Tianyi Huang
Research Mentor(s): Dongshan Yang
Mentor Department: Internal Medicine
Authors: Tianyi Huang, Dongshan Yang
Session: Session 2 (10:00am – 10:50am)
Presentation Type: Poster 62
Abstract
CRISPR/Cas9 gene-editing technology holds immense potential for generating genetically engineered animal models with pathogenic mutations and for correcting genetic defects as a therapeutic strategy, including those associated with inherited retinal diseases (IRDs). However, off-target effects—unintended edits at non-target genomic loci—pose significant challenges to the validation of animal models and the safe application of gene therapies. This study aims to evaluate the off-target effects in rabbit models and ensure gene-editing accuracy. We analyzed a rabbit model of IRDs generated using CRISPR/Cas9, targeting 2 mutations in USH2A. Potential off-target sites were predicted based on computational algorithms and guide RNA specificity analysis. PCR-based approaches allow precise amplification of these regions, followed by Sanger or next-generation sequencing to identify unintended insertion/deletions (indels) mutations at single-nucleotide resolution. Rabbits with off-target mutations were excluded from breeding programs to ensure the models accurately mimic disease pathogenesis. Our results demonstrate that while CRISPR/Cas9 is a powerful tool for generating rabbit models of IRDs, off-target mutations remain a concern, requiring careful validation and screening. By identifying and excluding rabbits with off-target effects, we ensure that our models more accurately represent the genetic mutations associated with the diseases. This study underscores the importance of rigorous off-target analysis for advancing gene-editing technologies and enhancing the safety and efficacy of gene therapies for inherited retinal diseases.
