CRISPR/Cas9-mediated viral interference in plants
© Ali et al. 2015
Received: 29 July 2015
Accepted: 7 October 2015
Published: 11 November 2015
The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species.
In this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses.
These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.
KeywordsPlant genome engineering CRISPR/Cas9 system Synthetic site-specific nucleases Viral-mediated genome editing Virus resistance Tomato yellow leaf curl virus Tobacco rattle virus
In bacteria and archaea, the clustered regularly interspaced palindromic repeat (CRISPR)/ CRISPR-associated (Cas) 9 (CRISPR/Cas9) system confers molecular immunity against nucleic acids of invading conjugative plasmids or phages [1–6]. The CRISPR/Cas9 system has recently been harnessed in diverse eukaryotic species, including plants, for the purpose of targeted genome editing and regulation [7, 8]. The CRISPR/Cas9 molecular immunity system comprises the Cas9 endonuclease of Streptococcus pyogenes and a synthetic single guide RNA (sgRNA), which directs the Cas9 endonuclease to a target sequence complementary to the 20 nucleotides preceding the protospacer-associated motif (PAM) NGG, which is required for Cas9 activity [9, 10]. Thus, engineering of the system for a user-selected target requires only the addition of 20 nucleotides to the sgRNA molecule, allowing facile targeted genome editing and regulation. Furthermore, simultaneous targeting of several genomic loci (multiplexing) is feasible using multiple sgRNAs .
Geminiviruses, a large family of plant DNA viruses, cause severe crop losses and economic consequences that threaten food security worldwide, especially in underdeveloped regions like sub-Saharan Africa [11, 12]. Members of the Geminiviridae possess a twin icosahedral capsid structure that encapsidates a circular single-stranded DNA (ssDNA) genome between 2.3 and 3 kb in length. Geminiviridae viruses replicate their genome either by a rolling-circle amplification (RCA) mechanism via a double-stranded DNA (dsDNA) replicative form (similar to that of Φ174 phage), or by recombination-mediated replication . Geminiviruses do not encode their own DNA polymerase, but instead reactivate S phase and the cellular machinery to facilitate replication of their own genome . Upon infection of plant cells, the viral Rep protein binds to the origin of replication, thereby initiating viral replication in the nucleus . Based on their genome organization, host range, insect vectors, and genome-wide pairwise sequence identity, members of Geminiviridae are classified into seven genera: Begomovirus, Mastrevirus, Curtovirus, Becurtovirus, Eragrovirus, Turncurtovirus and Topocuvirus. Members of the genus Begomovirus, which infect dicotyledonous plants via whitefly transmission vectors, include bipartite and monopartite viruses . Bipartite viruses, such as African cassava mosaic virus and cabbage leaf curl virus, have genomes comprising two components (A and B) that differ in sequence (except for the common region, a 200–250-bp sequence that is nearly identical) . The common region forms part of the highly conserved intergenic region (IR), which contains the origin of replication and promoter sequences for RNA polymerase II . By contrast, monopartite viruses, such as tomato yellow leaf curl virus (TYLCV), have a single genomic component. A conserved nucleosome-free IR is present in the minichromosome of all geminiviruses [17, 18].
Targeted DNA mutagenesis using site-specific endonucleases has been used against a variety of mammalian DNA viruses . Very recently, the CRISPR/Cas9 system was used to target different mammalian viruses. For example, this technology was used to efficiently eradicate HIV proviral DNA from the host genome and prevent HIV infection . It was also used to disrupt the hepatitis B virus, treat latent infection by Epstein-Barr virus, and engineer the large genome of the herpes simplex virus [31–34]. A recent report showed the feasibility of targeting a eukaryotic RNA virus using the CRISPR/Cas machinery . In this work, we demonstrate the use of CRISPR/Cas9 for in planta viral interference against TYLCV. Both the TYLCV ORFs and the IR sequence could be targeted for cleavage and mutagenesis by the CRISPR/Cas9 system. Targeting of the TYLCV genome resulted in significant reduction or attenuation of disease symptoms. Further, the CRISPR/Cas9 system was able to target multiple virus sequences simultaneously. Therefore, it may be generally possible to use the CRISPR/Cas9 system to develop plants that are resistant to TYLCV and other DNA viruses.
