ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 6, pp. 786-803 © Pleiades Publishing, Ltd., 2025.
Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 6, pp. 847-866.
786
REVIEW
Progress in CRISPR/Cas13-Mediated Suppression
of InfluenzaA and SARS-CoV-2 Virus Infection
in in vitro and in vivo Models
Alisa A. Kazakova
1
, Elena I. Leonova
2,a
*, Julia V. Sopova
2
,
Angelina V. Chirinskaite
2
, Ekaterina S. Minskaya
1
, Ivan S. Kukushkin
1
,
Roman A. Ivanov
1
, and Vasiliy V. Reshetnikov
1,3,b
*
1
Sirius University of Science and Technology, 354340 Federal Territory “Sirius”, Russia
2
Saint-Petersburg State University, 199034 Saint-Petersburg, Russia
3
Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences,
630090 Novosibirsk, Russia
a
e-mail: e.leonova@spbu.ru
b
e-mail: reshetnikov.vv@talantiuspeh.ru
Received April 17, 2025
Revised April 17, 2025
Accepted April 18, 2025
Abstract— The worldwide number of deaths from complications caused by severe influenza and COVID-19
is about 1 million cases annually. Development of the effective antiviral therapy strategies for the disease
treatment is one of the most important tasks. Use of the CRISPR/Cas13 system, which specifically degrades
viral RNA and significantly reduces titer of the virus, could be a solution of this problem. Despite the fact
that Cas13 nucleases have been discovered only recently, they already have shown high efficiency in sup-
pressing viral transcripts in cell cultures. The recent advances in mRNA technology and improvements in
non-viral delivery systems have made it possible to effectively use CRISPR/Cas13 in animal models as well.
In this review, we analyzed experimental in vitro and in vivo studies on the use of CRISPR/Cas13 systems as
an antiviral agent in cell cultures and animal models and discussed main directions for improving the CRISPR/
Cas13 system. These data allow us to understand prospects and limitations of the further use of CRISPR/Cas13
in the treatment of viral diseases.
DOI: 10.1134/S0006297925601212
Keywords: CRISPR/Cas13, influenza virus, COVID-19, SARS-CoV-2, antiviral drugs
* To whom correspondence should be addressed.
INTRODUCTION
The CRISPR/Cas systems (Clustered Regularly In-
terspaced Short Palindromic Repeats/CRISPR-associated
nuclease) for targeted genome editing revolutionized
modern molecular biology [1]. At present numerous
types of such systems have been discovered, with ma-
jority of them using Cas-nuclease in complex with the
guide RNAs to introduce double-strand breaks to the
complementary DNA target [2]. A new class of Cas-nu-
cleases has been discovered recently that targets sin-
gle-stranded RNAs – Cas13 [3]. At present 6 types of
Cas13 proteins have been identified (a-d, x, y) that
differ in size [4, 5]. All Cas13 nucleases investigated
so far exhibit ribonuclease activity mediated by two
HEPN-domains. Cas13 protein in complex with guide
CRISPR RNA (crRNA) binds to the complementary se-
quence of the RNA target followed by its cleavage by
the activated Cas13. Unlike in the case of Cas9, the use
of Cas13 does not cause any changes in the eukaryote
genome and allows cleaving viral RNA inside the in-
fected cells thus preventing translation of viral mRNA.
Absence of the effects on genome provides advantage
to this strategy in antiviral therapy.
The first studies on inactivation of the target
RNA with the help of Cas13 were aiming at activa-
tion of apoptosis in pancreatic cancer cell line [6].
Thestudies on the use of nucleases of the Cas13 family
CRISPR/Cas13 IN INFLUENZA AND SARS-CoV-2 THERAPY 787
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
in suppression of infection in mammalian cell cul-
ture were published in 2019 [7]. American scientists
developed a CARVER platform (Cas13-assisted restric-
tion of viral expression and readout), which combines
the Cas13-mediated cleavage of viral RNA with rap-
id Cas13-based diagnostics (SHERLOCK) [7, 8]. The
Cas13a nucleases of Leptotrichia wadei (LwaCas13a)
and Cas13b of Prevotella sp. P5-125 (PspCas13b) were
successfully used for inactivation of the following
viral single-stranded RNA (ssRNA): Lymphocytic Cho-
riomeningitis Virus (LCMV), influenza A virus (IAV),
and vesicular stomatitis virus (VSV) in different cell
cultures. Use of LwaCas13a and PspCas13b resulted in
significant decrease of the level of viral RNA and titer
of the LCMV, IAV, and VSV viruses; it was shown in
the course of analysis of the virus genome after treat-
ment with nucleases that there were no mutations in
the target sequences of the guide RNAs [7]. These re-
sults opened new perspectives for the development
of innovative preparations for antiviral therapy. As a
result, many scientific groups worldwide joined the
research including studies devoted to the search of
new Cas13 orthologs capable of fighting infections.
In 2020 Li et al. [9] discovered that cleavage of the
NS3 gene in Dengue virus by the LwaCas13a nuclease
could effectively inhibit virus replication in the Afri-
can green monkey kidney cells (Vero cells).
The coronavirus pandemic caused by the SARS-
CoV-2 emphasized the need for improvement of anti-
viral strategies, which could arrest spread of infection
as soon as possible and without additional vaccination
of the population. For this purpose, Abbott et al. in
2020 [10] tested the possibility of using the RfxCas13d
nuclease for suppressing development of IVA infection
in the human lung adenocarcinoma cell line A549.
Lentiviruses were used for nuclease gene delivery,
while guide RNAs were transfected with the help of
lipofectamine. This technique was termed PAC-MAN.
Following this work in 2022 in the same laboratory
the PAC-MAN technique was successfully used for
suppression of different variants of coronaviruses,
including SARS-CoV-2, in cell culture. Co-localization
of nuclease and guide RNAs was shown to be import-
ant: nuclear localization in the case of added nucle-
ar localization signal (NLS) to the RfxCas13d protein
and lentivirus-mediated delivery of guide RNAs; and
in the case of added nuclear export signal (NES) to
the RfxCas13d protein and crRNA delivery mediated
by lipid nanoparticles[11]. In 2021 Nguyen etal.[12]
also used RfxCas13d with nuclear localization signal
for suppression of development of the human immu-
nodeficiency virus HIV-1 in the human cell culture.
In addition to the used delivery method, presence
of modified nucleosides in the crRNA could signifi-
cantly affect efficiency of the Cas13-mediated sup-
pression of expression. In particular, it was shown
by Chaveset al. [13] that the crRNAs containing four
phosphorothioate modifications reduced the number
of copies of PB2 (polymerase basic 2) of the influenza
virus A to a greater extend in comparison with the
non-modified crRNA. It was demonstrated in another
study [14] that the presence of 2′-O-methylated bases
or phosphorothioate modifications in the crRNA re-
sults in the enhanced knockdown of the endogenous
transcripts in human cells, furthermore, modifications
at the 3′-ends were preferable. It should be noted that
the simultaneous presence of two types of modifica-
tions in crRNA results in a lesser extent of expression
suppression [13, 14].
The results of the use of CRISPR/Cas13 in anti-
viral therapy of SARS-CoV-2 and influenza virus us-
ing cell culture demonstrated its high efficiency in
suppression of viral transcripts. However, number of
such studies using CRISPR/Cas13 in animal models is
limited. In this review we discuss recent advances
in this research area and provide detailed analysis
of the results of experimental studies devoted to the
development of CRISPR/Cas13-based preparations for
treatment of influenza virus A and SARS-CoV-2 viral
infections using cell cultures and animal models.
BIOLOGY OF INFLUENZA VIRUS A
ANDSARS-CoV-2
SARS-CoV-2 is a positive (+) single-stranded RNA-
containing virus belonging to the family of corona-
viruses (Coronaviridae). Coronaviruses have large
genomes (~30kb), which is 2-3-fold larger than the ge-
nomes of the majority of RNA viruses, and it encodes
a large set of non-structural (nsp) and structural pro-
teins as well as accessory proteins. Replication–tran-
scription complex of this virus contains 16 nsp(1-16),
which interact with each other and perform various
functions required for effective transcription and rep-
lication [15] (Fig. 1). The key element of this complex
is the RNA-dependent RNA polymerase(RdRp), which
performs synthesis of the viral RNA genome, howev-
er, replication and transcription would be impossible
without the presence of key non-structural proteins
assisting these processes and, hence, representing ef-
fective targets for the Cas13-based antiviral therapy,
same as RdRp, which catalyses RNA synthesis with
the help of nsp7 and nsp8. Previous investigations
of the SARS-CoV showed presence in the genome of
nsp8 (which is primase and 3′-end adenylyl transfer-
ase [16]), nsp13 (which is RNA helicase containing
Zn-binding domain [17] and RNA 5′-phosphatase[18]),
nsp14 – (which is 3′→5′ editing exonuclease (proof-
reading) [19]), nsp15 (which is uridylyl-specific endo-
nuclease [20]), and nsp16 (which is 2′-O-methyl trans-
ferase) [21]. These proteins are capable to perform
KAZAKOVA et al.788
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
Fig. 1. Structural and non-structural proteins encoded in the genome of SARS-CoV. Non-structural proteins, nsp (1-16), per-
form important roles in virus replication and are encoded by the ORF1ab. Polyproteins PP1a and PP1ab are cleaved by
the viral proteases PL
pro
(papain-like protease) and 3CL
pro
(3C-like basic protease) into active viral proteins. Structural and
accessory proteins are encoded in the 3′-end part of the genome.
their functions only after the replicase polyproteins
PP1a and PP1ab are proteolytically cleaved by the
viral papain-like proteases PL
pro
and 3CL
pro
(3C-like
protease) into individual active components. Synthesis
of the PP1ab variant proceeds due to appearance of
the programmed ribosomal shift of the open reading
frame (ORF), which occurs at the end of ORF1a at the
particular “slippery” sequence (5′-U UUA AAC-3′). As a
result of the open reading frame shift, there could be
the shift of the ribosome translating codons UUA and
AAC back (−1) by one nucleotide, which is followed
by the re-start of translation at the CGG codon in the
ORF1b[22]. This process occurs due to the presence of
the specific pseudoknot structure in the RNA located
after the “slippery” sequence with tertiary structure
preventing correct translation of the ribosome [23].
