Evaluation of the effect of RNA secondary structure on Cas13d-mediated target RNA cleavage

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13d system was adapted as a powerful tool for targeting viral RNA sequences, making it a promising approach for antiviral strategies. Understanding the influence of template RNA structure on Cas13d binding and cleavage efficiency is crucial for optimizing its therapeutic potential. In this study, we investigated the effect of local RNA secondary structure on Cas13d activity. To do so, we varied the stability of a hairpin structure containing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target sequence, allowing us to determine the threshold RNA stability at which Cas13d activity is affected. Our results demonstrate that Cas13d possesses the ability to effectively bind and cleave highly stable RNA structures. Notably, we only observed a decrease in Cas13d activity in the case of exceptionally stable RNA hairpins with completely base-paired stems, which are rarely encountered in natural RNA molecules. A comparison of Cas13d and RNA interference (RNAi)-mediated cleavage of the same RNA targets demonstrated that RNAi is more sensitive for local target RNA structures than Cas13d. These results underscore the suitability of the CRISPR-Cas13d system for targeting viruses with highly structured RNA genomes.

Keywords: CRISPR-Cas13d; MT: RNA/DNA Editing; RNA interference; RNA structure; SARS-CoV-2; cleavage activity; gene editing; targeting efficiency.

Graphical abstract

Figure 1

Design of target RNA structures (A) Schematic representation of the expression plasmid used for Cas13d and crRNA expression (top) and the firefly luciferase reporter (bottom). The RNA polymerase III (RNA Pol III) human U6 promoter drives crRNA transcription, while Cas13d expression is driven by the RNA Pol II EF1α core promoter. The SARS-CoV-2 target sequences were inserted downstream of the firefly luciferase gene under the control of the SV40 promoter in the pGL3 reporter plasmid. (B and C) Predicted RNA structures of the wild-type (WT) RdRp (B) and 5′ UTR-leader (C) target sequences, along with the mutated hairpins (1L–5L and 1R–5R). The 23-nt target sequence complementary to the crRNA is highlighted in gray, and the mutated nucleotides are encircled. The thermodynamic stability (ΔG in kcal/mol) of both the WT target sequence and the artificial hairpins is indicated below the corresponding RNA structures.

Figure 2

CRISPR-Cas13d targeting efficacy The efficiency of CRISPR-Cas13d targeting of (A and B) RdRp- and (C and D) 5′ UTR-leader-encoding sequences was assessed in HEK293T cells. Cells were simultaneously transfected with the luciferase reporter constructs and Cas13d- and crRNA-expressing constructs (as shown in Figure 1), and the luciferase level was measured at 48 h post-transfection. (A and C) For every target variant, the luciferase activity measured with a non-targeting crRNA was set at 100%, and the relative luciferase level obtained with the targeting crRNA was calculated. The mean values (±SD) of three experiments performed in duplicate (N = 6) are presented. Statistical analysis using two-way ANOVA followed by Tukey’s post hoc test was performed to identify statistically significant differences between the WT and mutant reporter constructs (∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗∗p ≤ 0.0001). (B and D) The mean relative luciferase expression levels as measured in (A) and (C) are plotted against the thermodynamic stability (ΔG in kcal/mol) of the WT (black triangles) and mutant target RNA structures (black and white circles representing 1L–5L and 1R–5R variants, respectively). Curves were generated with GraphPad Prism 9.1.0.

Figure 3

In vitro Cas13d cleavage assay (A) In vitro cleavage of RdRp target RNAs using Cas13d protein and a crRNA targeting the RdRp sequence (crRdRp). WT and mutant (1R–4R) target RNAs were incubated with only the crRNA or with both the crRNA and Cas13d protein. The target RNAs and crRNAs were visualized by denaturing agarose gel electrophoresis. A representative gel image is shown, with the size of the RNAs shown on the left. (B) The target RNA bands observed without and with Cas13d addition were quantified with ImageJ software, and the percentage of uncleaved RNA observed upon Cas13d addition is shown (target RNA level with Cas13d/target RNA level without Cas13d × 100%). The bars represent the mean values (±SD) of 4 independent assays. Statistical analysis using two-way ANOVA followed by Tukey’s post hoc test was performed to identify statistically significant differences between the WT and mutant target RNAs (∗p < 0.05 and ∗∗∗p < 0.001).

Figure 4

RNAi targeting efficacy The efficiency of RNAi targeting of the WT and mutant RdRp (A) and 5′ UTR-leader (B) target RNA structures was assessed in HEK293T cells by co-transfection of the luciferase reporter constructs and shRNA expressing plasmids. Luciferase levels were measured 2 days after transfection. For every target variant, the luciferase level obtained with a non-targeting shRNA was set at 100%, and the relative luciferase level obtained with the targeting shRNA was calculated. The mean values (±SD) of three experiments performed in duplicate are presented (N = 6). Statistical analysis using two-way ANOVA followed by Tukey’s post hoc test was performed to identify statistically significant differences between the WT and mutant reporter constructs (∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗∗p ≤ 0.0001).