CRISPR/Cas9-mediated interference with TYLCV
In this study, we investigated whether the CRISPR/Cas9 system can be used in plants to impart molecular immunity against DNA viruses. To this end, we used our recently developed system for genome editing, which involves systemic delivery of sgRNA molecules via tobacco rattle virus (TRV) into N. benthamiana plants overexpressing Cas9 endonuclease (NB-Cas9OE) . We designed sgRNAs specific for different TYLCV coding and non-coding sequences (Fig. 1a), and inserted them into the TRV RNA2 genome. Next, we delivered the sgRNAs via agroinfection of TRV into NB-Cas9OE plants. Seven days post-infiltration (dpi) with TRV, we challenged the NB-Cas9OE plants with an infectious TYLCV clone via agroinfection (Fig. 1b) . Ten days later, we isolated total RNA and DNA from the NB-Cas9OE plant systemic leaves for various molecular studies. To determine TYLCV titer, we performed semi-quantitative PCR using primers encompassing the IR region (Table S1 in Additional file 1). The titer was lower in samples co-infiltrated with sgRNA targeting the IR region than in those infiltrated with TRV vector controls and TYLCV (Fig. 1c). TYLCV replicates through a RCA mechanism that exploits the plant machinery . An RCA assay revealed that targeting of the IR via the CRISPR/Cas9 system prevented accumulation of the TYLCV genome (Fig. 1d). Because TYLCV is a ssDNA virus that is converted to dsDNA inside the plant cell nucleus, interference with TYLCV replication by targeting the IR within replicating viral dsDNA should significantly reduce the accumulation of both the ssDNA and dsDNA forms. To test for interference with TYLCV replication, we performed dot blot assays. The results showed that the titer of TYLCV in IR-sgRNA plants was lower than that of the TRV vector control (Figure S1 in Additional file 2). In addition, we validated our dot blot results by Southern blotting, which confirmed that targeting the IR of TYLCV prevented accumulation of both ssDNA and dsDNA (Fig. 1e).
We further confirmed our findings by using a different method for TYLCV inoculation, namely, the sap transmission method (Additional file 3). Sap from young leaves of TYLCV-infected wild-type N. benthamiana plants was directly applied to N. benthamiana Cas9OE plants 7 days after infection with TRV-sgRNA. DNA was extracted from the systemic leaves after 21 days of sap application and then subjected to different types of molecular analysis. Non-specific sgRNA (possessing no sequence similarity to the TYLCV genome; Supplementary sequence 9 in Additional file 4) rather than an empty TRV vector was used in the sap transmission experiments. The RCA results revealed a reduction in TYLCV genome accumulation in both samples treated with IR-sgRNA or CP-IR-sgRNA compared with samples treated with non-specific sgRNA or TYLCV alone (Figure S2a in Additional file 2). To confirm the RCA results, we next performed semi-quantitative PCR to amplify a 560-bp fragment encompassing the TYLCV IR. The results revealed lower amplification of TYLCV with specific sgRNAs than with controls (Figure S2b in Additional file 2), thereby confirming the RCA results. Both the RCA and semi-quantitative PCR assays are based on the amplification of available TYLCV genome. To further confirm these data, we next performed Southern blotting, which confirmed lower accumulation of TYLCV in the presence of specific sgRNAs than in the presence of controls (Figure S2c in Additional file 2).
