RNA and Single-Stranded DNA Phages: Unveiling the Promise from the Underexplored World of Viruses

Abstract

RNA and single-stranded DNA (ssDNA) phages make up an understudied subset of bacteriophages that have been rapidly expanding in the last decade thanks to advancements in metaviromics. Since their discovery, applications of genetic engineering to ssDNA and RNA phages have revealed their immense potential for diverse applications in healthcare and biotechnology. In this review, we explore the past and present applications of this underexplored group of phages, particularly their current usage as therapeutic agents against multidrug-resistant bacteria. We also discuss engineering techniques such as recombinant expression, CRISPR/Cas-based genome editing, and synthetic rebooting of phage-like particles for their role in tailoring phages for disease treatment, imaging, biomaterial development, and delivery systems. Recent breakthroughs in RNA phage engineering techniques are especially highlighted. We conclude with a perspective on challenges and future prospects, emphasizing the untapped diversity of ssDNA and RNA phages and their potential to revolutionize biotechnology and medicine.

Full text

Citation: Nguyen, H.M.; Watanabe,
S.; Sharmin, S.; Kawaguchi, T.; Tan,
X.-E.; Wannigama, D.L.; Cui, L. RNA
and Single-Stranded DNA Phages:
Unveiling the Promise from the
Underexplored World of Viruses. Int.
J. Mol. Sci. 2023,24, 17029. https://
doi.org/10.3390/ijms242317029
Academic Editor: Alicja Wegrzyn
Received: 1 November 2023
Revised: 26 November 2023
Accepted: 28 November 2023
Published: 1 December 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Review
RNA and Single-Stranded DNA Phages: Unveiling the Promise
from the Underexplored World of Viruses
Huong Minh Nguyen 1, Shinya Watanabe 1, Sultana Sharmin 1, Tomofumi Kawaguchi 1, Xin-Ee Tan 1,
Dhammika Leshan Wannigama 2and Longzhu Cui 1,*
1Division of Bacteriology, Department of Infection and Immunity, Jichi Medical University,
Shimotsuke 329-0498, Tochigi, Japan; nguyen.mh@jichi.ac.jp (H.M.N.); swatanabe@jichi.ac.jp (S.W.);
sultana.sharmin@jichi.ac.jp (S.S.); kwgctmfm@jichi.ac.jp (T.K.); xinee@jichi.ac.jp (X.-E.T.)
2Department of Infectious Diseases and Infection Control, Yamagata Prefectural Central Hospital,
Yamagata 990-2292, Yamagata, Japan; leshanwannigama@gmail.com
*Correspondence: longzhu@jichi.ac.jp
Abstract:
RNA and single-stranded DNA (ssDNA) phages make up an understudied subset of
bacteriophages that have been rapidly expanding in the last decade thanks to advancements in
metaviromics. Since their discovery, applications of genetic engineering to ssDNA and RNA phages
have revealed their immense potential for diverse applications in healthcare and biotechnology. In
this review, we explore the past and present applications of this underexplored group of phages,
particularly their current usage as therapeutic agents against multidrug-resistant bacteria. We also
discuss engineering techniques such as recombinant expression, CRISPR/Cas-based genome editing,
and synthetic rebooting of phage-like particles for their role in tailoring phages for disease treatment,
imaging, biomaterial development, and delivery systems. Recent breakthroughs in RNA phage
engineering techniques are especially highlighted. We conclude with a perspective on challenges and
future prospects, emphasizing the untapped diversity of ssDNA and RNA phages and their potential
to revolutionize biotechnology and medicine.
Keywords:
RNA phages; ssDNA phages; metaviromics; phage-based applications; genetic engineer-
ing; CRISPR/Cas-based genome editing; synthetic rebooting; phage therapy
1. Introduction
First discovered independently by F. W. Twort in 1915 [
1
] and F. d’Herelle in 1917 [
2
] as
a bacteria-eating virus, bacteriophages (or phages) are generally considered the most abun-
dant biological entity on Earth, with an estimation of approximately 10
31
particles in the
biosphere [3]. Found in virtually all environments, phages exhibit unprecedented diverse
morphology (polyhedral, filamentous, or tailed), genomic composition (DNA or RNA,
double-stranded or single-stranded), and life cycle (lytic, lysogenic, or chronic productive
infection) [
4
]. The use of phages to treat bacterial infections, called phage therapy, started in
Eastern Europe from the early days of phage discovery but was later forgotten following the
discovery of antibiotics [
5
]. The last few decades have seen antibiotic-resistant bacteria rise
and persist as one of the major public health concerns worldwide [
6
], leading to renewed in-
terest in the once-forgotten phage therapy. In recent years, treatments of bacterial infections
using natural or engineered phages have restarted in Europe and America with notable
successes and continue to gain momentum [
7
,
8
]. Together with antibacterial treatments,
these triumphs have spurred phage research and engineering attempts to explore broader
applications of phages in gene therapy, diagnostics and detection, drug delivery, cancer
treatment, and vaccine development [9,10].
Until recently, most known phages were classified into one order, 14 families, and
37 genera, mainly based on their morphology [
11
]. With current advances in metagenomic
and metatranscriptomic studies, however, in the most recent 2022 taxonomy update of
Int. J. Mol. Sci. 2023,24, 17029. https://doi.org/10.3390/ijms242317029 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023,24, 17029 2 of 23
the International Committee on Taxonomy of Viruses (ICTV), several major changes in
phage classification have been officialized to put more focus on the genomic basis of
these viruses. This includes the abolishment of the morphology-based families Myoviridae,
Podoviridae, and Siphoviridae, and the replacement of the order Caudovirales by the class
Caudoviricetes to include all tailed viruses of bacteria and archaea with icosahedral capsids
and double-stranded DNA genomes [
12
]. Although the first single-stranded DNA (ssDNA),
double-stranded RNA (dsRNA), and single-stranded RNA (ssRNA) phages were isolated
back in the 1930s and 1970s [
13
–
15
], more than 90% of known phages to date are phages
with double-stranded DNA (dsDNA) genomes [
11
]. By June 2019, only 12 representative
ssRNA phage genome sequences were available from the NCBI Genome database [
16
]. A
microvirus (ssDNA phage) dataset compiled in a study in 2022 included only 2147 genomes
from GenBank; among these representative genome sequences of isolated virions were
542 [
17
]. Thus, it is unsurprising that dsDNA phages are the most thoroughly studied group
and have played a pivotal, if not exclusive, role in many major phage-based discoveries
and applications until now.
Despite accounting for less than 10% of all known phages to date, RNA and ssDNA
phages possess multiple distinct features from dsDNA phages, such as their structure
and genetic makeup, encoded lytic proteins, host range and receptors, or their life cycle,
making them an attractive addition to dsDNA phages in various therapeutic applications.
An exhaustive review of the hugely diverse RNA and ssDNA phages and their many
practical applications is beyond the scope of this paper. In this review, we will attempt to
deliver a concise account of the usages of this underexploited group of phages in various
applications, focusing on their therapeutic usages as antimicrobial agents. To conclude, we
will present our viewpoint on the future promise of engineering this group of phages for
antimicrobial treatments, highlighting their potential advantages and distinctiveness.
2. A Brief History of ssDNA and RNA Phages
Although RNA and ssDNA phages were discovered not long after the isolation of the
first dsDNA phage, this group has been historically understudied and underrepresented.
Thanks to the recent rapid advancement of metagenomics and metatranscriptomics, the
number of novel phage sequences, especially ssDNA and RNA phage sequences, has been
significantly expanded. Substantial data on the evolution, hosts, life cycles, and molecular
structure of ssRNA phages [
18
,
19
], dsRNA phages [
20
], and ssDNA phages [
21
,
22
] are
available elsewhere. Here, we will briefly summarize relevant and current information
about ssDNA and RNA phages to provide an overview of their important biological
features and most recent taxonomical expansion (Table 1).
2.1. ssDNA Phages
The current classification separates ssDNA phages into five families with two dis-
tinct shapes: the tubular Inoviridae,Paulinoviridae, and Plectroviridae, and the icosahedral
capsid Microviridae and Finnlakeviridae. These ssDNA phages have a circular genome of
about
4.5–10.6 kb
that encodes for 4–15 proteins; approximately half of these carry out
morphogenesis and structural functions such as adhesion, replication, virion, and assembly.
Besides these, unclassified ssDNA filamentous phage isolates (such as Vibrio phage K05K4,
Genbank ID CP017905) or prophages [
23
] with genomes larger than 20 kb have also been
identified. Except for a few well-studied ssDNA phages, such as M13 or phiX174, the
functions of the remaining encoded proteins remain unknown [
24
,
25
]. Genomes of all
ssDNA phages replicate through the rolling-circle mechanism with the involvement of host
polymerase, converting the ssDNA into covalently bound dsDNA, known as the replicative
form [
24
]. ssDNA phages are pili specific, binding to the tip of the host cell’s male pilus,
such as Hfr, F+, and F’, via their coat protein (e.g., g3p in the case of Ff phages). The binding
causes a conformational change of the coat protein, revealing the domain that can interact
with another membrane-bound co-receptor of the host, TolA, facilitating phage genome
entry [26].

