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.
Highlights
The era of genomics has demanded the development of more efficient and timesaving approaches to validate gene function in disease
We examined 10 surviving F0 mutant mice to investigate the incidence of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas[9] produced mosaicism
Consistent with published data[6,7,19,25], the CRISPR-Cas[9] generated mosaicism was commonly detected in two separate zygote injections with a high efficiency of 80% in total: 8 out of 10 examined Kcnj[13] F0 mutant mice showed KCNJ13 mosaic expression. These animals showed different ranges of KCNJ13 mosaic expression; of particular note, KCNJ13 expression was not observed in k713110103 retinal pigment epithelium (RPE) cells (Fig. 3e) one wild-type allele was detected in F1 progeny together with 3 other mutant alleles (Fig. 1)
Summary
The era of genomics has demanded the development of more efficient and timesaving approaches to validate gene function in disease. Homozygous lethal alleles have required researchers to adopt the Cre-loxP system to produce conditional loss-of-function mosaics in which deletion of the gene of interest is limited to only the cell types or tissues of interest This approach enables animals to survive beyond the lethal phase but at the same time permits study of the null phenotype in specific groups of cells. Recent reports indicate that compound mosaicism, where substantial numbers of cells in F0 animals carry mutations in both copies of a gene, can be detected in CRISPR-Cas[9] generated mice[6,7,19]. Our results demonstrate that CRISPR-Cas[9] generated mosaicism can be efficiently utilized to overcome early lethality and subsequently perform phenotypic analysis and dissect gene function in disease by comparing mutant and wild-type cells in the same F0 animal
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