ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 6, pp. 804-817 © Pleiades Publishing, Ltd., 2025.
Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 6, pp. 867-883.
804
REVIEW
Biotechnological Approaches to Plant
Antiviral Resistance: CRISPR-Cas or RNA Interference?
Natalia O. Kalinina
1,2,a
*, Nadezhda A. Spechenkova
1
, and Michael E. Taliansky
1
1
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
117997 Moscow, Russia
2
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,
119234 Moscow, Russia
a
e-mail: kalinina@belozersky.msu.ru
Received January 21, 2025
Revised February 27, 2025
Accepted February 28, 2025
Abstract— Established genome editing technologies, such as CRISPR-Cas and RNA interference (RNAi), have
significantly advanced research studies in nearly all fields of life sciences, including biotechnology and medi-
cine, and have become increasingly in demand in plant biology. In the review, we present the main principles
of the CRISPR-Cas and RNAi technologies and their application in model plants and crops for the control of
viral diseases. The review explores the antiviral effects they provide, including direct suppression of genomes
of DNA- and RNA-containing viruses and inhibition of activity of host genes that increase plant susceptibil-
ity to viruses. We also provide a detailed comparison of the effectiveness of CRISPR-Cas and RNAi methods
in plant protection, as well as discuss their advantages and disadvantages, factors limiting their application
in practice, and possible approaches to overcome such limitations.
DOI: 10.1134/S0006297925600139
Keywords: plant viruses, RNA interference, CRISPR-Cas, dsRNA, plant antiviral resistance
* To whom correspondence should be addressed.
INTRODUCTION
Viral diseases of plants present a serious danger
to global agriculture by adversely affecting yield, food
safety, and economic stability [1]. There is an urgent
need to control epidemics caused by the spread of
plant viruses and their new variants appearing due to
the genetic evolution, transmission from natural plant
reservoirs, changes in agriculture, mixed infections,
and impact of global warming [1]. Almost half (47%)
of all repeatedly emerging outbreaks of plant diseases
are caused by viruses (i.e., more than by any other
plant disease agent) [1, 2].
Currently, the major approach to combating vi-
ral diseases is the use of agrochemicals and resistant
crop varieties. Although application of pesticides to
eliminate natural vectors of viral infections (mites,
nematodes, aphids, thrips, cicadas, and whiteflies) is
quite effective, it is nonselective and can be harmful
to other (beneficial) organisms, leading to the disrup-
tion in the ecological balance [3].
The use of genetically resistant plant varieties is
commonly believed to be the most efficient, cost-ef-
fective, and consumer-friendly approach to controlling
viral diseases. However, many crops lack genetic re-
sistance to viruses due to a deficiency of resistance
genes in genetically compatible relatives. Selection of
resistance genes even by modern molecular methods,
such as quantitative trait locus (QTL) mapping, mark-
er-assisted selection, and whole-genome sequencing,
is often time-consuming, labor intensive (i.e., due to
the difficulties in crossing elite lines with wild plant
species), and requires monitoring of large plant pop-
ulations over a number of generations, which may
take several years in crops with long-term breeding
cycles [4].
Other plant protection technics include preventive
measures, such as quarantine, certification, cross-pro-
tection, removal of infected plants, and microprop-
agation to obtain virus-free planting material [5];
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BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
however, they are often insufficient to reduce the
harmful effects of viral diseases on agricultural plants.
Achievements in genetic engineering and plant
transformation methods in the early 1980s have made
it possible to obtain virus-resistant lines of transgenic
plants [6] by producing plants expressing viral pro-
teins (envelope proteins, replicases, transport pro-
teins, etc.) or their fragments, as well as non-coding
viral nucleotide sequences [7-10]. These techniques
also have significant limitations due, in particular, to
the fact that the acquired resistance is usually highly
specific to a particular virus strain and is eventually
overcome because of a uniquely great capacity of vi-
ruses for mutagenesis and their ability (especially of
RNA-containing viruses) to rapidly evolve [11]. Anoth-
er approach was to induce plant resistance by trans-
genic expression of cellular genes, including natural
resistance genes, as has been done by many research
groups [6]. However, legal restrictions on the use of
genetically modified organisms (GMOs) significantly
reduce or even prohibit cultivating transgenic plants
in many countries.
An expanding knowledge on the molecular mech-
anisms of plant interaction with viruses along with
the rapid progress of genetic technologies in recent
decades, have opened up new and completely dif-
ferent prospects for the development and imple-
mentation of effective and environmentally friendly
approaches to plant defense against viral infections.
Currently, the most promising genetic tool is genome
editing mediated by the CRISPR-Cas system [12-16].
CRISPR-Cas enables introduction of directed changes
into the target genes. It is based on the RNAi mech-
anism [17, 18] that allows to specifically inhibit viral
replication by cleaving the viral RNA. The CRISPR-Cas
technology has already led to significant advance-
ments in nearly all fields of life sciences, including
biotechnology and medicine, and has become increas-
ingly common in plant biology. RNAi has long been
used as a transgenic tool (host-induced gene silenc-
ing, HIGS) for the degradation of viral RNAs or inac-
tivation of genes responsible for viral susceptibility
in agricultural plants [6, 19-21]. Recently developed
RNAi-based methods (spray-induced gene silenc-
ing, SIGS) that use exogenous double-stranded RNAs
(dsRNAs), small interfering RNAs (siRNAs), and short
hairpin RNAs not only cause the degradation of viral
RNA, but also appear to be more stable, safe, and so-
cially acceptable alternative to transgenic methods [17,
18, 22].