Int. J. Mol. Sci. 2023,24, 17029 3 of 23
Among the three families of tubular phages, Inoviridae and Paulinoviridae viral particles
exist in a long, filamentous form, while Plectroviridae appears as a rigid rod-shape structure.
As with all filamentous phages, members of these families lead a chronic productive
infection life cycle where phage particles are released by extrusion without lysing their
host cells. Interestingly, these three families infect completely different types of hosts.
Inoviridae infect Gram-negative bacteria with a lipopolysaccharide (LPS)-containing outer
membrane, while Paulinoviridae infect both Gram-negative and Gram-positive hosts and are
found associated with mostly LPS-lacking Gram-negative hosts (ICTV-approved taxonomy
proposals, Paulinoviridae,https://ictv.global/filebrowser/download/5770 (accessed on
26 November 2023)). Plectroviridae infect cell-wall-less bacteria, such as members of the
Mollicutes order [27].
Microviridae and Finnlakeviridae comprise the other two families of ssDNA phages
with small icosahedral capsids. The Microviridae family of ssDNA phages can be divided
into two phylogenetic lineages, Microvirus and Gokushovirinae, based on the presence
or absence of spike proteins on their capsids. Microvirus has 12 spikes on the 5-fold
vertices, while Gokushovirinae lacks spikes but has mushroom-like protrusions at the
3-fold
vertices [
22
]. Initially, all cultured phages of the Microviridae family were isolated from
Enterobacteriaceae (in the case of Microvirus) and intracellular parasites (in the case of
Gokushovirinae), leading to the belief that this group of phages has a narrow host range. The
culturing technique, however, can lead to underestimation, as prophages of gokushoviruses
were later found in Bacteroidetes genomes using the prophage detection method [
28
]. Recent
metagenomics surveys revealed that microviruses are much more prevalent and present in
most environments, highlighting their ecological importance [29].
Finnlakeviridae is a recently recognized ssDNA phage family that includes a single
genus, Finnlakevirus, with only one species, Finnlakevirus FLiP, isolated from a boreal
freshwater habitat in Central Finland in 2010 [
30
]. It is the first described ssDNA phage
that contains an internal membrane enclosed in the icosahedral capsid [
31
]. The striking
similarity between the capsid proteins of Finnlakevirus FLiP and dsDNA viruses of the
PRD1-adenovirus lineage suggested a possible evolutionary relatedness between some
ssDNA and dsDNA viruses.

Int. J. Mol. Sci. 2023,24, 17029 4 of 23
Table 1. Current classification of RNA and ssDNA phages.
Group Family Virion Genome Replication Host Range Life Cycle Members 1Example
[References]
ssDNA
Inoviridae Non-enveloped
flexible filaments Circular +ssDNA Rolling circle LPS+Gram-negative
bacteria
Chronic
infection
25 genera,
43 species Phage M13 [24]
Paulinoviridae Non-enveloped
flexible filaments Circular +ssDNA Rolling circle LPS−Gram-negative
and -positive bacteria
Chronic
infection
2 genera,
2 species
Phage B5; phage
OH3 [27,32]
Plectroviridae Non-enveloped
rigid rods Circular +ssDNA Rolling circle or
transposition Cell wall-less bacteria Chronic
infection
4 genera,
6 species Phage MV-L1 [33]
Microviridae
Non-enveloped
icosahedral virions,
spikes −/+
Circular +ssDNA Rolling circle and
other mechanism(s)
Enterobacteria, intracel-
lular parasitic bacteria,
cell wall-less bacteria
Lytic 7 genera,
22 species
Phage ϕX174;
phage 4 [34]
Finnlakeviridae
Icosahedral virion
with spikes, internal
lipid membrane
Circular +ssDNA Possibly rolling
circle
Gram-negative
Flavobacterium Lytic 1 genus,
1 species Phage FLiP [30]
dsRNA Cystoviridae
Enveloped multi-layer
icosahedral virions
with spikes
Segmented, linear
dsRNA ssRNA →dsRNA
Gram-negative
bacteria, mostly
Pseudomonas
Lytic 1 genus,
7 species Phage phi6 [35]
ssRNA
(before 2021) Leviviridae Non-enveloped
icosahedral virions Linear +ssRNA −ssRNA →
+ssRNA
Gram-negative
bacteria Lytic 2 genera,
4 species
Phage MS2 [36],
phage Qβ[34]
ssRNA
(since 2021) 3
Atkinsviridae NA 2NA 2NA 2NA 2NA 256 genera,
91 species
Uncultured viral
genomes [37]
Duinviridae NA 2NA 2NA 2NA 2NA 26 genera,
6 species
Uncultured viral
genomes [37]
Fiersviridae
(formerly Leviviridae)
Non-enveloped
icosahedral virions Linear +ssRNA −ssRNA →
+ssRNA
Gram-negative
bacteria Lytic 185 genera,
298 species
Phage MS2 [36],
phage Qβ[37]
Solspiviridae NA 2NA 2NA 2NA 2NA 224 genera,
31 species
Uncultured viral
genomes [37]
Blumeviridae NA 2NA 2NA 2NA 2NA 231 genera,
35 species
Uncultured viral
genomes [37]

Int. J. Mol. Sci. 2023,24, 17029 5 of 23
Table 1. Cont.
Group Family Virion Genome Replication Host Range Life Cycle Members 1Example
[References]
ssRNA
(since 2021) 3
Steitzviridae NA 2NA 2NA 2NA 2NA 2117 genera,
412 species
Uncultured viral
genomes [37]
Unassigned NA 2NA 2NA 2NA 2NA 29 genera,
9 species
Uncultured viral
genomes [37]
1
https://ictv.global/msl (ICTV_Master_Species_List_2022_MSL38.v3.xlsx, created 9 November 2023—11:17) (accessed on 26 November 2023).
2
NA: not available.
3
Due to the
hyperextension of discovered ssRNA phage sequences from metatranscriptomes, since 2021, this group includes 882 species and their taxonomy has been completely restructured. Noted
that many species are sequence-only or uncultured viral genomes.

