For the last two decades, several genetic studies were performed to identify the NIHL susceptibility genes and among them various NIHL susceptibility genes have been known to involved in different cellular pathways such as the genes involved in the potassium recycling pathway (Kcnql, Kcnq4, kcnel, Kcnj10, Gjbl, Gjb2, and Gjb4) [51, 52], oxidative stress gene (Sod2, Cat, Gstml, and Pon2) [53, 54], heat shock protein genes (Hsp70) , and monogenic deafness genes (Myh14
and Pcdh15) .
Most cases of ADNSHHI are characterized by postlingual progressive sensorineural hearing loss, with the age of onset mostly being in the second or the 3 [sup]rd year, such as is the case with KCNQ4 , [sup] GJB3 , [sup] and MYH14 .
Nonmuscle myosin heavy-chain gene MYH14 is expressed in cochlea and mutated in patients affected by autosomal dominant hearing impairment (DFNA4).
An extended analysis of 644 SNPs in 53 candidate genes was performed in two independent (Swedish and Polish) populations; and two SNPs (rs667907 and rs588035) in MYH14 resulted in a positive association in the Polish sample set and significant interaction with noise exposure level in the Swedish sample set [19, 20].
MYH14, also known as nonmuscle II heavy chain (NMHCII-C), together with two other nonmuscle chains (MYH9 and MYH10), is a member of the myosin family, which have been implicated in many motile processes such as ion-channel gating, organelle translocation, and cytoskeleton rearrangement [21, 22].
In our study, we applied the CRISPR/Cas9 technology to establish a Myh14 knockout mouse line ([Myh14.sup.-/-]) using the CBA/CaJ background strain.
The genomic DNA fragment around the gRNA target site was amplified by PCR using two sets of primers: Myh14 forward, 5'-ACCTCGTGCTTGTTCAG-3', and Myh14 reverse 5'-TGTCTTCAGCAGGGTGT-3'.
Five potential off-target sites for the Myh14 gene were found.
ANOVA was performed to evaluate the difference in ABR threshold shifts, one-way ANOVA or t-test was selected to test the MYH14 expression and cochlear hearing loss, and statistical analysis was performed using GraphPad software.