Fig. 2

Single- and dual-color labeling endogenous mRNAs using CRISPR-dCas13 systems. a A pipeline to identify endogenous transcripts for dCas13 targeting. See also Additional file 1: Fig. S4a, b. b A schematic of the eppk1 locus, which contains two exons. The transcript of eppk1 is predicted to be about 16,900 nt and exon 2 contains 12 repeated units of about 1000 nt each. Three modified geppk1s were designed to target each unit. c Representative images of eppk1 expression examined by WISH at 5.3 hpf, shown as the lateral (left) and dorsal (middle) views. A schematic eppk1 expression in enveloping layer (EVL) cells is shown on right. d Representative images of the CRISPR-dPspCas13b system-labeled eppk1 at 6 hpf in live embryos. White arrowheads indicate the colocalization between dPspCas13b-EGFP and Cy3-geppk1s. e smFISH confirms the CRISPR-dPspCas13b system-labeled eppk1 at 8 hpf in fixed embryos. White arrowheads indicate the colocalization of eppk1 smFISH, dPspCas13b-EGFP and Cy3-geppk1s. In some cells, four transcription sites (two pairs of signal spots) could be detected at two alleles. Blue box, zoomed in f. f Analysis of the blue box region in e. Line scan of the relative fluorescence intensity of signals (white dot line arrow in upper panel) shows the colocalization of Cy3-geppk1s, dPspCas13b-EGFP, and eppk1 smFISH. Scale bar 2 μm. g–i Representative images of the CRISPR-dRfxCas13d system-labeled eppk1 (white arrowheads) with geppk1s-MS at 6 hpf in live embryos (g), confirmed by smFISH (white arrowheads) in h and i. See d–f for details. j Mean SNR statistics of eppk1 signals labeled by Cy3-geppk1s with dPspCas13b-EGFP and geppk1s-MS with dRfxCas13d-EGFP. Data from four independent experiments. Of note, the spacer on the gRNA of PspCas13b and RfxCas13d is at the 5′ and 3′ end, respectively [43]. k A schematic view of CRISPR-dPspCas13b-EGFP and CRISPR-dRfxCas13d-mScarlet systems to label two endogenous mRNAs in dual color with MS-modified gRNAs. l Representative images of muc5.1 mRNA (green arrowheads) and eppk1 mRNA (white arrowheads) labeled by dPspCas13b and dRfxCas13d systems at 10 hpf in live embryos, respectively. m Representative images of 100537515 mRNA (green arrowheads) and eppk1 mRNA (white arrowheads) labeled by dPspCas13b and dRfxCas13d at 10 hpf in live embryos, respectively. In d, e, g, and h, data scale bar 20 μm; the white box indicates magnified area, scale bar 5 μm. In (l and m), data scale bar 5 μm. In j, data is represented as mean ± SD, unpaired two-tail Student’s t test, ns, not significant