A schematic screen to identify robust CRISPR-dCas13 systems to visualize RNAs in zebrafish embryos. 5.6 μM dCas13-EGFP protein assembled with 8.4 μM in vitro transcribed gRNA ( gGCN4 ) at a molar ratio 1:1.5, plus 5.9 nM (50 ng/μL) β-actin-48× GCN4 plasmid were injected into zebrafish embryos at the 1-cell stage. For some lower stability dCas13 constructs, we increased the concentration (see “ Methods ”). After injection, fluorescent signals in the nucleus were detected at 6–10 hpf (hours post fertilization). The CRISPR-dCas13 detected signals were confirmed by smFISH. β-actin-48× GCN4 , zebrafish β-actin promoter derives 48× GCN4 expression.
