Detection of Multiple Genome Modifications Induced by the CRISPR/Cas9 System

Area of Science:

• * Molecular Biology
• * Genetics
• * Developmental Biology

Background:

• * The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a revolutionary tool for precise genome modification.
• * CRISPR/Cas9 technology has been successfully applied to various model organisms, including zebrafish, for targeted genetic alterations.
• * Efficiently inducing and detecting multiple genomic modifications simultaneously in zebrafish presents a significant challenge.

Purpose of the Study:

• * To describe a procedure for inducing multiple CRISPR/Cas9-mediated genome modifications in zebrafish embryos.
• * To present a convenient and efficient method for detecting CRISPR/Cas9-induced insertion and/or deletion (indel) mutations.

Main Methods:

• * Simultaneous injection of multiple guide RNAs and Cas9 nuclease into zebrafish embryos.
• * Application of the heteroduplex mobility assay (HMA) for mutation detection.
• * Analysis of insertion and/or deletion (indel) mutations at distinct genomic loci.

Main Results:

• * Successful induction of multiple genome modifications at different genomic loci in zebrafish using CRISPR/Cas9.
• * The heteroduplex mobility assay (HMA) proved effective in detecting CRISPR/Cas9-induced indel mutations.
• * The described method allows for simultaneous genome editing and straightforward mutation screening.

Conclusions:

• * CRISPR/Cas9 technology enables efficient multiplex genome editing in zebrafish.
• * The heteroduplex mobility assay (HMA) is a valuable and simple tool for detecting CRISPR/Cas9-induced indel mutations in zebrafish.
• * This combined approach facilitates the study of gene function through multiplex genome engineering in zebrafish models.