Fig 1.
Structure and use of ribozyme to generate gRNA.
(A) Sequence of HH ribozyme. Map of HH-gRNA. First 6 bases (grey text) of the ribozyme are complementary to the first six bases of the gRNA (red text) and cleavage occurs at the 5’ end of the gRNA (arrow). (B) Denaturing PAGE gel of IVT RNA. RNA containing HH and HDV ribozyme show spontaneous cleavage for generation of gRNA. HH and gRNA correspond to the HH ribozyme and gRNA respectively. (C) Sequencing of Smo 5’ UTR shows that the gRNA target site is mutagenized.
Fig 2.
gRNA generated containing HH and HDV ribozyme is efficient at inducing mutagenesis of its target site.
(A) mCherryHH-gRNA-HDV construct used to generate gRNA. H2a mCherry is used to label cells expressing gRNA and MALAT is used to stabilize the RNA after cleavage by HH and HDV ribozyme. (B) Injection of Smo gRNA together with Cas9 mRNA causes mutation of its target sequence. (C) Images of smyhc1: GFP embryos at 2dpf show that the majority of GFP expressing cells are lost upon injection of GFP gRNA and Cas9 mRNA. On average, there are ~24 GFP positive slow muscle fibers per somite in uninjected smyhc1: GFP embryos whereas only 6 GFP positive slow muscle fibres remained in each somite of injected smyhc1: GFP embryos (n = 5). Scale bars: 40 μm.
Fig 3.
gRNA generated using ribozymes is as efficient as gRNA generated by transcription alone.
PAGE analysis of smo 5’ UTR shows the formation of heteroduplex after injection of gRNA and Cas9 mRNA compared to the uninjected control. Comparison of intensity of heteroduplex and homoduplex shows that gRNA with or without ribozymes have similar efficacy in inducing mutagenesis. Red vertical bar shows the region of heteroduplex that was used for analysis. Percentage of heteroduplex vs homoduplex is shown.
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).