Efficiency of the open reading frame shift is 45-70%,
hence, the level of expression of the protein encoded
by the ORF1a is 1.5-2-fold higher in comparison with
the protein encoded by the ORF1b[24].
Synthesis of RNA by the replication–translation
complex from the viral (+) genomic RNA starts with
the synthesis of the complete chain of antigenomic
(−) RNA, which serves as a template for new genom-
ic RNA and set of subgenomic mRNAs encoding four
structural proteins required for the virion assembly,
and accessory proteins exhibiting various functions
such as modulation of innate immune responses of
the host cells [25]. Replication produces full-size vi-
ral genomic (+) RNA, which could be translated into
additional polyproteins of the replicative complex, as
well as it serves as a template for additional synthe-
sis of antigenomic (−) RNA or is packed into virions.
Transcription produces a set of subgenomic mRNAs
used for expression of structural and accessory pro-
teins. Replication and transcription require specific
templates of antigenomic (−) RNA.
The structural proteins encoded after the ORF1ab
include the spike (S), membrane (M), envelope (E), and
nucleocapsid (N) proteins. In addition to the above-
mentioned proteins with investigated functions, small
open reading frames have been identified overlapping
with several described ORFs. For some SARS-CoV pro-
teins presence of the leaky scanning mechanism has
been reported, when the ribosome starts transcription
at the second inner methionine rather than at the first
terminal methionine [26, 27].
Influenza virus A belongs to the family of or-
thomyxoviruses (Orthomyxoviridae) and, similarly to
SARS-CoV-2, causes seasonal respiratory diseases in
humans. Influenza virus genome comprises a negative
(−) genomic ssRNA. Unlike the (+)RNA virus SARS-CoV
with the genomic RNA comprising functional mRNA,
the genomic (−) RNA of these viral pathogens is not
capable of initiating infection on its own when intro-
duced into the permissive cells because the viral pro-
teins are translated from the complementary mRNA
(Fig. 2a). The transcriptionally active complex is a
viral ribonucleoprotein (vRNP) consisting of the viral
(−) genomic RNA in complex with nucleoprotein (NP)
CRISPR/Cas13 IN INFLUENZA AND SARS-CoV-2 THERAPY 789
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
Fig. 2. Schematic representation of the influenza virus A genome. a)Replication of influenza virus and of other RNA viruses
(–) RNA genome. Ribonucleoprotein complex (RNP) consisting of (–) genomic RNA in complex with NP cannot be translat-
ed and produce viral proteins. Viral polymerase RdRp interacting with RNP first transcribes (+) mRNA, from which viral
proteins are translated that are required for the virus replication, and next participate in replication of a new genomic
(–) RNA. b) 8 segments of RNA encode viral proteins. Cas13 targets are marked with red.
and RdRp [28]. Viral RNA polymerase is required for
transcription of both mRNA and complementary to the
(−) genomic RNA antigenomic (+) RNA, because mam-
malian cells do not have such enzyme. Moreover, the
viral mRNA is truncated at 3′-end and could serve only
as a template for translation of viral proteins. The in-
fluenza virus A genome consists of eight RNA chains
encoding the following proteins: PB1 (polymerase ba-
sic 1), PB2, PA (polymerase acidic), HA (hemaggluti-
nin), NP, NA(neuraminidase), M(matrix proteins), and
NS (non-structural proteins) [29] (Fig. 2b). Each seg-
ment of the viral RNA (1-6) encodes one protein: PB2,
PB1, PA, HA, NP, and NA. The seventh RNA encodes
2 matrix proteins (M1 and M2), which are formed as
a result of splicing of the initial RNA [30]. The eighth
segment encodes interferon agonist NS1 [31]; this vi-
ral RNA also could be subjected to alternative splic-
ing and could encode the NEP/NS2 protein [32], which
participates in export of vRNP from the nucleus of the
infected cells, where replication occurs, unlike in the
case of the SARS-CoV virus, where replication occurs
in cytoplasm. The active viral RdRp is composed of
three subunits: proteins PB1, PB2, and PA[33]. Similar
to the case of SARS-CoV-2, sequence of the RdRp RNA
includes highly conserved regions in the majority of
strains, which makes them very attractive targets for
the CRISPR/Cas13 system [34]. On the contrary, the vi-
ral glycoproteins NA and HA are surface proteins with
high degree of variability. Eighteen subtypes of HA
and 11 of NA have been identified, however, only 3 HA
subtypes (H1, H2, and H3) and 2NA subtypes (N1 and
N2) caused epidemics in humans [35]. Virion assem-
bly requires interaction of three integral membrane
proteins, HA, NA, and M2, with M1, which encloses
virion core. Nuclear export protein (NEP, also known
as non-structural protein 2, NS2) and RNP are located
inside the M1 matrix. It is worth mentioning that, un-
like the SARS-CoV-2, the influenza virus does not have
exonuclease with 3′→5′ editing activity, which is the
cause of emergence of a large number of mutations
in the virus genome and, hence, reduced efficiency
of vaccines.
CLASSIFICATION OF CRISPR/Cas13 SYSTEMS
Systems of ‘adaptive immunity’ in bacteria, CRISPR/
Cas, are widely used in laboratory practice, and, re-
cently, started to be introduced into therapy. Cas-nu-
cleases are commonly divided into class I nucleases (in
which cleavage of the target nucleic acids is mediated
by multisubunit protein complex) and class 2 nucleas-
es (in which a full multidomain protein functions as an
effector nuclease). In the class 2 nucleases 3 main nu-
clease families have been identified: Cas9, Cas12, and
Cas13. The first two families include mainly DNA-spe-
cific nucleases, while the family of Cas13 nuclease in-
clude predominantly RNA-specific enzymes [36].
The following variants have been recognized in
the family of Cas13 nucleases: Cas13a (previously
known as C2c2), Cas13b (previously known as C2c6),
Cas13c (previously known as C2c7), Cas13ct, Cas13d
(synonymic name CasRx), Cas13X, and Cas13Y (previ-
ously assigned to the group Cas13bt) [37, 38]. The so-
called ‘ancestral’ Cas13an nuclease has been recently
discovered [39] (Fig. 3a). Furthermore, metagenomic
analysis predicts existence of at least 5 more groups
ofCas13 enzymes, which have not been found yet[40].
KAZAKOVA et al.790
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
Fig. 3. Diversity of Cas13 nucleases. a)Discovered and characterized enzymes of the Cas13 family with the degree of phy-
logenetic relatedness (based on the information reported in [37-39]). b)Enzymes of the Cas13 family used for inactivation
of viruses in cell culture and under invivo conditions (based on the information reported in [3,37,38, 41, 42]).
The Cas13 nucleases are capable of cleaving RNA
in complex with crRNA consisting of a spacer comple-
mentary to the target RNA and direct repeat forming
a hairpin through which the guide RNA binds to nu-
clease and activates it[41]. Depending on the nuclease
type spacer could be located at the 5′-end relative the
direct repeat (such as in the case of PspCas13b nucle-
ase), or at the 3′-end of the direct repeat (such as in
the case of LbuCas13a) (Fig. 3b).
As has been mentioned above, Cas13 nucleases
perform cleavage of the target RNA at two HEPN sites
[42]. In addition to the target RNA, these sites are re-
sponsible for cleaving pre-crRNA in the process of
maturation of guide RNA [43]. Unlike the enzymes of
the Cas12 or Cas9 families, the PAM sequence (Proto-
spacer adjacent motif) is not required for cleaving the
target sequence, however, the enzymes of the Cas13
family exhibit loose preference for 5′- or 3′-flanking
protospacer sequence (PFS): for example, the enzyme
Cas13X.1 is more active towards the sequences with
5′-flanking guanine [38].
Characteristic feature of the Cas13 nucleases is
the so-called collateral activity– non-specific cleavage
of non-target RNA after introduction of the targeted
break; the SHERLOCK system for detection of nucleic
acids is based on this property [8]. It has been also
demonstrated that this activity could cause arrest of
the host cell growth to prevent spread of the viral
infection in the culture [44].
The family of Cas13 proteins is very diverse and
includes enzymes with size from ~500 aa (ancestral
Cas13) to ~1250 aa (Cas13a enzymes). Efficiency and
specificity of the targeted RNA cleavage for different
enzymes also vary: it was shown that the cleavage by
miniature nucleases (Cas13X.1 or RfxCas13d) is more
effective and specific than by the larger LwaCas13a
or PspCas13b [37].
APPLICATION OF CRISPR/CaS13
IN SARS-CoV-2 THERAPY
The SARS-CoV-2 coronavirus attracts the highest
interest in the context of using the CRISPR/Cas13 sys-
tem against viral infection: the first study evaluating
antiviral efficiency of the CRISPR/Cas13 system was
published in 2020 (Table1)[10]. The highly conserved
genes RdRp and N were used as targets, and the Rfx-
Cas13d system was selected as an effector. The study
was carried out using the human lung adenocarcinoma
cell line A549, which was transduced with lentivirus
to generate a transgenic cell line Cas13d-A549 stably
expressing Cas13d. It was shown that the use of one
from the five pools of crRNA specifically recognizing
sequence of the RdRP gene resulted in the decrease
of expression of the reporter GFP protein fused with
RdRp and N by 86%. The results of quantitative PCR
(qPCR) demonstrated that the therapy with different
pools of crRNA resulted in the decrease of the amount
of viral mRNA by more than 80%. In other studies,
with crRNA targeting mRNA of nucleocapsid, the re-
searchers also demonstrated significant success in
the cell cultures [11, 37, 45,46]. In particular, the use
of crRNA together with Cas13d effectively inhibited
CRISPR/Cas13 IN INFLUENZA AND SARS-CoV-2 THERAPY 791
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
Table 1. Use of the CRISPR/Cas13 system for protection against viral infection in vitro
Pathogen
Target
sequence
Enzyme Delivery method
Experimental
model
Main results References
SARS-CoV-2 RdRp, N
RfxCas13d-NLS
Cas13 – lentiviral
transduction;
crRNA –
lipofection
A549
pool of crRNAs targeting RdRp ↓ expression
of viral proteins by 86%, reporter proteins – by 83%;
pool of crRNAs targeting N ↓ expression of viral protein
by 71%, reporter proteins – by 79%
[10]
IAV H1N1
highly
conserved
regions in
RdRp, HA,
NA, NP, M1,
M2, NS1, NEP
genes
crRNA targeting NA ↓ expression of the reporter
by 72% at MOI 2.5;
crRNA targeting NA ↓ expression of the reporter
by 52% at MOI 5;
crRNA targeting HA ↓ expression of the reporter
by 26% at MOI 2.5;
crRNA targeting HA ↓ expression of the reporter
by 55% at MOI 2.5
SARS-CoV-2 S, NCP pspCas13b27-NES lipofection
HEK293T
crRNA targeting S ↓ level of S-transcripts by 99%;
crRNA targeting NCP ↓ level of nucleocapsid transcripts
by 99%
[49]
Vero
crRNA targeting S ↓ level of S-transcripts by 90%;
crRNA targeting NCP ↓ level of nucleocapsid transcript
by 90%, ↓ virus load by ~90% and by 60% 24 and 48 h
after infection
Calu-3 crRNA targeting NCP ↓ viral titer by 98%
IAV WSN/33 PB1, PB2
LbuCas13a/
LbuCas13a-NLS
lipofection
A549
crRNA targeting
PB1 + LbuCas13a ↓ number of copies
of PB1 gene by 83%;
crRNA targeting PB1 + LbuCas13a-NLS ↓
number of copies of PB1 gene by 78%;
crRNA targeting PB2 + LbuCas13a/LbuCas13a-NLS ↓
number of copies of PB2 gene by 75%;
crRNA targeting PB1 + crRNA targeting PB2+
LbuCas13a-NLS ↓ number of copies of PB1 and PB2 genes
by 10% in comparison withmonotherapy
[53]
SARS-CoV-2 RdRp, N Vero E6
pool of crRNAs targeting N + LbuCas13a ↓ cell death by 72%;
crRNA targeting RdRp + LbuCas13a ↓ number of copies
of RdRp genes by 93.7%;
crRNA targeting N + LbuCas13a ↓ number of copies
of N genes by 94.1%.
