Megashortscript kit

The sequences target the g.C1901T (p.R96C) variant in the ovine SOCS2 gene is listed in Supplementary Table S1. Two oligonucleotides (Supplementary Table S2) used for the transcription of sgRNA in vitro were precisely synthesized and annealed to form double-stranded oligos. These double-stranded oligos were subcloned into the pUC57-T7-gRNA vector as described previously (Shen et al., 2013 (link)). The clones containing the desired sequence were selected, expanded by cultivation, and the plasmid was extracted using a plasmid extraction kit (AP-MN-P-250G; Axygen, Union City, CA, United States), sgRNA was transcribed in vitro using the MEGAshortscript Kit (AM1354; Ambion, Foster City, CA, United States) and purified using the MEGAclear Kit (AM1908; Ambion). Subsequently, the BE3 mRNA in vitro transcription vector (No. 44758; Addgene, Cambridge, MA, United States) was used as a template to produce BE3 mRNAs following a previously published protocol (Shen et al., 2013 (link)).

The Cas9/sgRNA expression vector was purchased from Nuolanxin Bio-Chemistry Co. Ltd., Beijing, China. Mouse MPC1 gene sequence was retrieved from the gene bank. Two MPC1 exon 1 targets were selected and named as MPC1-g1 and MPC1-g2 (Figure 7A). Two plasmids were constructed as one circle plasmid, and the structure of the constructed plasmids is shown in Figure 7B. Then, by using the circle plasmid, sgRNAs and Cas9 mRNA were transcribed by sgRNA and Cas9 expression vectors and the MEGAshortscript™ Kit (Ambion, AM1345). Following transcription completion, poly (A) tailing reaction and DNase I (TURBO, AM2238) treatment were performed. The sgRNA and the Cas9-encoding mRNA were then purified by MEGAclear™ Kit (Ambion, AM1908) and re-dissolved in RNase-free water. The luciferase SSA analyses were carried out to confirm the plasmids' activity, which showed an activity of 7.18 and 6.25 high (Figure 7C). Finally, the one-cell stage C57BL/6 embryos were injected by the mixture of 25 ng/μl of sgRNA and 50 ng/μl of Cas9 mRNA, and then the gene-edited embryos were transferred into two pseudo pregnant foster mothers' endometrium.
Due to the random mutation feature of this technology, there were several heterozygous MPC1 mutation mice in F0. Five homozygous mutation mice fostered by the NO.17 mouse were obtained with the heterologous F0 mice mating experiments.

MELOE‐1 intercistronic region IR_1215‐1490 (275 nt IRES region) [20 (link)] was cloned into a pBSSK vector under the control of a T7 promoter. It was linearized with NotI and in vitro transcribed according to the MegaShort Script Kit (Ambion, Thermo Fisher Scientific) protocol. RNA (0.2 nmol) was biotinylated using the 5’end modification kit and biotin maleimide (Vector Laboratories, Eurobio Scientific, Les Ulis, France) following the manufacturer’s guidelines.

The sgRNA design and in vitro transcription (IVT) of Cas9 mRNA and sgRNA were described in our previous study [17 (link)]. Briefly, four sgRNAs (Supplementary Table S1) targeting sheep MSTN exon 3 were designed with CRISPR tools (https://benchling.com/, accessed on 15 September 2020). The oligos for each sgRNA (Supplementary Table S1) were annealed and cloned into the pX330 plasmid. The IVT templates for Cas9 and sgRNAs were amplified using the T7 promotor-appended primers (Supplementary Table S2) and were gel-purified using a QiaQuick Spin Column (28104Qiagen, Shanghai, China). The Cas9 IVT template (400 ng for a 20 µL reaction) was subjected to a T7 Ultra Kit (AM1345, Ambion, Shanghai, China), and sgRNA IVT templates (200 ng for a 20 µL reaction) were transcribed using the MEGAshortscript Kit (AM1354, Ambion) in vitro. The Cas9 mRNA and sgRNAs were purified using the MEGAclear Kit (AM1908, Ambion). The purified RNAs were quantified using NanoDrop 2000 and then subjected to electrophoresis in 1.5% agarose gel (Supplementary Figure S1). The high-quality RNAs were stored at −80 °C before use.