Preparation of the all-in-one CRISPR-Cas9/gRNA plasmid DNA was performed as described previously (Ran et al., 2013 (link)). Briefly, the single complementary DNA oligonucleotides corresponding to the gRNA sequence were commercially synthesized (Integrated DNA Technologies). After phosphorylation and annealing, the paired double strand DNA oligo was cloned into the BbsI linearized plasmid pSpCas9(BB)-2A-GFP. Positive clones containing the gRNA-encoded DNA sequences were confirmed by DNA sequencing. For EC-specific genomic editing, the CAG promoter in pSpCas9(BB)-2A-GFP plasmid (CRISPRCAG) was replaced with the human CDH5 promoter (Gory et al., 1999 (link)) using Kpn I and Age I restriction enzyme sites (CRISPRCDH5). All gRNA sequences are listed in Table S1 .
To generate CRISPRCDH5 plasmid expressing both Pik3cg gRNA and FOXM1, the P2A and FOXM1 cDNA fragment was inserted into BsrGI linearized CRISPRCDH5 plasmid DNA expressing Pik3cg gRNA using NEBuilder HiFi DNA assembly (New England Biolabs).
Dual targeting CRISPR plasmid DNA expressing Pik3cg gRNA and Vegfr2 gRNA was generated by inserting cassette of U6-Vegfr2 gRNA into CRISPRCDH5 plasmid DNA expressing Pik3cg gRNA using Xba I and Kpn I sites. All plasmids were confirmed by DNA sequencing.
To generate CRISPRCDH5 plasmid expressing both Pik3cg gRNA and FOXM1, the P2A and FOXM1 cDNA fragment was inserted into BsrGI linearized CRISPRCDH5 plasmid DNA expressing Pik3cg gRNA using NEBuilder HiFi DNA assembly (New England Biolabs).
Dual targeting CRISPR plasmid DNA expressing Pik3cg gRNA and Vegfr2 gRNA was generated by inserting cassette of U6-Vegfr2 gRNA into CRISPRCDH5 plasmid DNA expressing Pik3cg gRNA using Xba I and Kpn I sites. All plasmids were confirmed by DNA sequencing.