The use of CRISPR-Cas and RNAi technologies to
provide plants with virus resistance, as well as the
underlying mechanisms, have been described in de-
tail in many reviews [12, 14, 16, 17, 22-25], however,
comparative analysis of these technologies has re-
ceived very little attention in the published literature.
In this article, we aimed to fill the gap in the in-
formation on the applicability of CRISPR-Cas and
RNAi tools with special emphasis on the outcome of
their use for the generation of virus-resistant plants.
We also discussed advantages and disadvantages of
the CRISPR-Cas and RNAi methods, as well as their
prospects in crop production and plant protection.
CRISPR-Cas: ENGINEERING TOOLKIT
FOR ANTIVIRAL PROTECTION
The structure, properties, and principles of the
CRISPR-Cas genome-editing system have been de-
scribed in many publications [12-14, 25, 26]. The
CRISPR-Cas system includes clustered, regularly in-
terspaced short palindromic repeats (CRISPRs) and
CRISPR-associated proteins (Cas) and has originated
from the adaptive immune system of bacteria and ar-
chaea that prevents intrusion of foreign plasmids and
bacteriophages by cleaving their DNA [27]. The active
CRISPR-Cas complex consists of the Cas endonuclease
and guide RNA (gRNA) which directs the Cas protein
to the target DNA or RNA. gRNA contains a scaffold
for the Cas protein binding and a spacer sequence of
approximately 20 nucleotides (nt) for recognizing the
target sequence in the phage or plasmid DNA [28].
Researchers have adapted this bacterial immune sys-
tem for DNA editing in eukaryotes. When introduced
into cells, gRNA recognizes the target DNA sequence
and the Cas9 enzyme cuts DNA at this site, similar
to the natural process taking place in bacteria. After
DNA cleavage, cell apparatus repairs the breaks using
either homologous recombination or non-homologous
end joining mechanisms, resulting in the insertion
or deletion of genetic material or DNA modification
accompanied by the replacement of the native DNA
segment with a new sequence, which eventually leads
to the loss of function of the targeted gene.
The CRISPR-Cass-based methods of antiviral de-
fense are classified into two categories: (1) direct
cleavage or degradation of the viral genome and
(2) modification (mutation) of the host plant genes
required for the virus life cycle.
DIRECT EFFECT OF THE CRISPR-Cas SYSTEM
ON THE VIRAL GENOME
DNA-containing viruses. The original CRISPR-Cas
genome-editing system was derived from Strep-
tococcus pyogenes and included Cas9 endonucle-
ase responsible for the DNA cleavage. Because of
this fact, the CRISPR-Cas9 technique was first test-
ed against a variety of DNA-containing geminivirus-
es (Geminiviridae family). Thus, transgenic tobacco
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BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
and Arabidopsis thaliana plants expressing CRISPR-
Cas components (Cas9 endonuclease and gRNAs
against several coding and non-coding regions of the
geminivirus genome) were more resistant to the viral
infection due to the direct action of CRISPR-Cas on
the geminivirus genome [29, 30]. Successful inhibition
of geminivirus propagation in model plants has been
replicated in agricultural crops, such as cotton, toma-
toes, potatoes, peppers, watermelon, soybeans, beans,
barley, etc. [16]. The CRISPR-Cas9 system has been
also used to induce immunity to other DNA-contain-
ing plant viruses. For example, transgenic A. thaliana
plants expressing Cas9 and gRNAs targeting the ge-
nome of cauliflower mosaic virus (CaMV; Caulimovi-
ridae family) were highly resistant to this virus [31].
However, in some cases, DNA-containing viruses gen-
erated mutants that overcame resistance mediated by
CRISPR-Cas9 and were capable of rapidly spreading in
the environment. Hence, the CRISPR-Cas system pos-
es some potential risk by stimulating generation and
spread of new pathogenic virus variants [32].
RNA-containing viruses represent the most seri-
ous threat to agricultural plants by causing significant
agronomic losses, including reduced crop yields, lower
product quality, and shorter shelf life. The discovery
of RNA-specific endonucleases associated with the
CRISPR system, such as Cas9 from Francisella novicida
(FnCas9), Cas13 from Leptotrichia shahii (LshCas13a),
and Cas13 from Ruminococcus flavefaciens (RfCas13d),
has allowed to develop systems that directly cleave
viral RNA genomes [12, 14, 16]. Transgenic plants ex-
pressing RNA-specific Cas endonucleases together with
gRNAs directed at viral RNA targets exhibited a signifi-
cant resistance to RNA-containing viruses. Interesting-
ly, deactivated forms of Cas proteins lacking the RNA
endonuclease activity retained the antiviral effect.
Itwas hypothesized that in this case the infection was
inhibited at the levels of viral RNA translation and/or
replication [33]. The high efficiency of this approach
has been confirmed by successful suppression of more
than 15 RNA-containing viruses (both with positive
and negative genomes) in a wide range of model and
agricultural plants [12, 14, 16].
Although Cas endonucleases are important tech-
nological tools capable of providing plant resistance
to viruses by directly affecting the viral genome,
this approach requires constant presence of these
enzymes and associated gRNAs in plant cells, which
can be achieved only through their transgenic expres-
sion. In this regard, the practical application of the
CRISPR-Cas system may be strongly limited by the
regulations on the use of GMOs. Another factor re-
stricting implementation of this method is accelerated
generation of new mutant viral variants capable of
overcoming the CRISPR-Cas-based resistance and re-
lease of these viruses into the environment.
