Available online at www.sciencedirect.
com Current Opinion in
ScienceDirect
Advancing the design and delivery of CRISPR
antimicrobials
Jennie R. Fagen, Daphne Collias, Atul K. Singh and
Chase L. Beisel
Abstract
CRISPR-Cas systems are prokaryotic immune systems whose
RNA-guided nucleases have been co-opted for applications
ranging from genome editing and gene regulation to in vitro
diagnostics and DNA imaging. Here, we review the current
efforts toward repurposing CRISPR nucleases as program-
mable antimicrobials. Antimicrobial activity is achieved by
targeted cleavage of multidrug-resistance plasmids or the
bacterial chromosome, resulting in antibiotic sensitivity or cell
death. As part of the review, we discuss the different types of
nucleases available for CRISPR antimicrobials, the use of
bacteriophages as delivery vehicles, and opportunities to
enhance antimicrobial activity, delivery, and specificity.
Through further advances, these programmable DNA-targeting
antimicrobials may help quell the spread of antimicrobial
resistance and provide a tool for the manipulation of complex
microbial communities.
Addresses
Department of Chemical and Biomolecular Engineering, North Car-
olina State University, Raleigh NC 27695, USA
Corresponding author: Beisel, Chase L. (cbeisel@ncsu.edu)
Current Opinion in Biomedical Engineering 2017, 4:57–64
This review comes from a themed issue on Synthetic Biology and
Biomedical Engineering
Edited by Charlie A. Gersbach
Received 29 June 2017, revised 29 September 2017, accepted 2
October 2017
https://doi.org/10.1016/j.cobme.2017.10.001
2468-4511/© 2017 Elsevier Inc. All rights reserved.
Keywords
Bacteriophage, Cas9, CRISPR-Cas systems, sgRNA, Tail-fiber
proteins.
Introduction
CRISPR-Cas systems are adaptive immune systems in
bacteria and archaea whose CRISPR nucleases and
guide RNAs have formed the foundation of powerful
tools in biotechnology and medicine. The guide RNAs
direct the nuclease to bind and cleave complementary
DNA or RNA sequences often flanked by a protospacer-
adjacent motif (PAM). The ease of designing guide
RNAs, the portability of the nuclease-guide RNA pair,
and the broad applicability of programmable DNA
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Biomedical Engineering
binding and cleavage has given rise to a diverse set of
applications ranging from genome editing and gene
regulation to in vitro diagnostics, real-time DNA and
RNA imaging, and gene drives [1,2].
The standard mechanism of CRISPR-based genome
editing is the directed repair of cleaved DNA. In
eukaryotic cells, the cleaved DNA is efficiently repaired
through ubiquitous mechanisms such as homology-
directed repair or non-homologous end joining. How-
ever, in bacteria, multiple studies have indicated that
DNA cleavage by CRISPR nucleases often cannot be
repaired and is therefore lethal. One line of evidence is
the dearth of naturally occurring genome-targeting
spacers in active CRISPR-Cas systems, even though
incorporation of such sequences regularly occurs during
naive spacer acquisition [3,4]. Separately, the intro-
duction of genome-targeting guide RNAs in bacteria
with an active CRISPR-Cas system arrested the cell
cycle, leading to eventual death or loss of large genomic
segments containing the target sequence [5,6]. These
insights helped inspire the use of CRISPR nucleases as
programmable antibacterial agents that could program-
mably and irreversibly destroy targeted DNA only in
selected bacteria. This mechanism has been developed
primarily to address the rising challenge of antibiotic
resistance and the diminishing supply of new small-
molecule drugs [7]. Here, we review how CRISPR nu-
cleases have been harnessed as programmable antimi-
crobial agents to combat human disease and the rise of
antibiotic resistance, and we discuss opportunities for
the further development of this promising antimicrobial
strategy.
CRISPR nuclease selection
CRISPR-Cas systems commonly act as immune systems
by cleaving foreign DNA or RNA, yet a diversity of
system types have been reported (Figure 1A) [8e10].
These system types are differentiated based on the
associated proteins responsible for acquisition and
targeting as well as their modes of action. For instance,
Type II systems generate blunt-ended cuts in target
DNA, Type III systems synergistically cleave target
RNA and DNA, and Type VI systems cleave target RNA
followed by the non-discriminate cleavage of other
RNAs [10e12]. Given that CRISPR-Cas systems are
present in roughly half of bacteria, these endogenous
