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

Area of Science:

• * Molecular Biology
• * Genetics
• * Reproductive Biology

Background:

• * CRISPR-Cas9 technology offers precise genome editing capabilities.
• * Efficient gene editing in livestock is crucial for agricultural advancements and disease resistance.

Purpose of the Study:

• * To evaluate the efficacy of single-step CRISPR-Cas9 zygote electroporation for genome editing in small ruminants.
• * To compare electroporation with microinjection for gene editing efficiency.
• * To assess mutation rates and biallelic editing in targeted genes (SOCS2, PDX1, OTX2).

Main Methods:

• * CRISPR-Cas9 zygote electroporation was performed on sheep and goat embryos.
• * Dual sgRNA approach targeted specific genes (SOCS2, PDX1 in sheep; OTX2 in goats).
• * Electroporation parameters and timing were optimized and compared to microinjection.

Main Results:

• * CRISPR-Cas9 electroporation achieved high mutation rates (95.6% for SOCS2) with predominantly biallelic edits (95.4%).
• * Electroporation efficiency surpassed microinjection for SOCS2 gene editing.
• * Successful disruption of PDX1 in sheep (81% biallelic) and OTX2 in goats (85% biallelic) was confirmed.

Conclusions:

• * Single-step CRISPR-Cas9 zygote electroporation is an effective method for genome editing in small ruminants.
• * This technique facilitates high rates of biallelic gene disruption in sheep and goat embryos.
• * Electroporation presents a viable alternative to microinjection for efficient gene editing in these species.