EDITING OF HOST PLANT
SUSCEPTIBILITY FACTORS
Since viruses have to use and modify host plant
systems for successful infection (replication and prop-
agation through the plant) and depend on many host
cellular mechanisms (for example, interaction between
the virus and plant proteins is necessary to perform
certain viral functions). Neutralization of these pro-
tein partners should inevitably lead to the inhibition
of viral infection and can be achieved by introducing
mutations into the corresponding plant genes using
the CRISPR-Cas system, as it was demonstrated for
the recessive genes encoding eukaryotic translation
initiation factors eIF4E and eIF4G and their isoforms
eIF(iso)4E and eIF(iso)4G [16, 34]. These factors, also
known as cap-binding proteins, are key elements of
protein synthesis in eukaryotes. As parts of the com-
plex also including eIF4G and eIF4A proteins, they
bind methylated guanine residue added post-tran-
scriptionally to the 5′-end of eukaryotic mRNAs, which
triggers assembly of the translation initiation complex,
ribosome binding, and initiation of protein synthesis
[34]. It was shown that many plant viruses also re-
quire interaction with eIF4E/eIF4G to ensure a suc-
cessful infection.
Mutagenesis of the eIF4E gene using the
CRISPR-Cas system induced resistance of cucumber
plants against zucchini yellow mosaic virus (ZYMV),
papaya ring spot mosaic virus W (PRSV-W), and cu-
cumber vein yellowing virus (CVYV) [35]. Rice plants
with the modified eIF4G gene allele introduced using
CRISPR-Cas9, were resistant to Rice tungro spherical
virus (RTSV) [36]. A mutation in the eIF(iso)4E gene
induced by CRISPR-Cas9 in cassava and A. thaliana
plants, provided full resistance to cassava brown stripe
virus (CBSV) [37] and turnip mosaic virus (TuMV) [38],
respectively. It should be noted that the genome of
cassava encodes additional eIF4E-like proteins (nCBP-1
and nCBP-2). Mutations introduced with CRISPR-Cas9
into the nCBP-1 and nCBP-2 genes established plant re-
sistance to cassava brown steak virus [37]. The use of
Cas9 fused with cytidine deaminase enabled a highly
efficient editing of the target codons and introduction
of the N176K mutation into the eIF4E1 gene allele in
A. thaliana, resulting in the generation of plants re-
sistant to the clover yellow vein virus (ClYVV) [39].
Despite obvious achievements in the induction
of antiviral resistance by introducing mutation in the
alleles of translation initiation factor genes, the fol-
lowing aspects should be taken into account: (1) sig-
nificant redundancy of such factors, which allows vi-
ruses to use unmodified factors for their replication;
(2) a possible effect of mutations on the translational
apparatus of the host plant and, as a result, on plant
physiology; (3) a high frequency of overcoming induced
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BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
resistance by RNA-containing viruses capable of rapid
evolutionary changes [34].
Resistance to viral infections can be enhanced
by knocking out other susceptibility genes (S-genes)
in addition to mutations in the eIF4E/eIF4G genes. For
example, the knockout of the SlPelo gene led to the in-
creased plant resistance to the tomato yellow curl vi-
rus [40]. Similarly, disruption of the chloride channel
(CLC-Nt1) gene in Nicotiana benthamiana plants sup-
pressed potato virus Y (PVY) replication [41]. Editing
at least one allele of the coilin (structural protein of
Cajal subnuclear bodies) gene dramatically improved
resistance of potato plants to the PVY infection, as
well as their tolerance to salt and osmotic stresses
[42]. Other S-genes whose editing enhanced the antivi-
ral resistance include soybean GmF3H1, GmF3H2, and
GmFNSII-1, wheat TaPDIL5-1, Arabidopsis Tom1, and
N. benthamiana NbUbEF1B and NbCCR4/NOT3 [25].
These data reveal the potential of employing S-genes
to create new plant varieties with a wide range of
tolerance to viruses using the CRISPR-Cas9 system.
However, it should be remembered that beside con-
trolling viral infections, many S-genes are involved in
important endogenous processes, such as plant growth
and development, so that their editing can have an
unexpected negative effect on the plants. For exam-
ple, the ssi2 mutation in A. thaliana significantly pro-
moted accumulation of salicylic acid (phytohormone
involved in the antiviral defense), but also induced
phenotypic abnormalities in the growing plants [43].
Hence, the search of promising targets for the
CRISPR-Cas-mediated editing is one of the most prin-
cipal tasks in the creation of virus-resistant plants.
Recent achievements in the functional genomics, in-
cluding the CRISPR-Cas system itself, has brought this
search to a new level. The programmability of the
CRISPR system has proven to be very useful for the
high-throughput identification of genes with specific
functions [44]. Development of simple methods for
the synthesis of gRNA libraries has made it possible
to obtain large populations of designed plants and to
screen them for genes with specific function, which in
turn can be effective in developing new strategies for
the regulation of antiviral resistance. Cas9 can be used
for the analysis of plant–virus interactions, particular-
ly, in terms of the host gene functions. Moreover, the
ability of Cas13 to cleave RNA opens up new prospects
for studying the functions of long non-coding RNAs,
which are involved in many processes, including re-
sistance to pathogens [45] and, therefore, may provide
a new basis for the antiviral resistance.