systems could be co-opted as antimicrobials through the
delivery of a guide RNA targeting the bacterial genome
Current Opinion in Biomedical Engineering 2017, 4:57–64
58 Synthetic Biology and Biomedical Engineering
Figure 1
CRISPR antimicrobial design. (A) Diversity of CRISPR-Cas systems with their respective nucleic-acid targets and mechanisms of attack. Each
displayed PAM is from a representative, well-characterized system. Type IV systems have not been experimentally characterized and are therefore
excluded here. (B) Potential accessory factors for augmenting CRISPR antimicrobials by blocking DNA break repair.
[5,13]. However, the utility of this approach is limited to
bacteria with functionally active CRISPR-Cas systems
and is sensitive to the expression and genetic stability of
the CRISPR-Cas locus. A more robust and self-
contained antimicrobial design consists of introducing
the designed guide RNA and the CRISPR nuclease, the
strategy used for virtually all CRISPR antimicrobials to-
date.
While each type of nuclease has potential as an anti-
microbial agent, the primary nuclease of choice has been
Cas9, the effector protein from Type II CRISPR-Cas
systems. Cas9 is the most thoroughly investigated
RNA-guided nuclease and has been the trailblazer for
current CRISPR technologies [14e18]. Cas9 has also
been the primary nuclease used in CRISPR antimicro-
bials, where it has been successfully used to target
antibiotic-resistance genes encoded on plasmids or in
the genome [19,20] as well as essential and non-
essential chromosomal genes [5,13,21,22].
One critical feature of Cas9 is that it generates a blunt-
end double-stranded break that is amenable to DNA
repair, potentially compromising Cas9 as an
Current Opinion in Biomedical Engineering 2017, 4:57–64
Current Opinion in Biomedical Engineering
antimicrobial. Cui and Bikard directly reported this
phenomenon in Escherichia coli, where cleavage events
were often repaired by homologous recombination with
extra copies of the genome [21]. The repair was
dependent on RecA and allowed the cells to survive
attack when Cas9 targeted a broad range of non-
essential and even a few essential genes. What remains
unclear is why some sites led to potent killing and
whether repair will be a barrier to Cas9-based antimi-
crobials in other bacteria. Non-specific cytotoxicity from
Cas9 has also been reported [23,24], raising concerns
about potential non-specific killing in any cells that
express exogenous Cas9.
Despite the intense focus on Cas9, Type I CRISPR-Cas
systems have also shown promise as antimicrobial
agents. The Cas3 nuclease for these systems nick and
processively degrade the non-bound strand of DNA in
the 30 -to-50 direction, leading to immediate destruction
of large portions of DNA upstream of the target
sequence [25,26]. Accordingly, multiple groups have co-
opted the I-E CRISPR-Cas system from E. coli for
potent and selective killing. Gomaa et al. demonstrated
the utility of the E. coli I-E system as a programmable
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antimicrobial and demonstrated activity when express-
ing the system endogenously or heterologously [13].
Furthermore, Gomaa et al. used the I-E system
expressed heterologously to kill individual or multiple
strains based on a unique or shared genomic sequence.
Yosef et al. took a similar approach to immunize E. coli
against the uptake of antibiotic resistance plasmids [27].
Finally, Caliando and Voigt engineered E. coli with a
chromosomally integrated, inducible I-E CRISPR-Cas
system to conditionally degrade regions of the genome
that are proprietary [26]. Critically, Gomaa et al.
observed low escape rates regardless of the genomic
target site, suggesting that Type I systems could offer
more flexible antimicrobials than those reliant on Cas9
[13]. The drawback to Type I systems is that they
require the coordinated expression of four to seven
proteins, potentially complicating their delivery and
expression in diverse bacteria. Instead, given that Type I
systems are the most prevalent type of CRISPR-Cas
system in nature [10], active endogenous systems
could be co-opted through the delivery of a genome-
targeting guide RNA [13].
Recently, new types of CRISPR nucleases have been
discovered that could also serve as antimicrobial
agents. The Type V-A CRISPR effector protein Cpf1
(Cas12a) functions similarly to Cas9 but tends to be
smaller and leaves a staggered DNA break [28,29]. It
remains to be seen whether this staggered cut is
