Skip to Main content Skip to Navigation
Journal articles

Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair

Abstract : 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.
Document type :
Journal articles
Complete list of metadatas

Cited literature [24 references]  Display  Hide  Download

https://www.hal.inserm.fr/inserm-02561070
Contributor : Sylvie Le Bihan <>
Submitted on : Sunday, May 3, 2020 - 10:48:45 AM
Last modification on : Friday, October 16, 2020 - 11:26:06 AM

File

s41598-020-62879-y.pdf
Publisher files allowed on an open archive

Identifiers

Collections

Citation

A. Menchaca, P. dos Santos-Neto, M. Souza-Neves, F. Cuadro, A. Mulet, et al.. Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair. Scientific Reports, Nature Publishing Group, 2020, 10 (1), pp.5995. ⟨10.1038/s41598-020-62879-y⟩. ⟨inserm-02561070⟩

Share

Metrics

Record views

95

Files downloads

97