Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes
A) Exon 1 of the ovine MSTN gene. The sequence recognized by sgRNA is in italic and bold, from nt 110–130, the PAM sequence TGG is underlined and the rest of exonic sequences in smaller font. B) Ovine cells were transfected with pX330-cas9-MSTN plasmid co-expressing Cas9 and sgRNA and three days later cellular DNA was isolated and subjected to T7EI assay for detecting mutation at the targeted MSTN exon 1. The PCR amplifies a band of 634 bp and uncleaved (asterisk) and cleaved (arrows, 255 bp and 379 bp) products are indicated. Numbers below each lane indicate the percentage of targeted indels. NT: non-transduced cells, T: transfected cells, L: 1Kb DNA. The experiment is representative of two replicates performed with similar results.