Another problem in genome editing of host plant
genes using the CRISPR-Cas system is that many ed-
iting techniques are based on transgenic CRISPR-Cas
components. In recent years, alternative plasmid-free
methods have been developed for the delivery of
gRNAs and Cas9 protein to plant cells, that have elim-
inated the need for the transgenic system application.
For example, Cas9 and gRNA can be directly delivered
to the cells as a preassembled ribonucleoprotein (RNP)
complex. RNP complexes can be introduced into plant
protoplasts by transfection or can be transferred into
immature plant embryonic cells, callus cells, and epi-
dermis cells by particle bombardment using gold and
tungsten microparticles or mesopore silicon nanopar-
ticles [12]. The main advantage of DNA-free technolo-
gy is the lack of need for the DNA introduction, since
in conventional DNA-based methods, such DNA inte-
grates into the genome at random sites and constitu-
tive expression of the Cas9 gene from this DNA likely
leads to the off-target editing. Another advantage of
the Cas9–gRNA complex delivery is that it ensures
fast genome editing followed by rapid degradation of
the editing complex in the cell, which also reduces
the likelihood of adverse off-target effects [12]. Plas-
mid-free methods for the transfer of genome-editing
complexes are both promising and preferable, since
they facilitate the generation of plants exhibiting
specified properties without being the subjects of an-
ti-GMO regulations.
Another approach is removal (deletion) of foreign
sequences encoding Cas and gRNAs from initially ed-
ited (transgenic) plants by crossing them with their
wild-type counterparts [37, 38]. Plants obtained by this
method are resistant to the selected viruses without
being transgenic.
The above approaches enable to obtain nontrans-
genic plants with the required characteristics. How-
ever, it remains unclear whether plants produced us-
ing the CRISPR-Cas technology should be classified as
GMOs. Moreover, crossing with the wild-type plants
to obtain plants with certain properties but lacking
transgenic sequences is impossible for some species,
in particular, plants reproducing by vegetative prop-
agation (e.g., potatoes), since production of seeds can
alter the characteristic cultivar properties.
We discovered another interesting feature of the
CRISPR-Cas-mediated genome editing when chitosan
nanoparticles were used to deliver preformed gRNA–
Cas9 complexes targeting genes for phytoene desat-
urase (PDS) and coilin to the apical meristem cells
that were then regenerated into viable potato plants
[42, 46, 47]. Typically, editing plant genome with the
CRISPR-Cas system leads to the appearance of inser-
tions or deletions (indels) 10-20 base pairs (bp) in
length. However, we observed large deletions (up to
600 bp) in the region flanking the gRNA-binding site
in the target gene, but no short indels [42, 46, 47].
Therefore, when using this approach, it is important
to choose the targeted site so that any large deletion
would be located entirely within the knocked-out
gene. Large deletions caused by CRISPR-Cas have also
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BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
been previously detected in animal cells with a high
mitotic activity [48], which correlates with our data,
since meristem cells are rapidly dividing cells.
CRISPR DIAGNOSTICS
Unlike in eukaryotic cells, where Cas13 mediates
exclusively highly specific RNA cleavage directed by
gRNA, in prokaryotes and in vitro reaction mixtures,
specific cleavage of a target RNA enables Cas13 to
engage in the collateral (nonspecific) degradation of
other single-stranded RNAs [49]. This side effect has
been used in the development of highly sensitive vi-
rus diagnostic methods that employ the SHERLOCK
(specific high-sensitivity enzymatic reporter unlocking)
approach for high-accuracy detection of nucleic acids,
in particular, viral RNA and DNA [50]. In SHERLOCK,
analyzed samples are amplified to enrich the target
DNA (if present) using recombinase polymerase reac-
tion (RPA). If the identified molecule is RNA, it is re-
verse transcribed before the RPA. The products of RPA
are transcribed into RNA-by-RNA polymerase and the
obtained transcripts are cleaved by Cas13 in the pres-
ence of a quenched single-stranded RNA fluorescent
reporter. The concomitant cleavage by Cas13 generates
fluorescence signal, thus indicating the presence of the
target viral DNA or RNA. Recently, this method has
been significantly simplified and adapted for practical
diagnostics in a form of fast, inexpensive, and highly
sensitive paper-strip test [50] applicable for rapid and
reliable detection of plant viruses in the field.
RNAi: RNA-DIRECTED TECHNOLOGY
FOR PLANT ANTIVIRAL RESISTANCE
RNAi and plant protection. The concept of
pathogen-derived resistance postulated in the 1980s
[51] has initiated a breakthrough in the field of plant
protection biotechnology by proposing the use of viral
genes or their fragments to suppress viral infections.
First, transgenic plants with an increased resistance to
viruses were created via expression of viral structur-
al and replicative proteins (replicases) [6, 22, 52]. The
following discovery of RNAi has marked a new phase
in plant biotechnology, as it enabled more specific and
efficient plant antiviral defense [6, 22, 52].