KAZAKOVA et al.792
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
Table 1 (cont.)
Pathogen
Target
sequence
Enzyme Delivery method
Experimental
model
Main results References
SARS-CoV-2 RdRp, N
LbuCas13a/
LbuCas13a-NLS
lipofection Huh7
crRNA targeting RdRp + LbuCas13a ↓ number of copies
of RdRp genes by 94.5%;
crRNA targeting N + LbuCas13a ↓ number of copies
of N genes by 99.1%
[53]
SARS-CoV-2 N
RfxCas13d-NLS
lentiviral
transduction
Vero E6 crRNA targeting N ↓ viral titer by 96% during 72 h
[11]
Cas13d-NES
lentiviral
transduction and
LNP with crRNA
hPBECs ↓ viral titer by 54% after 24 h and by 97%– after 48 h
HCoV-229E
N, RdRp Cas13d-NLS
lentiviral
transduction
MRC-5
crRNA targeting N ↓ virus replication by 95%;
crRNA targeting N ↓ viral titer by 99%
N Cas13d-NES
lentiviral
transduction and
LNP with crRNA
hPBECs ↓ of viral titer by 78% after 48 h and by 92%– after 72h
HCoV-OC43 N, RdRp Cas13d-NLS
lentiviral
transduction
MRC-5 crRNA targeting N ↓ viral titer by 99%
HCoV-OC43 N, RdRp CasRx
transfection
with calcium
phosphate
method
Vero
crRNA targeting RdRp ↓ content of viral RNA by ~80%
during 96 h with peak at 72 h (90%)
[50]
HCoV-OC43
N, RdRp CasRx
transfection
with calcium
phosphate
method
Vero
↓ of viral titer by 99.97% after 2 days
[45]HCoV-229E ↓ of viral titer from 78.44% to 99.39% after 2 days
SARS-CoV-2
↓ of viral titer from 69.65% to 94.07% after 4 days
SARS-CoV-2
pseudoknot
in ORF1b,
RdRp
Cas13b-NES transfection
Vero E6
crRNA targeting ORF1b ↓ number of copies
of SARS-CoV-2 genes and viral titer by99%;
crRNA targeting RdRp ↓ number of copies
of SARS-CoV-2 genes by 98%, and viral titer by 90%
[55]
Calu-3
crRNA targeting ORF1b ↓ number of copies
of SARS-CoV-2 genes by 99.9%
CRISPR/Cas13 IN INFLUENZA AND SARS-CoV-2 THERAPY 793
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
Table 1 (cont.)
Pathogen
Target
sequence
Enzyme Delivery method
Experimental
model
Main results References
SARS‐CoV‐2
ORF1ab,
nsp13,
nsp14, N
Cas13d transfection HeLa‐ACE2
crRNA targeting N ↓ expression of N by 99%;
crRNA targeting ORF1ab ↓ amount of RNA by 99%
[47]
SARS‐CoV‐2
Ctsl RfxCas13d lipofection
HEK293FT crRNA targeting Ctsl ↓ expression of Ctsl by 75%
[54]
Vero-ACE2-
TMPRSS2
63% blocking of pseudotyped infection;
↓ expression of N by 60%;
↓ infection 10-fold
Caco-2 65% blocking of pseudotyped infection
SARS-CoV-1 Caco-2 45% blocking of pseudotyped infection
SARS‐CoV‐2 RdRp, E
Cas13X.1 lipofection
HEK293T ↓ expression of reporter by 70%
[37]
H1N1 NP MDCK
↓ expression of viral proteins by 70%;
↓ viral load 4-fold
IAV H1N1
WSN
PB, NP, M Cas13a-NLS lipofection DF1
↓ 4-fold of WSN titer after 24 h
[62]
IAV H1N1
PR8
↓ 2-fold titer of PR8 after 24 h
IAV
mRNA and
complementary
viral RNA
PspCas13b
electroporation MDCK
↓ of viral RNA 7–22-fold
[7]
PspCas13b-NLS
crRNAs does not decrease level of viral RNA;
pool of crRNA ↓ viral RNA 8-fold
PspCas13b-NES
infectivity of IAV ↓ 300-fold;
↓ of viral RNA 5-fold
SARS-CoV-2 S Cas13a
lentiviral
transduction
for Cas13a;
lipofection
for crRNA
HepG2 ↓ expression of S by 99.9% after 48 h;
[48]
AT2
↓ expression of S by 93% after 48 h;
↓ total expression by 90%
KAZAKOVA et al.794
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
Table 1 (cont.)
Pathogen
Target
sequence
Enzyme Delivery method
Experimental
model
Main results References
SARS-CoV-2
M, N, E, S,
nsp3, nsp5,
RdRp
Cas13b-NES transfection HEK293-ACE2
crRNA targeting RdRp ↓ infectivity of SARS-CoV-2
by 63%
crRNA targeting RdRp + crRNA targeting nsp15 ↓
amount of viral RNA 1.25–1.5-fold in comparison
with monotherapy;
crRNA targeting RdRp + crRNA targeting S ↓
viral RNA 1.8-fold in comparison with crRNA
targeting RdRp;
crRNA targeting RdRp + crRNA targeting N ↓
viral RNA 2.2-fold in comparison with crRNA
targeting N;
testing efficiency of crRNA targeting RdRp
against SARS-CoV demonstrated decrease
of SARS-CoV RNA level by 35%,
while the decrease in the case
of crRNA targeting N was 22%
[46]
SARS-CoV
crRNA targeting RdRp ↓ the level
of SARS-CoV RNA by 35%;
crRNA targeting N↓ the level
of SARS-CoV RNA by 22%
SARS-CoV-2
5′-UTR,
nsp3, nsp4,
nsp6, RdRp,
N, nsp13,
pseudoknot
in ORF1b,
“slippery”
sequence,
s2m
Cas13d-NLS lipofection HEK293T
crRNA targeting s2m ↓ expression
of the reporter by 83%;
crRNA targeting s2m + crRNA targeting nsp3 ↓
expression of the reporter by 92%;
crRNA targeting 5′-UTR ↓ replication
of the virus by 82%
[52]
SARS-CoV
crRNA targeting 5′-UTR ↓ replication
of the virus by 90%
IAV/H1N1
Cal04/09
PB2
LbuCas13a +
LbuCas13a-NLS
transfection
A549
90% of viral knockdown;
↓ of viral titer by 90%;
↓ of the level of PB2 by 90%
[46]
IAV H3N2
A549 ↓ of viral titer 10–50-fold
HBEC3-KT ↓ of viral titer 5–25-fold
CRISPR/Cas13 IN INFLUENZA AND SARS-CoV-2 THERAPY 795
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
replication of different variants of N-transcript in the
SARS-CoV-2, thus reducing the virus titer by 96%[11].
Decrease in inhibition of expression of viral proteins
with time has been demonstrated for some crRNAs,
however, individual combinations of crRNAs retain
their efficiency of inhibition from 24 to 72 h. The
authors emphasize that targeting the N-transcript
allows inhibiting different strains of SARS-CoV-2 in-
cluding D614G, Alpha, Zeta, Epsilon (B.1.427), and Ep-
silon (B.1.429) with close efficiency of more than 90%.
Moreover, increase of the degree of SARS-CoV-2 inhibi-
tion has been observed on addition of low molecular
weight preparations (camostat mesylate, EIDD-1931,
remdesivir, clofazimine, E-64d, elbasvir, and velpat-
asvir) in combination with Cas13d and crRNA. Using
of the low molecular weight compounds EIDD-1931
or of the Cas13d + crRNA-N preparation separately
ensured 5.9- and 2.9-fold decrease of the virus titer,
respectively, while the use of their combination result-
ed in 32.2-fold decrease of the virus titer. Hence, the
CRISPR/Cas13-based antiviral therapy could be used in
combination therapy [11].
First successes in the SARS-CoV-2 therapy demon-
strated with cell cultures served as a starting point
for initiation of investigation of the use of different
variants of Cas13 in the fight against the SARS-CoV-2
coronavirus. Main attention of the studies was focused
on selection of nuclease, delivery methods, selection
of the crRNA target, design of crRNAs and their mul-
tiplexing, as well as development of optimal regime
of therapy.
In their study Xuetal.[37] selected transcripts of
the RdRp and E genes as targets for crRNAs, which are
conserved among the SARS-CoV viruses. The HEK293T
cells were co-transfected with the reporter vector con-
taining a hybrid gene combining sequence of the GFP
gene and fragments of the RdRp and E genes, and the
vector with Cas13X.1/crRNA. It was shown that 48 h af-
ter co-transfection almost all tested crRNAs (27of 30)
inhibited GFP fluorescence in the cells by ~70%. One
other study devoted to the use of Cas13d against the
SARS-CoV-2 used the most conserved regions of the
ORF1ab, nsp13 and nsp14, which perform important
functions in all SARS-CoV-2 strains, as targets [47].
