Fig. 2.

The Cas9 vector pMWCas9 (GenBank accession number: MH683611). (A) Cloning strategy of sgRNA. The original two nucleotides AG (shaded) of the sgRNA scaffold were changed into GT in order to introduce a unique XbaI site. The sticky ends generated by EcoRI and XbaI digestion (X/E) can be ligated with the annealed double stranded synthetic oligonucleotides to form the functional sgRNA. N₂₀ is the target sequence should be 3 bp upstream of a PAM sequence. (B) Map of pMWCas9, the backbone is a segregationally unstable pIJ101 replicon, cas9 is controlled by the thiostrepton inducible tipA promoter, the sgRNA cassette is under control of the permE* promoter, apramycin (aac(3)-IV) and codA serve as the selection and counter-selection markers, respectively. The unique cloning sites StuI, SpeI, HpaI and HindIII are for insertion of the homologous template sequence. This plasmid can shuttle between E. coli and Actinomycetes.