Efficient introduction of an isogenic homozygous mutation to induced pluripotent stem cells from a hereditary hearing loss family using CRISPR/Cas9 and single-stranded donor oligonucleotides

Hearing loss (HL) is the most common sensory disorder of humans,1,2 with an estimated 299 million men and 239 million women affected worldwide.3 Genetic etiology plays an important role in the pathogenesis of deafness, accounting for around 50% of HL cases.4 With the development of biological informatics and next-generation sequencing technology, increasing numbers of genes involved in hereditary HL are being rapidly mapped and cloned. The identification of mutations that contribute to deafness and an understanding of the molecular mechanisms underlying HL will undoubtedly give new insights into genetic therapy, genetic counseling, and molecular diagnosis for the disease. However, for many deafness genes, the detailed pathogenesis of HL remains unknown. Furthermore, various limitations preclude deciphering of the pathological mechanism, for example an ability to acquire a patient’s cochlea and typical limitations of rodent modeling of human disorders. Therefore, we propose that the use of genetically modified human induced pluripotent stem cells (hiPSCs) differentiated into auditory neuron-like cells could provide a promising complementary tool for the study of hereditary HL. This would extend the possibilities of how we investigate the mechanisms of deafness-related genes in HL, as well as how we can pursue new cellular therapies for treating hereditary HL. A large Chinese family with autosomal dominant deafness-41, a progressive sensorineural HL, was described in 2002.5 After locus refinement in 2005,6 the causative purinergic receptor p2x gene (P2RX2) was finally discovered in two Chinese families.7 Mutations in P2RX2 are inherited in an autosomal dominant manner, and have been implicated in age-related and noise-induced HL.7 However, the detailed mechanism underlying pathophysiological changes in relation to HL is unknown. Previous studies showed that P2RX2 p.V60L abolished the response of P2RX2 to ATP by patch clamp recoding of HEK293 cells transfected with a green fluorescent protein (GFP)-tagged P2RX2 p.V60L vector, and P2RX2 was considered to be responsible for the development of a temporary threshold shift in P2RX2 knockout mice.7,8 P2RX2 c.178G>T is a rare heterozygous allele that cosegregated with fully penetrant HL in a six-generation kindred living in Sichuan, China.7 Three patients derived from this family were recruited for our present trial. It is extremely difficult to study human temporal bone pathology in nonlethal diseases because biopsy is precluded by cochlear anatomy. Moreover, although transgenic mice are useful tools for hearing research, many studies have suggested that human deafness is not recapitulated in rodent models.9 Additionally, the generation of mouse models carrying specific transgenes is costly and time-consuming. Furthermore, the differences between human and rodent P2RX2 gene and protein sequences mean that it is also necessary to develop novel complementary models for P2RX2 pathophysiological studies. In the present study, we first generated patient-specific hiPSC lines carrying the heterozygous P2RX2 c.178G>T mutation. To better understand the genotype–phenotype relationship on the basis of HL pathogenesis, we introduced an isogenic mutation to the site of interest, thereby generating a unique homozygous P2RX2 c.178G>T hiPSC line for pathological research by clustered regularly interspaced palindromic repeats (CRISPR)/ CRISPR-associated protein (Cas)9 and single stranded oligonucleotide (ssODN)-based gene editing.