Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish.

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• Targeted genome editing tools significantly advance animal model research.
• The CRISPR/Cas9 system shows promise but requires further validation in model organisms regarding efficiency and specificity.

Purpose of the Study:

• To evaluate the efficiency, heritability, and off-target effects of CRISPR/Cas9 mutagenesis in zebrafish.
• To assess the utility of CRISPR/Cas9 for targeted knock-in of protein tags in zebrafish.

Main Methods:

• CRISPR/Cas9 system application in zebrafish for mutagenesis.
• Analysis of mutagenesis efficiency and germline transmission rates.
• Assessment of off-target mutation rates at potential genomic sites.
• Evaluation of knock-in efficiency using a donor oligonucleotide for protein tagging.

Main Results:

• CRISPR/Cas9 mutagenesis achieved high efficiency (up to 86.0%) and was heritable in zebrafish.
• Targeted protein tag knock-in efficiencies ranged from 3.5% to 15.6%.
• Off-target mutation rates were low, ranging from 1.1% to 2.5%, indicating high specificity.

Conclusions:

• CRISPR/Cas9 is a highly efficient and specific genome engineering tool for zebrafish.
• The system facilitates heritable gene modifications and targeted knock-ins, valuable for in vivo studies.
• Limited off-target effects underscore the system's safety and reliability in model organisms.