Different mutations of the OTOF gene, encoding for otoferlin protein expressed 
in the cochlear inner hair cells, induces a form of deafness that is the major 
cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. 
We report the generation of the first large animal model of OTOF mutations using 
the CRISPR system associated with different Cas9 components (mRNA or protein) 
assisted by single strand oligodeoxynucleotides (ssODN) to induce 
homology-directed repair (HDR). Zygote microinjection was performed with two 
sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at 
different concentrations in a mix with an ssODN template targeting HDR in exon 5 
containing two STOP sequences. A total of 73 lambs were born, 13 showing indel 
mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher 
concentrations of Cas9-RNP induced targeted mutations more effectively, but 
negatively affected embryo survival and pregnancy rate. This study reports by 
the first time the generation of OTOF disrupted sheep, which may allow better 
understanding and development of new therapies for human deafness related to 
genetic disorders. These results support the use of CRISPR/Cas system assisted 
by ssODN as an effective tool for gene editing in livestock.