Publication Date:
2022-05-26
Description:
Author Posting. © The Author(s), 2017. This is the author's version of the work. It is posted here by permission of Genetics Society of America for personal use, not for redistribution. The definitive version was published in Genetics 208 (2018): 673-686, doi:10.1534/genetics.117.300468.
Description:
Studying genes involved in organogenesis is often difficult because many of these
genes are also essential for early development. The allotetraploid frog, Xenopus laevis,
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 Xenopus. The goal of this study is to determine
whether CRISPR/Cas9-mediated gene knockout can be targeted to the Xenopus 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 lhx1 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 Xenopus by editing a gene (slc45a2) that when knocked out results in
albinism without altering kidney development. This study establishes the feasibility of
tissue-specific gene knockout in Xenopus, 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.
Description:
These studies were supported by a National Institutes
of Health (NIH) KO1 grant (K01DK092320 to R.K.M.), startup funding from the
Department of Pediatrics 424 Pediatric Research Center at the University of Texas
McGovern Medical School (to R.K.M.), an NIH National Xenopus Resource Center
grant (P40OD010997 to M.E.H.), and an NIH R01 grant (R01HD084409 to M.E.H.).
Repository Name:
Woods Hole Open Access Server
Type:
Preprint
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