Tissue-Specific Gene Inactivation in Xenopus laevis: Knockout of lhx1 in the Kidney with CRISPR/Cas9

Genetics: A Periodical Record of Investigations Bearing on Heredity and Variation

Norihiro Sudou^5¹  Masanori Taira^6²  Rachel K. Miller^7³  Jian Min Deng^4⁴  Marko E. Horb^3⁵  Mark E. Corkins^1⁵  Bridget D. DeLay^1⁶  Matthew Salanga^3⁷  Hannah L. Hanania^2⁸ 

[1] Department of Anatomy, School of Medicine, Tokyo Women’s Medical University, 162-8666, Japan^5;Department of Biological Sciences, Graduate School of Science, University of Tokyo, 113-8654, Japan^6;Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, Texas 77030;Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, Texas 77030^4;Department of Pediatrics, Pediatric Research Center, University of Texas Health Science Center McGovern Medical School, Houston, Texas 77030;Department of Pediatrics, Pediatric Research Center, University of Texas Health Science Center McGovern Medical School, Houston, Texas 77030^1;National Xenopus Resource and Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, Massachusetts 02543^3;Program in Biochemistry and Cell Biology, Rice University, Houston, Texas 77251^2;Program in Biochemistry and Cell Biology, The University of Texas MD Anderson Cancer Center University of Texas Health Science Center Graduate School of Biomedical Sciences, Houston, Texas 77030^7;Program in Genetics and Epigenetics, The University of Texas MD Anderson Cancer Center University of Texas Health Science Center Graduate School of Biomedical Sciences, Houston, Texas 77030

关键词: CRISPR;    kidney;    targeted injection;    Xenopus laevis;    lhx1;   

DOI  :  10.1534/genetics.117.300468

学科分类：医学（综合）

来源: Genetics Society of America

【 摘 要 】

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.

【 授权许可】

CC BY