Development of scalable methods for rapid phenotyping and functional testing of variants

Project Summary Advances in sequencing have greatly improved our capacity to discover the genetic causes of human diseases. Many genome-wide association studies (GWAS) and exome sequencing projects have uncovered thousands of candidate genes linked to human diseases. However, how these genes contribute to disease pathology is still unclear. Researchers use zebrafish to validate these candidate genes and identify pathologically relevant genes to understand the pathophysiology better. CRISPR/Cas9 technology has significantly impacted functional studies in zebrafish, enabling the generation of loss-of-function alleles. Gene knockout generation is efficient, but point mutations remain challenging to create in a high-throughput manner. Understanding disease pathophysiology through knockout alleles is informative but hypomorphic, and gain-of-function alleles require advanced methods like CRISPR base editors. These tools are underutilized due to limited targeting coverage and low efficiency, but expanding their coverage would benefit the research community. In the first aim, we will use a knockout library of 80 mutants to develop a high-throughput phenotyping pipeline. Then in the second aim, we will optimize and expand the targeting coverage of CRISPR-based base editors that can induce single nucleotide changes in the genome in a targeted manner. We will then use the candidate genes and phenotype data from the Aim 1 to test these base editors on a larger number of loci. These aims will generate 80 disease models and robust base editing tools tested on a diverse set of genes. Completing these aims will result in 80 disease models and improved base editing tools that will be accessible to the research community via the Zebrafish International Resource Center (ZIRC).