207 EFFICIENT GENERATION OF MYOSTATIN PROMOTER MUTATIONS IN BOVINE EMBRYOS USING THE CRISPR/Cas9 SYSTEM

Abstract

The myostatin gene or growth differentiation factor 8 is a member of the transforming growth factor-β superfamily that acts as a negative regulator of muscle growth. Mutations inactivating this gene occur naturally in Piedmontese and Belgian Blue cattle breeds, resulting in a dramatic increase in muscle mass, albeit with unwanted consequences of increased dystocia and decreased fertility. Modulation of muscle mass increase without the unwanted effects would be of great value for improving livestock growth and economic value of livestock. The objective of our work was to use the CRISPR-Cas9 genetic engineering tool to generate deletions of different elements in the myostatin promoter in order to decrease the level of expression and obtain an attenuated phenotype without the detrimental consequences of an inactivating mutation. To achieve this objective 4 different small guide RNA (sgRNA) targeting the promoter near the mutation were designed with PAM positions from transcription starting site of −1577, −689, −555, and −116. These sgRNA were cloned individually into the Cas9 plasmids (px461, and px462; Addgene®). These plasmids allow for a dual puromycin resistance (px462) and green fluorescent protein (px461) selection. We first tested the functionality of these sgRNA in vitro by co-transfecting bovine fetal fibroblasts with a combination of both plasmids (Set 1 = sgRNA 1–4; Set 2 = sgRNA 2–3). Cells were exposed to puromycin (0.2 µg mL⁻¹) for 72 h, then single and mixed colonies positive for green fluorescent protein expression were separated for propagation. The DNA was extracted for PCR amplification of the targeted region. Multiple deletions and a few insertion events were observed after PCR, bands were cloned into TOPO® vector (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced. Sequencing results confirmed the PCR products as insertions or deletions in the myostatin promoter region. We proceeded to modify the myostatin promoter directly in bovine zygotes. For this, IVF-derived zygotes were randomly assigned to 3 different treatment groups Set 1, Set 2, or Null (no sgRNA) for microinjections. Each zygote was injected with ~100 pL of trophectoderm buffer containing 50 ng µL⁻¹ of total sgRNA, 10 ng µL⁻¹ of Cas9 mRNA, and 30 ng µL⁻¹ of Cas9 protein with 1 mg mL⁻¹ of fluorescent dextran. Day 7 post-IVF blastocysts were lysed and DNA was extracted for PCR amplification of the target region. In Set 1, 16 of 19 embryos (94.12%) were successfully edited, whereas in Set 2 there were 11 of 17 embryos (64.7%) edited. In both sets of sgRNA there was a high degree of mosaicism, with only 1 embryo demonstrating a homozygous deletion. In conclusion, CRISPR/Cas9 acts over the course of the first few cleavage divisions Further research is necessary to refine the CRISPR/Cas9 system for inducing genetic mutations in bovine embryos.