Guide sequence for sgRNA targeting the ZBED6 binding motif in intron 3 of porcine IGF2 were designed using the open-source tool, CHOPCHOP (http://chopchop.cbu.uib.no/). The BE3 (a fusion protein of deaminase Apobec-1, Cas9 (D10A) nickase, and Uracil Glycosylase Inhibitor (UGI)) coding sequence was digested from the pCMV-BE3 (Addgene #73021, Cambridge, MA, USA), and cloned into the pX458 (Addgene #48138, Cambridge, MA, USA) to replace the 3 × FLAG-Cas9 coding sequence to generate the pX458-BE3 plasmid, which contains the enhanced green fluorescent protein (EGFP) reporter. Oligos of gRNA targeting porcine IGF2-intron3-3071 were synthesized (Sangon Biotech, Shanghai, China) and cloned into the plasmid pX458-BE3 through Bbs I restriction site to create the plasmid pX458-BE3-gRNA.
Duo T., Liu X., Mo D., Bian Y., Cai S., Wang M., Li R., Zhu Q., Tong X., Liang Z., Jiang W., Chen S., Chen Y, & He Z. (2023). Single-base editing in IGF2 improves meat production and intramuscular fat deposition in Liang Guang Small Spotted pigs. Journal of Animal Science and Biotechnology, 14, 141.
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