High-throughput Validation of Human Candidate Disease Genes in Zebrafish
Since the completion of the Human Genome Project and the advent of low-cost whole-genome sequencing technologies, hundreds of genome-wide association studies (GWAS) and whole-exome sequencing projects have been revealing the full spectrum of mutations associated with human disease genes. Human geneticists are now facing the immense challenge of validating these candidate disease genes. For the vast majority of genes the clinical data is only correlative, i.e. it is insufficient to demonstrate causality of the disease state.
Our lab is focused on tackling the huge challenge of systematically validating the flood of human candidate disease genes identified in various genome- wide association (GWAS) and whole-exome sequencing studies with the additional goal of generating animal models for the validated disease genes.
We have developed and validated a high-throughput strategy for mutagenizing the zebrafish genome using CRISPR/Cas9 (Varshney et al. Gen Res 2015), making targeted screening of hundreds of gene knockouts possible with relatively modest resource investments.
Previous forward and reverse genetic screens in zebrafish, while labor intensive, have generated thousands of papers and tens to hundreds of useful disease models in zebrafish, but with the transformative development of gene targeting approaches using CRISPR/Cas9, completely new screening paradigms can be developed. We will use the high-throughput, targeted mutagenesis techniques that I developed at the National Human Genome Research Institute to systematically mutagenize genes in the zebrafish genome related to human deafness. As a proof-of- principle, We selected 50 human, non-syndromic deafness genes from the carefully curated list at hereditaryhearingloss.org, and have already generated knockouts of all 50 orthologs in zebrafish. We are systematically testing systematically these knockouts for the phenotypic characteristics of deaf fish, e.g. failed startle responses, aberrant swimming or developmental defects in the ear or lateral line.
Development of Novel CRISPR-based Strategies for Functional Genomics in Zebrafish
We believe that it is essential to continually push technology development that will break open new avenues of research, so we will continue to develop new cutting edge techniques such as CRISPR/Cas9 based methods to integrate fluorescent tags (knock-in) to study the expression patterns and subcellular protein dynamics of candidate genes, or artificial transcription factors using modified Cas9 proteins. It is critical to develop the efficient techniques for “humanizing” zebrafish mutants (it is currently possible but difficult) to test the wide variety of missense alleles believed to result in altered gene function.