CRISPR/Cas9 mediates targeted cleavage of the TYLCV genome
CRISPR/Cas9 system mediates specific and multiple targeting of viral genomes
The CRISPR/Cas9 system attenuates or represses TYLCV symptoms
Interference with TYLCV replication by the CRISPR/Cas9 machinery is predicted to eliminate or reduce TYLCV symptoms, reminiscent of the CRISPR/Cas9 system’s originally evolved function in natural immunity of bacteria against phages. Accordingly, we assessed and evaluated TYLCV symptoms in NB-Cas9OE plants expressing sgRNAs against TYLCV coding and non-coding sequences. In these experiments, we challenged three groups of NB-Cas9OE plants, which expressed sgRNAs specific to the IR, CP, or Rep regions, with an infectious TYLCV clone. NB-Cas9OE plants expressing sgRNA targeting the IR exhibited significantly reduced TYLCV symptoms relative to the TRV vector control (Figure S6 in Additional file 2; Table S2 in Additional file 1). Moreover, NB-Cas9OE plants expressing sgRNA targeting the CP or Rep ORF also exhibited a reduction of TYLCV symptoms, but the magnitude of this reduction was smaller than that achieved by targeting the IR-sgRNA (Figure S7 in Additional file 2; Table S2 in Additional file 1). In a second set of experiments, we investigated whether targeting more than one sequence of the TYLCV genome would lead to greater reduction of TYLCV symptoms. To test this, we co-infiltrated a pair of TRV RNA2 genomes carrying sgRNAs targeting the CP region and IR. Targeting two different sequences did not have an additive effect on the reduction of TYLCV symptoms (Figure S8 in Additional file 2; Table S2 in Additional file 1).
Because co-infiltration of two RNA2 genomes carrying two different sgRNAs does not ensure the delivery of both sgRNAs into a single cell (and their subsequent activities against single molecules of the viral genome), we multiplexed our sgRNA delivery using the recently developed polycistronic tRNA–gRNA (PTG) system . Subsequently, to determine the activity of both gRNAs in this system, we delivered a single RNA2 genome carrying both IR-sgRNA and CP-sgRNA. This TRV system was capable of expressing two sgRNAs that can target both IR and CP sequences. Restriction site loss assays (Figure S9 in Additional file 2) and Sanger sequencing confirmed the targeted modification of both IR and CP sequences (Figure S10 in Additional file 2). Furthermore, simultaneous targeting of two sequences by PTG-based expression led to greater reduction of virus titer and recovery of disease symptoms in NB-Cas9OE plants (Figure S11 in Additional file 2; Table S2 in Additional file 1). Next, we performed Southern blotting to confirm the absence or reduced accumulation of the TYLCV genome in IR-CP-sgRNA infiltrated plants relative to that in plants infiltrated with the vector control. The molecular analyses showed a significant reduction in viral genome levels when the IR and CP region were simultaneously targeted for cleavage. This confirmed the phenotypic data regarding the TYLCV symptoms (Figure S12 in Additional file 2).
The CRISPR/Cas9 system confers molecular immunity in bacterial and archaeal species, enabling these prokaryotes to fend off viral infections . In this study, we demonstrated the portability of the CRISPR/Cas9 system to plants, where it is capable of conferring molecular immunity against invading DNA viruses. To evaluate the feasibility of using CRISPR/Cas9-mediated interference against DNA viruses in plants, we performed TRV-mediated systemic delivery of sgRNAs into NB-Cas9OE plants. Targeting of the coding and non-coding sequences of TYLCV resulted in specific modification of viral sequences, and subsequently attenuated viral replication and accumulation. Because binding of the CRISPR/Cas9 system to the IR can attenuate viral replication, we investigated the presence and efficiency of genomic modification at the IR. Our data demonstrate considerable levels of genomic modification at the IR, indicating that the Cas9 endonuclease was catalytically active. However, it remains to be determined whether binding of catalytically inactive Cas9 (dCas9) could interfere with virus replication and accumulation.
We tested sgRNAs targeting the CP, the RCRII motif of Rep, and the IR sequences, and compared their efficiency to interfere with TYLCV. All sgRNAs were capable of mediating targeted cleavage of the TYLCV genome. Extensive molecular analysis using RCA, semi-quantitative PCR, and Southern blotting assays revealed that targeting the IR led to a significant reduction in viral replication and accumulation relative to other TYLCV targets. Targeting of CP and RCRII attenuated symptoms to a lesser extent than targeting of the IR, possibly because both CP and RCRII encode proteins, and a small amount of protein may be sufficient for TYLCV to complete its cycle and develop symptoms. Furthermore, because the IR contains the stem-loop invariant sequence (TAATATTAC) of the TYLCV IR, occupation or modification of this region by Cas9 makes it inaccessible to Rep and/or other binding proteins responsible for viral replication.
