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. 2021 Dec 23;3(1):101058. doi: 10.1016/j.xpro.2021.101058

Figure 2.

Figure 2

Enhanced gRNAs are functional in vivo

(A) Schematic representation of the WT-gRNA with 22 nt of binding site and the optimized gRNA, SLD-gRNA, with the Stem loop disruption and 23 nt of binding sequence, both modifications are highlighted in red.

(B) qRT-PCR analysis showing the levels of tbxta mRNA in zebrafish embryos at 5 h post fertilization (hpf). Embryos were injected with 3 ng of RfxCas13d protein (Cas13d) and 300 pg of 3 gRNAs (see Table 1) targeting the ORF of tbxta mRNA. Different in vitro transcribed gRNAs were used: while gNTL-WT has the wild-type stem loop and a spacer of 22 nt, gNTL-SLD has the stem loop disruption described in Wessels et al. (2020) and a spacer of 23 nt. Results are shown as the averages ± standard deviation of the mean from two independents experiments with two biological replicates (n = 10 embryos) per experiment ∗∗∗ p< 0.001, ∗∗∗∗ p< 0.0001; (One-way anova).

(C) No-tail phenotype severity after knockdown of tbxta mRNA in similar conditions described in panel (B). Tbxta is involved in the formation of the notochord and the posterior part of the zebrafish embryo. Zebrafish embryo phenotypes were classified at 28 h post fertilization. Class I: Short tail (least extreme). Class II: Absence of notochord and short tail (medium level). Class III: Absence of notochord and extremely short tail (most extreme). ∗∗ p< 0.01; (Chi-Square). Number of embryos evaluated (n) is shown for each condition. Results from 2 independent experiments.

(D) In vitro transcription efficiency of different gRNAs formulations. Results are shown as the averages ± standard deviation of the mean from 11 RNA quantifications, after in vitro transcription of gRNAs differing in the stem loop formulation: wild-type (gRNA-WT) or with the stem loop disruption (gRNA-SLD). ∗ p< 0.05; (two tailed t test).

(E) Phenotypes obtained after injection of the Cas13d and the gRNAs (WT or optimized) targeting tbxta in zebrafish embryos (Lateral views). Levels of mosaicism compared to wild type (WT) were evaluated at 28 hpf. Class I: Short tail (least extreme). Class II: Absence of notochord and short tail (medium level). Class III: Absence of notochord and extremely short tail (most extreme). Scale bar, 0.25 mm.