RNA virus interference via CRISPR/Cas13a system in plants

Abstract

Background: CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants.

Results: CRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. CRISPR RNA (crRNAs) targeting the HC-Pro and GFP sequences exhibit better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs.

Conclusions: Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.

Keywords: CRISPR/Cas systems; CRISPR/Cas13a; Molecular immunity; RNA interference; RNA knockdown; Transcriptome regulation; Virus interference.

Fig. 1

Reconstitution of the CRISPR/Cas13a machinery in plants. a Schematic assembly of the plant codon-optimized Cas13a (pCas13a). pCas13a was custom synthesized as four fragments. The F1 (with attL1 and 3x-HA), F2, F3, and F4 (with nls and attL2) fragments were assembled in the cloning vector using a restriction-ligation system. The respective restriction enzymes sites are represented on the top. By LR reaction, pCas13a was moved into the Gateway-compatible binary vector pK2GW7 to make pK2GW7-pCas13a. HA human influenza hemagglutinin epitope tag, nls nuclear localization signal, attL1 and attL2, Gateway cloning recombination sites. b Confirmation of pCas13a expression in transgenic lines. Total protein was extracted from three independent lines of N. benthamiana transformed with pK2GW7-pCas13a. Anti-HA antibody was used to detect HA-tagged pCas13a. The arrow indicates the presence of pCas13a. Wild type (WT) was used as a negative control. c The TuMV-GFP genome with selected targets. The arrowheads indicate the four selected sites. The lower panel represents the target RNA sequences paired with their respective crRNAs. PFS protospacer flanking site. d Expression of the crRNA from the TRV system. The repeat-guide DNA sequences were cloned under the PEBV promoter in RNA2 of TRV for constitutive and systemic expression. e Diagrammatic representation of the pCas13–crRNA–target complex. The targeting complex pCas13a–crRNA (repeat sequence-n28, guide RNA-n28) bound to the RNA target n28 for interference

Fig. 2

The pCas13a–crRNA complex interferes with TuMV-GFP in planta. a pCas13a mediates interference with the GFP-expressing TuMV virus in plants. N. benthamiana plants expressing pCas13a were infiltrated with TRV expressing crRNAs and TuMV-GFP. At 7 dpi, plants were imaged under UV light for GFP. The GFP signal of the plants having target crRNAs were compared to plants having no crRNA or a ns-crRNA. b GFP protein detection was used to validate the TuMV-GFP interference. Protein blots from (a) were developed with anti-GFP antibody. The arrow indicates the size of the GFP band. c Northern blot confirms that Hc-crRNA and GFP2-crRNA give better interference with TuMV. RNA blots from (a) 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 RNA genome. In (b) and (c) the lower panels were used as loading controls

Fig. 3

pCas13a-processed poly-crRNA mediates interference with TuMV-GFP. a Schematic of the cleavage of poly-crRNA by pCas13a. Rectangles denote the spacer sequences and solid lines denote repeat elements. The predicted cleavage sites are depicted. b Northern blot for pCas13a-mediated cleavage of poly-crRNA. RNA blot from two independent transgenic lines of pCas13a (L-9 and L-6) infiltrated with TRV RNA1 and RNA2 expressing specific poly-crRNA and a ns-poly-crRNA and TuMV-GFP. The RNA blot was probed and detected with a DIG-labeled 28-nt RNA fragment against the repeat sequence. The arrow indicates the processed crRNA (56 nt) in L-9 from both ns-poly-crRNA and poly-crRNA compared to WT N. benthamiana. In L-6, the processed crRNA level from poly-crRNA remained under the detection level. The synthetic crRNA (56 nt) was used as an experimental control. c GFP signal data demonstrating the poly-crRNA-mediated interference against TuMV-GFP. pCas13a transgenic N. benthamiana plants infiltrated with TRV expressing poly-crRNA (three crRNAs) and TuMV-GFP were imaged under UV light for GFP at 7 dpi. The GFP signal of the plants expressing poly-crRNAs were compared to plants expressing ns-poly-crRNA or empty vector control. d GFP quantification of poly-crRNA-mediated viral interference. The GFP signal (n = 5) data were used for the generation of a percentile graph. The GFP signal of the plants inoculated with TuMV-GFP and empty vector control were used as a reference. e Northern blot showing the poly-crRNA-based interference against TuMV-GFP. RNA blots were probed with the DIG-labeled RNA fragment and developed with anti-DIG antibody. The arrow indicates the accumulation of the TuMV RNA genome. f Western blot to confirm the TuMV-GFP interference by poly-crRNA. Protein blots from the poly-crRNA and ns-poly-crRNA were detected for GFP with anti-gfp antibody. ns-poly-crRNA and plants inoculated with only TuMV-GFP were used as control. In (e) and (f), the lower panels were used as loading controls