Subsequently, we investigated whether the co-delivery of multiple sgRNAs via the TRV system would have an additive effect, leading to higher interference levels than obtained with single sgRNAs. The results demonstrated that targeting CP and IR using separate RNA2 genomes reduced viral replication and accumulation to levels similar to those obtained with a single sgRNA targeting either the IR or CP. Although we did not observe any additive effect on the accumulation of the TYLCV genome, this might be due to the nature of the TRV infection; i.e., because the two sgRNAs are not made in the same cell, they are unable to simultaneously cleave the TYLCV. On the other hand, construction of a TRV RNA2 genome containing multiple sgRNAs led to multiplexed editing in single cells, resulting in an additive effect.
One important criterion for in planta viral interference is specificity against particular viral strains. We investigated the specificity of our CRISPR/Cas9-mediated viral interference system using sgRNAs targeting either TYLCV or BCTV. When we used sgRNA targeting TYLCV sequences, only the TYLCV genome was modified, whereas when we used sgRNA targeting the BCTV, only the BCTV genome sequence was modified, demonstrating the specificity of interference. Such specificity is of particular importance when targeting newly evolved viral variants.
Engineered resistance of the plant host to viral infection might place selection pressure on the virus, leading to generation of variants with better adaptation, e.g., by altering the PAM tri-nucleotide sequence or abrogating recognition of the target sequence by the CRISPR/Cas9 system. For example, TYLCV recombinant variants that overcome resistance have emerged in Ty-I resistant plants . Because the CRISPR/Cas9 system targets viral DNA for destruction, DNA repair can lead to the generation of viral variants with different PAM sequences that can evade the activity of the CRISPR/Cas9 system. Moreover, because particular DNA sequences (e.g., the IR) are key to viral replication, mutagenesis of these sequences will lead to significant reduction in viral replication . To maintain the replication of a new viral variant, the target sequence must mutate to escape recognition by the CRISPR/Cas9 system, and the key replication enzymes that bind to this sequence must also mutate to recognize the new sequence. Targeting two viral sequences for cleavage will lead to the destruction of the viral genome, reducing the likelihood of DNA repair and generation of infectious viral variants. The emergence of variants can be combated through the design and application of sgRNA molecules specific for the new variant sequences.
We subsequently tested whether the CRISPR/Cas9 system is capable of targeting multiple viral strains by co-infiltration of sgRNAs targeting TYLCV, BCTV, and MeMV. The results showed that it was possible to target more than one virus with a single sgRNA. Recent work demonstrated the feasibility of expressing several sgRNAs simultaneously . Such a system could be used to express sgRNAs with specificity for multiple DNA viruses, enabling engineering of plants resistant to multiple viruses or viral strains. This strategy should be effective in developing resistance to mixed infections. It should be noted that when using TRV-mediated delivery of sgRNAs targeting coding and noncoding sequences, the CRISPR/Cas9 system resulted in efficient interference of TYLCV. However, we observed less accumulation of the TYLCV genome when empty TRV vector was used in NB-Cas9OE plants challenged with TYLCV compared with NB-Cas9OE plants challenged with TYLCV but carrying no TRV vectors (Fig. 1d, e; Figure S12 in Additional file 2). Therefore, we do not entirely exclude other contributing silencing factors caused by the presence of TRV vector due to more complex phenomena at play, which requires further studies.
In summary, CRISPR/Cas9-mediated virus interference in plants has numerous important features, including: 1) the ability to target multiple DNA viruses simultaneously using a single sgRNA targeting a conserved sequence preceding the PAM tri-nucleotide sequence; 2) the capacity for multiplexed editing of single or multiple viruses using multiple sgRNAs; 3) the potential to overcome resistance by targeting newly evolved viral revertants with new sgRNAs; 4) applicability to all plant DNA viruses; and 5) applicability to all transformable plant species. Establishing the efficacy and extending the utility of the CRISPR/Cas9 system for viral interference in plants will create a platform for dissecting natural resistance and immune functions. At the same time, it will provide biotechnologists with a powerful tool for producing crop plants resistant to multiple viral infections.