Int. J. Mol. Sci. 2023,24, 17029 6 of 23
2.2. dsRNA Phages
RNA (dsRNA and ssRNA) phages constitute the smallest portion of known bacte-
riophages. Currently, ICTV registers seven species of dsRNA phages, all belonging to
the single family Cystoviridae. The family Cystoviridae includes enveloped viruses with a
tri-segmented dsRNA genome enclosed in one or two concentric, icosahedrally symmetric
protein shells. The innermost protein layer is a polymerase complex responsible for genome
packaging, replication, and transcription [35].
All known dsRNA phages infect Gram-negative bacteria, mainly plant-pathogenic
Pseudomonas syringae strains [
35
]. Until 1999, the Pseudomonas phage phi6 was the sole
species of the Cystoviridae family [
38
]. Additional lipid-containing, three-segmented dsRNA
phages were subsequently isolated into two groups: Those closely related (phi 7, phi9,
phi10, and phi11) and those distantly related (phi8 and phi12) to phi6. Phi13 lies between
the two groups [
39
]. Phi6 and its closely related relatives utilize type IV pili as the host
receptor, limiting their infection range to P. syringae and several mutants of P. pseudoalcali-
genes ERA [
39
]. In contrast, phi8 and phi12 infect P. syringae with rough LPS [
38
]. Phi8,
phi12, and phi13 could infect other rough-LPS-containing Gram-negative bacteria, though
plaque formation was not observed when phi12 was used to infect JM109 [
39
]. Two other
P. syringae
dsRNA phages, phi2954 and phiNN, were later isolated from radish leaves [
40
]
and freshwater habitat [
41
], respectively; both utilize type IV pili as the host receptor. In
2016, the first dsRNA phage infecting the human pathogen P. aeruginosa was successfully
isolated from hospital sewage in China [
42
]. These studies demonstrate a wider distribution
and broader host range than previously anticipated for these dsRNA viruses.
2.3. ssRNA Phages
In previous ICTV taxonomy releases, the ssRNA phages were grouped into a single
family, the Leviviridae, consisting of two genera and four species: Levivirus with two species
(MS2 and BZ13) and Allolevivirus with two species (Q
β
and F1) [
34
]. All ssRNA phages have
small icosahedral virion encapsulating a linear, plus-strand RNA genome that encodes for
four proteins: the coat protein, the replicase, the maturation protein, and the lysis protein
(read-through protein in the case of Allolevirus) [
43
]. Similar to ssDNA phages, RNA phages
also utilize the host’s pili as their binding site, although instead of binding to the tip of the
pilus, both dsRNA and ssRNA phages bind to the side of the pilus to start their infection
cycle [
44
–
46
]. ssRNA phages bind to their hosts via its maturation protein [
47
], which also
interacts with the 3
0
end of the genomic RNA and enters the host cell together with the
genomic RNA following adsorption [48].
By 2019, only 12 genome sequences of ssRNA phages were available from the NCBI
database, leading to the assumption that this group of phages is scarce. Recent metagenomic
and metatranscriptomic studies, however, have revealed that ssRNA phages are much more
diverse and abundant than previously thought, expanding the number of known genomes
from this group from tens to over a thousand [
16
], resulting in their complete taxonomical
restructuring [
37
]. ssRNA phage sequences were detected on three continents, showing
their widespread global distribution. ssRNA phages are now ratified into the Leviviricetes
class, encompassing two orders (Norzivirales and Timlovirales) with six families, 428 genera,
and 882 species (https://ictv.global/msl (ICTV_Master_Species_List_2022_MSL38.v3.xlsx,
created 9 November 2023—11:17) (accessed on 26 November 2023)). It is safe to say that
RNA phages, including both dsRNA and ssRNA phages, are the fastest-growing group of
phages nowadays.
2.4. Current Opportunities and Challenges in Metaviromic Studies
Over the last decades, enabled by high-throughput sequencing technologies, an un-
precedented diversity of microbial and viral communities has been revealed from various
environmental and biological samples [
16
,
17
,
29
,
49
–
52
]. For a long time, however, sam-
ple preparation and analysis methods for metagenomic studies were mainly developed

Int. J. Mol. Sci. 2023,24, 17029 7 of 23
and optimized with a focus on dsDNA. This led to a significant underpresentation of
ssDNA samples. Similarly, metatranscriptomic studies of the RNA virome also face var-
ious challenges concerning sample preparation and analysis. Most metaviromic studies
proceed through the basic steps of isolating and concentrating viral samples, followed by
nucleic acid extraction and library preparation. Without careful consideration, biases can
be introduced in each step [
53
]. Szekely and colleagues [
22
] listed several potential biases
in sample preparation due to the fundamental differences between dsDNA and ssDNA
phages, identifying the two most prominent biases that could lead to underpresentation of
ssDNA phages: Different buoyant density and library amplification methods. The authors
recommended recovering viral samples at a lower buoyant density and preparing the
metagenomic library using multiple displacement amplification (MDA) method to ensure
better enrichment of ssDNA samples. There is also promise in the use of library preparation
kits that employ ssDNA as starting material to overcome biases from the MDA method.
Following nucleic acid extraction, metatranscriptomic samples often proceed with
ribosomal RNA (rRNA) removal and then complementary DNA (cDNA) generation by
reverse transcription. Subsequently, random primers are used to enrich the resultant cDNA
library, followed by adapter ligation and sequencing [
54
]. rRNA-reduced libraries are
shown to produce more viral contigs than polyadenylation-selected libraries [
55
], proving
their suitability for RNA phage samples. The problem of low abundancy and incomplete
dsRNA viral genome retrieval in metatranscriptomes was addressed by Urayama and
colleagues using their new sequencing method named fragmented and primer-ligated
dsRNA sequencing (FLDS) [
56
]. The remaining major bottleneck in metatranscriptome
analysis is the lack of reference sequences in less well-studied viral groups, such as bacterial
RNA viruses. To this end, the strategy employed by Neri and colleagues combined RNA-
dependent RNA polymerase (RdRP)-based discovery, matching bacterial and viral CRISPR
spacers, and identification of bacteriolytic proteins was shown to be effective [
52
]. Despite
multiple challenges, the field of metaviromic studies is expected to continue expanding
rapidly, unveiling broader and deeper knowledge of the virosphere, including the ssDNA
and RNA phageome.
3. Genetic Engineering of RNA and ssDNA Phages
RNA and ssDNA phages have been pivotal models in molecular biology since the
early days, playing a significant role in understanding genetic code, RNA translation, and
virus-host interactions [
57
]. These studies have paved the way for the development of
multiple practical applications such as bio-imaging [
58
], affinity purification [
59
], bacterial
detection and control [
60
], gene editing [
61
], gene and drug delivery, vaccine development,
and cancer treatment [
62
]. Most recently, the use of ssDNA and RNA phage-derived
components in synthetic biology, a younger branch of modern biotechnology that arose
roughly two decades ago, has led to the development of various useful tools such as DNA
origami [
63
], riboswitches [
64
], and multiple other therapeutic RNA modalities. In this
section, we highlight examples of the major contributions of ssDNA and RNA phages to
biotechnology (Table 2).
In addition to providing tools and technologies for modern biology, ssDNA and RNA
phages have also been employed to control bacteria and treat infections. Recent devel-
opments in phage engineering bring novel therapeutic values to phage therapy, creating
new prospects for its applications in medical treatments. Our group, among others, has
pioneered the development of phage-based RNA-cleaving antibacterials, encompassing
sequence-specific bacterial gene detection, selective elimination of drug-resistant bacteria,
and targeted manipulation of the bacterial flora [
65
]. Although not yet as widely utilized
as dsDNA phages, ssDNA and RNA phages also make substantial contributions to an-
timicrobial therapy, a topic we will delve into in the next section, along with our related
research results.

