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

A Menchaca,M Gantier,F Cuadro,I Anegón,M Crispo,A P Mulet,J M Heslan,M Souza-Neves,L Tesson,P C Dos Santos-Neto,V Chenouard,A Guiffès

doi:10.1038/s41598-020-62879-y

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.

Highlights

Since the first report of CRISPR edited mammals was published in 2013 in mice[1], the CRISPR/Cas system has revolutionized the field of genome editing for a number of species
The current study reports for the first time the use of the CRISPR/Cas[9] system to develop a KI sheep model using single strand oligodeoxynucleotides (ssODN) to induce two stop codons to produce a KO for OTOF in exon 5 or 6
Capillary electrophoresis performed in embryos showed heteroduplexes in many blastocyst samples due to either non-homologous end joining (NHEJ) reparation after CRISPR-mediated DNA cleavage or to different SNPs present in different embryos

Summary

Introduction

Since the first report of CRISPR edited mammals was published in 2013 in mice[1], the CRISPR/Cas system has revolutionized the field of genome editing for a number of species. CRISPR/Cas[9] system can trigger the homology directed repair (HDR) pathway, reports employing this strategy to mediate the knock-in (KI) of exogenous donor DNA have generally been limited to mice or rats, with little success in livestock[9] Another promising approach that requires further study in livestock is to use single-stranded oligodinucleotides (ssODNs) to induce HDR of DNA by single-strand annealing[8]. Certain mutations in the OTOF gene are reported to be the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans[14] Studying these mutations in a large animal model could provide a better understanding of the role of OTOF and to further test different therapies and possibly rescue the function of hair cells. We compare the efficiency of both the Cas[9] mRNA or RNP strategies

Methods

Results

Conclusion