Engineering RNA Virus Interference via the CRISPR/Cas13 Machinery in Arabidopsis

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are key immune mechanisms helping prokaryotic species fend off RNA and DNA viruses. CRISPR/Cas9 has broad applications in basic research and biotechnology and has been widely used across eukaryotic species for genome engineering and functional analysis of genes. The recently developed CRISPR/Cas13 systems target RNA rather than DNA and thus offer new potential for transcriptome engineering and combatting RNA viruses. Here, we used CRISPR/LshCas13a to stably engineer Arabidopsis thaliana for interference against the RNA genome of Turnip mosaic virus (TuMV). Our data demonstrate that CRISPR RNAs (crRNAs) guiding Cas13a to the sequences encoding helper component proteinase silencing suppressor (HC-Pro) or GFP target 2 (GFP-T2) provide better interference compared to crRNAs targeting other regions of the TuMV RNA genome. This work demonstrates the exciting potential of CRISPR/Cas13 to be used as an antiviral strategy to obstruct RNA viruses, and encourages the search for more robust and effective Cas13 variants or CRISPR systems that can target RNA.

Keywords: CRISPR/Cas systems; CRISPR/Cas13a; RNA interference; RNA knockdown; molecular immunity; transcriptome regulation; virus interference.

Figure 1

Cas13a interference with TuMV-GFP in A. thaliana plants. (A) Schematic representation of the engineered system for in planta expression. The T-DNA consists of pCas13a and crRNA driven by the 35s and AtU6-26 promoter, respectively. In addition, it also contains Kan^r (kanamycin resistance) driven by the Nos promoter for selection of transgenic plants. (B) Confirmation of pCas13a protein expression in T₁ A. thaliana plants. Western blot analysis with anti-HA antibody was used to detect the expression of Cas13a in T₁ A. thaliana lines. NB-pCas13a represents protein extracted from transiently expressed pCas13a in Nicotiana benthamiana. (C) Interference of TuMV-GFP in pCas13a expressing transgenic Arabidopsis lines. Stably transformed Arabidopsis lines expressing pCas13 and crRNA were inoculated with TuMV-GFP sap from N. benthamiana. Plants were imaged for GFP fluorescence to examine TuMV-GFP systemic spread under UV light in the dark. (D) Western blot analysis to confirm TuMV-GFP accumulation in Arabidopsis systemic leaves. Protein resolved on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was blotted with GFP antibody to detect virus accumulation. Coomassie brilliant blue (CBB) stained membrane (lower panel) was used as loading control. (E) Northern blot to analyze TuMV-GFP virus titer in plants. Northern blot confirms that Hc-crRNA and GFP-T2 crRNAs give better interference with TuMV-GFP followed by GFP-T1 and CP crRNAs. RNA blots from (C) were probed with a DIG-labeled TuMV complementary (250-nt) RNA fragment and detected with anti-DIG antibody. The arrow indicates the accumulation of the TuMV-GFP RNA genome. (F) Quantification of TuMV-GFP RNA genome. The graph represents the relative expression of the TuMV-GFP RNA virus as calculated on the bases of three independent biological replicates of northern blot. Error bars represents STDEV.

Figure 2

A schematic representation of CRISPR/Cas13-mediated RNA virus interference in plants. The diagram illustrates the mechanism of molecular immunity against RNA viruses in A. thaliana plants expressing CRISPR/Cas13a. When plants are infected with virus, Cas13 guided by virus-targeting crRNA recognizes and degrades the RNA virus genome, providing immunity against the virus.