The data presented here show that the CRISPR/Cas9 system can be used for targeted interference and cleavage of the TYLCV genome. Targeting the TYLCV IR led to a significant reduction in TYLCV accumulation and disease symptoms. CRISPR/Cas9-mediated interference is virus strain-specific, and can therefore be used to target multiple viruses. Our results confirm the efficacy of the CRISPR/Cas9 system for virus interference, providing new possibilities for engineering plants resistant to DNA viruses.
Materials and methods
To clone sgRNAs targeting the TYLCV genome in the TRV RNA2 vector, we used a PCR-based restriction ligation procedure. A fragment containing the 20-nucleotide target sequence, the 84-bp Cas9 binding loop for sgRNA, and a 7 T repeat (as a terminator) was amplified by PCR. A forward primer containing an XbaI recognition site, 20-nucleotide target sequence, and 23-nucleotide Cas9-binding sgRNA scaffold was used with a reverse primer containing an XmaI recognition site to amplify a 116-bp PCR fragment. Primer sequences are provided in Table S1 in Additional file 1. The 116-bp PCR fragment of the sgRNA for each target was cloned into the TRV RNA2 vector under the control of the pea early browining virus promoter using the XbaI and XmaI restriction enzymes. Sanger sequencing was used to confirm all clone sequences.
Mutation detection by restriction site loss
To test for targeted modification in the TYLCV genome by the CRISPR/Cas9 system, we subjected PCR products encompassing the target sequence to the restriction site loss assay. Genomic DNA was isolated from samples collected at 10 or 15 dpi. A fragment flanking the target region of TYLCV was PCR amplified using a specific primer set and Phusion high-fidelity polymerase (Supplementary sequence 1 in Additional file 4). DNA of PCR products was gel purified, and 200 ng was subjected to restriction enzyme protection analysis to detect the presence of indels. The digested products were separated on 2 % agarose gel. The PCR product was cloned into the pJet2.1 vector and subjected to Sanger sequencing.
T7EI mutation detection assay
To determine and quantify the activity of the CRISPR/Cas9 system on the TYLCV genome, we measured mutations resulting from double strand break repair through the non-homologous end joining pathway, as described previously . Briefly, genomic DNA was prepared from samples collected at 10 and 15 dpi and used as a template for PCR amplification of fragments encompassing the target sequences (Supplementary sequence 1 in Additional file 4). PCR amplicons were denatured, renatured, and treated with T7EI. To calculate the frequency of modification, the PCR amplicons were cloned into the pJET2.1 cloning vector and successful cloning was confirmed by colony PCR and BglII restriction digestion of the extracted plasmids. The percentage of modified clones was calculated after Sanger sequencing.
Total genomic DNA extracted from plants was quantified using a NanoDrop spectrophotometer, adjusted to a concentration of 50 ng/μl, and analyzed using the RCA amplicon kit (GE Healthcare). Genomic DNA (50 ng) was incubated for 3 min at 95 °C in sample buffer and placed on ice for 5 min. Enzyme mix and reaction buffer were added, and the samples were incubated at 30 °C for 18 h for amplification, followed by incubation at 65 °C for 15 min to inactivate the enzyme. NcoI was added to the samples and incubated for 1 h at 37 °C, and the digested samples were resolved on 1 % agarose gels.
beet curly top virus
clustered regularly interspaced palindromic repeat
Merremia mosaic virus
open reading frame
polymerase chain reaction
single guide RNA
tobacco rattle virus
tomato yellow leaf curl virus
We wish to thank members of the Laboratory for Genome Engineering at King Abdullah University of Science and Technology for helpful discussions and comments. The study was supported by King Abdullah University of Science and Technology.
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