Fig. 3
Gene disruption using CRISPR-Cas9 system. A pTHA(Cas9) vector was constructed using S. pyogenes cas9 gene, pTrc99a, and pUCpHAw. Expression of cas9 was under trc promoter regulated by lacIq and induced by the addition of IPTG. The detailed flow of the vector construction is shown in Additional file 1: Fig. S5. B Cas9 protein in KM-1 was detected by western blotting. Positions of the two largest subunits of KM-1 endogenous RNA polymerase are indicated on the left. C Expression cassette of guide RNA was prepared on pgRNA-bacteria. It consisted of an artificial promoter (PJ23119), base-pairing region of pyrF gene, Cas9 handle, and S. pyogenes terminator. D pgRNAHA_pyrF vector was constructed based on pHA1AT_32 and the pgRNA-bacteria including the base-pairing region of pyrF. The detailed flow of construction is shown in Additional file 1: Fig. S6. E Agarose gel electrophoresis was conducted with linearized plasmid vectors extracted from KM-1 with pTHA(Cas9) (lane 1), E. coli with pgRNAHA_pyrF (lane 2), and KM-1 with pTHA(Cas9) and pgRNAHA_pyrF (lane 3). BglII digestion was conducted to linearize the vectors (right half). Two triangles indicate the positions of linearized vectors