Fig. 4

Dissection of nonspecific labeling foci associated with gRNA-labeling systems. a ChIP-qPCR results demonstrate that the specific binding of gRNAs to their endogenous targets is dependent on dCas9. Relative enrichment levels of GFP-tagged PUFc at the targeted loci by EGFA-T1-gRNA (left), EMX1-gRNA (right), and control loci were compared in the conditions with and without dCas9. b Targeted gene transcription activation by Casilio system is also dependent on dCas9. PUFc-VP64 and gRNA-5 × PBSc targeting IL1RN or Oct4 promoter were transfected into HEK293T cells with or without dCas9. RT-qPCR was performed to evaluate the fold changes of IL1RN and Oct4 expression. c Schematic of the “one vector” and “two vectors” settings to dissect the nonspecific foci observed in gRNA labeling systems. d Nonspecific labeling foci came from accumulation of gRNA transcripts surrounding the gRNA transcription cassettes. In “one vector,” a single plasmid containing expression cassettes for both MUCI and MUC4e gRNAs was used. In “two vectors,” two plasmids containing individual MUCI and MUC4e gRNA expression cassettes were transfected. The representative images and quantifications are shown. The data are displayed as mean ± standard deviation (SD) from at least three independent experiments. Unpaired t test was used. ***p < 0.001; ns not significant