Novel CRISPR/Cas12a-based genetic diagnostic approach for SLC26A4 mutation-related hereditary hearing loss.

Hereditary hearing loss is a common defect of the auditory nervous system with high-incidence, seriously affecting the quality of life of the patients. The clinical manifestations of SLC26A4 mutation-related hearing loss are congenital sensorineural or mixed deafness. Sensitive and specific SLC26A4 mutation detection in the early clinical stage is key for the early indication of potential hearing loss in the lack of effective treatment. Using clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection technology, we designed a fast and sensitive detection system for SLC26A4 pathogenic mutations (c.919-2A > G, c.2168A > G and c.1229C > T). This recombinase-aided amplification-based detection system allows rapid target gene amplification and, in combination with the CRISPR-based nucleic acid testing (NAT) system, mutation site detection. Moreover, mismatches were introduced in CRISPR-derived RNA (crRNA) to increase signal differences between the wild-type genes and mutant genes. A total of 64 samples were examined using this approach and all results were verified using Sanger sequencing. The detection results were consistent with the polymerase chain reaction-Sanger sequencing results. Overall, this CRISPR-based NAT technology provides a sensitive and fast new approach for the detection of hereditary deafness and provides a crRNA optimization strategy for single-nucleotide polymorphism detection, which could be helpful for the clinical diagnosis of SLC26A4 mutation-related hereditary hearing loss.