RNAi is an evolutionarily conserved, sequence-spe-
cific mechanism for suppressing gene expression in
most eukaryotes, including plants. It controls expres-
sion of endogenous genes and leads to the degradation
of foreign nucleic acids. RNAi is triggered in the pres-
ence of dsRNA precursors originating from the host
plant hairpin RNA structures or from foreign dsRNA
intermediates (e.g., replicative forms of viral RNA).
dsRNA molecules are cleaved by Dicer-like endonu-
cleases (DCLs) into siRNAs 21-24 bp in length or mi-
croRNAs. This process is further amplified with the
involvement of host RNA-dependent RNA polymerases
(RDR1 and RDR6), leading to the formation of second-
ary siRNAs. These siRNAs are loaded into a complex
formed by ARGONAUTE (AGO) family proteins with
the generation of activated RISC (AGO-containing
RNA-induced silencing complex) and direct the deg-
radation or translational repression of complementa-
ry (specific) target RNA molecules [53, 54]. RNAi has
become one of the most efficient approaches to ob-
tain virus-resistant plants by expressing virus-specific
dsRNA.
Specific antiviral effect of exogenous dsRNAs.
The commercial use of transgenic plants with artifi-
cially induced disease resistance is limited by the reg-
ulations targeting GMOs, as well as negative public
perception, which creates the need for more sustain-
able, efficient, environmentally friendly, and socially
accepted alternative approaches to plant protection.
The SIGS technology based on spraying plants with ex-
ogenous dsRNA meets such requirements, and, more-
over, has already been successfully applied to induce
antiviral resistance in a wide range of crops [12, 13,
17, 18, 55-57]. Mechanical inoculation with dsRNAs
and high-pressure spraying have also been proven
to protect plants from viruses. Exogenous technolo-
gies have been used in laboratories to suppress the
replication of more than 20 economically important
DNA- and RNA-containing plant viruses from various
taxonomic groups in more than 10 plant species [17,
18, 58].
It is generally believed that the mechanism of the
antiviral action of exogenous dsRNA is similar to the
classical RNAi mechanism, including involvement of
DCL, RDR, AGO, and other RNAi system components,
since the effect of exogenous dsRNA in plants is spe-
cific to the targeted DNA/RNA sequence [17, 59-66].
However, there is no direct evidence supporting this
concept. Our recent detailed comparative analysis
of short RNAs formed in potato plants infected with
PVY revealed that exogenous treatment of plants with
the PVY-specific dsRNA was accompanied by the for-
mation of non-canonical RNA fragments 18-30 nt in
length vs. classical 21- and 22-nt siRNAs induced by
the PVY infection in control plants [67]. Formation of
the 21- and 22-nt siRNAs was consistent with the data
on the size of siRNAs formed during infection with
other RNA-containing viruses [67]. Such siRNAs are
produced with the involvement of CL4 and DCL2, re-
spectively, and then interact with AGO1 and AGO2 to
form the RISC, which ensures hydrolysis of viral RNA
(RNA silencing). Interestingly, similar size distribution
of short RNAs was described by Tabein et al. [63]
and Rego-Machado et al. [64] for the external plant
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treatment with dsRNAs against tomato spotted wilt
virus (TSWV) and tomato mosaic virus, respectively.
Therefore, despite the fact that the anti-PVY dsRNA
specifically inhibited the replication of PVY (and not
of unrelated viruses) [67], these results may indicate
that exogenous dsRNA can be processed through a
previously unknown DCL-independent mechanism.
It is also possible that the antiviral effect of exoge-
nous dsRNA is mediated by some mechanisms other
than the classical RNAi, but this suggestion requires
further investigation.
Nonspecific antiviral effect of exogenous
dsRNAs. Beside the ability to induce RNAi, dsRNAs
can serve as effectors of pattern-triggered immunity
(PTI) [68, 69]. RNA-based pathogen-associated molec-
ular patterns (PAMPs) are well-known inducers of
immunity in animals [70, 71] and plants [68, 72]. De-
fensive responses caused by viruses and dsRNAs are
canonical for PTI and include generation of reactive
oxygen species (ROS), induction of phytohormonal sig-
naling, activation of mitogen-activated protein kinas-
es, and triggering of expression of protective genes
[68, 73, 74]. Unlike RNAi, dsRNA-induced PTI does not
depend on the RNA sequence and can also be activat-
ed by non-viral dsRNAs, such as synthetic polyinosin-
ic-polycytidylic acid [poly(I:C)] or GFP-specific dsRNA
[68]. For example, poly(I:C), which induced the expres-
sion of PTI marker genes, also caused strong antiviral
protection against oilseed rape mosaic virus (ORMV)
infection [68].
Considering that dsRNA can function as a signal
inducing antiviral plant defense by triggering both
specific RNAi and non-specific PTI-type defense re-
sponses, we hypothesized that these two pathways
could also be induced by the application of exoge-
nous dsRNAs, resulting in changes in the susceptibility
of the dsRNA-treated potato plants to PVY. We ana-
lyzed the impact of exogenously applied PVY-specific
dsRNA on both defense mechanisms (RNAi and PTI)
and studied its effect on the accumulation of homol-
ogous (PVY) and unrelated (potato virus X. PVX) vi-
ruses [66]. The use of dsRNA against PVY in potato
plants induced accumulation of siRNAs (RNAi mark-
ers) and transcripts of genes coding for PTI-associ-
ated proteins, such as WRKY29 (transcription factor,
molecular marker of PTI), RbohD (respiratory burst
oxidase homolog D), EDS5 (increased susceptibility to
diseases 5), SERK3 (somatic embryogenesis receptor
kinase), and PR-1b (pathogenesis-related protein 1b)
[66]. At the same time, the treatment suppressed
only the PVY replication, but produced no effect on
the PVX infection, despite the PTI induction in the
presence of PVX [66]. Since the RNAi-mediated an-
tiviral immunity is the main resistance mechanism,
it can be assumed that the dsRNA-induced PTI alone
was not sufficient to suppress viral infection under
these conditions. However, it should be mentioned
that Necro et al. [75] demonstrated the ability of a
non-specific (anti-PVY) dsRNA to suppress replication
of the non-homologous PVX, although with a lower
efficiency than the specific anti-PVX dsRNA.
