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.

Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9

“…It has been exhibited a numerous percentage of about 70% of polymorphisms, especially deletions in Populus genome editing by the Cas system while knocking down the PDS gene family (An et al, 2020). Menchaca et al (2020) recorded 17.8% indel mutations in sheep genome editing, with 61.5% knock-in mutations happened by HDR. Also, it has been shown that silencing of NHEJ factors such as Ku70 and Ku80 caused to reduce indels significantly from 64% to 38% and 39.4%, respectively (Li et al, 2018).…”

“…Aslan et al (2017) increased HDR efficiency to 25.7% in the Xenopus tropicalis genome by inserting mall pieces of DNA, while Danilo et al (2018) succeeded in knocking in a DDT with 400 bp into the tomato genome with low efficiency of 1.29%. Jang et al (2018) attempted to develop HDR efficiency to 38% in mouse lines by applying multiple sgRNAs, and Menchaca et al (2020) tried to enhance the HDR efficiency to 61.5% in sheep utilizing single strand oligodeoxynucleotides. Still, there are some reports on Populus genome editing, but they are limited only to knock out genes, and mutations happened by Cas9 and Cas12a (An et al, 2020; Di Fan et al, 2015; Liu et al, 2015), but no report has been carried out on improving the HDR efficiency in poplar.…”

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes

“…However, the primary success with targeting a knock-in of embryos using ssDNA has been through attempting allelic conversions, such as small insertions, deletions or single nucleotide polymorphisms. Each of these cases was performed using ssODNs of varying length ranging from 35 to 120 bp [26][27][28]. The largest integration performed using ssDNA was a 1368 bp insert using a~1.5 kb ssODN in mouse embryos in a method called Easi-CRISPR [29].…”

Gene editing and its applications in biomedicine

“…The donor may be single-stranded oligodeoxynucleotides (ssODNs) or double-stranded DNA (dsDNA) (Zhang et al, 2021). The editing system could also be used in different formats, including Cas9-sgRNA RNP (Menchaca et al, 2020), RNA (Yang et al, 2013b), virus particles (Swiech et al, 2015), plasmids (Xue et al, 2014), cationic lipid nucleic acids (Zuris et al, 2015), and lipid nanoparticles (Musunuru et al, 2021). Because of the different applications and animal species, there are three main methods to generate genetically modified animals (Figure 2).…”