Int. J. Mol. Sci. 2023,24, 17029 8 of 23
Table 2. Tools and technologies based on genetic engineering of ssDNA and RNA phages.
Phage Components 1Tools and Technologies Applications 1Reference(s)
ssDNA phages
Phage display Library screening [66–69]
Cancer treatment [70–72]
Cell adhesive substrates using
electrospinning Drug delivery [73–75]
Carbon nanotubes Biosensing and imaging [76–81]
dsRNA phages dsRNA production RNAi-based crop protection [82]
Surrogate model dsRNA virus research [83–85]
CP and TR of ssRNA phages
Protein–RNA tethering
Single tagging In vivo RNA imaging [58,86]
Riboswitch screening [64]
Dual tagging In vivo
two-color RNA imaging
[87–89]
Affinity purification
RNA–protein In vitro complex [59]
In vivo complex [90]
RNA–RNA In vivo complex [91]
CRISPR/Cas9-based gene regulation In vivo transcription activation [61,92]
Recombinant VLPs of ssRNA phages
Therapeutic display
Peptide Cell targeting and penetrating [93,94]
Glycan Cell targeting [95]
DNA aptamer Cell targeting [96]
Antibody Cell targeting [97]
Therapeutic delivery
RNAs Small interfering RNA delivery [98]
Toxins Protein toxin delivery [99]
Small molecules Chemotherapeutic delivery [100]
Antigen display Vaccine development [101,102]
RNA cargo RNA vaccine development [103]
Armored RNA RNA virus detection [104,105]
Nanoreactor Controlled enzymatic reaction [106,107]
1CP: coat protein, TR: translation repression, VLP: virus-like particles, RNAi: RNA interference.
3.1. Engineering ssDNA Phages
Among ssDNA phages, filamentous ssDNA M13 phage is commonly employed for
practical and therapeutic applications, utilizing engineering platforms such as genetic
engineering for phage display, electrospinning for bionanomaterial development, and
phage-directed nanomaterial combinations [
75
]. Phage display, a genetic engineering pro-
cess, involves the insertion of foreign peptide coding sequences into phage capsid genes,
resulting in the display of corresponding peptides on the phage surface [
66
–
68
]. Libraries
of displayed peptides are often constructed, followed by rounds of affinity selection (called
biopanning) to select the most suitable therapeutic candidates [
69
]. Using M13 phage,
phage display technology has been applied to identify cancer cell surface markers, leading
to the development of functional anticancer peptides for therapeutic treatment [
70
]. In this
study, Wang and colleagues used M13 phages to display the antitumor cytokine GM-CSF, a
potent activator of STAT5 signaling in macrophages of rodents, to target colorectal cancer

Int. J. Mol. Sci. 2023,24, 17029 9 of 23
cells. Post-GM-CSF M13 administration, the tumor size was significantly attenuated, and
the number of CD4+ lymphocytes increased. Combining GM-CSF M13 phage therapy and
radiation resulted in a 100% survival rate and a 25% complete mitigation rate in mice. In
another study, Jin and colleagues applied engineered M13 phage to selectively bind to
different types of collagens using collagen-mimetic peptide motifs. The engineered M13
phages could target and label abnormal collagens, which were then easily detected by
fluorescence imaging, enabling monitoring and diagnosis of various pathological condi-
tions [
72
]. Furthermore, in the case of cardiovascular disease, Lee and colleagues developed
an M13 phage-based double functional peptide-carrying system where RGD peptides (a
cell adhesive motif) were displayed in the pIII minor coat proteins to bind with integrin-
expressing cells for constructing an artificial niche [
108
]. The engineered M13 nanofiber
dramatically enhanced ischemic neovascularization by activating intracellular and extra-
cellular processes such as proliferation, migration, and tube formation in the endothelial
progenitor cells (EPCs) after transplanting M13-phage-treated EPCs into a mouse hindlimb
ischemia model.
“Electrospinning” is the construction technique of fibers ranging from nanometers
to micrometers by passing polymer solutions through a highly electrically charged en-
vironment to generate fibrous membranes. Shin and colleagues used electrospinning to
create hybrid nanofiber sheets from a mixture of poly-lactic-co-glycolic acid (PLGA) and
peptide-displaying M13 phages, generating an ideal cell-adhesive substrate [
73
]. These
nanofibers can be used as efficient drug delivery systems for various therapies, incorporat-
ing bioactive molecules such as antibiotics, anti-inflammatories, anti-cancer drugs, genomic
DNA, proteins, probiotics, and enzymes [74,75].
Phage-directed nanomaterial combinations involve compounds’ genetic and chemical
integration to create phage-directed fusion substances for biosensing and scaffold build-
ing [
79
]. Carbon nanotubes have been assembled on M13 phage surfaces for detecting
bacteria and tumors through fluorescence imaging [
76
,
77
]. Researchers constructed M13-
SWNT (single-walled carbon nanotube) conjugates and demonstrated comparable effects
on monitoring specific bacteria, prostate cancer, and ovarian cancer, respectively [
76
–
78
].
Combining M13 phage with metal nanoparticles has also been demonstrated to improve
fluorescence bioimaging for detecting bacteria [
80
] and cancer cells [
81
]. Dong and col-
leagues used silver nanoparticle-conjugated M13 phages that caused deconstruction of
bacterial cell walls and intracellular biomolecules, inducing oxidative stress that killed
pro-tumoral bacteria for gut microbiota regulation [
71
]. Thus, engineered M13 phage has
versatile applications in different aspects of bionanomaterial that is useful in drug delivery,
biodetection, tissue regeneration, and targeted cancer therapy.
3.2. Engineering dsRNA Phages
Phi6 has found utility in the production of antiviral dsRNA for crop protection [
82
].
Loss by crop pathogens and pests is estimated at around USD 100 billion annually, and
resistance against chemical pesticides is rising. A promising alternative approach is the
application of double-stranded RNA (dsRNA), which triggers RNA interference (RNAi), an
antiviral defense mechanism in eukaryotes that cleaves invading viral RNA genomes and
represses translation of viral transcripts [
109
]. Previously, antiviral dsRNAs were produced
through post-synthetic hybridization of
in vitro
or
in vivo
transcribed ssRNAs from DNA
templates, but this approach is costly and inefficient. By using the recombinant protein P2
of phi6, Makeyev and colleagues could efficiently produce double-stranded RNA
in vitro
from positive-sense RNA substrates [
110
]. Niehl and colleagues later developed an
in vivo
dsRNA production system in P. syringae using constructs carrying the phi6 replication
complex and different targets inserted into the phi6 genomic S (small) and M (medium)
segments, observing significant dsRNA-mediated inhibition of tobacco mosaic virus (TMV)
propagation in the tested disease model [
82
]. The established platform thus provides
an economical and efficient large-scale production of multiple long dsRNAs for various
therapeutic applications. Future research may uncover additional effective methods for