The transgenic HeLa-ACE2 cells line producing ACE2
receptor was used to evaluate efficiency of the sys-
tem. This cell line was co-transfected with the plasmid
containing Cas13d, and pool of the crRNA plasmids
targeting non-structural proteins nsp13 and nsp14.
Twenty-four hours after co-transfection the cells were
infected with the SARS-CoV-2, and one day after infec-
tion the amount of N protein was assessed with the
help of Western blotting. The obtained result showed
that the crRNA/Cas13d system indeed was capable of
dealing with high virus load [MOI (multiplicity of in-
fection) of 2] in the course of SARS-CoV-2 infection,
and all tested crRNAs decreased significantly the levels
of viral proteins and RNA. The Cas13d + crRNA system
was capable of suppressing the emerging epidemic
strains of SARS-CoV-2 (including strains B.1, B.1.351,
B.1.1.7, and B.1.617) due to targeting the highly con-
served regions [47].
In 2021 Wang et al. [48] used LwaCas13a in
their study with crRNA targeting the sequence of re-
ceptor-binding domain in the S-protein, which plays
a key role in interaction with ACE2. This study did
not involve infection of the cells with virus, and ef-
ficiency of the CRISPR/Cas13 system was determined
in the model cell line stably expressing the coronavi-
rus S-protein. Human alveolar type 2 cells (AT2) and
human hepatocellular carcinoma cells (HepG2) with
high level of ACE2 expression were selected as tar-
gets. The results of qPCR demonstrated that the effi-
ciency of suppression of the expression of S-protein
mRNA reached >99.9% in the HepG2 and AT2 cells.
In another study using S-protein mRNA as a target
the variant of PspCas13b with NES was used with
increased spacer sequence in the crRNA, which, sup-
posedly, increases specificity of this system [49]. High
efficiency of suppression of the S-protein expression
was demonstrated for all tested crRNAs. Similar re-
sults were obtained using the Vero cell line with the
best crRNA demonstrating >90% decrease of the lev-
el of S-protein transcripts in the HEK293T and Vero
cells. Dose-dependent effect was observed indicating
that the availability of crRNA in the cell is a key factor
for degradation of viral RNA, moreover, it has been
noted that presence of only few copies of the plasmid
encoding crRNA is sufficient for effective suppression
of the S-protein expression.
Potential flexibility of the template and its effi-
ciency even in the case of presence of mutations in
the viral RNA target could play an important role
in the overall efficiency of the CRISPR/Cas13 system.
The study by Fareh et al. [49] evaluated efficiency of
using PspCas13b together with crRNAs carrying 3, 6,
and 9 substitutions. The 3-nt mismatch in the central
part of the crRNA spacer at positions 14-16 or at the
3′-end of the spacer (positions 28-30) affected only
insignificantly cleavage of the target RNA, while the
3-nt mismatch at the 5′-end of crRNA resulted in the
decrease of cleavage of the target transcript by ~50%.
The 6-nt mismatch introduced at different positions of
the crRNA decreased significantly efficiency of inhibi-
tion, and the 9-nt mismatch resulted in the absence of
degradation of the S-protein transcript. It is important
to note that the ability of PspCas13b to recognize the
target sequence even in the presence of unpaired nu-
cleotides, especially in the inner regions of the spacer,
indicates its potential efficiency against majority of
the variants of the target sequences with single-nu-
cleotide polymorphisms, thus providing protection
KAZAKOVA et al.796
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
against mutant strains. Similar results were obtained
in the Vero cell line in the experiments with coro-
navirus HCoV-OC43, with highly conserved RdRp and
N genes used as targets [50]. Single-substitutions or
triplet substitutions in the crRNA sequence only slight-
ly affected the decrease of the viral RNA in compari-
son with the crRNA fully complementary to the RdRp
mRNA.
Efficiency of crRNAs multiplexing during infection
with SARS-CoV-2 was also evaluated [49]. Vero cells
were transfected using different crRNA pools with
each of them including 4 different crRNAs targeting
either structural proteins S and N, or non-structural
proteins nsp7 and nsp8 [51]. All 4 pools of crRNAs
decreased significantly the level of viral RNA and ti-
ter of the virus in the supernatant at MOI both 0.1
and 0.01; however, the efficiency was higher at lower
dose and it was retained over the entire observation
period, while at higher dose of the virus the inhibition
effect noticeably and gradually decreased with time.
In the process of infection of Vero cells with SARS-
CoV-2 at MOI 0.1 and 0.01 and 72h after transfection
with PspCas13b and crRNA, expression of viral RNA
was suppressed by ~80% and ~90%, respectively [49].
The degree of the virus titer decrease also depends
on the type of the target RNA chain – (+) or (−) [52].
Efficiency of cleavage of the target RNA in the HEK293T
cells was tested using PfxCas13d and crRNA target-
ing different sequences of the SARS-CoV-2 genome in
both RNA chains. It was shown in the experiments
with HEK293T cells using SARS-CoV-2 replicons that
the most efficient cleavage of the target RNA was ob-
served when the crRNA targeting (+)-chain was used,
furthermore, all crRNAs targeting (−)-chain of RNA
were ineffective except the crRNA targeting the “slip-
pery” sequence.
The studies evaluating efficiency of Cas13a both
in in vitro system and under in vivo conditions are
of particular interest (Table 2) [53-55]. In the study
by Blanchard et al. [53] crRNAs targeting highly con-
served areas in the replicase and nucleocapsid regions
of the SARS-CoV-2 genome were tested. For this pur-
pose, Vero E6 and Huh7 cells were transfected with
the LbuCas13a mRNA and various combinations of
crRNAs. It was found out that combination of the crR-
NAs targeting both N and RdRp used together with
the LbuCas13a mRNA decreases cell death by more
than 50%. The crRNA targeting N or combination of
two best crRNAs targeting N ensured decrease of the
Vero E6 cells by more than 72%; moreover, the sim-
ilar result was observed for the Huh7 cell line [53].
Therapy based on the use of Cas13a + crRNA target-
ing RdRp resulted in the decrease of the number of
copies of the RdRp gene by 93.7% and 94.5% in the
Vero E6 and Huh7 cells, respectively; and the system
based on Cas13a + crRNA targeting N decreased the
number of N transcript by 94.1% and 99.1% in the
VeroE6 and Huh7 cells, respectively. Golden hamsters
(Mesocricetus auratus) were used as an invivo model
in the study by Blanchardetal. [53]: 125 µg of mRNA
encoding Cas13a and crRNA targeting N together with
the PBAE polymer were administered intranasally to
4-week-old male hamsters. After 20 h hamsters were
intranasally infected with 10
3
plaque forming units
of SARS-CoV-2 (USA-WA1/2020). It was shown that the
Cas13a/crRNA-based therapy resulted in the decrease
of the number of copies of the viral N RNA in the
hamster lungs by 57%.
The CRISPR/Cas13-based strategy for fighting vi-
rus infection in the case of coronavirus could be also
not associated with suppression of viral transcripts. In
their elegant study, Cuietal.[54] suggested a non-typ-
ical approach to CRISPR/Cas-based antiviral therapy,
which targets not the viral protein, but the lung prote-
ase cathepsin L (Ctsl). Ctsl is an important endosomal
cysteine protease, which facilitates priming of S-pro-
tein and penetration of the virus into the cell via the
virus–host cell endosome membrane fusion. Ctsl in-
hibitors block penetration of coronaviruses (such as
SARS-CoV-1 and SARS-CoV-2) in vitro and development
of pseudotyped SARS-CoV-2 infection in vivo [56-59].
Investigation on suppression of Ctsl expression in
the cell culture was performed using the HEK293FT
cells with the help of co-transfection of different crRNAs
and expression plasmid carrying the RfxCas13d gene
[54]. The most effective crRNA facilitated 75%-sup-
pression of the Ctsl expression; it was shown in fur-
ther experiments that this effect not only resulted in
inhibition of the of pseudotyped SARS-CoV-2 infection,
but also of the of pseudotyped SARS-CoV-1 infection
in the Vero-ACE2-TMPRSS2 and Caco-2 cells. In addi-
tion, the RfxCas13d-mediated specific knockdown of
the Ctsl gene resulted in the effective inhibition of
different virus strains, such as, for example, B.1.617.2
Delta. These results on successful suppression of the
Ctsl gene were confirmed in the experiments with
animal models [54]. Delivery of the CasRx mRNA and
Ctsl-targeting crRNA to the mouse lungs was carried
out with the help of LNP-particles based on MC3 with
addition of supplementary cationic lipids. Upon infec-
tion with SARS-CoV-2 (USA-WA1/2020) the K18-hACE2
mice in three control groups demonstrated graduate
weight loss and 100% mortality within 8 days after
virus administration. On the contrary, administra-
tion of LNP-CasRx-Ctsl-crRNA to the mice 2 days pri-
or and 1 day after infection with SARS-CoV-2, result-
ed in the delayed manifestation of the disease, and
survival level reached 50%. It was also detected that
the amount of mRNAs of the Ctsl protein, and of the
viral proteins N and E decreased in the animals af-
ter the LNP-CasRx-Ctsl-crRNA injection. In addition to
evaluation of the level of expression of the Ctsl gene
CRISPR/Cas13 IN INFLUENZA AND SARS-CoV-2 THERAPY 797
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
Table 2. Use of the CRISPR/Cas13 system for protection against viral infection in vivo
Pathogen
Target
sequence
Enzyme
Delivery
method
Experimental
model
Main results References
IAV WSN/33 PB1
LbuCas13a PBAE + mRNA
BALB/c mice ↓ of viral RNA by 96.2%
[53]
SARS-CoV-2 N
LVG Syrian
gold
hamsters
↓ number of copies
of N by 57%
SARS-CoV-2
pseudoknot
in ORF1b,
RdRp
Cas13b
intranasal
administration
invivo-jetRNA
with siGLO
K18-hACE2
mice
crRNA targeting ORF1b ↓
viral titer by 99%
[55]
SARS‐CoV‐2 Ctsl RfxCas13d
injection into
tail vein of
LNP-mRNA
K18-hACE2
mice
↑ survival by 50%;
↓ weight loss,
level of Ccl5, Ccl2.
and Isg15 cytokines,
inflammation in lungs;
↓ expression of Ctsl 100-fold;
↓ expression
of proteins N and E;
↓ infectivity
of the virus by 2log
[54]
IAV/H1N1
Cal04/09
PB2 LbuCas13a
intranasal
administration
of mRNA
Syrian gold
hamster
prophylactics:
↓ of number of copies
of PB2 and of viral titer
in lungs by 1log;
therapy:
↓ of number of copies
of PB2 by 93%,
viraltiter– by 88%;
use of two-dose regime:
↓ of number of copies
of PB2 and viral titer
in lungs by 1log and 2log,
respectively
[46]
the authors also assessed the levels of expression of
proinflammatory cytokines and chemokines associat-
ed with the development of cytokine storm. Expres-
sion of Cxcl10, Tnf, Ccl5, Ccl2, and Isg15 decreased
in response to the LNP-CasRx-Ctsl-crRNA therapy.
