Fig. 4

RfxCas13d Protein Enhances Maternal RNA Targeting (A) Schematic illustration of the experimental setup to use CRISPR-Cas13d purified protein. (B) Stacked barplots showing percentage of observed phenotypes in embryos injected with RfxCas13d mRNA (mCas13d) or protein (Cas13d Prot.), alone or together with in-vitro-transcribed (gNANOG) or chemically synthesized (gNANOG-Synt) gRNAs. The amount of gRNA, mRNA, or protein injected per embryo is indicated. Number of embryos evaluated (n) is shown for each condition. Representative pictures of epiboly-stage zebrafish embryos displaying gastrulation and epiboly defects in embryos injected with Cas13d protein plus gNANOG are shown in the top panel. 30% epiboly, 50% epiboly, germ ring, and shield stages correspond to 4.6, 5.3, 5.7, and 6 hpf in WT embryos growing in standard conditions, respectively (scale bar, 0.5 mm). (C) qRT-PCR analysis of nanog mRNA in embryos at 2 and 4 hpi injected with mCas13d or protein alone or together with gRNAs targeting nanog mRNA. Results are shown as the averages ± standard error of the mean from two independent experiments with at least 2 biological replicates per experiment (n = 10 embryos/biological replicate). Taf15 mRNA was used as normalization control. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 t test, comparing each knockdown condition versus its corresponding control) (D) Fluorescence pictures of zebrafish embryos (30 hpi) injected with Cas13d protein (3 ng) and GFP with nanos 3′UTR (40 pg) without or with gDND1 (400 pg). Insets show magnification of the region where the germ line forms. The ratio of embryos displaying intact germ cells (mCas13d alone) or without germ cells (mCas13d plus gDND1) versus total number of analyzed embryos (at least from two independent experiments) are indicated (scale bar, 0.5 mm). (E) Representative pictures of zebrafish embryos (7 hpi) injected with Cas13d protein alone (3 ng) or Cas13d protein plus gSZRD1s (400 pg) (scale bar, 10 μm). (F) Scatterplot showing mRNA level (RNA-seq) of zebrafish embryos injected with Cas13d protein plus dnd1 or szrd1 gRNAs compared with that of embryos injected with Cas13d protein alone at 6 hpi. Dnd1 and szrd1 mRNAs are indicated in red in their respective panels (>7-fold and >6.3-fold decrease, respectively). Dashed lines indicate a 4-fold difference between RNA levels. (G) qRT-PCR analysis showing levels of brd3a, brd3b, and brd4 mRNAs in embryos at 4 hpi injected with Cas13d protein alone (3 ng/embryo) or together with gRNAs (300 pg/embryo) targeting the different brds mRNAs. Results are shown as the averages ± standard error of the mean from two independent experiments with at least 2 biological replicates per experiment (n = 10 embryos/biological replicate). Taf15 mRNA was used as normalization control (∗p < 0.05, ∗∗p < 0.01, t test comparing each knockdown condition versus its corresponding control). (H) Stacked barplots showing percentage of observed phenotypes using gRNAs targeting brd3a, brd3b, and brd4 mRNAs co-injected with Cas13d protein at the same concentrations. Number of embryos evaluated (n) is shown for each condition. The phenotype selection criteria were the same as in (B).