Int. J. Mol. Sci. 2023,24, 17029 10 of 23
producing dsRNA from the replication machinery of other dsRNA phages, a topic that has
not yet been thoroughly explored.
dsRNA phages have also been used as a surrogate model in studies of human
pathogenic RNA viruses such as coronavirus, SARS-CoV-2, influenza, and Ebola [
83
–
85
]
due to their similar size and structural organization. The non-enveloped ssRNA phage
MS2 and the ssDNA phage PhiX174 often served as additional controls in these studies.
With their segmented dsRNA genome, dsRNA phages share similarities with the family
Reoviridae, a diverse family consisting of plant, fungi, invertebrate, and vertebrate viruses.
Therefore, dsRNA phages provide opportunities to study the genome packaging and as-
sembly mechanisms of dsRNA-segmented genome viruses in a simple and easy-to-handle
prokaryotic system [38].
3.3. Engineering ssRNA Phages
Prominent applications of ssRNA phage involve various protein-RNA tethering sys-
tems and recombinant virus-like particles (VLPs) (see [
111
] for a detailed review). Protein-
RNA tethering systems were established by exploiting the distinctive ability of different
ssRNA phage coat proteins (CP) to recognize and bind to their specific cognate RNA stem
loop operators known as TR (translation repression) [
112
–
114
]. An efficient
in vivo
imaging
system for mRNA was developed by Tyagi, utilizing reporter-fused CPs and TR-tagged
RNAs [
86
]. This methodology has since been proven effective in tracking the processing,
import and export, localization, translation, and degradation of tagged mRNA [
58
]. Wu
and colleagues conducted a high-throughput experiment on 2875 sensor molecules by
utilizing the binding of fluorescently tagged MS2 CPs to the MS2 TR segment within
designed riboswitches. This approach, combined with the recently developed RNA on a
massively parallel array (RNA-MaP) platform, allowed for the testing of diverse riboswitch
designs [
64
]. Additionally, dual tagging systems have been devised, employing either fluo-
rescent protein fragments to reduce background signal [
87
,
88
] or two different fluorescent
proteins to enable two-colored labeling of a single-molecule RNA [89].
The CP–TR interaction was also employed to develop affinity purification methods
for RNA–protein [
59
] and RNA–RNA [
91
] complexes. Bardwell and colleagues utilized
resin-bound R17 CP to capture tandem-TR-tagged mRNA along with its binding factors.
Tsai and colleagues [
90
] implemented MS2 BioTRAP, incorporating histidine and biotin
(HB)-tagged MS2 CPs co-expressed with TR-tagged RNA targets, to capture and quantify
in vivo
interactions between target RNA and protein factors. A similar approach was
used to identify miRNA interactions with their target mRNAs [
91
], thus expanding the
application of CP as an in vivo RNA–RNA tether molecule.
The MS2 CP–TR tethering system has found application in CRISPR/Cas9 technology
for orthogonal gene knockout and transcriptional activation in human cells [
61
]. Dahlman
and colleagues achieved this by incorporating the MS2 TR loops into the shortened sgRNA,
effectively inhibiting Cas9
0
s nuclease activity while preserving its binding to the target
promoter sequence. This strategic manipulation facilitated recruitment of the transcription
activator VP64 through MS2 TR loops, resulting in potent upregulation of gene expression.
Beyond its role in simultaneous gene knockout and transcriptional activation, this plat-
form has also been employed for genome-scale gain-of-function screening, particularly in
studying drug resistance in a melanoma model [92].
Recombinant VLPs of ssRNA phages can be generated by expressing the CP gene in
bacteria or yeast [
115
,
116
]. Due to their single protein composition, these VLPs offer a more
straightforward engineering process compared to most VLPs derived from other phages
and viruses. They can be decorated with peptides [
93
,
94
], glycans [
95
], DNA aptamers [
96
],
or antibodies [
97
] with cell-targeting and cell-penetrating capabilities. These decorated
VLPs, in turn, serve as carriers for therapeutic agents such as RNAs [
98
], toxins [
99
],
nanoparticles, or small molecules [
100
] directed towards desired targets. In the realm of
vaccine development, VLPs play a dual role—displaying immunogenic components on
their surface [
101
] or carrying them internally [
103
]. Moreover, VLPs themselves contribute

Int. J. Mol. Sci. 2023,24, 17029 11 of 23
as adjuvants thanks to their potent immunogenic properties (for an up-to-date review of
RNA phage VLP-based vaccine platforms, refer to [102]).
Two other noteworthy applications employing ssRNA phage VLPs are armored
RNA [
104
] and nanoreactors [
106
,
107
]. In the first application, VLPs serve as protective
cages, safeguarding control RNAs in various RT-qPCR-based RNA virus detection systems
from unwanted RNase degradation [
105
]. In the second application, VLPs act as carriers
for encapsulating functional enzymes, facilitating controlled biochemical reactions [
107
].
The commonly used strategy for packaging therapeutic cargoes within ssRNA phage VLPs
involves the conjugation with the TR RNA stem loop, achieved either through genetic
engineering in the case of RNA cargoes [
104
] or through chemical conjugation methods for
cargoes of different natures [106].
3.4. Techniques for Genetic Engineering of ssDNA and RNA Phages
The continuous evolution of technology to manipulate phages for addressing health-
care and economic challenges is expected. Engineered phages hold promise for carrying
exogenous DNA or RNA, as well as displaying functional molecules for application in
disease treatment and prevention, imaging and detection, biomaterial development, and
diverse delivery systems. In this section, we will briefly outline the fundamental tech-
niques commonly employed in phage engineering. These include recombinant expression
of phage-derived products, engineering phage genomes through homologous and non-
homologous recombination, CRISPR/Cas-based phage genome editing, and synthetic
rebooting of phage-like particles. Additionally, we will delve into recently developed
techniques for engineering RNA phages, as illustrated in Figure 1.
Recombinant expression of phage proteins is typically conducted in natural host
cells or closely related counterparts. Unlike dsDNA phages, ssRNA phage VLPs exhibit
self-assemble capabilities when recombinant coat proteins are present alongside suitable ge-
nomic DNA or RNA fragments. Therefore, a dual-plasmid system is commonly employed
for co-expressing the phage’s coat protein and copies of relevant viral genomic segments.
This approach facilitates the generation of diverse functional phage particles, allowing the
insertion of desired cargoes within the genomic fragment or their display on the virion
surface (for a comprehensive review, refer to [117]).
Thanks to the small genome size, engineering of ssDNA and RNA phage genomes can
be performed through standard molecular cloning and plasmid transformation techniques
into host cells. The reverse genetic system for RNA viruses uses complementary DNA
(cDNA) to introduce desired mutations into the RNA genomes [
118
]. Modifications of
cDNA, serving as templates for phage protein synthesis and RNA replication, in turn
allow for studies of the viral life cycle and engineering of these viruses for practical
applications [
119
,
120
]. For dsRNA phages such as phi6 and phi8, a carrier state can be
established by electroporating non-replicative plasmids containing cDNA copies of viral
genomes into host cells [
121
]. This construction allows for the incorporation of marker
genes such as kan or lac
α
inside the phage virions. Phagemids, carrying phage origin of
replication and packaging sites, are efficiently replicated and packaged by suitable phage
machinery provided in trans from a separate helper phage [
122
]. Phagemids are widely
employed in generating both dsDNA and ssDNA phage particles, encapsulating and
displaying exogenous therapeutic elements [123,124].

Int. J. Mol. Sci. 2023,24, 17029 12 of 23
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 10 of 22
such as kan or lacα inside the phage virions. Phagemids, carrying phage origin of replica-
tion and packaging sites, are efficiently replicated and packaged by suitable phage ma-
chinery provided in trans from a separate helper phage [122]. Phagemids are widely em-
ployed in generating both dsDNA and ssDNA phage particles, encapsulating and dis-
playing exogenous therapeutic elements [123,124].
Figure 1. Strategies to engineer RNA phages for practical and therapeutic purposes. (1) Co-expres-
sion of phage coat proteins and suitable RNA templates from transformed plasmids. Recombinant
VLPs can be assembled through the direct interaction between coat proteins and RNA molecules
[125]. (2) RNA recombination through co-infection. When two ssRNA genomes replicate (step ①),
template switching can occur, leading to the production of hybrid genomes (step ②) and subse-
quently, hybrid phages [126]. A similar recombination phenomenon can occur during dsRNA phage
replication (step ③), after which genome reassortment can lead to various combinations of pack-
aged genomic segments (step ④) [127]. (3) Programmable RNA editing using guide RNA (crRNA)
and CRISPR-guided nuclease (CRISPR-Cms) cleavage followed by RNA ligase-mediated strand re-
pair in the presence of a synthetic DNA splint (adapted from [128]). (4) Synthetic platform for the
production of viral VLPs from metaviromic data. Predicted genes encoding coat proteins are chem-
ically synthesized, cloned, and transformed into bacterial hosts. Successful expression will lead to
the assembly and release of virus-like particles [129].
Natural recombination, whether homologous or heterologous, is frequently observed
in positive-sense single-stranded RNA viruses, including ssRNA phages. Viable hybrid
progenies between different RNA phage species can be generated through a two-plasmid
Figure 1.
Strategies to engineer RNA phages for practical and therapeutic purposes. (1) Co-expression
of phage coat proteins and suitable RNA templates from transformed plasmids. Recombinant VLPs
can be assembled through the direct interaction between coat proteins and RNA molecules [
125
].
(2) RNA recombination through co-infection. When two ssRNA genomes replicate (step
1