In addition to the induction of RNAi and PTI, an-
ti-PVY dsRNA was able to regulate poly(ADP-ribosyl)
ation (PARylation), which is a protein post-transla-
tional modification during which ADP-ribose chains
are added to the target protein by poly(ADP-ribose)
polymerases (PARPs) [66]. PARylation plays an import-
ant role in plant resistance to the genotoxic stress,
DNA repair and transcription, cell cycle control, and
response to biotic and abiotic stresses, including pro-
cesses associated with programmed cell death and
regulation of plant immunity [76, 77]. Poly(ADP- ribose)
(PAR) residues can be removed by poly(ADP-ribose)
glycohydrolase (PARG). Interestingly, we found that
anti-PVY dsRNA increased the content of PARG in po-
tato plants, which correlated with a decrease in the
PAR accumulation and ultimately contributed to the
suppression of PVY infection [66].
Therefore, exogenous dsRNAs can be used as “mul-
titools” that mainly trigger RNAi as the key mecha-
nism of antiviral resistance, as well as induce other
mechanisms involved in PTI/PARylation, which pro-
duce a cumulative antiviral effect and represent an
extra protective strategy when RNAi alone proves in-
effective against viral infection.
Taken together, these data suggest the further de-
velopment of technologies combining dsRNA-induced
protective responses [RNAi, PTI, and modulation of
poly(ADP-ribose) metabolism] in a coordinated man-
ner will be able to ensure a high level of crop protec-
tion against viruses.
TECHNICAL FACTORS
LIMITING THE USE OF dsRNAs
Viral suppressors of RNAi. Despite the above
examples of successful use of RNAi-based technology,
this approach has certain limitations because of the
existence of viral RNA silencing suppressors [12, 78].
Many plant viruses encode silencing suppressors that
interfere with RNAi through various mechanisms and
at different stages. For example, viral suppressors can
bind tRNAs or siRNAs, thus preventing the functioning
of DCL or AGO, respectively [12, 17] and inevitably re-
ducing the antiviral effect.
Delivery of dsRNAs to plant cells and tissues
is another factor that limits the use of exogenous
dsRNAs. For exogenous nucleic acid to enter plant
cells, it should maintain its integrity on the leaf sur-
face against the effects of environmental factors, such
as ultraviolet radiation, wind, rain, pH, and attack
KALININA et al.810
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
of bacterial nucleases. Next, dsRNA should penetrate
numerous natural barriers, such as leaf cuticle, cell
wall, and plasma membrane, as well as compensate
for a low efficiency of absorption by the cell [79, 80].
Moreover, for producing a reliable and stable RNA
phenotype, dsRNAs (or siRNAs formed from them)
should spread throughout the plant via the inter-
cellular transport system (plasmodesmata) and the
long-distance transport system (phloem) [81]. Final-
ly, inside the cell, dsRNA should be integrated into
the RNAi mechanism occurring as a series of events,
e.g., dsRNA processing by DCL with the generation
of primary siRNA duplexes, amplification of primary
miRNAs by RDR, loading of siRNAs into the RISC, and
recognition of the target RNA site and its cleavage.
These stages are strictly compartmentalized and de-
termine the biological function of RNAi. An intuitive
assumption would be that the sites of dsRNA genera-
tion (e.g., virus replication sites) can be mechanically
linked to the compartments associated with the RNAi
machinery, and therefore, endogenously produced
dsRNAs would be able to naturally integrate into the
RNAi mechanism, which can then operate as a convey-
or system, in which the products of the previous stage
are moved for the use at the subsequent stage (for
example, the primary siRNAs obtained by the cleav-
age of dsRNA are transferred to the stage of siRNA
amplification, etc.). Hence, to ensure an efficient RNAi
operation, exogenous dsRNA should be incorporated
into the RNAi “conveyor”.
Solving the problem of dsDNA delivery has cur-
rently become a priority. At least in part, this problem
can be managed by using additives and carriers that
can improve the stability of dsRNA molecules and/or
facilitate their adhesion and penetration into plants.
Such function can be performed by cationic oligopep-
tides, various nanoparticles, surfactants, liposomes,
artificial extracellular vesicles, and chromosome-free
minicells (developed by AgroSpheres; https://www.
agrospheres.com/) [82]. Thus, it has been found that
nanocarriers can function as extremely efficient de-
livery systems that are currently commonly used for
dsRNA delivery [24, 79, 80, 83]. A combination of RNAi
technology and nanotechnology opens very encour-
aging prospects in plant protection. One example is
the use of degradable dsRNA-containing clay particles
(BioClay) that not only significantly facilitate the deliv-
ery of dsRNAs to plants, but also considerably prolong
the time of their antiviral action (from 7 to 24 days)
[62]. Further application of laboratory developments
in crop production will depend on the cost and effec-
tiveness of developed nanocarriers.
Methods for dsRNA synthesis and their cost-ef-
ficiency. Exogenous DNAs are currently produced by
invitro transcription, microbial expression in bacteria
and fungi, and cell-free synthesis [24, 83].
