The self-cleaving T3H48 ribozyme induces pre-mRNA cleavage in zebrafish.

(a) Schematic of the dual ubb promoter vector that controls the expression of an eGFP containing a human β-globin (hHBB) intron and mCherry; the active and inactive ribozymes are inserted in the hHBB intron; red x indicates the location of the ribozyme inactivating mutation. (b) Activity of the N79, N107, N117, T3H48, and 3xT3H48 ribozymes represented as the ratio of eGFP mRNA levels in embryos injected with the active or inactive ribozyme construct; mCherry was used as a reference gene in this experiment to account for the variable number of plasmid copies in each injected embryo; n=3 biologically independent samples; Ct values are listed in S1 Table. (c-d) Merge of brightfield and fluorescence images of 25 hpf (hours post-fertilization) embryos injected at the one-cell stage with the dual ubb vector, comprising an inactive (c) or active (d) T3H48-HHR. The proportion of embryos matching the image shown is indicated in the top right corner of each image. The diagram indicates the Anterior-Posterior (A-P) and Dorsal-Ventral (D-V) axes.

Intronic insertion of the T3H48 ribozyme in the alb gene recapitulates the mutant phenotype.

(a) Schematic of the alb locus showing the position of the T3H48-HHR integration, the target CRISPR site, and the genomic sequences before and after insertion. (b) Relative alb mRNA levels in 36 hpf wild-type and alb^HHR/HHR embryos; n=3 biologically independent samples; Ct values are listed in S1 Table. (c-f) Brightfield images of 36 hpf wild-type (c), alb^b4/b4 (d), alb^HHR/b4 (e), and alb^HHR/HHR (f) embryos. (g) Schematic of the integrated T3H48-HHR in the alb gene, the new CRISPR site created by the insertion, the A>G editing window of the ABE-Umax, and the A>G inactivating mutation in the ribozyme. (h-k) Brightfield imaging (h-i) and sanger sequencing (j-k) of part of the ribozyme and flanking element from 36 hpf alb^HHR/b4 non-injected (h,j) or injected with ABE-Umax mRNA and a sgRNA that inserts an inactivating mutation in the T3H48-HHR (i,k). The proportion of embryos matching the image shown is indicated in the top right corner of each image. The diagram indicates the Anterior-Posterior (A-P) and Dorsal-Ventral (D-V) axes.

RiboFlip is a Flp- and Cre-inducible knockdown cassette.

(a) Schematics of the alb locus showing the position of the RiboFlip integration and the details of the cassette in the T3H48-HHR OFF orientation, Flp-ON orientation, and Cre-ON orientation; T3H48-HHR antisense orientation is in red and sense orientation in green; RiboFlip includes six unique primer sites (P1-6), three universal CRISPR sites (U1-3), a β-globin terminator (bGH) downstream of the TagBFP, and recombination sites (LOX/FRT/ROX). (b) Relative alb mRNA levels in 36 hpf alb^R-OFF/R-OFF, alb^{R-Flp-ON/R-Flp-ON}, and alb^{R-Cre-ON/R-Cre-ON} embryos; n=3 biologically independent samples; Ct values are listed in S1 Table. (c-f) Brightfield images of 36 hpf alb^R-OFF/R-OFF (c), alb^{R-Flp-ON/R-Flp-ON} (d,f), and alb^{R-Cre-ON/R-Cre-ON} (e) embryos non-injected (c-e) or injected at the one-cell stage with Cre mRNA (f). The proportion of embryos matching the image shown is indicated in the top right corner of each image. The diagram indicates the Anterior-Posterior (A-P) and Dorsal-Ventral (D-V) axes.