), template
switching can occur, leading to the production of hybrid genomes (step
2

) and subsequently, hybrid
phages [
126
]. A similar recombination phenomenon can occur during dsRNA phage replication
(step
3

), after which genome reassortment can lead to various combinations of packaged genomic
segments (step
4

) [
127
]. (3) Programmable RNA editing using guide RNA (crRNA) and CRISPR-
guided nuclease (CRISPR-Cms) cleavage followed by RNA ligase-mediated strand repair in the
presence of a synthetic DNA splint (adapted from [
128
]). (4) Synthetic platform for the production of
viral VLPs from metaviromic data. Predicted genes encoding coat proteins are chemically synthesized,
cloned, and transformed into bacterial hosts. Successful expression will lead to the assembly and
release of virus-like particles [129].
Natural recombination, whether homologous or heterologous, is frequently observed
in positive-sense single-stranded RNA viruses, including ssRNA phages. Viable hybrid
progenies between different RNA phage species can be generated through a two-plasmid co-
transformation system into bacterial hosts [
130
].
In vitro
recombination of non-replicating
genomic RNA fragments with overlapping ends in the presence of Q
β
replicase has been
observed, resulting in replicative Q
β
genomes [
131
]. However, it is important to note
that recombination in RNA viruses often yields multiple outcomes, reducing the precision

Int. J. Mol. Sci. 2023,24, 17029 13 of 23
of modification. A technique known as CRE-REP, established by Lowry and colleagues,
utilizes well-defined non-viable “donor” and “receptor” RNA genomic fragments to enable
the selection of viable recombinants from cell-based recombination upon co-transfection.
This approach has significantly improved the detection and quantification of recombination
events [
132
]. Originally developed for poliovirus, the technique has been adapted for use
with other viruses [133,134].
In September 2023, Nemudryi and colleagues unveiled an innovative CRISPR/Cas-
based RNA editing technique facilitating rapid and programmable deletions, insertions,
and substitutions in RNA without the need for DNA intermediates [
128
]. By combining
type III CRISPR/Cas-based RNA cleavage with splinted RNA ligation, the authors achieved
targeted modifications in RNA sequences. This method, free from DNA intermediates,
offers a workaround for the reliance on known hosts, a prevalent bottleneck in the study
and engineering of most RNA phages. This platform holds great potential for robust
engineering of a wide range of RNA viruses with precision and efficiency.
Recently, chemical synthesis of novel phage sequences or genomes from metavi-
rome data, followed by cloning and heterologous expression, or rebooting, has become a
powerful technique to study and engineer the richly diverse novel RNA phages without
extensive prior knowledge. For instance, Lieknina and colleagues synthesized, cloned, and
overexpressed 110 coat protein genes from 150 novel, uncultured ssRNA phage sequences
identified from metagenomic data. This effort successfully yielded 80 assembled VLPs [
129
].
The obtained VLPs exhibited variations in size, shape, stability, and assembly temperature,
with some demonstrating specific interactions with different candidate RNA structures.
This synthetic platform can be applied to virtually all annotatable protein sequences of
RNA phages retrieved from the metaviromic data. Furthermore, rebooted dsRNA phages
from synthetic cDNA clones have been used to broaden the host range of the studied
phages [
135
], presenting an additional avenue for engineering RNA phages with flexibility
and robustness for diverse purposes.
4. Current Applications of RNA and ssDNA Phages as Therapeutic Agents against
Multi-Drug Resistant Bacteria
Antibiotics play a crucial role in treating bacterial infections, but their overuse and
misuse have led to the emergence of antimicrobial resistance (AMR), resulting in reduced
drug efficiency and persistent infections. Given the rapid evolution of AMR and the
challenges of developing novel drugs, exploring alternative strategies becomes impera-
tive. Phage therapy, utilizing phage-derived products and both natural and engineered
phages for infection treatment, along with infection prevention through vaccines targeting
antimicrobial-resistant pathogens, stands out as a promising frontier in the battle against
AMR [
136
]. In the following sections, we discuss the contribution of ssDNA and RNA
phages as antibacterial therapeutics, highlighting their advantages and unique features
(see Figure 2).
4.1. Phage-Derived Lytic Enzymes as Antibacterial Agents
Phage-derived lysins, also known as “enzybiotics,” present a promising alternative to
conventional antibiotics. These enzymes exert their antibacterial effects by lysing host bac-
teria through the cleavage of peptidoglycan (PG), the primary structural component of the
bacterial cell wall. Phage-derived lytic enzymes fall into two categories: virion-associated
peptidoglycan hydrolases (VAPGHs) and endolysins. VAPGHs initiate degradation of PG
at the onset of the phage infection [
137
,
138
], whereas endolysins act at the conclusion of
the phage infection cycle, facilitating the release of mature phages [
139
]. In most cases,
dsDNA phages encode distinct VAPGHs and endolysins, whereas dsRNA phages feature a
single lytic protein that serves as both VAPGH and endolysin [
140
]. In contrast, ssDNA
and ssRNA phages employ a single-gene lysis (Sgl) protein—an impactful degradative
protein that induces cytolysis without enzymatically breaking down PGs [141,142].

Int. J. Mol. Sci. 2023,24, 17029 14 of 23
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 12 of 22
cases, dsDNA phages encode distinct VAPGHs and endolysins, whereas dsRNA phages
feature a single lytic protein that serves as both VAPGH and endolysin [140]. In contrast,
ssDNA and ssRNA phages employ a single-gene lysis (Sgl) protein—an impactful degra-
dative protein that induces cytolysis without enzymatically breaking down PGs [141,142].
Figure 2. Strategies for phage therapy using ssDNA and RNA phages and their products. (1) Utili-
zation of phage-derived components as antibacterial agents. For instance, the degradation of bacte-
rial cell wall by dsRNA phage lytic enzyme [143] or inhibition of cell wall synthesis using single-
gene lysis (Slg) protein from ssDNA and ssRNA phages [141,142,144]. (2) Phage cocktail employing
natural ssDNA and RNA phages as antimicrobial therapeutics against AMR bacteria and phage-
resistant mutants [145,146]. (3) Engineering ssDNA and RNA phages for antibacterial therapies. Ex-
amples include ① engineered phages displaying specific antigens as vaccine against drug-resistant
bacteria [147,148]; ② engineered phages to enhance antibiotic efficacy in phage-antibiotic combina-
tory therapy [149]; ③ engineered antibacterial capsids carrying CRISPR/Cas13a for sequence-spe-
cific-based bacterial gene detection, bacterial flora modification, and treatment for AMR bacterial
infections [65]. Abx: antibiotic.
Ply17, a lytic enzyme produced by Pseudomonas dsRNA phage phiYY, has demon-
strated the capability to reduce the number of viable Gram-negative bacteria such as P.
aeruginosa and E. coli, as well as Gram-positive bacteria including S. aureus and S. epider-
midis, by approximately 2 logs when treated with an outer membrane permeabilizer such
as EDTA [143]. Notably, recent genome analyses of the animal dsRNA viruses from the
Picobirnaviridae and Partitiviridae families have unveiled potential antibacterial and anti-
fungal lysis genes [52]. Upon cloning and expression of the lytic genes from Picobirnavirus
and Partitivirus in E. coli DH5a, the observed growth inhibition was comparable to that
induced by the Enterobacteria MS2 phage lysin L [150].
The bacteriolytic activity of ssRNA Leviviridae phages varies among different phages.
For example, Sgl
M
of phiM and Sgl
PP7
of Pseudomonas phage PP7 exhibit bacteriolytic ac-
tivity by inhibiting MurJ, the transporter responsible for moving lipid-binding pepti-
doglycan precursors from the inside to the outside of the plasma membrane [141,142]. On
Figure 2.
Strategies for phage therapy using ssDNA and RNA phages and their products. (1) Utiliza-
tion of phage-derived components as antibacterial agents. For instance, the degradation of bacterial
cell wall by dsRNA phage lytic enzyme [
143
] or inhibition of cell wall synthesis using single-gene
lysis (Slg) protein from ssDNA and ssRNA phages [
141
,
142
,
144
]. (2) Phage cocktail employing natural
ssDNA and RNA phages as antimicrobial therapeutics against AMR bacteria and phage-resistant mu-
tants [
145
,
146
]. (3) Engineering ssDNA and RNA phages for antibacterial therapies. Examples include
1

