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. 2015 Feb 10:5:8366.
doi: 10.1038/srep08366.

CRISPR-engineered mosaicism rapidly reveals that loss of Kcnj13 function in mice mimics human disease phenotypes

Hua Zhong  1 , Yiyun Chen  2 , Yumei Li  2 , Rui Chen  3 , Graeme Mardon  4
Affiliations

CRISPR-engineered mosaicism rapidly reveals that loss of Kcnj13 function in mice mimics human disease phenotypes

Hua Zhong et al. Sci Rep. .

Erratum in

Abstract

The era of genomics has demanded the development of more efficient and timesaving approaches to validate gene function in disease. Here, we utilized the CRISPR-Cas9 system to generate Kcnj13 mutant mice by zygote injection to verify the pathogenic role of human KCNJ13, mutations of which are thought to cause Leber congenital amaurosis (LCA), an early-onset form of blindness. We found that complete loss of Kcnj13 is likely postnatal lethal. Among surviving F0-generation mice examined, 80% show mosaic KCNJ13 expression in the retinal pigment epithelium (RPE). Mosaic expression correlates with decreased response to light and photoreceptor degeneration, indicating that Kcnj13 mutant mice mimic human KCNJ13-related LCA disease. Importantly, mosaic animals enable us to directly compare Kcnj13 mutant and wild-type RPE cells in the same eye. We found that RPE cells lacking KCNJ13 protein still survive but overlying photoreceptors exhibit cell degeneration. At the same time, wild-type RPE cells can rescue neighboring photoreceptor cells that overlie mutant RPE cells. These results suggest that KCNJ13 expression is required for RPE cells to maintain photoreceptor survival. Moreover, we show that CRISPR-Cas9 engineered mosaicism can be used to rapidly test candidate gene function in vivo.

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Figures

Figure 1
Figure 1. CRISPR-Cas9-induced mutations in Kcnj13 F0 mice.
E1, E2 and E3 in the schematic of the Kcnj13 gene structure indicate three known exons. P1 and P2 are genotyping primers for the CRISPR-Cas9 targeting site. The blue ATG is the putative start codon of Kcnj13. The red arrowhead indicates the potential Cas9 cleavage site. PAM: the protospacer-adjacent motif required for the binding and cleavage of DNA by CRISPR-Cas9. The underlined sequence is the sgRNA target we selected to delete the start codon. Small deletions (1–14 bp), small insertions (1 and 7 bp), and one large deletion (216 bp) were detected. All ATG-deleted alleles are defined as candidate Kcnj13 null alleles (N, named N2-15); alleles that leave the ATG intact are defined as wild-type alleles (W, named W1, W4 and W9). The wild-type reference allele is abbreviated as “WT”. Seven F0 mutant mice were selected to intercross or cross with wild-type C57BL/6 mice to test if their mutant alleles could be transmitted to F1 animals; transmitted alleles are labeled in green.
Figure 2
Figure 2. Decreased responses to light in CRISPR-Cas9 F0 mutant mice as measured by dark-adapted ERG tests.
ERGs were tested at 13 weeks (a,b), 22 weeks (c,d), and 33 weeks (e,f) of age. ‘#’ indicates F0 mutant mouse k813110115; ‘##’ indicates F0 mutant mouse k713110104. ‘***’ indicates a two-way ANOVA p-value < 0.001. Compared to F0 wild-type mice, decreased ERG a- and b-waves suggest functional deficiencies in F0 mutant retinas.
Figure 3
Figure 3. CRISPR-Cas9-induced F0 mosaic loss of KCNJ13 in RPE cells causes photoreceptor disease phenotypes.
Protein expression of KCNJ13 is shown in green, rhodopsin and RPE65 are shown in red. DAPI (blue) marks nuclei. The loss of photoreceptor cells was determined by comparing the thickness of the ONL between mutant and wild-type mice. (a) A cross-section of a F0 wild-type eyecup stained with H&E is shown. (b) KCNJ13 expression in wild-type RPE cells and rhodopsin localization in the outer segment (OS) of photoreceptor cells are shown. Images a and b are from adjacent serial sections of the same F0 wild-type eyecup prepared from an animal at 13 weeks of age. (c,d,e,f,g,h) KCNJ13 mosaic expression and rhodopsin localization in retinas prepared from animals at 13 weeks (c,d), 17 weeks (e), 22 weeks (f), and 33 weeks of age (g,h). Each image represents one mosaic mutant mouse (each unique ID is included with each image). (i,j,k) Loss of photoreceptor cells (i,k) and accompanied rhodopsin mislocalization, indicated by white arrowheads (i,j). Images e and e’, as well as j and j’, are taken from similar regions of adjacent serial sections. k and j’ are from the same H&E stained section and show that retinal morphology and loss of photoreceptor cells correlates with regions lacking KCNJ13 protein. (l,m) Co-staining of KCNJ13 and RPE65 to demonstrate that mutant RPE cells are surviving. RPE, retinal pigment epithelium. OS, outer segment. IS, inner segment. ONL, outer nuclear layer. OPL, outer plexiform layer. INL, inner nuclear layer. IPL, inner plexiform layer. GC, ganglion cell. Scale bar is 50 µm.
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