Plant Viruses Already Carry Weapons That Can Blind CRISPR-Cas13

CRISPR-Cas13 has rapidly become one of the most versatile tools in plant biotechnology, offering researchers the ability to target RNA molecules directly rather than editing the genome itself. Because Cas13 cleaves RNA rather than DNA, it promises flexible, reversible control of gene expression and a powerful new line of defense against plant RNA viruses. But a new study published in Stress Biology by Veerendra K. Sharma, Sandeep Marla, and David E. Cook of Kansas State University and Oregon State University reveals an unexpected vulnerability: proteins that plant viruses evolved long ago to defeat one RNA-silencing system can also defeat another. The viral suppressors of RNA interference known as HcPro and P19, the researchers found, block not only the plant’s native RNAi machinery but also CRISPR-Cas13-mediated transcript interference, effectively acting as anti-CRISPR factors.

The motivation behind the study was practical. Cas13 depends on a short guide RNA to find its target, but the team had previously discovered that these guides can silence transcripts even when the Cas13 protein is absent, a phenomenon they termed guide-induced gene silencing, or GIGS. This Cas13-independent silencing appears to hijack the plant’s endogenous RNA interference pathway, which processes small RNAs and uses them to direct silencing complexes to complementary sequences. Similar guide-only effects have been observed in human cell lines and mosquito cells, suggesting that Cas13-independent silencing is a widespread feature of eukaryotic cells rather than a plant-specific quirk. For biotechnologists, this is a problem: silencing that runs through RNAi rather than Cas13 is harder to control and more prone to off-target effects, undermining the precision that makes Cas13 attractive in the first place.

The researchers’ initial hypothesis was elegant in its simplicity. Plant viruses face relentless pressure from host RNAi, the primary antiviral defense in plants, and in response nearly all characterized plant viruses encode viral suppressors of RNAi, or VSRs. These specialized proteins neutralize the silencing machinery through diverse mechanisms, including sequestering small interfering RNAs so they cannot assemble into active RISC complexes, or directly interacting with core RNAi components such as Dicer-like proteins, Argonautes, and RNA-dependent RNA polymerases. The team reasoned that if they co-expressed a VSR alongside Cas13 and its guide, the VSR would mop up the RNAi-mediated, Cas13-independent silencing while leaving the Cas13 protein itself untouched. The result would be clean, Cas13-dependent transcript knockdown free of background noise.

Reality proved messier. The team tested two well-characterized VSRs: HcPro, the helper component protease from Turnip mosaic virus (TuMV), and P19 from Tomato bushy stunt virus (TBSV). Their assay system used Nicotiana benthamiana leaves infiltrated with Agrobacterium carrying the relevant constructs, with a GFP-expressing TuMV as the target. A multiguide construct carrying three targeting sequences against the TuMV genome effectively reduced viral accumulation, visible as diminished GFP fluorescence, in both Cas13-independent and Cas13-mediated assays when a GUS control protein was co-expressed. But when HcPro was substituted for GUS, the picture changed dramatically. HcPro suppressed the expected Cas13-independent silencing, consistent with its known anti-RNAi function, yet it also crippled Cas13-mediated targeting, restoring GFP fluorescence to levels comparable to controls in which no targeting occurred at all.

The quantitative data underscored how potent this interference is. Western blotting and densitometric analysis showed that HcPro expression restored GFP accumulation by approximately 20 percent in the Cas13-independent assays, but produced a nearly sixfold increase in GFP accumulation when Cas13 was present. P19 told the same story: co-expression increased GFP accumulation by 33 percent in Cas13-independent assays and by 78 percent in Cas13-mediated assays. Control experiments confirmed that the effect was specific to targeting guides rather than a general effect on viral accumulation, and RT-PCR verified that Cas13 transcripts were present only in the Cas13-mediated assays, ruling out contamination as an explanation. Both suppressors, in other words, were not merely dampening background RNAi but actively dismantling the CRISPR effector’s ability to silence its target.