readily repaired similar to the blunt cut from Cas9. A
separate, recently discovered Type VI-A nuclease with
antimicrobial potential is C2c2 (Cas13a). This protein
specifically binds and cleaves complementary RNA but
then undergoes a conformational change that leads to
promiscuous RNA cleavage [30,31]. Abudayyeh
showed that this activity could arrest cell growth [30],
although the effect may be bacteriostatic rather than
bactericidal. Given that other CRISPR nucleases likely
await discovery, the best nuclease for antimicrobial
applications remains to be determined and could be
dependent on the mode of delivery and the physiology
of the target bacterium.
While DNA cleaved by Cas9 may undergo repair, addi-
tional effectors may be added to increase its lethality
(Figure 1B). For instance, Cui and Bikard showed that
the expression of Cas9 and the Mu phage protein GamS,
an inhibitor of the RecBCD DNA repair machinery,
blocked DNA repair and resulted in no bacterial survi-
vors [21]. Other viral proteins that inhibit DNA repair
such as the Ref protein from P1 phage could similarly
improve the efficacy of Cas9 and other CRISPR nucle-
ases by hindering DNA break repair [32,33]. Finally, the
co-dominant negative RecA56 mutant has been shown
to inhibit certain functions of the bacterial DNA repair
pathway [34], and could help to further prevent recov-
ery from CRISPR-induced DNA cleavage. Going for-
ward, the selection of a potent CRISPR nuclease and
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Advancing CRISPR antimicrobials Fagen et al. 59
any accessory factors that will enhance antimicrobial
activity will be an important part of the overall CRISPR
antimicrobial design.
CRISPR antimicrobial delivery
Arguably the most pervasive challenge to the develop-
ment of CRISPR antimicrobials is efficient delivery to
diverse bacteria. To-date, CRISPR antimicrobials have
been principally encoded as DNA and delivered using
bacteriophage particles [20e22,27] (Figure 2A). Bacte-
riophage (or phage for short) are adept at injecting their
genetic material across the bacterial cell wall and into
the cytoplasm of its host. CRISPR antimicrobials have
been encoded in two forms to ensure packaging by the
phage particles: phagemids and the phage genome
(Figure 2B). Phagemids are plasmids that contain
packaging signals along with a selection marker and
origin-of-replication and therefore do not encode any
additional phage components that could inadvertently
affect cell physiology. As plasmids, phagemids can be
equipped with CRISPR antimicrobials through standard
in vitro assembly techniques. Accordingly, multiple
groups have used phagemids for phage-mediated de-
livery of CRISPR antimicrobials to E. coli and Staphylo-
coccus aureus [19,20,22]. The second option is
introducing the CRISPR antimicrobial DNA into the
phage genome. DNA manipulation is more laborious for
phage genomes than for phagemids, and phage genomes
can introduce unwanted phage components. However,
phage genomes regularly outcompete phagemids for
packaging and encode factors that can block bacterial
immune systems such as restriction-modification sys-
tems that degrade foreign DNA. Yosef et al. successfully
introduced a Type I CRISPR-Cas system into the
genome of l phage that integrates into the bacterial
genome, thereby enabling heritable targeting of
multidrug-resistance plasmids and stymying the spread
of multidrug resistance through a bacterial population
[27]. CRISPR antimicrobials have been encoded in the
genomes of temperate phage, where CRISPR is the sole
agent of cell killing. However, lytic phage could also be
promising delivery vehicles, as CRISPR could augment
the natural killing abilities of the phage. In the long
term, it will be interesting to see whether lytic or lyso-
genic phage equipped with killing mechanisms such as
CRISPR prove to serve as superior antimicrobials
[22,28].
For phage to deliver their genetic cargo, they must
efficiently bind to the surface of the target microbeda
process known as adsorption. Phage traditionally adsorb
to a narrow range of bacteria, with most phage limited to
a subset of bacterial strains within a single species. This
narrow adsorption range presents an immediate chal-
lenge for utilizing phage as generalized DNA delivery
vehicles. A potential solution is the use of a phage
cocktail or broad-host phage such as P1 [35,36]. Another
solution being explored is to alter or exchange the tail
Current Opinion in Biomedical Engineering 2017, 4:57–64
60 Synthetic Biology and Biomedical Engineering

