Supplementary Figure 2: Comparison of the stability of the CRISPR RNA scaffolds with insertion of aptamers at different positions.
From: CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging
(a) Diagram of the CRISPR sgRNA-Broccoli systems. Nuclease-dead Cas9 (dCas9) was fused at the C-terminus to mCherry and expression was under the control of the CMV-TetO promoter. The sgRNA tagged with Broccoli RNA aptamer either at the 3’ end or the tetraloop was constitutively transcribed from the U6 promoter but becomes fluorescent only upon addition of DFHBI-1T. This same plasmid contained the tetracycline repressor (TetR) and BFP via a 2A self-cleaving peptide (P2A) linker constitutively expressed via the hPGK promoter. BFP is used as a report for guide RNA expression. (b) Visualization of guide RNA along with dCas9 in live U2OS cells. Localization of BFP report (blue), dCas9-mCherry (red), and C19–1-sgRNA–Broccoli (green) in U2OS cells. Scale bar, 5 μm. The images with the same color were scaled to the same minimal and maximal levels. Data in all panels are representative of experiments performed at least three times.
