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. 2019 Dec 2;20(1):263.
doi: 10.1186/s13059-019-1881-2.

CRISPR-Cas13d Mediates Robust RNA Virus Interference in Plants

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Free PMC article

CRISPR-Cas13d Mediates Robust RNA Virus Interference in Plants

Ahmed Mahas et al. Genome Biol. .
Free PMC article

Abstract

Background: CRISPR-Cas systems endow bacterial and archaeal species with adaptive immunity mechanisms to fend off invading phages and foreign genetic elements. CRISPR-Cas9 has been harnessed to confer virus interference against DNA viruses in eukaryotes, including plants. In addition, CRISPR-Cas13 systems have been used to target RNA viruses and the transcriptome in mammalian and plant cells. Recently, CRISPR-Cas13a has been shown to confer modest interference against RNA viruses. Here, we characterized a set of different Cas13 variants to identify those with the most efficient, robust, and specific interference activities against RNA viruses in planta using Nicotiana benthamiana.

Results: Our data show that LwaCas13a, PspCas13b, and CasRx variants mediate high interference activities against RNA viruses in transient assays. Moreover, CasRx mediated robust interference in both transient and stable overexpression assays when compared to the other variants tested. CasRx targets either one virus alone or two RNA viruses simultaneously, with robust interference efficiencies. In addition, CasRx exhibits strong specificity against the target virus and does not exhibit collateral activity in planta.

Conclusions: Our data establish CasRx as the most robust Cas13 variant for RNA virus interference applications in planta and demonstrate its suitability for studying key questions relating to virus biology.