To determine whether this anti-Cas13 activity was limited to viral RNA or represented a broader antagonism of Cas13 function, the researchers turned to an endogenous plant transcript. They targeted phytoene desaturase, or PDS, a gene whose silencing produces a visually distinctive photobleaching phenotype and whose transcript levels can be measured precisely by quantitative PCR. When GUS was co-expressed with a PDS-targeting multiguide and Cas13, PDS transcripts dropped by roughly 60 percent. When HcPro or P19 was co-expressed instead, PDS transcript levels remained statistically indistinguishable from those seen with a non-targeting guide. Northern blotting with a PDS-specific probe independently confirmed that the transcript was stabilized in the presence of the viral suppressors. The inhibitory effect, therefore, extends beyond antiviral contexts to general mRNA targeting, establishing HcPro and P19 as genuine antagonists of Cas13 interference.

The mechanistic key came from a mutant. HcPro suppresses RNAi by binding and sequestering small interfering RNAs, preventing the assembly of active RISC complexes, and this binding depends on amino acids in the protein’s central domain. The AS9 mutations, previously characterized in HcPro, abolish siRNA-binding activity without destroying the protein outright. When the team expressed wild-type HcPro alongside Cas13 and the PDS-targeting guide, Cas13-mediated silencing was blocked as before. But the HcPro AS9 mutant failed to inhibit Cas13-mediated silencing, and PDS transcripts were knocked down just as efficiently as in the GUS controls. This result ties HcPro’s anti-CRISPR activity directly to its RNA-binding, silencing-suppression function, and points to a plausible mechanism: both RNAi and Cas13 rely on short guide RNAs complexed with a protein effector, and a suppressor that sequesters small double-stranded RNAs indiscriminately will intercept Cas13 guide RNAs just as readily as viral siRNAs.

The findings resonate with a broader story about anti-CRISPR proteins. Bacteriophages long ago evolved proteins that disable bacterial CRISPR-Cas immunity, through mechanisms ranging from blocking PAM recognition to mimicking crRNA structure, and recent work has identified anti-CRISPR factors specific to RNA-targeting Cas13 systems. Plant viruses have almost certainly never encountered selective pressure from bacterial CRISPR-Cas systems, yet this study shows they already possess Cas13-inhibitory capacity as a byproduct of their anti-RNAi arsenal. Intriguingly, previous reports of successful Cas13-mediated virus resistance in plants often targeted the viral suppressor sequences themselves, which may have skewed the arms race toward the plant by limiting VSR protein accumulation. Guides directed against HcPro provided stronger TuMV interference in the team’s earlier work than guides against other regions of the viral genome, and other studies found that targeting viral open reading frames other than VSRs yielded weaker resistance.

There is an important caveat, and it concerns timing. A previous study reported that transgenic potato plants expressing Cas13 targeting the Potato virus Y coat protein resisted PVY infection effectively, seemingly contradicting the idea that VSRs can defeat Cas13 during infection. But that same line lost its resistance under mixed infections with PVY plus Potato virus X or Potato virus S, which carry their own heterologous suppressors. In stably transformed plants, Cas13 and its guide are present before the virus arrives, allowing the Cas13-crRNA complex to form and strike early, before suppressors accumulate. In the transient assays of the new study, Cas13, guide, and VSR are delivered simultaneously, giving the suppressor a window to accumulate and block complex formation. The authors suggest that any scenario reducing Cas13-crRNA accumulation, whether through stress, developmental regulation, or co-infecting viruses, could similarly compromise resistance. The sobering takeaway is that plant viruses did not evolve to counter Cas13, yet they may already be primed to overcome engineered CRISPR-Cas13 antiviral defenses through suppressors refined over millions of years of warfare with RNAi, and viral variants with enhanced Cas13-inhibiting function could be selected in the field.

Subject of Research: Inhibition of CRISPR-Cas13-mediated RNA targeting by plant viral suppressors of RNA interference

Article Title: Plant viral suppressors HcPro and P19 inhibit CRISPR-Cas13-mediated transcript interference

Article References: Sharma, V. K., Marla, S., & Cook, D. E. (2026). Plant viral suppressors HcPro and P19 inhibit CRISPR-Cas13-mediated transcript interference. Stress Biology, 6(1), Article 69. https://doi.org/10.1007/s44154-026-00339-9

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00339-9

Keywords: CRISPR-Cas13, viral suppressors of RNAi, HcPro, P19, guide-induced gene silencing, RNA interference, Turnip mosaic virus, Tomato bushy stunt virus, anti-CRISPR, plant virology, transcript engineering, Nicotiana benthamiana