In vitro transcription involves the use of a tar-
get sequence flanked by two convergent (i.e., orient-
ed toward each other) 5′-RNA polymerase promoters
(for example, T7 bacteriophage RNA polymerase pro-
moter). Such DNA template enables transcription of
both sense and antisense RNA strands, which rapidly
anneal in the same reaction mixture with the forma-
tion of dsRNA [83].
In vivo dsRNA synthesis in microbial systems is
mainly carried out in Escherichia coli HT115 (DE3)
strain defective by RNase III (enzyme that specifical-
ly degrades dsRNA). The methods used for the dsRNA
production in E. coli are similar to those employed
for the synthesis of recombinant proteins. After trans-
formation with a plasmid containing a fragment cod-
ing for the dsRNA sequence against a specific RNA
target and placed under the control of the T7 RNA
polymerase promoters, the cells are grown to the ex-
ponential phase and induced with IPTG. After incuba-
tion for ~4-6 h, the cells are collected and lysed, and
dsRNA is purified [83].
Cell-free transcription/translation systems use cell
lysates for the mRNA transcription coupled with the
in vitro protein translation. Cell-free extracts are op-
timized to contain most of the cellular cytoplasmic
components necessary for transcription and transla-
tion and have several advantages over in vivo bacte-
rial systems. For example, elimination of secondary
processes necessary to maintain cell viability and
growth makes it possible to fully utilize the activity
of RNA polymerase and the pool of ribonucleotides in
the reaction mixture. The absence of cell walls makes
it easier the control the synthesis and facilitates the
process of sample preparation. Cell-free systems are
currently successfully used for the production of
RNA vaccines. Since for the application in the field,
dsRNAs have to be produced in large amounts at a
relatively low cost, the purity of such dsRNAs might
be less than the purity of nucleic acid preparations
intended for the medical use. GreenLight Bioscienc-
es Company (https://www.greenlightbiosciences.com/)
[84] has developed a unique cell-free biotechnological
platform that provides large-scale dsRNA production
at a low cost ($0.5/g) compared to fermentation ($1/g),
in vitro transcription ($1000/g), and chemical synthe-
sis ($100,000/g) [24, 83].
CRISPR-Cas OR RNAi:
HOW TO MAKE THE RIGHT CHOICE?
As discussed above, both dsRNA-based RNAi and
CRISPR-Cas technologies are powerful tools for the
production of virus-resistant plants. However, which
of these seemingly competing approaches is most ap-
propriate for a specific application? Before making
BIOTECHNOLOGIES OF PLANT RESISTANCE TO VIRUSES 811
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
Fig. 1. Advantages and disadvantages of CRISPR-Cas and RNAi in protecting plants from viral infections.
thechoice, it is necessary to consider several technical
and methodological aspects (Fig. 1).
Direct impact on viruses. GMOs. As noted
above, some aspects of the CRISPR-Cas functioning,
in particular, direct action on RNA- and DNA-contain-
ing plant viruses, require a constant presence of the
CRISPR-Cas system components in plants, which can
only be achieved by transgenic methods, while the
RNAi-based SIGS technology does not require gener-
ation of transgenic organisms (GMOs). Exogenously
applied dsRNAs (“biopesticides”) offer a clear ad-
vantage due to less strict GMO regulations and less
public concern. In this regard, it is still necessary to
establish an evidence base to support the approval of
the application of biopesticides (dsRNAs) in the field.
At the same time, it is essential to monitor the fate of
dsRNAs in the environment, their impact on non-tar-
geted organisms, and overall safety.
Plant regeneration. Inactivation of genetic sus-
ceptibility to viruses in host plant does not require
a constant presence of Cas and gRNA, therefore, re-
moval of transgenes encoding Cas9 and gRNA or de-
livery of editing reagents in a form of mRNAs or RNP
complexes can help to avoid the use of transgenic
plants. However, regeneration of whole plants from
the edited cells and further identification of edited
plant lines are typically time-consuming, technically
complex, and expensive. Moreover, many agricultural
plants and varieties cannot be regenerated from cells.
So far, transgenic-independent CRISPR methods have
been implemented only in a limited range of plant
species and varieties. In contrast, the SIGS approach
does not include the regeneration stage.
Reversibility. Classical DNA-editing techniques
usually result in irreversible genome modification and
cause complete loss of gene function. This significantly
restricts the editing of essential (e.g., housekeeping)
genes, because the knockout of such genes will lead
to plant death. The dsRNA-based SIGS method induces
reversible changes in the gene expression over a giv-
en period of time, which is important because some
plant genes can be deactivated only at certain stages
of plant development, as they play an important func-
tional role at other stages. Moreover, the same genes
can simultaneously determine resistance to some ad-
verse factors and susceptibility to others.
Time to phenotype manifestation. Production of
genetically modified plants takes at least six months,
while a new dsRNA preparation can be obtained in
less than 2-4 weeks. The time aspect is especially
important in unforeseen circumstances, such as an
emergence of a new virus type or a new strain. Se-
quencing of its genome followed by the fragment clon-
ing to obtain dsRNA, fits well within the allotted time
frame.
Ploidy. The efficiency of genome editing depends
on the plant ploidy (genetic heterogeneity). Polyploi-
dy, or the presence of more than two sets of chromo-
KALININA et al.812
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
somes in the cell nucleus, is common in plants (for
example, all cultivated potato varieties are tetraploid
and wheat plants are hexaploid). The presence of
several homologous genes as editing targets requires
more meticulous work and specialized techniques to
assess the success of genome editing procedure [85].
