Dataset Information

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.

SUBMITTER: Menchaca A

PROVIDER: S-EPMC7138848 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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

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

Scientific reports 20200407 1

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). Zygo ...[more]

PMID: 32265471

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Project description:BackgroundMicroalgae are considered as a sustainable feedstock for the production of biofuels and other value-added compounds. In particular, Nannochloropsis spp. stand out from other microalgal species due to their capabilities to accumulate both triacylglycerol (TAG) and polyunsaturated fatty acids (PUFAs). However, the commercialization of microalgae-derived products is primarily hindered by the high production costs compared to less sustainable alternatives. Efficient genome editing techniques leading to effective metabolic engineering could result in strains with enhanced productivities of interesting metabolites and thereby reduce the production costs. Competent CRISPR-based genome editing techniques have been reported in several microalgal species, and only very recently in Nannochloropsis spp. (2017). All the reported CRISPR-Cas-based systems in Nannochloropsis spp. rely on plasmid-borne constitutive expression of Cas9 and a specific guide, combined with repair of double-stranded breaks (DSB) by non-homologous end joining (NHEJ) for the target gene knockout.ResultsIn this study, we report for the first time an alternative approach for CRISPR-Cas-mediated genome editing in Nannochloropsis sp.; the Cas ribonucleoproteins (RNP) and an editing template were directly delivered into microalgal cells via electroporation, making Cas expression dispensable and homology-directed repair (HDR) possible with high efficiency. Apart from widely used SpCas9, Cas12a variants from three different bacterium were used for this approach. We observed that FnCas12a from Francisella novicida generated HDR-based targeted mutants with highest efficiency (up to 93% mutants among transformants) while AsCas12a from Acidaminococcus sp. resulted in the lowest efficiency. We initially show that the native homologous recombination (HR) system in N. oceanica IMET1 is not efficient for easy isolation of targeted mutants by HR. Cas9/sgRNA RNP delivery greatly enhanced HR at the target site, generating around 70% of positive mutant lines.ConclusionWe show that the delivery of Cas RNP by electroporation can be an alternative approach to the presently reported plasmid-based Cas9 method for generating mutants of N. oceanica. The co-delivery of Cas-RNPs along with a dsDNA repair template efficiently enhanced HR at the target site, resulting in a remarkable higher percentage of positive mutant lines. Therefore, this approach can be used for efficient generation of targeted mutants in Nannochloropsis sp. In addition, we here report the activity of several Cas12a homologs in N. oceanica IMET1, identifying FnCas12a as the best performer for high efficiency targeted genome editing.

Dataset Information

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

Publications

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

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