Fig. 3: Multi-color and orthogonal genomic loci imaging with fCRISPR.

a fCRISPR enables multi-color imaging by modulating fluorescent proteins-tDeg. We showed that fCRISPR is able to image genomic loci with red fluorescence using tdTomato-tDeg reporter in Figs. 1 and 2. Here, we apply fCRISPR to image Chromosome 3 in living U2OS cells varying from blue to NIR color by fusing various colors of fluorescent proteins including mCerulean (blue), mNeonGreen (green), YPet (yellow), tdTomato (red), and iRFP670 (NIR) with tDeg. These results (white arrowheads) confirmed that fCRISPR can be developed to detect genomic loci with multiple colors. Scale bar, 5 μm. n = 3 biologically independent experiments. b Schematic of dual genomic loci imaging with fCRISPR using tdTomato-tDeg reporter (lower figure), and CRISPR using Broccoli-BI reporter (upper figure). Both CRISPR systems are orthogonal. Figure 3b was created with BioRender.com. c Orthogonal imaging of low-copy genomic loci in Chromosome 3 and Chromosome 13 in U2OS cells. To simultaneously image low-copy genomic loci, such as Chromosome 3 (25 copies, green arrowheads) and Chromosome 13 (14 copies, red arrowheads), we co-expressed fCRISPR using tdTomato-tDeg reporter, and Broccoli-fused CRISPR using green fluorescent BI reporter. Both red and green fluorescent puncta were readily detected in the presence of BI dyes (10 μM) and co-localized with the blue fluorescent dCas9-BFP reporter in the merged image. These results demonstrated that fCRISPR can provide an orthogonal imaging platform with other CRISPR imaging systems in living cells. The white dotted circle line represents the nuclear. Scale bar, 5 μm. n = 3 biologically independent experiments.