Tissue-Targeted CRISPR-Cas9-Mediated Genome Editing of Multiple Homeologs in F0 Generation Xenopus Laevis Embryos

Authors

Mark E Corkins
Bridget D DeLay
Rachel K Miller

Publication Date

3-1-2022

Journal

Cold Spring Harbor Protocols

Abstract

Xenopus laevis frogs are a powerful developmental model that enables studies combining classical embryology and molecular manipulation. Because of the large embryo size, ease of microinjection, and ability to target tissues through established fate maps, X. laevis have become the predominant amphibian research model. Given that their allotetraploid genome has complicated the generation of gene knockouts, strategies need to be established for efficient mutagenesis of multiple homeologs to evaluate gene function. Here we describe a protocol to utilize CRISPR-Cas9-mediated genome editing to target either single alleles or multiple alloalleles in F0 X. laevis embryos. A single guide (sg) RNA is designed to target a specific DNA sequence encoding a critical protein domain. To mutagenize a gene with two alloalleles, the sgRNA is designed against a sequence that is common to both homeologs. This sgRNA, along with the Cas9 protein, is microinjected into the zygote to disrupt the genomic sequences in the whole embryo or into a specific blastomere for tissue-targeted effects. Error-prone repair of CRISPR/Cas9 generated DNA double strand breaks leads to insertions and deletions creating mosaic gene lesions within the embryos. The genomic DNA isolated from each mosaic F0 embryo is sequenced, and software is applied to assess the nature of the mutations generated and degree of mosaicism. This protocol enables the knockout of genes within the whole embryo or in specific tissues in F0 X. laevis embryos to facilitate the evaluation of resulting phenotypes.

Keywords

Animals, CRISPR-Associated Protein 9, CRISPR-Cas Systems, Gene Editing, RNA, Guide, CRISPR-Cas Systems, Xenopus laevis

Comments

PMID: 34911820