Development of Precision Genome Editing Tools for the Functional Characterization of Genetic Variants

About This Grant

Summary Over half of the genetic variants identified through clinical sequencing are variants of uncertain significance (VUS), posing a significant challenge to accurate genetic diagnosis and effective patient care due to the unknown functional impact of these single-nucleotide variants (SNVs). Current genome editing methods lack the efficiency, specificity, and scalability required for high-throughput functional validation in vivo, presenting challenges such as bystander mutations, insertions/deletions, and limited targeting coverage in zebrafish, an ideal model organism for human disease genetics. This proposal aims to establish a high-throughput, cost-effective platform for in vivo functional validation of human disease-associated genetic variants using the zebrafish model. The central hypothesis is that optimized CRISPR editors, when paired with zebrafish-specific computational tools, will enable efficient and precise modeling of single-nucleotide variants (SNVs). Three integrated aims will be pursued: (1) Develop high-fidelity base editors with expanded genomic targeting to model SNVs with minimal bystander mutations and enhanced editing efficiency; (2) Create a deep learning model to design optimized guide RNAs for CRISPR base editing, improving variant editing accuracy; and (3) Engineer next-generation prime and Click Editors for precise in vivo editing. This innovative approach introduces zebrafish-optimized CRISPR tools, a novel deep learning model, and pioneering Click Editing, overcoming specificity and scalability barriers. The platform will accelerate VUS interpretation, reduce diagnostic delays, and be extended to other aquatic models, advancing therapeutic development and supporting the goals of PAR-23-039.