CRISPR-Cas9-Mediated Genomic Deletions Protocol in Zebrafish.

STAR protocols (2020-12-31)
João Pedro Amorim, Renata Bordeira-Carriço, Ana Gali-Macedo, Chiara Perrod, José Bessa
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).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Acetic acid, glacial, ReagentPlus®, ≥99%
Sigma-Aldrich
Magnesium chloride, anhydrous, ≥98%
Sigma-Aldrich
Sodium acetate, anhydrous, for molecular biology, ≥99%
Sigma-Aldrich
Diethyl pyrocarbonate, ≥97% (NMR)
Sigma-Aldrich
Calcium chloride dihydrate, ACS reagent, ≥99%
Sigma-Aldrich
Yeast Extract, for use in microbial growth medium
Sigma-Aldrich
Magnesium chloride hexahydrate, BioXtra, ≥99.0%
Sigma-Aldrich
Phenol red solution, 0.5%, liquid, sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Ethyl 3-aminobenzoate methanesulfonate, 98%