The similar results were demonstrated when the
LNP-CasRx-Ctsl-crRNA preparation was administered
2h and 1 day after infection with 10
5
plaque forming
units of SARS-CoV-2.
Biodistribution of the preparation and its safety
were also examined in the study by Cui et al. [54].
Expression of the CasRx mRNA in lungs reached max-
imum 4 h after infection followed by significant de-
creased after 24 h. Remarkably, the Ctsl expression
did not change in liver or spleen, and expression of
other representatives of the cathepsin family, Ctsd,
Ctsb, and Ctss, did not change in lungs after therapy.
These results demonstrate high specificity of the used
system. Moreover, there were no significant chang-
es in the expression of cytokines/chemokines in the
lungs following treatment with the LNP-CasRx- Ctsl-
crRNA. And finally, the authors did not observe sig-
nificant changes in the liver and kidney functions
of the mice, as well as in hematological parameters.
Taken together these data demonstrate that the tar-
geted delivery of the CasRx-Ctsl-crRNA to lungs with
the help of lipid nanoparticles allows for effective,
specific, and safe suppression of the targeted tran-
scripts. Moreover, customized targeting and fast bio-
distribution of lipid nanoparticles with mRNA over
the entire organism within a few hours after admin-
istration of the preparation opens up new possibili-
ties for emergency therapy of the severe cases of viral
infection [60, 61].
KAZAKOVA et al.798
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
In another study with animals the PspCas13b-NES
system targeting the RdRp gene in SARS-CoV-2 that in-
cluded the pseudoknot region of the ORF1b was used
[55]. Cas13b and crRNA were delivered as mRNAs,
Cas13b expression was detected already 2 h after
transfection, degradation of both RdRp mRNA and of
the RdRp protein was also observed 2 h after transfec-
tion; this effect increased 6 h after transfection. Analy-
sis of the viral load with the TCID50 assay showed that
the titer of SARS-CoV-2 in the lungs of mice treated
with targeting crRNA decreased by 99% in compar-
ison with the virus titer in the lung of non-treated
mice. Similarly, the levels of viral RNA of the RdRp
gene and N protein were also significantly reduced in
the lungs. These results could indicate that targeting
the region of pseudoknot in ORF1b followed by the
Cas13b- mediated knockdown is a promising strategy
for treatment of the SARS-CoV-2 infection [55].
USE OF CRISPR/Cas13 IN THERAPY
OF INFLUENZA A VIRUS
CRISPR/Cas13 system is used for inhibition of a
wide spectrum of viruses including influenza A virus
with single-stranded negative-sense RNA. In one of
the first studies devoted to this issue the PspCas13b
nuclease was used with the Madin–Darby canine kid-
ney cells (MDCK); next, efficiency of five different
crRNAs targeting genes encoding proteins NP and M
was examined (Table1) [7]. The cells were transfected
using electroporation with two plasmids, with one of
them containing sequence of crRNA, and another– se-
quence of the PspCas13b gene, and 24 h after infection
of the cell culture with the IAV amount of viral RNA
in the supernatant was determined using PCR. It was
show that the amount of viral RNA decreased 7-22-fold
in comparison with the control group [7]. In another
study the Cas13X.1 was used in the MDCK cells with
crRNAs targeting mRNA of nucleoprotein required for
IAV replication and transcription [37]. For this purpose,
the cells were transfected with the vector containing
Cas13X.1 and crRNA, which was followed by infection
with the influenza strain H1N1 A/Puerto Rico/8/1934.
Three of the four tested crRNAs demonstrated high
efficiency of the nucleoprotein transcript knockdown
and significantly decreased titer of the virus in the
mammalian cells.
Detailed investigation of the antiviral effect in cell
cultures also included experiments on the effects of
localization of PspCas13b, type of cells, and multiplex-
ing [7]. It was revealed that the Cas13b localization
indeed could affect efficiency of crRNA, because one
of the crRNAs targeting the gene encoding NP signifi-
cantly decreased the level of viral RNA only in the
cases, when Cas13b was localized in cytoplasm and
not in the nucleus. At the same, change of the cell
line did not affect significantly efficiency of the sys-
tem– amount of the viral RNA decreased 20.6-fold in
the A549 cells, which was comparable with the results
obtained in the MDCK cells. Multiplexing was also
shown to be efficient: using simultaneously 4 crRNAs
targeting the gene encoding NP protein resulted in the
8-fold decrease of viral RNA in comparison with the
effects of mono-therapy. Similar results of multiplex-
ing of crRNAs targeting transcripts of the PB, NP, and
M genes in the virus genome were obtained with the
chicken embryo fibroblasts cell line (DF1)[62].
Another study performed with the help of
RfxCas13d in the A549 cell line used crRNAs target-
ing highly conserved regions, which affect the virus
assembly [10]. Selection of this type of targets could
potentially facilitate inhibition of a wide spectrum
of IVA strains using the CRISPR/Cas13d system. In to-
tal 48 crRNAs were used in the study, 6 for each of
the 8 segments of the influenza virus A genome. The
Cas13d A549 cells stably expressing Cas13d nuclease
were transfected with the pool of 6 crRNAs targeting
highly conserved regions of the viral genome. Two
days after transfection the cells were infected with the
virus at MOI 2.5 or 5.0; 18 h after infection the cells
were analyzed for IVA infection using flowcytometry
and microscopy. Among all tested crRNAs, the pool
of crRNAs targeting the segment 6, encoding the NA
protein, demonstrated most consistent and reproduc-
ible results at both MOI levels (decrease by 72% for
MOI = 2.5 and decrease by 52% for MOI = 5). Further-
more, the pool of crRNAs targeting the segment 4,
which encodes the HA protein, also demonstrated
moderate inhibition of the viral infection. Interesting-
ly enough, it was shown that expression of the Cas13d
nuclease affected the levels of RNAs of those regions
of IVA that were not directly targeted. In total, the
results of this study demonstrated that Cas13d is ca-
pable of affecting highly conserved regions in the viral
genome and inhibit IVA replication in the human lung
epithelial cells.
In the later study, in addition to in vitro exper-
iments, in vivo experiments were also conducted
(Table 2) [53]. The used crRNAs targeted the sequenc-
es of the PB1 and PB2 genes. The A549 cells were in-
fected with the influenza virus A/WSN/33 at MOI 0.01;
24 h after infection the cells were transfected with the
mixture of mRNAs of LbuCas13a with or without NLS
and crRNA targeting PB1, and 48 h after infection the
levels of PB1 RNA were assessed with the help of PCR.
One of the crRNAs demonstrated high efficiency de-
creasing expression of the viral RNA within 24 h by
83% when the cytosolic form of Cas13a was used, and
by 78%, when Cas13a with nucleus localization was
used. In addition, efficiency of the crRNA targeting
the conserved region of the PB2 gene was evaluated.
CRISPR/Cas13 IN INFLUENZA AND SARS-CoV-2 THERAPY 799
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
The crRNA was introduced 24 h after infection, the
degree of viral infection was evaluated 48 h after in-
fection. Three of six tested sequences of crRNAs caused
decrease of the level of viral RNA by more than 50%,
however, efficiency of the antiviral effect varied de-
pending on the variant of the used nuclease – with
or without NLS.
In the in vivo study with mice, RNA was intro-
duced intranasally with the help of nebulizer using
the PBAE polymer [53]. The mice were infected with
3 LD50 of the influenza virus A/WSN/33 via intra-
nasal administration, and 6 h after mRNA encoding
LbuCas13a without NLS was administrated, as well as
crRNA targeting PB1. The CRISPR/Cas13-based therapy
resulted in the decrease of amount of the viral RNA
96.2% 3 days after infection.
In the experiments involving Syrian golden ham-
ster [46], the LbuCas13a nuclease mRNA was admin-
istered intranasally together with the crRNA target-
ing PB2 with incorporated 2′-O-methyl-nucleotides.
The animals were infected with the influenza virus
A/H1N1/California/04/09 (Cal04/09). In the case of pro-
phylactic approach administration of one dose of the
preparation decreased the level of RNA and of the
infectious virus (PFU/ml) in lungs by approximately
one order of magnitude. Therapeutic administration
with the help of Cas13 resulted in 93%-reduction of
the number of the PB2 gene copies and88%-reduction
of the virus titer. In the case of two-dose regime (one
injection prior to infection and one– after) the level of
RNA and of the infectious virus (PFU/ml) in the lungs
decreased by almost 2 orders of magnitude. Hence,
the results of experiments with mice and hamsters
that used the CRISPR/Cas13-based treatment against
the influenza virus indicate that this system causes
efficient suppression of the viral RNA and decrease
of the virus titer.
DISCUSSION
Viral infections represent a serious problem for
health care, primarily due to the fact that the specif-
ic therapeutic approaches exist for only few types of
viruses. Recent coronavirus pandemics and influenza
virus A pandemics (H1N1) in 2009 demonstrated that
the development of innovative antiviral treatments is
a very urgent task. The idea for using bacterial system
of adaptive immunity, CRISPR/Cas13, that specifically
targets viral RNA as an antiviral therapy is currently
realized in the experiments with cell cultures; how-
ever, promising data on the efficiency of this system
in vivo in model animals start to emerge.
One of the advantages of the CRISPR/Cas13 sys-
tem is its activity towards the viral RNA, hence, its
use does not cause any changes in the cell genome.
