Dual-Targeting Rosa26 CRISPR Vector

As illustrated in Figure 1A-a, the pCas9gG-Rosa26 vector was constructed on the base of our homemade vector pU6CGR, which contains U6 promoter to drive small guide RNA (sgRNAs) expression. First, the CAG-spCas9 expression cassette was PCR amplified from pX330 (Addgene plasmid #42230), resulting in pCas9(CAG-spCas9-U6-EF1-eGFP-PA). Gibson Assembly reaction [75 (link)] was carried out to knockout the BmsI site in spCas9 cDNA sequence. To express dual sgRNAs that target the Rosa26 locus, we subcloned the fragments containing U6 promoter and sgRNA scaffold into the above vector. Two previously characterized Rosa26-targeting sgRNAs [49 (link)] were chosen and subcloned into the BsmI and BbsI sites, respectively: sgRNA1, GCG CAC TAG ACG TTG AGG TCagg and sgRNA2, GAA GAT GGG CGG GAG TCT TCtgg, where the PAM sequences are in lower case. Lastly, a CMV-driven eGFP expression cassette was subcloned into the vector for monitoring transfection efficiency. The final construct was designated as pCas9gG-Rosa26, which is used to deliver constitutive expression of spCas9 and a pair of gRNAs to produce dual gRNA-guided double-nicking in mouse Rosa26 locus. All cloning junctions and critical sequences were sequencing verified. Details about the vector construction are available upon request.