3 Embryo knockout efficiency improved when targeting ovine suppressor of cytokine signalling 2 with 2 small guide RNA

Abstract

Suppressor of cytokine signalling (SOCS2) is a key regulator of growth. Growth hormone (GH) and insulin growth factor-1 induce SOCS2 expression, which acts as a suppressor of GH and insulin growth factor-1 signalling by preventing the activation of the signal transducer and activator of transcription proteins. In mice, SOCS2 knockout results in a gigantic phenotype with elevated GH levels, longer bones, enlargement of internal organs, and 30 to 40% heavier body weight in comparison with wild-type animals. High growth was also noted in naturally occurring SOCS2 mutant pigs. The aim of this project was to produce high-growth sheep by introducing sense mutations to SOCS2 by direct injection of CRISPR/Cas9 in ovine embryos. In this study, we compared the editing efficiency of 2 small guide RNA (sgRNA), which were designed to target the first exon of SOCS2. Ovine ovaries were collected from a local slaughterhouse and cumulus-oocyte complexes were aspirated from 2- to 6-mm antral follicles, matured in vitro for 22 h, and then denuded from the surrounding cumulus cells. Approximately 6 pL of CRISPR/Cas9 solution (80 ng µL⁻¹ of sgRNA, 80 ng µL⁻¹ of Cas9 mRNA, and 160 ng µL⁻¹ of Cas9 protein in low-TE buffer) was microinjected into the cytoplasm of metaphase II oocytes using a laser-assisted microinjection technique (Bogliotti et al. 2016 J. Vis. Exp. 116, e54465). Groups of 50 oocytes were injected with the CRISPR/Cas9 mix containing sgRNA1 (group G1), sgRNA2 (group G2), sgRNA1 and sgRNA2 (group G1/2), or low-TE buffer alone (control). After microinjection, IVF was carried out using fresh semen, and oocytes were co-incubated with the sperm for 16 h. Embryos were cultured in groups of 25 in 50-µL drops of KSOM (Evolve, Zenith Technologies, Cork, Ireland) with 4 mg mL⁻¹ of BSA under oil at 38.5°C, 5% CO₂, 5% O₂, and 90% N₂. Blastocysts were collected on Day 7 post-fertilization to assess mutation efficiency. The CRISPR/Cas9 target sequence was amplified using nested PCR from single-blastocyst whole lysates and analysed by Sanger sequencing. Mutations in each embryo were identified by visual inspection of the sequence traces aided by trace deconvolution software (SnapGene, GSL Biotech, Chicago, IL, USA). The experiment was replicated 3 times. Blastocyst rates were 22.6, 21.3, 21.3, and 29.3% for G1, G2, G1/2, and control, respectively. Targeting the exon with 2 sgRNA (G1/2) resulted in the highest mutation rate (87.3%), with an average indel size of 55.6 bp, leading to 100% nonfunctional SOCS2, and with 87.6% of the sequenced embryos presenting bi-allelic mutations. Using a single sgRNA caused mutations in 47.3 and 38.6% of embryos for G1 and G2, respectively, with averages indel size of 8.2 bp in G1 and 13.8 bp in G2. In G1, 88.6% of the mutations were biallelic and 90% would result in nonfunctional SOCS2. In G2, 50% of the mutations were biallelic and 40% translated to nonfunctional SOCS2. Based on these results, the percentage of SOCS2 knockout embryos was 37.7, 7.7, and 76.4% in G1, G2, and G1/2, respectively. In conclusion, the knockout efficiency improved after targeting ovine SOCS2 with 2 sgRNA.