This system is capable of recognizing RNA tar-
get even in the presence of unpaired bases in the
crRNA and target, hence, high mutation rate in the
viral genomes does not prevent effective cleavage
of the viral RNA. For therapeutic purposes it is ad-
vantageous to use Cas13 nucleases with the smallest
size, as it facilitates their delivery to the cell, hence,
the Cas13d and Cas13X nucleases, as well as the re-
cently discovered Cas13an nuclease seems to be most
promising for treatment of influenza and SARS-CoV-2
infections. In the experiments with cell cultures all
used Cas13 nucleases demonstrates their efficiency by
significantly decreasing the level of viral RNA and ti-
ter of the virus (Table 1). The results of recent studies
with model animals on the use of the CRISPR/Cas13
system in antiviral therapy of SARS-CoV-2 and influ-
enza virus infection are encouraging (Table 2). Despite
the fact that the number of such studies is limited,
they generally demonstrate level of efficiency similar
to the results obtained in cell cultures. Small number
of studies with model animals is likely related to the
fact that the use of CRISPR/Cas13-based preparations
for antiviral therapy is associated with two problems:
selection of the effective delivery method and possible
excessive activation of the innate and adaptive im-
mune response to the individual components of the
CRISPR/Cas13 system. In 2022 Tang et al. [63] exam-
ined the levels of IgG-antibodies specific to RfxCas13d
and evaluated the RfxCas13d-induced proliferation of
the CD4
+
and CD8
+
T-lymphocytes in healthy donors.
It was revealed that majority of the donors have
IgG-antibodies to RfxCas13d- and RfxCas13d-reacting
T-cells capable of producing proinflammatory cyto-
kines IFN-γ, TNF-α, and IL-17. Development of such
immune response could be explained by coloniza-
tion of the human gut by the Ruminococcus bicir-
culans bacteria producing proteins of the Cas13 fam-
ily, which are similar to the RfxCas13d nuclease.
Potential for the development of unwanted side re-
actions limits possibilities of clinical application of
the preparations based on CRISPR/Cas13, but devel-
opment of new administration formats (such as in
form of mRNA) and delivery systems, including sys-
tems for targeted delivery based on lipid nanoparti-
cles, opens wide perspectives to minimize potential
side effects and increase efficiency of the future
CRISPR/Cas13-based preparations [54, 61].
The results of presented studies also indicate
that localization of the Cas13 nuclease and selection
of the optimal crRNA sequences, as well as the use of
multiplexing involving several crRNAs allowed to in-
crease significantly efficiency of the system. Moreover,
effective system for the delivery of the components of
CRISPR/Cas13 system to the infection foci also has not
been fully developed. In the recent study on the use
of CRISPR/Cas13 the delivery systems based on lipid
KAZAKOVA et al.800
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
nanoparticles are considered most promising, they
have been shown to ensure effective and selective de-
livery with sufficiently high safety level [54]. Delivery
of mRNA with the help of lipid nanoparticles has been
proven to be reliable in the case of the known and
approved mRNA SARS-CoV-2 vaccines: RNA-1273 vac-
cine (Moderna) and BNT162b2 vaccine (Comirnaty®,
BioNTech and Pfizer). Considering that the mRNA plat-
form allows to accelerate significantly development
of preparations with changed sequences of the target
antigen/crRNA, this platform is highly suitable for pro-
phylaxis and therapy of seasonal viral diseases. Inthis
context, the idea of complex development of two
mRNA preparations with delivery system in the form
of lipid nanoparticles against the seasonal strains of
the influenza virus A and SARS-CoV-2 including mRNA
vaccine encoding sequence of viral antigens as a pro-
phylactic preparation and mRNA–CRISPR/Cas13 as an
emergency therapy in the case of severe disease seems
very interesting. As has been mentioned above, mRNA
packed into the lipid nanoparticles ensured fast bio-
distribution in the tissues and translation of the target
protein already in the first hours after administration
of the preparation [60].
Another strategy to increase efficiency of antivi-
ral therapy is successful use of combination of the
CRISPR/Cas13-based therapy with treatment with small
molecules [37]. Conducting complex studies on the use
of different combination of Cas13 variants, RNA tar-
gets, and delivery methods, as well as search for the
compounds without antiviral activity, but capable of
potentiation of the CRISPR/Cas13 activity, seems as a
promising direction for future studies on the strate-
gies for fighting viral diseases.
Abbreviations. Structural proteins: E– envelope
protein, N – nucleocapsid protein, S – spike protein;
crRNA, CRISPR RNA, portion of CRISPR guide RNA; Ctsl,
lung cathepsin protease L; HA, hemagglutinin; IAV, in-
fluenza A virus; MDCK, Madin–Darby canine kidney
cells; MOI, multiplicity of infection; A, neuraminidase;
NES, nuclear export signal; NLS, nuclear localization
signal; NP, nucleoprotein; nsp, non structural proteins;
ORF, open reading frame; PA, polymerase acidic; PB1,
polymerase basic 1; PB2, polymerase basic 2; RdRp,
RNA dependent RNA polymerase.
Contributions. A. A. Kazakova, Yu. V. Sopova,
A. V. Chirinskaite, E. S. Minskaya, and I. S. Kukushkin–
literature search, preparation of figures, whiting
text of the paper. E. I. Leonova, R. A. Ivanov, and V. V.
Reshetnikov – concept of the review article, writing
and editing text of the paper.
Funding. Work of A. A. Kazakova, E. S. Minskaya,
I. S. Kukushkina, R. A. Ivanova, and V. V. Reshetnikov
was financially supported by the State Budget Pro-
gram Sirius: Scientific Technological development of
the federal district ‘Sirius’ (Agreement no.03-03, Feb-
ruary 18, 2025). Work of E. I. Leonova, Yu. V. Sopova,
and A. V. Chrinskaite was financially supported by the
St. Petersburg State University (grant ID E129658320).
Ethics approval and consent to participate.
This work does not contain any studies involving hu-
man and animal subjects.
Conflict of interest. The authors of this work de-
clare that they have no conflicts of interest.
REFERENCES
1. Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott,
D.A., and Zhang,F. (2013) Genome engineering using
the CRISPR-Cas9 system, Nat. Protoc., 8, 2281-2308,
https://doi.org/10.1038/nprot.2013.143.
2. Charpentier, E., Richter, H., van der Oost, J., and
White, M. F. (2015) Biogenesis pathways of RNA
guides in archaeal and bacterial CRISPR-Cas adaptive
immunity, FEMS Microbiol. Rev., 39, 428-441, https://
doi.org/10.1093/femsre/fuv023.
3. Abudayyeh, O. O., Gootenberg, J. S., Konermann, S.,
Joung,J., Slaymaker, I.M., Cox, D.B., Shmakov,S., Ma-
karova, K.S., Semenova,E., Minakhin,L., Severinov,K.,
Regev, A., Lander, E. S., Koonin, E. V., and Zhang, F.
(2016) C2c2 is a single-component programmable
RNA-guided RNA-targeting CRISPR effector, Science,
353, aaf5573, https://doi.org/10.1126/science.aaf5573.
4. Shmakov,S., Smargon,A., Scott,D., Cox,D., Pyzocha,N.,
Yan, W., Abudayyeh, O. O., Gootenberg, J. S.,
Makarova, K. S., Wolf, Y. I., Severinov, K., Zhang, F.,
and Koonin, E. V. (2017) Diversity and evolution of
class 2 CRISPR-Cas systems, Nat. Rev. Microbiol., 15,
169-182, https://doi.org/10.1038/nrmicro.2016.184.
5. Xue, Y., Chen, Z., Zhang, W., and Zhang, J. (2022)
Engineering CRISPR/Cas13 system against RNA virus-
es: from diagnostics to therapeutics, Bioengineering,
9, 291, https://doi.org/10.3390/bioengineering9070291.
6. Zhao, X., Liu, L., Lang, J., Cheng, K., Wang, Y., Li, X.,
Shi, J., Wang, Y., and Nie, G. (2018) A CRISPR-Cas13a
system for efficient and specific therapeutic target-
ing of mutant KRAS for pancreatic cancer treatment,
Cancer Lett., 431, 171-181, https://doi.org/10.1016/
j.canlet.2018.05.042.
7. Freije, C. A., Myhrvold, C., Boehm, C. K., Lin, A. E.,
Welch, N. L., Carter, A., Metsky, H. C., Luo, C. Y.,
Abudayyeh, O. O., Gootenberg, J. S., Yozwiak, N. L.,
Zhang, F., and Sabeti, P. C. (2019) Programmable in-
hibition and detection of RNA viruses using Cas13,
Mol. Cell, 76, 826-837.e11, https://doi.org/10.1016/
j.molcel.2019.09.013.
8. Kellner, M.J., Koob, J.G., Gootenberg, J.S., Abudayyeh,
O.O., and Zhang,F. (2019) SHERLOCK: nucleic acid de-
tection with CRISPR nucleases, Nat. Protoc., 14, 2986-
3012, https://doi.org/10.1038/s41596-019-0210-2.
CRISPR/Cas13 IN INFLUENZA AND SARS-CoV-2 THERAPY 801
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
9. Li, H., Wang,S., Dong,X., Li,Q., Li,M., Li,J., Guo,Y.,
Jin,X., Zhou,Y., Song,H., and Kou,Z. (2020) CRISPR-
Cas13a cleavage of dengue virus NS3 gene efficiently
inhibits viral replication, Mol. Ther. Nucleic Acids, 19,
1460-1469, https://doi.org/10.1016/j.omtn.2020.01.028.
10. Abbott, T.R., Dhamdhere,G., Liu,Y., Lin,X., Goudy,L.,
Zeng, L., Chemparathy,A., Chmura,S., Heaton, N.S.,
Debs, R., Pande, T., Endy, D., La Russa, M. F., Lewis,
D.B., and Qi, L. S. (2020) Development of CRISPR as
an antiviral strategy to combat SARS-CoV-2 and influ-
enza, Cell, 181, 865-876.e12, https://doi.org/10.1016/
j.cell.2020.04.020.