Keywords: CRISPR-Cas; Cas13; CasRx; RNA interference; Virus interference; Virus resistance.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
CRISPR/Cas13 orthologues for RNA virus interference. a Schematic representation of CRISPR/Cas13 variants and their respective crRNA structures. Structural representations of different Cas13 subtypes with their corresponding crRNA structures are shown with the estimated average size of the Cas13 protein under each subtype. HEPN, high eukaryotic and prokaryotic nucleotide binding domains; aa, amino acid; nt, nucleotides. b Schematic of different Cas13 protein variants used in this study. The different Cas13 variants are shown with their different fusions. NLS, nuclear localization signal; NES, nuclear export signal; GFP, green fluorescent protein. c Schematic of TRBO-based Cas13 mediated RNA virus interference. The highly replicating plant TRBO RNA virus expressing GFP protein was used as a reporter system to screen for efficient Cas13 activity in transient assays via crRNA delivered through TRV system, targeting two different regions within the GFP sequence of the virus RNA genome. 35S, CaMV 35S promoter; pPEBV, pea early browning virus promoter; RNA1/RNA2, genomes of the tobacco rattle virus (TRV)
Fig. 2
Fig. 2
TRBO-GFP-based screening of different Cas13 orthologues for efficient virus interference. a GFP monitoring to assess the Cas13-mediated virus interference activities in Agro-infiltrated wild type N. benthamiana leaves in transient assays. Images were taken 3 days post-infiltration. NS, non-specific crRNA. b Relative fluorescence intensity quantification of leaf images. For each Cas13 variant, GFP signal intensity of each targeting crRNA (GFP-T1 and GFP-T2) is shown relative to the non-targeting (NS) crRNA. Values shown as mean ± SEM (n = 3). c Western blot analysis of the abundance of the virus expressed GFP protein to confirm the Cas13-mediated TRBO-GFP virus interference. Protein blots were developed with anti-GFP antibody. α-GFP, anti-GFP antibody. Ponceau staining served as loading control. d Quantification of the western blot data. For each Cas13 variant, the abundance of the GFP protein with the targeting crRNAs (GFP-T1 and GFP-T2) is shown relative to the non-targeting (NS) crRNA. Error bars indicate SEM (n = 3). e RT-qPCR analysis of TRBO-GFP knockdown with different Cas13 variants using the two position-matched crRNAs. For each Cas13 variant, knockdown efficiency of each targeting crRNA (GFP-T1 and GFP-T2) is shown relative to the non-targeting (NS) crRNA. Values shown as mean ± SEM (n = 3)
Fig. 3
Fig. 3
Characterization of LwaCas13a, PspCas13b, and CasRx activity against essential and conserved genomic region of RNA viruses. a GFP monitoring to assess the Cas13-mediated virus interference activities in Agro-infiltrated wild type N. benthamiana leaves in transient assays. Images were taken 3 days post-infiltration. NS, non-specific crRNA; Rep, replicase. b Relative fluorescence intensity quantification of leaf images. For each Cas13 variant, GFP signal intensity of each targeting crRNA (Rep-T1, Rep-T2, and Rep-T3) is shown relative to the non-targeting (NS) crRNA. Values shown as mean ± SEM (n = 3). c Western blot analysis of the abundance of the virus expressed GFP protein to confirm the Cas13-mediated TRBO-GFP virus interference. Protein blots were developed with anti-GFP and anti-HA antibodies. α-GFP, anti-GFP antibody, α-HA, anti-HA antibody. Ponceau staining served as loading control. d Quantification of the western blot data. For each Cas13 variant, the abundance of the GFP protein with the targeting crRNAs (Rep-T1, Rep-T2, and Rep-T3) is shown relative to the non-targeting (NS) crRNA. Error bars indicate SEM (n = 3). e RT-qPCR analysis of TRBO-GFP knockdown with different Cas13 variants using the three position-matched crRNAs. Transcript levels are shown relative to leaves inoculated with only TRBO-GFP vector. Values shown as mean ± SEM (n = 3)
Fig. 4
Fig. 4
CasRx mediates efficient interference against TuMV-GFP virus by preventing its systemic spread. a Cas13-mediated interference against the GFP-expressing TuMV virus in plants. N. benthamiana plants expressing LwaCas13a, PspCas13b, and CasRx proteins were co-infiltrated with TRV (expressing crRNAs targeting the TuMV-GFP virus and NS crRNA) and TuMV-GFP. At 7 dpi, plants were imaged under UV light for GFP signal monitoring. b RT-qPCR analysis of TuMV-GFP knockdown with different Cas13 variants using the different position-matched crRNAs. For each Cas13 variant, knockdown efficiency of each targeting crRNA is shown relative to the non-targeting (NS) crRNA. Values shown as mean ± SEM (n = 3)
Fig. 5
Fig. 5
CasRx catalytic activity is required for RNA virus interference. a Illustration of the targeting activity of the catalytically active and catalytically deactivated (dCasRx) CasRx variants against the targeted TRBO-GFP virus. b GFP monitoring to assess the Cas13-mediated virus interference activities in Agro-infiltrated wild type N. benthamiana leaves in transient assays. Images were taken 3 days post-infiltration. NS, non-specific crRNA. c Western blot analysis of the abundance of the virus expressed GFP protein to confirm the Cas13-mediated TRBO-GFP virus interference. Protein blots were developed with anti-GFP and anti-HA antibodies. α-GFP, anti-GFP antibody, α-HA, anti-HA antibody. Ponceau staining served as loading control. d RT-qPCR analysis of TRBO-GFP knockdown with the catalytically active and catalytically deactivated CasRx variants. For each CasRx variant, knockdown efficiency of each targeting crRNA (GFP-T1 and GFP-T2) is shown relative to the non-targeting (NS) crRNA. Values shown as mean ± SEM (n = 3)
Fig. 6
Fig. 6
Specific virus targeting via CasRx with no observed collateral activity. a Schematic of CasRx targeting of TRBO-BFP and PVX-GFP viruses in synchronous co-infected leaves with the two different viral vectors, and the possible outcomes. b BFP monitoring to assess the Cas13-mediated virus interference activities in Agro-infiltrated wild type N. benthamiana leaves in transient assays (left). Images were taken 3 days post-infiltration. NS, non-specific crRNA; Rep, replicase. RT-qPCR analysis of TRBO-BFP knockdown with Rep-T3 crRNA used against TRBO-BFP virus (right). The transcript levels of TRBO-BFP are shown relative to the transcript level of TRBO-BFP virus targeted with (NS) crRNA. Values shown as mean ± SEM (n = 3). c BFP and GFP monitoring to assess the Cas13-mediated virus interference activities in Agro-infiltrated wild type N. benthamiana leaves in transient assays (left). Images were taken 3 days post-infiltration. NS, non-specific crRNA; Rep, replicase. RT-qPCR analysis of TRBO-BFP and PVX-GFP knockdown with Rep-T3 crRNA (right). The transcript levels of TRBO-BFP are shown relative to the transcript level of the non-targeted virus (PVX-GFP), and the transcript level of both viruses is shown relative to the transcript level of both viruses targeted with (NS) crRNA. Values shown as mean ± SEM (n = 3). d GFP monitoring to assess the Cas13-mediated virus interference activities in Agro-infiltrated wild type N. benthamiana leaves in transient assays (left). Images were taken 3 days post-infiltration. NS, non-specific crRNA. RT-qPCR analysis of PVX-GFP knockdown with GFP-T2 crRNA used against PVX-GFP virus (right). The transcript levels of PVX-GFP are shown relative to the transcript level of PVX-GFP virus targeted with (NS) crRNA. Values shown as mean ± SEM (n = 3). e BFP and GFP monitoring to assess the Cas13-mediated virus interference activities in Agro-infiltrated wild type N. benthamiana leaves in transient assays (left). Images were taken 3 days post-infiltration. NS, non-specific crRNA. RT-qPCR analysis of TRBO-BFP and PVX-GFP knockdown with GFP-T2 crRNA (right). The transcript levels of PVX-GFP are shown relative to the transcript level of the non-targeted virus (TRBO-BFP), and the transcript level of both viruses is shown relative to the transcript level of both viruses targeted with (NS) crRNA. Values shown as mean ± SEM (n = 3)

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