(A) In vitro R-loop formation shown in an ethidium bromide stained agarose gel using sglacZ targeting of pUC19 (150 ng). Proteins were as indicated at 40, 75, 150 and 300 nM, or with no protein in lane 1 – casposase (lanes 2–5), dCas9 (lanes 6–9) and Casp–dCas9 (lanes 10–13). Reactions were incubated at 37°C for 20 min. (B) A Western blot using anti-(His)₆ antibody confirmed that the Casp–Cas9/dCas9 protein were stably expressed for lacZ targeting in E. coli MG1655 K-12 ΔlacZ::T7 RNAP cells transformed with pRC7 and plasmids for expressing proteins and sglacZ. A single gel is shown, with a blank lane removed, indicated by the thin vertical black line. The uncropped image was shown in Supplementary Figure S12. (C). Schematic illustration of the Miller assay and the effect of sglacZ-dCas9 on the Miller assay. Miller assay measures the activity of β-galactosidase that cleaves ONPG to yield o-nitrophenol, which has a yellow color. sglacZ-dCas9 forms a R-loop at the transcription start site in the lacZ gene, thus reducing β-galactosidase expression and the Miller unit. (D and E) Miller assays to measure Casp–dCas9 targeting of the lacZ gene on (D) plasmid pRC7, or (E) the chromosome. Miller units were normalised to control ‘empty plasmid’ that lacks Casp–dCas9 or casposase encoding genes.