engineered phages displaying specific antigens as vaccine against drug-resistant bacteria [
147
,
148
];
2

engineered phages to enhance antibiotic efficacy in phage-antibiotic combinatory therapy [
149
];
3

engineered antibacterial capsids carrying CRISPR/Cas13a for sequence-specific-based bacte-
rial gene detection, bacterial flora modification, and treatment for AMR bacterial infections [
65
].
Abx: antibiotic.
Ply17, a lytic enzyme produced by Pseudomonas dsRNA phage phiYY, has demon-
strated the capability to reduce the number of viable Gram-negative bacteria such as P.
aeruginosa and E. coli, as well as Gram-positive bacteria including S. aureus and S. epider-
midis, by approximately 2 logs when treated with an outer membrane permeabilizer such
as EDTA [
143
]. Notably, recent genome analyses of the animal dsRNA viruses from the
Picobirnaviridae and Partitiviridae families have unveiled potential antibacterial and antifun-
gal lysis genes [
52
]. Upon cloning and expression of the lytic genes from Picobirnavirus and
Partitivirus in E. coli DH5a, the observed growth inhibition was comparable to that induced
by the Enterobacteria MS2 phage lysin L [150].
The bacteriolytic activity of ssRNA Leviviridae phages varies among different phages.
For example, Sgl
M
of phiM and Sgl
PP7
of Pseudomonas phage PP7 exhibit bacteriolytic activ-
ity by inhibiting MurJ, the transporter responsible for moving lipid-binding peptidoglycan
precursors from the inside to the outside of the plasma membrane [
141
,
142
]. On the other
hand, Sgl
Qβ
from Q
β
phage exhibits bacteriolytic activity by non-competitively inhibiting
MurA, the first enzyme in the PG biosynthetic pathway [
151
]. MS2 Sgl
MS2
(lysin L) pos-
sesses an N-terminal heat shock-responsive chaperone, DnaJ, but its specific mechanism
of action remains unclear. Other known Sgls include Sgl
PRR1
(Pseudomonas phage PRR1),

Int. J. Mol. Sci. 2023,24, 17029 15 of 23
Sgl
KU1
(Enterobacteria phage KU1), and Sgl
Hgal1
(Enterobacteria phage Hgal1), whose
mechanisms of action have not yet been elucidated [142].
Sgl
ϕ174X
, a bacteriolytic enzyme produced by the ssDNA phage phiX174, inhibits
MraY, an enzyme catalyzing the initial step of the lipid cycle reaction in PG biosynthesis.
This inhibition results in bacteriolysis [
142
,
144
]. As the cellular targets and modes of action
of many ssRNA and ssDNA phage Slgs are still unidentified, future genome analyses hold
great capacity for the discovery of new therapeutic candidates.
4.2. Natural ssDNA and RNA Phages as Antibacterial Agents
Pseudomonas phages phiYY [
42
] and phiZ98 [
152
], which belong to the dsRNA Cys-
toviridae family, utilize the lipopolysaccharide (LPS) core oligosaccharide of P. aeruginosa as
the binding receptor. Smooth-colony-type P. aeruginosa strains exhibit resistance to phiYY
due to the presence of the galU gene, which confers O-antigen to the LPS core oligosac-
charide [
145
]. However, LPS-deficient P. aeruginosa, a strain that emerged during dsDNA
phage therapy and lacks the galU gene [
153
], exposes its LPS core oligosaccharide on the
cell surface, making it susceptible to phiYY [
145
]. Phage therapy employing phiYY has been
reported to reduce bacterial load and alleviate clinical symptoms in patients diagnosed
with interstitial lung disease (ILD) and chronic lung infections [
146
]. Moreover, a phage
cocktail, including phiYY among five phages, has demonstrated effectiveness against a
broad spectrum of P. aeruginosa clinical isolates and significantly impedes the emergence of
phage-resistant mutants [145].
dsRNA phages have also been effectively applied as plant therapeutics. Pseudomonas
phage phi6 has been employed to manage various plant diseases, including controlling
P. syringae pv. phaseolicola
(Pph), the causal agent of halo disease in common bean (Phaseolus
vulgaris) [
154
]. Additionally, phi6 has been utilized against P. syringae pv. actinidiae (Psa),
responsible for kiwifruit psyllid [
155
], and P. syringae pv. syringae (Pss), which causes
early leaf symptoms of bean brown spot [
154
]. Other Pseudomonas phages, including phi8,
phi12, phi13, phi2954, phiNN, and phiYY, have also shown efficacy against Pph [
156
].
Collectively, the application of dsRNA phages holds significant promise for complementing
and enhancing phage therapy for both human and plant diseases.
4.3. Engineering ssDNA and RNA Phages for Antimicrobial Therapy
RNA phages exhibit significant potential as antigen carriers in the development of
vaccines targeting drug-resistant pathogens. Huo and colleagues pioneered the develop-
ment of Q
β
phage presenting Q
β
-glycan 1, a synthetic tetrasaccharide from Salmonella
enteritidis. This construct successfully induced robust IgG antibody responses in both mice
and rabbits [
147
]. Notably, carbohydrate-based antigens, such as tetrasaccharide, do not
directly interact with helper T cells, leading to limited immune memory. However, the
conjugation of glycan antigens to a potent carrier such as Q
β
enables engagement with
both T-helper and B cells, resulting in a vigorous antibody response. Mice treated with
post-immunization serum recovered from rabbits vaccinated with Q
β
-glycan 1 demon-
strated increased survival rates following administration of lethal doses of S. enteritidis. In
another study, Rashidijahanabad and colleagues reported the production of Q
β
-OSP, a Q
β
phage presenting O-specific polysaccharide (OSP) 1 of Vibrio cholerae. Employed in mouse
immunization, the Q
β
-OSP conjugate elicited strong IgG antibody responses against V.
cholerae O1 Inaba, reaching sufficient IgG antibody levels after just two administrations.
Remarkably, the titer of IgG antibodies remained detectable up to day 265 [148].
Phagemid vectors derived from the filamentous ssDNA M13 phage serve as a highly
efficient engineering platform for diverse therapeutic applications, including engineering
phages for enhanced antibacterial activity. The delivery of antibacterial therapeutics can
be achieved either through surface display on the virion or incorporated into the M13
phagemid itself. These displayed therapeutics may target vital cell components, virulent
factors, or entire bacterial pathogens. Notably, several potential candidates have been
identified for combating S. aureus,P. aeruginosa,H. pylori, and other pathogens. In another

