Analysis of MSTN off-target effect
The potential off-target effects caused by CRISPR–Cas9 in the threeMSTN mutant calves were assessed using Cas-OFFinder software (http://www.rgenome.net/cas-offinder/). This software offers a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. In theMSTN target site, candidates of five loci targeting the whole gene of cows were found by setting the mismatch number to 3. A primer targeting each sequence (Supplementary Table 1) was prepared to confirm the off-target effect through T7E1 assay.