Development and Characterization of CRISPR/Cas9 Mice with Candidate Causal Mutations for Human Deafness

Manipulating genomic sequences in model animals has always been a powerful tool in the study of genes and their function, but traditional approaches are laborious, expensive and slow. In recent years, the genome-editing tool CRISPR/Cas9, has proven flexible and efficient for studies of gene silencing and epigenetic modification and development of mouse models of human disease. We have applied this tool to our studies of human deafness. Hearing loss is affecting 466 million people worldwide. Hearing loss can be caused by genetic factors and by environmental factors; such as noise and drugs. Many genes responsible for deafness have been identified, but others remain unknown and may lie in pathways about which little is known. We have developed a pipeline for studying candidate causal mutations for deafness in human families based on mice created with these mutations using CRISPR/Cas9. With this pipeline, we can study the function of novel genes and regulatory networks in the inner ear. We design a guide RNA (gRNA) to create a double-strand break in the desired region of the mouse genome. The guide, donor DNA and Cas9 protein are then electroporated into single-cell embryos to induce genomic double-strand breaks and HDR by the donor DNA. Then mice are genotyped and potential off-target sites are assessed. Transgenic mice are phenotyped using auditory brainstem response for assessment of hearing function and various imaging techniques and molecular assays to dissect the mechanisms underlying the pathology. This work will expand the knowledge and understanding of the biology of hearing.