Fig. 4

RNAPol II dependent promoters can be used to generate gRNA in-vivo and induce mutagenesis.

(A) DNA Construct containing Cas9 and gRNA targeting GFP, both driven by ubiquitin promoter. (B) Transient expression of this construct in smyhc1:GFP embryos at 5dpf shows that some cells normally expressing GFP are lost. Cells expressing construct are labelled with H2a mCherry. On average 14 GFP positive slow muscle fibres remained in each somite after injections with this construct (n = 6), whereas there are ~24 GFP positive slow muscle fibers per somite in smyhc1: GFP embryos. Scale bars: 40 μm. (C) PAGE analysis of individual embryos injected with Ubi: Cas9 Ubi: GFPgRNA (left) and RNA of Cas9/GFP gRNA (right) show that GFP is mutagenized. (D) A heat shock promoter was used to drive the expression of two gRNA against Smo 5’ UTR. This construct was used to generate transgenic zebrafish (HSP: Smo gRNA). (E) Curved embryos seen after injection of AB with Cas9 mRNA and Smo gRNA targeting smo 5’ UTR and smo ATG (42 out of 117); and HSP: Smo gRNA embryos injected with Cas9 mRNA after heat shock (46 out of 131). Scale bars: 500 μm. (F) PAGE analysis of embryos from control (uninjected HSP: Smo gRNA) shows that Smo 5’ UTR and Smo ATG is mutagenized. Furthermore deletion of the region between the two gRNA targets can be detected (red asterisk).