The use of exogenous dsRNAs does not depend on the
plant ploidy, since in this case, RNAi is induced simul-
taneously for all alleles.
Plant varieties. When using the CRISPR technol-
ogy, each variety should be edited and tested inde-
pendently, whereas the SIGS-RNAi tools designed to
target conserved gene sequences can be applied in
multiple varieties.
Efficiency, sustainability, and duration of ac-
tion. In ~75% cases, SIGS causes a gene knockdown
and produces the phenotypic effect which sometimes
can be observed only in the treated leaves. The ef-
ficiency and duration of action vary and depend on
the environmental factors and/or the presence of viral
RNAi suppressors. These parameters can be improved
by using stabilizing nanoplatforms that facilitate the
delivery of dsRNAs into cells, as well as by repeated
treatment. The efficiency of the CRISPR-Cas editing is
10-40% per allele, but the effect is stable, permanent,
and inherited.
Non-specific (off-target) action is common to
both approaches. The off-target effect can be elimi-
nated (or at least minimized) by selection of specific
dsRNAs (RNAi) or gRNAs (CRISPR-Cas) that would not
interfere with the off-target genome regions, which
can be achieved by using advanced bioinformatic pro-
grams. At the same time, the RNAi technology has a
greater potential, since dsRNAs could be directed (at
least, theoretically) at any region of the targeted tran-
script, while gRNAs can be directed only to a specific
target site located next to the protospacer adjacent
motif (PAM) situated right after the DNA sequence
targeted by the Cas9 endonuclease.
CONCLUSION AND PROSPECTS
The CRISPR technology has already led to a
considerable progress in almost all fields of life sci-
ences, including biotechnology and medicine, and
is now becoming increasingly popular in plant biol-
ogy. However, despite its high popularity and great
technical capabilities, the application of the CRISPR
technology in agriculture may be somewhat limited.
In medicine, the CRISPR method is used to correct
pathological changes in the genomes of individual
patients, while plant biotechnology involves genome
alteration in all plants of the modified cultivar. In the
context of antiviral protection, the CRISPR system has
a number of disadvantages (see Fig. 1).
Direct editing (or degradation) of viral genomes
by CRISPR-Cas is achievable only with the use of trans-
genic plants expressing components of the CRISPR-Cas
system, which conflicts with current legislation and
public opinion in many countries. In addition, edit-
ing virus susceptibility genes can result in the forma-
tion of new, more pathogenic viral variants (super-
viruses) capable of overcoming plant resistance and
causing more pronounced infection symptoms [86,
87]. Since genome editing is carried out in isolated
cells (or tissues), the regeneration stage is required
to obtain intact plants, which may be technically dif-
ficult for some crops (or their varieties). In polyploid
cultures, genome modification can also be hindered
due to the presence of multiple alleles of the same
gene. As a result, plant genome editing is a time-con-
suming process, which is a disadvantage when new
infections emerge and urgent measures have to be
implemented to protect the plants. In this case, the
use of exogenous dsRNAs for the antiviral defense in-
duction seems to be a preferred technology. However,
to fully realize its potential, the following challenges
must be addressed:
• improvements in the delivery of exogenous dsRNAs
compatible with the RNAi machinery (e.g., by using
various polymers and nanoplatforms that facilitate
the penetration and controlled release of dsRNAs);
• optimization and scaling of dsRNA production;
• continuous monitoring of viral populations by
deep sequencing methods to ensure the rational
design of dsRNAs;
• identification of new target genes responsible for
the plant susceptibility to viruses, whose suppres-
sion by RNAi (and/or CRISPR-Cas) would enhance
plant antiviral resistance without producing side
effects;
• development of RNAi strategies that would min-
imize the effect of viral RNAi suppressors in the
dsRNA-directed antiviral defense.
Another important aspect that should be consid-
ered when developing RNAi and CRISPR-Cas approach-
es for plant protection is the existence of mixed in-
fections. In this case, it is necessary to suppress the
replication of all viruses simultaneously to avoid the
situation when a vacated niche can be filled by the
viruses whose replication has not been suppressed by
specific dsRNAs, which might facilitate the spread of
new infections.
Abbreviations. AGO, ARGONAUTE family pro-
teins; DCL, Dicer-like endonuclease; dsRNA, double-
stranded RNA; gRNA, guide RNA; PAR, poly(ADP-
ribose); PTI, pattern-triggered immunity; PVY, potato
virus Y; RNP complex, ribonucleoprotein complex;
RNAi, RNA interference; SIGS, spray-induced gene
silencing; siRNA, small interfering RNAs.
BIOTECHNOLOGIES OF PLANT RESISTANCE TO VIRUSES 813
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
Contributions. M.E.T. and N.O.K. developed the
review concept; N.O.K. prepared the “Introduction”
and “CRISPR-Cas: Engineering Toolkit for Antiviral
Protection” sections; M.E.T. prepared the “RNAi: RNA-
Directed Technology for Plant Antiviral Resistance”
and “Conclusions” sections; N.A.S. prepared the figures
and searched for the literature sources; N.O.K., M.E.T.,
and N.A.S. edited the manuscript.
Fundings. This work was supported by the Rus-
sian Foundation for Basic Research (project no.23-74-
30003).
Ethics approval and consent to participate. This
work does not contain any studies involving human
and animal subjects.
Conflict of interest. The authors of this work
declare that they have no conflicts of interest.
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