Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.

Area of Science:

• Genetics
• Molecular Biology
• Developmental Biology

Background:

• Targeted mutagenesis in zebrafish is crucial for genetic studies.
• Existing methods often result in monoallelic mutations, delaying homozygous analysis to the F2 generation.
• Efficient biallelic mutation generation in F0 zebrafish would enable direct phenotypic analysis.

Purpose of the Study:

• To develop a simple, robust, and efficient method for targeted mutagenesis in zebrafish.
• To create an improved clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system for zebrafish.
• To enable rapid generation of biallelic mutations and simultaneous targeting of multiple genes.

Main Methods:

• Utilized a zebrafish codon-optimized Cas9 protein and custom guide RNAs.
• Applied the CRISPR/Cas9 system to target a reporter transgene and four endogenous zebrafish loci (tyr, golden, mitfa, ddx19).
• Assessed mutagenesis rates, observed phenotypes, and evaluated germ-line transmission.

Main Results:

• Achieved high mutagenesis rates (75-99%) across targeted loci, indicating biallelic mutations in most cells.
• Observed recessive null-like phenotypes in four out of five targeted endogenous genes.
• Demonstrated efficient germ-line transmission of induced mutations.
• Successfully targeted five genomic loci simultaneously for multiplexed gene knockout.

Conclusions:

• The improved CRISPR/Cas9 system is highly effective for generating biallelic gene knockouts in zebrafish.
• This scalable method allows for direct phenotypic analysis in F0 animals and multiplexed gene targeting.
• The system holds significant potential for genetic research and applications in other model organisms.