Int. J. Mol. Sci. 2023,24, 17029 16 of 23
example, the dual display of two functional peptides enables M13 to (1) undergo endo-
cytosis by eukaryotic cells and (2) impede infection caused by the intracellular pathogen
Chlamydia trachomatis [
157
]. For an in-depth exploration of anti-infective development
using phage display, refer to [158].
Antibacterial efficacy can also be achieved through cargoes loaded onto M13 phagemid.
Lu and Collins cleverly designed an indirect antibacterial method using M13 phages
overexpressing lexA3, a repressor of the SOS DNA repair system [
149
]. The authors
demonstrated that suppressing the SOS network in E. coli with engineered lexA3-M13
significantly enhanced quinolone killing and increased the survival of infected mice. The
engineered phage holds promise for targeting antibiotic-resistant bacteria and biofilm cells
and modulating the bacterial population resistant to antibiotics post-treatment. In a parallel
approach, Prokopczuk and colleagues employed a filamentous phage Pf for an indirect
strategy to interfere with P. aeruginosa pathogenesis at burn wound sites [
159
]. Given that P.
aeruginosa is a major cause of burn-related infections and sepsis, often exhibiting multi-drug
resistance, the authors engineered a superinfective Pf phage (eSI-Pf). Administration of
eSI-Pf effectively attenuated P. aeruginosa virulence, reduced bacterial load at the wound
site, minimized bacterial dissemination from the burn site to internal organs, alleviated
septicemia symptoms, and ultimately improved mouse survival.
Direct bactericidal effects have been achieved through several successful therapies,
including the delivery of programmed CRISPR/Cas by recombinant M13 phages. In a proof-
of-concept study on phage therapy for strain-specific depletion and genomic deletions in the
gut microbiome, Lam and colleagues engineered M13 to deliver gfp-targeting CRISPR/Cas9
to bacteria residing in the gastrointestinal tract [
160
]. The engineered phage showed efficient
sequence-specific targeting of GFP-expressing E. coli in the gut, confirming the capability of
CRISPR/Cas9 to induce genomic deletions at the designated target site.
In our laboratory, utilizing M13 phagemids, we spearheaded the development of
a diverse range of CRISPR/Cas13a-based, sequence-specific antibacterial nucleocapsids,
referred to as CapsidCas13a(s) [
65
]. Our research showcased the versatility of M13-based
CapsidCas13a(s) in applications such as (1) bacterial gene detection, (2) modification of
bacterial flora, and (3) therapeutics for AMR bacterial infections. In the first instance,
we developed M13-based EC-CapsidM13Cas13a::KanR(s) for the detection of different
genotypes of carbapenem resistance genes (bla
IMP-1
,bla
OXA-48
, and bla
VIM-2
). In the pres-
ence of corresponding target genes, CRISPR/Cas13a will be activated, and the subsequent
non-specific RNase activity will result in host cell death. We observed specific activity
of CapsidCas13a against target genes on both plasmids and chromosomes. In the sec-
ond example, we demonstrated the potential of CapsidCas13a as a tool for modifying
bacterial flora. Individual treatment of nontargeting, bla
IMP-1
-, or mcr-2-targeting Cap-
sidCas13a with a mixed cell population of E. coli NEB5
α
F
0
I
q
(control) and NEB5
α
F
0
I
q
expressing bla
IMP-1
and mcr-2 at equal numbers resulted in a reduction in the corresponding
target cell population from over 34% to less than 4%. In the last example, we examined
the therapeutic effect of EC-CapsidCas13a-bla
IMP-1
using a Galleria mellonella infection
model. Administration of EC-CapsidCas13a-bla
IMP-1
at MOI 100 to G. mellonella larvae
infected with R10-61 (carbapenem-resistant clinical isolates of E. coli carrying bla
IMP-1
)
significantly improved host survival compared to controls, EC-CapsidCas13a-nontargeting,
and phosphate-buffered saline (PBS). Our work underscores the feasibility of harnessing
phages as antibacterial therapeutics for diverse applications to control AMR bacterial
infections and bacterial flora.
5. Concluding Thoughts and Future Outlook
Despite enormous advancements in biotechnology in the last century, nature’s true
diversity and richness remain largely unexplored. Regarding ssDNA and RNA phages,
their uncharted biodiversity is a rich resource awaiting to be uncovered. With the help of
precision gene editing technology and synthetic biology, modern phage therapy is distinct
from the once-forgotten conventional phage therapy in their usage of rationally designed

Int. J. Mol. Sci. 2023,24, 17029 17 of 23
engineered phages with enhanced properties. Engineered phages, benefiting from their
simplicity and compatibility with prokaryotic hosts, offer a more cost-effective and faster
production alternative compared to various systems, including eukaryotic viral ones.
The small, simple genomes of most RNA and ssDNA phages are amenable to genetic
engineering, presenting the potential to overcome transformation barriers. This simplicity
proves advantageous in synthetic engineering, with cell-free transcription-coupled transla-
tion (TX-TL) expected to operate efficiently. Despite these promising aspects, ssDNA and
RNA phages face technical hurdles limiting their use in antimicrobial therapy. A significant
challenge lies in the absence of an efficient isolation and culture method, stemming from
our limited understanding of their natural hosts. The non-lytic chronic infection life cycle
of certain ssDNA and RNA phages also poses challenges in developing isolation assays,
hindering the use of conventional plaque assays. Additionally, computational approaches
are needed to detect virus sequences in whole-genome shotgun sequencing data due to the
low number of known shared genes between viruses.
On the positive side, ssDNA and RNA phages possess distinct features that make
them valuable as antibacterial agents, particularly in combination therapy with dsDNA
phages. Their unique host range, employing distinct receptors for host binding and entry,
sets them apart. Their genetic nature, ssDNA or RNA, renders them immune to many
dsDNA-targeting host defenses. Furthermore, they are likely to engage different host
factors than dsDNA phages during their infection cycle, paving the way for therapeutic
cocktails that simultaneously target multiple vital host components.
The untapped biodiversity of ssDNA and RNA phages, coupled with their simplicity
and genetic amenability, positions them as valuable resources for future biotechnological
and medical developments. Their unique features, including distinct host receptors and
immunity to certain host defenses, open avenues for tailored therapeutic cocktails and
innovative engineering strategies. In navigating this uncharted territory, the impact of
RNA and ssDNA phages is poised to expand, offering solutions to current and emerging
challenges. In this review, we underscore the importance of exploring and harnessing the
diversity of nature, providing inspiration for future endeavors in synthetic biology and
phage therapy. With ongoing advancements in precision gene editing and synthetic biology,
the potential applications of these phages are limited only by our imagination, and the
journey of exploration continues to unfold.
Author Contributions:
Conceptualization, H.M.N., S.W. and L.C.; writing—original draft prepara-
tion, H.M.N., S.S. and T.K.; writing—review and editing, H.M.N., S.W., X.-E.T., D.L.W. and L.C.;
supervision, S.W. and L.C.; funding acquisition, H.M.N., S.W. and L.C. All authors have read and
agreed to the published version of the manuscript.
Funding:
This work was supported by the Takeda Science Foundation (to H.M.N.), the JSPS KAK-
ENHI (Grant No. 22K19386 to S.W.), the Japan Agency for Medical Research and Development
(Grant No. JP21fk0108497 to S.W., JP21ae0121045, JP22ae0121045, JP23ae0121045, JP21gm1610002,
JP22gm1610002, JP23gm1610002, and JP22fk0108134 to L.C.), and Cabinet Office, Government of
Japan (Grant No. JPJ009237 to L.C.).
Conflicts of Interest: The authors declare no conflict of interest.
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