@article{814f90324080407e87f255bff78c535e,
title = "Tissue-specific gene inactivation in xenopus laevis: Knockout of lhx1 in the kidney with CRISPR/Cas9",
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.",
keywords = "CRISPR, Kidney, Lhx1, Targeted injection, Xenopus laevis",
author = "Delay, {Bridget D.} and Corkins, {Mark E.} and Hanania, {Hannah L.} and Matthew Salanga and Deng, {Jian Min} and Norihiro Sudou and Masanori Taira and Horb, {Marko E.} and Miller, {Rachel K.}",
note = "Funding Information: We thank the instructors and teaching assistants of the 2015 and 2016 Cold Spring Harbor Laboratory Xenopus Course, in particular K.J. Liu, M.K. Khokha, and E.K. Mis, for training in Xenopus CRISPR gene editing and in situ hybridization techniques. The instructors of the 2017 National Xenopus Resource Xenopus Genome Editing Workshop also provided advice on genome editing techniques. We also thank N.R. De Lay for the gift of the T7 polymerase expression plasmid and Rosetta cells, as well as for advice on sgRNA production and purification. We are grateful to the members of the laboratories of R.K.M. and P.D. McCrea, as well as to M. Kloc, for their helpful suggestions and advice throughout this project. In particular, we thank V. Krneta-Stankic for early help with injections. Additionally, we thank S.-T. Yen for assessing the lhx1 CRISPR sgRNA sequences, as well as S. McNamara and N. Aryal for guidance on CRISPR sgRNA design. We especially thank R.R. Behringer for advice on CRISPR knockout of lhx1 and for critically reading this article and providing guidance throughout this project. We are grateful to the University of Texas Health Science Center Office of the Executive Vice President and Chief Academic Officer and the Department of Pediatrics Microscopy Core for funding and maintaining the Zeiss LSM800 confocal microscope used in this work. Funding Information: 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 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.). Publisher Copyright: {\textcopyright} 2018 by the Genetics Society of America.",
year = "2018",
month = feb,
doi = "10.1534/genetics.117.300468",
language = "English (US)",
volume = "208",
pages = "673--686",
journal = "Genetics",
issn = "0016-6731",
publisher = "Genetics Society of America",
number = "2",
}