Dataset Information

Single-Step Genome Editing of Small Ruminant Embryos by Electroporation.

ABSTRACT: We investigated the possibility of single-step genome editing in small ruminants by CRISPR-Cas9 zygote electroporation. We targeted SOCS2 and PDX1 in sheep embryos and OTX2 in goat embryos, utilizing a dual sgRNA approach. Gene editing efficiency was compared between microinjection and three different electroporation settings performed at four different times of embryo development. Electroporation of sheep zygotes 6 h after fertilization with settings that included short high-voltage (poring) and long low-voltage (transfer) pulses was efficient at producing SOCS2 knock-out blastocysts. The mutation rate after CRISPR/Cas9 electroporation was 95.6% ± 8%, including 95.4% ± 9% biallelic mutations; which compared favorably to 82.3% ± 8% and 25% ± 10%, respectively, when using microinjection. We also successfully disrupted the PDX1 gene in sheep and the OTX2 gene in goat embryos. The biallelic mutation rate was 81 ± 5% for PDX1 and 85% ± 6% for OTX2. In conclusion, using single-step CRISPR-Cas9 zygote electroporation, we successfully introduced biallelic deletions in the genome of small ruminant embryos.

SUBMITTER: Mahdi AK

PROVIDER: S-EPMC9499182 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Single-Step Genome Editing of Small Ruminant Embryos by Electroporation.

Mahdi Ahmed K AK Medrano Juan F JF Ross Pablo J PJ

International journal of molecular sciences 20220906 18

We investigated the possibility of single-step genome editing in small ruminants by CRISPR-Cas9 zygote electroporation. We targeted SOCS2 and PDX1 in sheep embryos and OTX2 in goat embryos, utilizing a dual sgRNA approach. Gene editing efficiency was compared between microinjection and three different electroporation settings performed at four different times of embryo development. Electroporation of sheep zygotes 6 h after fertilization with settings that included short high-voltage (poring) an ...[more]

PMID: 36142132

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Project description:The introduction of genome editing reagents into mammalian zygotes has traditionally been accomplished by cytoplasmic or pronuclear microinjection. This time-consuming procedure requires expensive equipment and a high level of skill. Electroporation of zygotes offers a simplified and more streamlined approach to transfect mammalian zygotes. There are a number of studies examining the parameters used in electroporation of mouse and rat zygotes. Here, we review the electroporation conditions, timing, and success rates that have been reported for mice and rats, in addition to the few reports about livestock zygotes, specifically pigs and cattle. The introduction of editing reagents at, or soon after, fertilization can help reduce the rate of mosaicism, the presence of two of more genotypes in the cells of an individual; as can the introduction of nuclease proteins rather than mRNA encoding nucleases. Mosaicism is particularly problematic in large livestock species with long generation intervals as it can take years to obtain non-mosaic, homozygous offspring through breeding. Gene knockouts accomplished via the non-homologous end joining pathway have been more widely reported and successfully accomplished using electroporation than have gene knock-ins. Delivering large DNA plasmids into the zygote is hindered by the zona pellucida (ZP), and the majority of gene knock-ins accomplished by electroporation have been using short single stranded DNA (ssDNA) repair templates, typically less than 1 kb. The most promising approach to deliver larger donor repair templates of up to 4.9 kb along with genome editing reagents into zygotes, without using cytoplasmic injection, is to use recombinant adeno-associated viruses (rAAVs) in combination with electroporation. However, similar to other methods used to deliver clustered regularly interspaced palindromic repeat (CRISPR) genome-editing reagents, this approach is also associated with high levels of mosaicism. Recent developments complementing germline ablated individuals with edited germline-competent cells offer an approach to avoid mosaicism in the germline of genome edited founder lines. Even with electroporation-mediated delivery of genome editing reagents to mammalian zygotes, there remain additional chokepoints in the genome editing pipeline that currently hinder the scalable production of non-mosaic genome edited livestock.

Dataset Information

Single-Step Genome Editing of Small Ruminant Embryos by Electroporation.

Publications

Single-Step Genome Editing of Small Ruminant Embryos by Electroporation.

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