Casp–Cas9 and Casp–dCas9 engineered fusion proteins are catalytically active in vitro
(A) The scheme illustrates the organisation of Casp-Cas9/dCas9 fusion proteins used in this work. (B) DNA fork disintegration assay to detect casposase-catalysed nucleophilic attack from a 3′-OH to remove a 5′ ssDNA flap, a reaction indicated to the left of the gel panel. Casposase activity of Casp–Cas9 and Casp-dCas9 (lanes 4 and 5) are compared with casposase, Cas9 and dCas9 (lanes 1–3). All proteins were used at 150 nM and incubated with 25 nM of DNA fork-3 for 1 h. The image detects the Cy5-end label (red star) on a single denaturing acrylamide gel – irrelevant lanes to this work (containing non-functional Casp–Cas9 fusions) are removed from the image, as indicated by the black lines between lanes 3-5. (C) Quantification of disintegration activity of Casp-dCas9 compared with casposase, proteins at 0, 25, 50, 75, 100, 250 nM, and fork-3 at 25 nM – a representative gel is given in Supplementary Figure S8. Error bars represent standard error of the mean. (D) EMSA of R-loop formation between sgran50 (300 nM) and dsran50 DNA (25 nM) catalysed by Casp–Cas9/dCas9 compared to Cas9/dCas9 controls (all proteins at 150 nM). Nucleic acids were migrated through TBE acrylamide gels prior to imaging to detect the position of Cy5 from dsran50. The image is taken from a single gel, but with irrelevant lanes removed as indicated by the black line between lanes 8 and 9. (E) Quantification of R-loop formation by Cas9 and Casp–Cas9/dCas9 using the same assay as in part (E), but proteins at 0, 75, 150, 300, 500 and 1000 nM – a representative gel of this assay is given in Supplementary Figure S10A. Error bars represent standard error of the mean.