Optimised genome editing for precise DNA insertion and substitution using prime editors in zebrafish

Area of Science:

• Molecular Biology
• Genetics
• Zebrafish Model Systems

Background:

• CRISPR/Cas9 is widely used for gene function studies and creating genetically modified organisms.
• Stochastic integration of insertions/deletions with CRISPR/Cas9 limits precise genome manipulation.
• Prime Editors (PEs) offer programmed integration of short DNA modifications but face challenges in animal models.

Purpose of the Study:

• To utilize nickase- and nuclease-based Prime Editors (PEs) for programmed DNA substitutions and insertions in the zebrafish genome.
• To evaluate the efficiency and precision of different PE variants for genome editing in zebrafish.
• To demonstrate the capability of PEs for incorporating longer DNA fragments and heritable gene modifications.

Main Methods:

• Application of nickase-based PE2 and nuclease-based PEn for targeted genome editing in zebrafish.
• Assessment of precise prime editing efficiency for substitutions and insertions at various genomic loci.
• Insertion of a nuclear localization signal into a reporter transgene using prime editing.

Main Results:

• Nickase-based PE2 showed a higher ratio of precise prime edits to total edits.
• Nuclease-based PEn achieved higher efficiency for short DNA modifications, with up to 27.3% precise insertion.
• Successful insertion of longer DNA fragments and transmission of edited genes to the next generation.

Conclusions:

• Prime editing offers an efficient method for precise genome manipulation in zebrafish.
• PE-mediated editing can be used to incorporate longer DNA fragments and achieve heritable genetic modifications.
• This approach enables efficient manipulation of zebrafish genome information without requiring exogenous donor DNA.