11. Zeng,L., Liu,Y., Nguyenla, X.H., Abbott, T.R., Han,M.,
Zhu, Y., Chemparathy, A., Lin, X., Chen, X., Wang,H.,
Rane, D.A., Spatz, J.M., Jain,S., Rustagi,A., Pinsky,B.,
Zepeda, A. E., Kadina, A. P., Walker, J. A., 3rd,
Holden,K., Temperton,N., Cochran, J.R., Barron, A.E.,
Connolly, M.D., Blish, C.A., Lewis, D.B., Stanley, S.A.,
La Russa, M. F., and Qi, L. S. (2022) Broad-spectrum
CRISPR-mediated inhibition of SARS-CoV-2 variants
and endemic coronaviruses in vitro, Nat. Commun.,
13, 2766, https://doi.org/10.1038/s41467-022-30546-7.
12. Nguyen, H., Wilson, H., Jayakumar, S., Kulkarni, V.,
and Kulkarni, S. (2021) Efficient inhibition of HIV
using CRISPR/Cas13d nuclease system, Viruses, 13,
1850, https://doi.org/10.3390/v13091850.
13. Chaves, L. C. S., Orr-Burks, N., Vanover, D., Mosur,
V.V., Hosking, S.R., Kumar, P.E.K., Jeong,H., Jung,Y.,
Assumpção, J. A. F., Peck, H. E., Nelson, S. L., Burke,
K. N., Garrison, M. A., Arthur, R. A., Claussen, H.,
Heaton, N.S., Lafontaine, E.R., Hogan, R.J., Zurla,C.,
and Santangelo, P. J. (2024) mRNA-encoded Cas13
treatment of influenza via site-specific degradation
of genomic RNA, PLoS Pathog., 20, e1012345, https://
doi.org/10.1371/journal.ppat.1012345.
14. Méndez-Mancilla, A., Wessels, H. H., Legut, M.,
Kadina, A., Mabuchi, M., Walker, J., Robb, G. B.,
Holden, K., and Sanjana, N. E. (2022) Chemically
modified guide RNAs enhance CRISPR-Cas13 knock-
down in human cells, Cell Chem. Biol., 29, 321-327.e4,
https://doi.org/10.1016/j.chembiol.2021.
15. Gorbalenya, A. E., Enjuanes, L., Ziebuhr, J., and
Snijder, E. J. (2006) Nidovirales: evolving the largest
RNA virus genome, Virus Res., 117, 17-37, https://
doi.org/10.1016/j.virusres.2006.01.017.
16. Imbert,I., Guillemot, J.C., Bourhis, J.M., Bussetta,C.,
Coutard, B., Egloff, M. P., Ferron, F., Gorbalenya,
A. E., and Canard, B. (2006) A second, non-canon-
ical RNA-dependent RNA polymerase in SARS coro-
navirus, EMBO J., 25, 4933-4942, https://doi.org/
10.1038/sj.emboj.7601368.
17. Seybert, A., Hegyi, A., Siddell, S. G., and Ziebuhr, J.
(2000) The human coronavirus 229E superfami-
ly 1 helicase has RNA and DNA duplex-unwinding
activities with 5′-to-3′ polarity, RNA, 6, 1056-1068,
https://doi.org/10.1017/s1355838200000728.
18. Ivanov, K.A., Thiel,V., Dobbe, J.C., van der Meer, Y.,
Snijder, E.J., and Ziebuhr,J. (2004) Multiple enzymat-
ic activities associated with severe acute respiratory
syndrome coronavirus helicase, J. Virol., 78, 5619-
5632, https://doi.org/10.1128/JVI.78.11.5619-5632.2004.
19. Minskaia, E., Hertzig, T., Gorbalenya, A. E.,
Campanacci, V., Cambillau, C., Canard, B., and
Ziebuhr,J. (2006) Discovery of an RNA virus 3′→5′ exo-
ribonuclease that is critically involved in coronavirus
RNA synthesis, Proc. Natl. Acad. Sci. USA, 103, 5108-
5113, https://doi.org/10.1073/pnas.0508200103.
20. Ivanov, K. A., Hertzig, T., Rozanov, M., Bayer, S.,
Thiel,V., Gorbalenya, A.E., and Ziebuhr,J. (2004) Ma-
jor genetic marker of nidoviruses encodes a replica-
tive endoribonuclease, Proc. Natl. Acad. Sci. USA, 101,
12694-12699, https://doi.org/10.1073/pnas.0403127101.
21. Benoni, R., Krafcikova, P., Baranowski, M. R.,
Kowalska, J., Boura, E., and Cahová, H. (2021) Sub-
strate specificity of SARS-CoV-2 Nsp10-Nsp16 methyl-
transferase, Viruses, 13, 1722, https://doi.org/10.3390/
v13091722.
22. Bhatt, P.R., Scaiola,A., Loughran,G., Leibundgut,M.,
Kratzel, A., Meurs, R., Dreos, R., O’Connor, K. M.,
McMillan,A., Bode, J.W., Thiel,V., Gatfield,D., Atkins,
J. F., and Ban, N. (2021) Structural basis of ribosom-
al frameshifting during translation of the SARS-
CoV-2 RNA genome, Science, 372, 1306-1313, https://
doi.org/10.1126/science.abf3546.
23. Brierley, I., Digard, P., and Inglis, S. C. (1989) Char-
acterization of an efficient coronavirus ribosomal
frameshifting signal: requirement for an RNA pseu-
doknot, Cell, 57, 537-547, https://doi.org/10.1016/
0092-8674(89)90124-4.
24. Finkel, Y., Mizrahi, O., Nachshon, A., Weingarten-
Gabbay, S., Morgenstern, D., Yahalom-Ronen, Y.,
Tamir,H., Achdout,H., Stein,D., Israeli,O., Beth-Din,A.,
Melamed, S., Weiss, S., Israely, T., Paran, N.,
Schwartz, M., and Stern-Ginossar, N. (2021) The cod-
ing capacity of SARS-CoV-2, Nature, 589, 125-130,
https://doi.org/10.1038/s41586-020-2739-1.
25. Wang, D., Jiang,A., Feng,J., Li,G., Guo,D., Sajid, M.,
Wu, K., Zhang, Q., Ponty, Y., Will, S., Liu, F., Yu, X.,
Li,S., Liu,Q., Yang, X.L., Guo,M., Li,X., Chen,M., Shi,
Z.L., Lan,K., Chen,Y., and Zhou,Y. (2021) TheSARS-
CoV-2 subgenome landscape and its novel regula-
tory features, Mol. Cell, 81, 2135-2147.e5, https://
doi.org/10.1016/j.molcel.2021.02.036.
26. Firth, A.E. (2020) Aputative new SARS-CoV protein, 3c,
encoded in an ORF overlapping ORF3a, J.Gen. Virol.,
101, 1085-1089, https://doi.org/10.1099/jgv.0.001469.
27. Schaecher, S. R., Mackenzie, J. M., and Pekosz, A.
(2007) The ORF7b protein of severe acute respira-
tory syndrome coronavirus (SARS-CoV) is expressed
in virus-infected cells and incorporated into SARS-
CoV particles, J. Virol., 81, 718-731, https://doi.org/
10.1128/JVI.01691-06.
KAZAKOVA et al.802
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
28. Eisfeld, A. J., Neumann, G., and Kawaoka, Y. (2015)
At the centre: influenza A virus ribonucleoproteins,
Nat. Rev. Microbiol., 13, 28-41, https://doi.org/10.1038/
nrmicro3367.
29. Fodor, E., and Te Velthuis, A. J. W. (2020) Structure
and function of the influenza virus transcription and
replication machinery, Cold Spring Harb. Perspect.
Med., 10, a038398, https://doi.org/10.1101/cshperspect.
a038398.
30. Lamb, R. A., Lai, C. J., and Choppin, P. W. (1981) Se-
quences of mRNAs derived from genome RNA seg-
ment 7 of influenza virus: colinear and interrupted
mRNAs code for overlapping proteins, Proc. Natl.
Acad. Sci. USA, 78, 4170-4174, https://doi.org/10.1073/
pnas.78.7.4170.
31. Dauber, B., Heins, G., and Wolff, T. (2004) The influ-
enza B virus nonstructural NS1 protein is essential
for efficient viral growth and antagonizes beta in-
terferon induction, J. Virol., 78, 1865-1872, https://
doi.org/10.1128/jvi.78.4.1865-1872.2004.
32. Lamb, R.A., Choppin, P. W., Chanock, R. M., and Lai,
C. J. (1980) Mapping of the two overlapping genes
for polypeptides NS1 and NS2 on RNA segment 8 of
influenza virus genome, Proc. Natl. Acad. Sci. USA,
77, 1857-1861, https://doi.org/10.1073/pnas.77.4.1857.
33. Te Velthuis, A.J., and Fodor,E. (2016) Influenza virus
RNA polymerase: insights into the mechanisms of vi-
ral RNA synthesis, Nat. Rev. Microbiol., 14, 479-493,
https://doi.org/10.1038/nrmicro.2016.87.
34. Marsh, G.A., Rabadán,R., Levine, A.J., and Palese,P.
(2008) Highly conserved regions of influenza a virus
polymerase gene segments are critical for efficient
viral RNA packaging, J. Virol., 82, 2295-2304, https://
doi.org/10.1128/JVI.02267-07.
35. Bouvier, N. M., and Palese, P. (2008) The biology
of influenza viruses, Vaccine, 26, D49-D53, https://
doi.org/10.1016/j.vaccine.2008.07.039.
36. Makarova, K.S., Wolf, Y.I., Iranzo,J., Shmakov, S.A.,
Alkhnbashi, O. S., Brouns, S. J. J., Charpentier, E.,
Cheng,D., Haft, D.H., Horvath,P., Moineau,S., Mojica,
F.J.M., Scott,D., Shah, S.A., Siksnys,V., Terns, M.P.,
Venclovas, Č., White, M. F., Yakunin, A. F., Yan, W.,
Zhang,F., Garrett, R.A., Backofen,R., van der Oost,J.,
Barrangou,R., and Koonin, E. V. (2020) Evolutionary
classification of CRISPR-Cas systems: a burst of class2
and derived variants, Nat. Rev. Microbiol., 18, 67-83,
https://doi.org/10.1038/s41579-019-0299-x.
37. Xu, C., Zhou, Y., Xiao, Q., He, B., Geng, G., Wang, Z.,
Cao,B., Dong,X., Bai,W., Wang,Y., Wang,X., Zhou,D.,
Yuan, T., Huo, X., Lai, J., and Yang, H. (2021) Pro-
grammable RNA editing with compact CRISPR-
Cas13 systems from uncultivated microbes, Nat.
Methods, 18, 499-506, https://doi.org/10.1038/s41592-
021-01124-4.
