Targeted knockout of lhx1 via CRISPR/Cas9 gene editing in the Xenopus laevis kidney
Targeted knockout of lhx1 via CRISPR/Cas9 gene editing in the Xenopus laevis kidney
Date
2017-09-29
Authors
DeLay, Bridget D.
Corkins, Mark E.
Hanania, Hannah L.
Salanga, Matthew C.
Deng, Jian Min
Sudou, Norihiro
Taira, Masanori
Horb, Marko E.
Miller, Rachel K.
Corkins, Mark E.
Hanania, Hannah L.
Salanga, Matthew C.
Deng, Jian Min
Sudou, Norihiro
Taira, Masanori
Horb, Marko E.
Miller, Rachel K.
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Alternative Title
Tissue-Specific Gene Inactivation in Xenopus laevis : Knockout of lhx1 in the Kidney with CRISPR/Cas9
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Abstract
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
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.