Figure 4.

In vitro transposition assays with the dCas9-transposase. Transposition reactions were 50 μl with 6.5 nM of supercoiled plasmid substrate (pRC650). The reactions were initiated by adding transposase and incubated for 4 h at 37°C. Reactions were deproteinated with proteinase K and SDS and 20 μl was electrophoresed on a TBE-buffered 1.1% agarose gel at 60 V overnight. Photographs of ethidium bromide stained gels are shown. When present, the sgRNA was preassembled in a 1:1.5 molar excess to the protein prior to the reaction. Where indicated, reactions contained 10 nM of a 70 bp oligoduplex complementary to the sgRNA.