Figure legends
Figure 1. Efficient production of MSTN mutant blastocysts. A)
Schematic design for MSTN target site (a) and graphical
illustration on analyzing microinjected embryos (b) The red letters and
line represent MSTN target site, and the green letters represent
PAM sequence. B) MSTN mutation sequence pattern on pooled cells
after transfection with sgRNA and Cas9 protein. C) Representative
pictures of bovine blastocyst on day 7 for each experiment condition
after microinjection (WT: microinjection with Tris-EDTA buffer, RNA1X;
Cas9 mRNA: 100 ng/μl, sgRNA: 50 ng/μl, RNA2X; Cas9 mRNA: 200 ng/μl,
sgRNA: 100 ng/μl, RNA 4X; Cas9 mRNA : 400 ng/μl, sgRNA: 200 ng/μl); D).
The blastocyst formation rate after microinjection with each condition.
*P < 0.05, **P < 0.01, ***P < 0.001. E)MSTN mutation rate of day 7 blastocysts for each condition.
Figure 2. Birth of MSTN mutated Korean beef calves. A) Mutation
rate of fetuses (labeled #1–17) on MSTN target site by deep
sequencing. B) Mutation assay using T7E1 assay for one calf (#17) (WT:
wild type, NC: negative control, PC: positive control, W- :T7E1 assay
without wild type genomic DNA, W+: T7E1 assay with wild type genomic
DNA). C) Analysis of off-target effects on five candidate genes. D)
Relative expression of MSTN mRNA. The bar graph represents the fold
changes in mRNA levels, and the error bars show SEMs (n=3). *P
< 0.05, **P < 0.01, ***P < 0.001. E)
Representative pictures of a biallelic knockout calf (#17) along with
age (a: 1 month, b: 2 months, c: 3 months, and d: 4 months).