CRISPR-Cas9-Mediated Genomic Deletions Protocol in Zebrafish

João Pedro Amorim; Renata Bordeira-Carriço; Ana Gali-Macedo; Chiara Perrod; José Bessa

doi:10.1016/j.xpro.2020.100208

CRISPR-Cas9-Mediated Genomic Deletions Protocol in Zebrafish

STAR Protoc. 2020 Dec 10;1(3):100208. doi: 10.1016/j.xpro.2020.100208. eCollection 2020 Dec 18.

Authors

João Pedro Amorim^{1

2}, Renata Bordeira-Carriço^{1

2}, Ana Gali-Macedo^{1

2}, Chiara Perrod^{1

2}, José Bessa^{1

2}

Affiliations

¹ i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.
² IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.

Abstract

Since its first application for site-directed mutagenesis, the CRISPR-Cas9 system has revolutionized genome engineering. Here, we present a validated workflow for the generation of targeted genomic deletions in zebrafish, including the design, cloning, and synthesis of single-guide RNAs and Cas9 mRNA, followed by microinjection in zebrafish embryos and subsequent genotype screening for the establishment of a mutant line. The versatility and efficiency of this pipeline makes the generation of zebrafish models a widely used approach in functional genetics. For complete details on the use and execution of this protocol, please refer to Amorim et al. (2020).

Keywords: CRISPR; Genetics; Genomics; Model Organisms.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Cas Systems / physiology
Gene Editing / methods*
Genetic Engineering / methods
Genome
Genomics
Microinjections / methods*
Mutagenesis, Site-Directed / methods*
RNA, Guide, CRISPR-Cas Systems / genetics
Sequence Deletion / genetics
Zebrafish / genetics

Substances

RNA, Guide, CRISPR-Cas Systems