GFP mice (Q344x/Q344x). B. Loss of rod cell nuclei in the ONL with age. The thickness of the ONL in μm is plotted against age to show the rate of nuclear loss. Images were taken from the mid-eccentricity area indicated by * in C. C. Spidergrams of ONL thickness in retinas from wild type, Q344X-hRho-GFP heterozygous, and Q344X-hRho-GFP homozygous mice. We examined retinas from at least three different 10-wk-old mice for each genotype. Eyecup images were divided into 10 segments of equal size on each side of the optic nerve head (ONH) and extending to the ora serrata (ORS), which marks the end of the retina. Thickness of the ONL was measured over each segment. Each data point corresponds to an average of 10 measurements. D. Length of the outer segment layer in wild type, Q344X-hRho-GFP heterozygous, and Q344X-hRho-GFP homozygous mice. OS layer lengths (μm) were measured along the long axis of the outer segments in images from 4-wk-old mice retina sections.</p

Transmission electron micrographs of rod photoreceptor cells in retinas of 4 week old mice.

<p>A. Structural organization at the base of the outer segment in wild type mice (+/+) B. Structural organization at the base of the outer segment in Q344X-hRho-GFP heterozygous mice (Q344X/+). In A and B, typical stacks of membranes disks are attached to the connecting cilium. C. Abnormal ultrastructure of rod cells in retinas from Q344X-hRho-GFP homozygous mouse (Q344X/Q344X). Isolated connecting cilia (asterisks) and disorganized membranes (indicated by arrows) are apparent, but no discernible rod-like structures are visible.</p

Rhodopsin transcript and protein levels in wild type and knockin mouse lines.

<p>A. Rhodopsin mRNA in wild type mice (mRho+/+), hRho-GFP homozygous mice (hRhoG/hRhoG), ID2-hRho-GFP homozygous (ID2/ID2) and heterozygous (ID2/+) mice, and Q344X-hRho-GFP homozygous (Q344X/Q344X) and heterozygous (Q344X/+) mice. Northern blot analysis used radiolabeled probes against human rhodopsin cDNA to detect human and mouse rhodopsin mRNA. Lane 1 shows the five species of mouse rhodopsin mRNA generated from the normal mouse rhodopsin locus. Lanes 2, 3, and 5 show the seven species of human rhodopsin mRNA generated from the knockin alleles. Lanes 4 and 6 show the mixture of species produced in heterozygous mice. Radioactive bands were quantified using a PhosphorImager and normalized for loading using the intensity of the 28S ribosomal rRNA bands in agarose gel before transfer (bottom). Marker sizes at the right of the gel are given in kilobases. B. Protein quantification by immunoblot of retina lysates probed with antibodies directed against the N-terminus of rhodopsin (mAb B630-N). Nine retinas were analyzed for each genotype. C. Total rhodopsin per retina as quantified by difference spectrophotometry. At least three retinas per genotype were analyzed.</p

Mutant human rhodopsin-EGFP knockin mice.

<p>A. Diagram illustrating the procedure used to replace endogenous mouse rhodopsin with the Q344X-hRho-GFP gene. Gene replacement was accomplished by homologous recombination between the endogenous mouse rhodopsin locus and the homologous mouse sequences (dark grey) flanking the HPRT-Q344X-hRho-GFP gene on the targeting vector. Expression of Cre recombinase in HPRT-Q344X-hRho-GFP ES cells eliminated the HPRT minigene. Final mouse lines contain the Q344X-hRho-GFP fusion gene (shown in black, with exons as rectangles and introns as lines), preceded by a single loxP site (inverted triangle). B. Southern-blot analysis of ES cells. Restriction enzymes used to digest genomic DNA for analysis of the 5′- and 3′- ends of the modified locus are shown in parentheses, and fragment sizes are indicated in kilobases. The pattern of bands in hRhoG(H) heterozygous (hRho-GFP/+) mice served as a marker for the correct pattern of bands in Q344X-hRho-GFP heterozygous mice. C. PCR analysis of tail genomic DNA from heterozygous and homozygous Q344X-hRho-GFP mice.</p

Spontaneous gene correction events in retinas of mutant mouse lines ID2-hRho-GFP/+ and Q344X-hRho-GFP/+.

<p>A. Schematic diagram of the structure of the ID2-hRho-GFP and Q344X-hRho-GFP genes and the mechanism leading to hRho-GFP expression. Exons are represented as black rectangles. In the ID2-hRho-GFP gene, the duplicated exon 2 is marked by horizontal arrows and shown in white. B. Projection of a stack of confocal images of a retinal wholemount, showing the GFP positive photoreceptors. The images of clusters of rod cells all came from ID2-hRho-GFP mice. The first image shows an extended supercluster of rod cells that was observed in a single mouse; the adjacent image shows an area of this retina at higher magnification. The next images show a variety of clusters arranged by decreasing number of rods per cluster The two images on the right are from Q344X-hRho-GFP retinas; they show individual rod photoreceptors that are overexposed to make the inner segments and nuclei visible. Scale bars are shown; the 20-μm scale bar applies to all the images without explicit scale bars. C. Age-dependent appearance of single green rods and clusters in ID2-hRho-GFP heterozygous mice. Retinas from several mice were examined at the indicated times and the values were averaged (age in weeks∶number of mice; 4∶2; 17∶6; 20∶3; 24∶2; 36∶4; 38∶4; 52∶6). Error bars indicate standard deviations. D. Age-dependent appearance of single green rods in Q344X-hRho-GFP heterozygous mice. Retinas were examined from several mice at the indicated times and the values were averaged (age in weeks∶number of mice; 2∶12; 4∶22; 10∶3; 11∶4; 15∶5; 18∶16; 35∶4; 65∶4; 70∶7). Error bars indicate standard deviations.</p

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

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

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

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

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

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