38. Kannan, S., Altae-Tran, H., Jin, X., Madigan, V. J.,
Oshiro, R., Makarova, K. S., Koonin, E. V., and
Zhang, F. (2022) Compact RNA editors with small
Cas13 proteins, Nat. Biotechnol., 40, 194-197, https://
doi.org/10.1038/s41587-021-01030-2.
39. Yoon, P.H., Zhang,Z., Loi, K.J., Adler, B.A., Lahiri,A.,
Vohra, K., Shi, H., Rabelo, D. B., Trinidad, M., Boger,
R. S., Al-Shimary, M. J., and Doudna, J. A. (2024)
Structure-guided discovery of ancestral CRISPR-
Cas13 ribonucleases, Science, 385, 538-543, https://
doi.org/10.1126/science.adq0553.
40. Hu, Y., Chen, Y., Xu, J., Wang, X., Luo, S., Mao, B.,
Zhou, Q., and Li, W. (2022) Metagenomic discovery
of novel CRISPR-Cas13 systems, Cell Discov., 8, 107,
https://doi.org/10.1038/s41421-022-00464-5.
41. Abudayyeh, O. O., Gootenberg, J.S., Essletzbichler,P.,
Han, S., Joung, J., Belanto, J. J., Verdine, V., Cox,
D.B.T., Kellner, M.J., Regev,A., Lander, E.S., Voytas,
D. F., Ting, A. Y., and Zhang, F. (2017) RNA target-
ing with CRISPR-Cas13, Nature, 550, 280-284, https://
doi.org/10.1038/nature24049.
42. Yan, W. X., Chong, S., Zhang, H., Makarova, K. S.,
Koonin, E. V., Cheng, D. R., and Scott, D. A. (2018)
Cas13d is a compact RNA-targeting type VI CRISPR
effector positively modulated by a WYL-domain-con-
taining accessory protein, Mol. Cell, 70, 327-339.e5,
https://doi.org/10.1016/j.molcel.2018.02.028.
43. O’Connell, M.R. (2019) Molecular mechanisms of RNA
targeting by Cas13-containing type VI CRISPR-Cas
systems, J. Mol. Biol., 431, 66-87, https://doi.org/
10.1016/j.jmb.2018.06.029.
44. Meeske, A. J., Nakandakari-Higa, S., and Marraffini,
L. A. (2019) Cas13-induced cellular dormancy pre-
vents the rise of CRISPR-resistant bacteriophage,
Nature, 570, 241-245, https://doi.org/10.1038/s41586-
019-1257-5.
45. Mayes, C.M., and Santarpia, J.L. (2023) Pan-coronavi-
rus CRISPR-CasRx effector system significantly reduc-
es viable titer in HCoV-OC43, HCoV-229E, and SARS-
CoV-2, CRISPR J., 6, 359-368, https://doi.org/10.1089/
crispr.2022.0095.
46. Andersson,K., Azatyan,A., Ekenberg,M., Güçlüler,G.,
Sardon Puig,L., Puumalainen,M., Pramer,T., Monteil,
V. M., and Mirazimi, A. (2024) A CRISPR-Cas13b sys-
tem degrades SARS-CoV and SARS-CoV-2 RNA invitro,
Viruses, 16, 1539, https://doi.org/10.3390/v16101539.
47. Liu, Z., Gao, X., Kan, C., Li, L., Zhang, Y., Gao, Y.,
Zhang, S., Zhou, L., Zhao, H., Li, M., Zhang, Z., and
Sun,Y. (2023) CRISPR-Cas13d effectively targets SARS-
CoV-2 variants, including Delta and Omicron, and
inhibits viral infection, MedComm, 4, e208, https://
doi.org/10.1002/mco2.208.
48. Wang, L., Zhou, J., Wang, Q., Wang, Y., and Kang, C.
(2021) Rapid design and development of CRISPR-
Cas13a targeting SARS-CoV-2 spike protein, Theranos-
tics, 11, 649-664, https://doi.org/10.7150/thno.51479.
49. Fareh,M., Zhao,W., Hu,W., Casan, J.M.L., Kumar,A.,
Symons, J., Zerbato, J. M., Fong, D., Voskoboinik, I.,
CRISPR/Cas13 IN INFLUENZA AND SARS-CoV-2 THERAPY 803
BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025
Ekert, P.G., Rudraraju,R., Purcell, D.F.J., Lewin,S.R.,
and Trapani, J. A. (2021) Reprogrammed CRISPR-
Cas13b suppresses SARS-CoV-2 replication and cir-
cumvents its mutational escape through mismatch
tolerance, Nat. Commun., 12, 4270, https://doi.org/
10.1038/s41467-021-24577-9.
50. Mayes, C. M., and Santarpia, J. (2022) Evaluating the
impact of gRNA SNPs in CasRx activity for reduc-
ing viral RNA in HCoV-OC43, Cells, 11, 1859, https://
doi.org/10.3390/cells11121859.
51. Hillen, H. S., Kokic, G., Farnung, L., Dienemann, C.,
Tegunov,D., and Cramer,P. (2020) Structure of repli-
cating SARS-CoV-2 polymerase, Nature, 584, 154-156,
https://doi.org/10.1038/s41586-020-2368-8.
52. Hussein, M., Andrade Dos Ramos, Z., Vink, M. A.,
Kroon,P., Yu,Z., Enjuanes,L., Zuñiga,S., Berkhout,B.,
and Herrera-Carrillo, E. (2023) Efficient CRISPR-
Cas13d-based antiviral strategy to combat SARS-CoV-2,
Viruses, 15, 686, https://doi.org/10.3390/v15030686.
53. Blanchard, E. L., Vanover, D., Bawage, S. S., Tiwari,
P. M., Rotolo, L., Beyersdorf, J., Peck, H. E., Bruno,
N.C., Hincapie,R., Michel,F., Murray,J., Sadhwani,H.,
Vanderheyden, B., Finn, M. G., Brinton, M. A.,
Lafontaine, E.R., Hogan, R.J., Zurla,C., and Santange-
lo, P.J. (2021) Treatment of influenza and SARS-CoV-2
infections via mRNA-encoded Cas13a in rodents,
Nat. Biotechnol., 39, 717-726, https://doi.org/10.1038/
s41587-021-00822-w.
54. Cui, Z., Zeng, C., Huang, F., Yuan, F., Yan,J., Zhao,Y.,
Zhou,Y., Hankey,W., Jin, V.X., Huang,J., Staats, H.F.,
Everitt, J. I., Sempowski, G. D., Wang, H., Dong, Y.,
Liu, S. L., and Wang, Q. (2022) Cas13d knockdown
of lung protease Ctsl prevents and treats SARS-CoV-2
infection, Nat. Chem. Biol., 18, 1056-1064, https://
doi.org/10.1038/s41589-022-01094-4.
55. Yu,D., Han, H.J., Yu,J., Kim,J., Lee, G.H., Yang, J.H.,
Song, B.M., Tark,D., Choi, B.S., Kang, S.M., and Heo,
W. D. (2023) Pseudoknot-targeting Cas13b combats
SARS-CoV-2 infection by suppressing viral replica-
tion, Mol. Ther., 31, 1675-1687, https://doi.org/10.1016/
j.ymthe.2023.03.018.
56. Ou, X., Liu, Y., Lei, X., Li, P., Mi, D., Ren, L., Guo, L.,
Guo, R., Chen, T., Hu, J., Xiang, Z., Mu, Z., Chen, X.,
Chen, J., Hu, K., Jin, Q., Wang,J., and Qian, Z. (2020)
Characterization of spike glycoprotein of SARS-
CoV-2 on virus entry and its immune cross-reactiv-
ity with SARS-CoV, Nat. Commun., 11, 1620, https://
doi.org/10.1038/s41467-020-15562-9.
57. Zhou,Y., Vedantham,P., Lu,K., Agudelo,J., Carrion,R.,Jr.,
Nunneley, J. W., Barnard, D., Pöhlmann,S., McKerrow,
J. H., Renslo, A. R., and Simmons, G. (2015) Protease
inhibitors targeting coronavirus and filovirus en-
try, Antiviral Res., 116, 76-84, https://doi.org/10.1016/
j.antiviral.2015.01.011.
58. Simmons,G., Gosalia, D.N., Rennekamp, A.J., Reeves,
J. D., Diamond, S. L., and Bates, P. (2005) Inhibi-
tors of cathepsin L prevent severe acute respirato-
ry syndrome coronavirus entry, Proc. Natl. Acad.
Sci. USA, 102, 11876-11881, https://doi.org/10.1073/
pnas.0505577102.
59. Zhao, M.M., Yang, W.L., Yang, F.Y., Zhang,L., Huang,
W.J., Hou,W., Fan, C.F., Jin, R.H., Feng, Y.M., Wang,
Y. C., and Yang, J. K. (2021) Cathepsin L plays a key
role in SARS-CoV-2 infection in humans and human-
ized mice and is a promising target for new drug
development, Signal Transduct. Target. Ther., 6, 134,
https://doi.org/10.1038/s41392-021-00558-8.
60. Pateev,I., Seregina,K., Ivanov,R., and Reshetnikov,V.
(2023) Biodistribution of RNA vaccines and of their
products: evidence from human and animal stud-
ies, Biomedicines, 12, 59, https://doi.org/10.3390/
biomedicines12010059.
61. Vasileva, O., Zaborova, O., Shmykov, B., Ivanov, R.,
and Reshetnikov, V. (2024) Composition of lipid
nanoparticles for targeted delivery: application to
mRNA therapeutics, Front. Pharmacol., 15, 1466337,
https://doi.org/10.3389/fphar.2024.1466337.
62. Challagulla, A., Schat, K. A., and Doran, T. J. (2021)
In vitro inhibition of influenza virus using CRISPR/
Cas13a in chicken cells, Methods Protoc., 4, 40, https://
doi.org/10.3390/mps4020040.
63. Tang, X.E., Tan, S.X., Hoon,S., and Yeo, G.W. (2022)
Pre-existing adaptive immunity to the RNA-editing
enzyme Cas13d in humans, Nat. Med., 28, 1372-1376,
https://doi.org/10.1038/s41591-022-01848-6.
Publisher’s Note. Pleiades Publishing remains
neutral with regard to jurisdictional claims in published
maps and institutional affiliations. AI tools may have
been used in the translation or editing of this article.
