2.4. gRNA cloning method

The genomic target sequence (5′-CTCATCAAACCTATGAAAGA-3′) of Mstn for the specificity of the Cas9 nuclease immediately precedes a 5′-NGG-3′ protospacer adjacent motif (PAM). Two partially complementary oligonucleotides (5′-CACCGTCATCAAACCTATGAAAGA-3′ and 5′-AAACTCTTTCATAGGTTTG ATGAC-3′) were synthesized, annealed, and ligated into pX330 after digestion using the BbsI restriction enzyme. This plasmid (designated as the Mstn KO plasmid) contained two expression cassettes, hSpCas9 and the chimeric guide RNA, which could guide hSpCas9 to the genomic target site in Mstn.