Developing robust genome editing tools for generating floxed alleles and editing amino acid change in zebrafish

PROJECT SUMMARY/ABSTRACT Zebrafish provide a valuable model system for studying developmental and regenerative biology. Recent advancements in genome editing techniques enable the generation of knock-out alleles in zebrafish, expanding the usefulness of this model organism to interrogate loss-of-function study. However, the majority of genome editing-mediated mutants in zebrafish consist of conventional loss- of-function mutants rather than more sophisticated alleles, such as conditional loss-of-function alleles. Thus, a critical obstacle in zebrafish research lies in the functional dissection of genes within specific cell types and during later development stages. A reliable and definitive conditional loss-of-function system that has been utilized extensively in other organisms is Cre/lox-mediated gene disruption. Although this Cre/lox system is functional in zebrafish, creating reliable floxed alleles has remained challenging. This proposal aims to address the existing limitations in conditional genetics within the zebrafish model by developing a robust and effective methodology. In Aim1, we will establish a synthetic exon-mediated integration approach, allowing the addition of two loxp sites into the target gene within a single generation. We will compare our strategy with the previously published method to assess efficiency. Our study will establish the effective method to generate reliable floxed alleles. Combined with CreER lines, these floxed alleles will enable conditional knock-out of target genes at a predefined time and/or in discrete cells, facilitating investigation of intricate cellular and molecular mechanisms. Advancements in base and prime editing techniques enable to introduce human disease- causing mutations into the zebrafish genome, significantly promoting the application of zebrafish for human disease modeling. However, the current base and prime editing approaches rely on PCR-based screening to identify lines carrying the edited genome. Given the low efficiency of base/prime editing and a reduced germline transmission rate, the current screening method may fail to select positive fish due to the complicated multiple steps for genotyping. In Aim 2, we will expand our synthetic exon- mediated genome editing approach to develop a proficient and highly effective method for editing amino acid changes in zebrafish. We will combine a synthetic exon approach with a robust fluorescence- based screening strategy to dramatically reducing screening effort for introducing amino acid change. Overall, our methods, lines, and reagents will save years of research time in developing diverse zebrafish genetic models that can open new research avenues within the zebrafish community.