Optimised genome editing for precise DNA insertion and substitution using Prime Editors in zebrafish

Abstract CRISPR/Cas9-mediated genome editing has rapidly become a popular tool for studying gene functions and generating genetically modified organisms. However, using this system, stochastic integration of random insertions and deletions restricts precise genome manipulation. Advanced CRISPR/Cas9 technologies using Prime Editors (PEs), Cas9 proteins fused with reverse transcriptase, enable programmed integration of short DNA modifications into the genome. However, its application in precise genome editing in animal models is challenging. Here, we utilise a nickase- and a nuclease-based PE to perform programmed short DNA substitution and insertion in various loci in the zebrafish genome. Whereas the nickase-based PE is advantageous for nucleotide substitutions, we find that the nuclease-based PE can be used to insert short DNA fragments precisely with high efficiency. To further evaluate our approach, we inserted a nuclear localisation signal into a reporter transgene to incorporate longer fragments by prime editing. These gene modifications were transmitted to the next generation. We demonstrate that PE-mediated prime editing can efficiently manipulate genome information in zebrafish without using exogenous donor DNA.