Tissue-Specific Gene Inactivation in : Knockout of in the Kidney with CRISPR/Cas9.

Studying genes involved in organogenesis is often difficult because many of these genes are also essential for early development. The allotetraploid frog, , is commonly used to study developmental processes, but because of the presence of two homeologs for many genes, it has been difficult to use as a genetic model. Few studies have successfully used CRISPR in amphibians, and currently there is no tissue-targeted knockout strategy described in The goal of this study is to determine whether CRISPR/Cas9-mediated gene knockout can be targeted to the kidney without perturbing essential early gene function. We demonstrate that targeting CRISPR gene editing to the kidney and the eye of F0 embryos is feasible. Our study shows that knockout of both homeologs of results in the disruption of kidney development and function but does not lead to early developmental defects. Therefore, targeting of CRISPR to the kidney may not be necessary to bypass the early developmental defects reported upon disruption of Lhx1 protein expression or function by morpholinos, antisense RNA, or dominant negative constructs. We also establish a control for CRISPR in by editing a gene () that when knocked out results in albinism without altering kidney development. This study establishes the feasibility of tissue-specific gene knockout in , providing a cost-effective and efficient method for assessing the roles of genes implicated in developmental abnormalities that is amenable to high-throughput gene or drug screening techniques.