Figure 2

A Phage anatomy B Forms of DNA encoding CRISPR antimicrobials

Capsid
Origin of replication

DNA packaging sites

Tail sheath Phagemid

CRISPR Phage genome
nuclease(s)
Tail fibers Genetic material
Genome
packaged within capsid
targeting spacer(s)

Baseplate

C Modulation of phage adsorption range

Tail fiber exchange Tail fiber mutation

T7 WT host T3 WT host
Phagemid encoding
A B tail fiber genes (black)

Random mutagenesis
of tail fiber genes

Transfer T3 tail fiber

genes on T7 phagemid Produce phage library
with mutant tail fiber genes

Produce T7/T3
Screen for DNA delivery
hybrid
to bacteria of interest

Screen for DNA delivery A B C Bacteria

to bacteria of interest

T7 delivery range
A B Bacteria

T7 delivery range Shifted delivery range

T3 delivery range Expanded delivery range

T3 delivery range Shifted delivery range

Current Opinion in Biomedical Engineering

Engineering phage as delivery vehicles for CRISPR antimicrobials. (A) Anatomy of a representative tailed phage. (B) DNA-encoded CRISPR
antimicrobials can be incorporated into a phagemid or the phage genome for viral packaging and intracellular delivery. (C) Strategies for modifying tail
fiber proteins to alter the phage host range. The model T7 lytic phage has been the focus of recent phage engineering efforts through tail fiber gene
exchange or mutation.

Current Opinion in Biomedical Engineering 2017, 4:57–64 www.sciencedirect.com


fiber proteins responsible for binding the bacterial cell
surface (Figure 2C). Recent studies using E. coli phage
T7 showed that this phage can accommodate the tail
fiber proteins of related phage, thereby allowing delivery
to varying enteric bacteria [37,38]. Yosef et al. further
applied directed evolution to the tail fiber proteins of
T7, resulting in increased infectivity in desired bacterial
strains [38]. A similar strategy could be employed to
quickly tailor the host range to target all strains for a
given pathogen or target a broad set of pathogens.
Phage aside, other delivery methods are being explored
and could hold promise for broad intracellular DNA
delivery. Kang et al. developed polymer-based nano-
particles that delivered Cas9-sgRNA ribonucleoprotein
complexes to bacterial cells [39]. While the delivery
efficiency was low, this DNA-independent approach has
the advantage of bypassing innate bacterial defenses
against mobile genetic elements as well as functioning
independently of the host’s transcription/translation
machinery. CRISPR antimicrobials have also been
introduced through conjugation [20]. While conjugation
is currently less efficient than phage-based delivery,
conjugation may be more broadly applicable as it can
occur between unrelated bacteria and requires minimal
modification of the conjugative donor. Furthermore,
conjugation could allow a CRISPR antimicrobial to
propagate through a mixed bacterial population,
potentially offering a means of community-wide sur-
veillance fordand eradication ofdundesirable strains or
genetic traits.
Figure 3
Targeted killing with
A CRISPR antimicrobial
Immune Unable to
Phage binding systems bind to host
to host
surface
receptor
Mutated
CRISPR or lost
antimicrobial host surface
DNA delivery Restriction receptor
enzyme
RNA-guided
endonuclease
complex
Chromosome
targeting and
cleavage
Cell death
Potential modes of heritable bacterial resistance or off-target killing. (A) Steps of effective delivery and targeted killing. (B) Potential modes of
heritable resistance from CRISPR antimicrobials through DNA degradation by the host’s immune systems, lost or mutated surface receptor for the
phage delivery vehicle, superinfection exclusion due to previous phage infection, expression of an antagonistic anti-CRISPR protein, lack of a complete
target site flanked by the appropriate PAM, or competitive binding of the CRISPR nuclease by host encoded guide RNAs. (C) Potential modes of
unintended bacterial killing through self-targeting using a native CRISPR array or cytotoxicity associated with nuclease overexpression.
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Advancing CRISPR antimicrobials Fagen et al. 61
Future challenges and opportunities
Even if DNA encoding a CRISPR antimicrobial is effi-
ciently delivered to the target cell, the DNA must avoid
the host’s immune systems to allow sufficient antimi-
crobial expression. Different types of immune systems
are known, such as restriction-modification, CRISPR-
Cas, and abortive infection among others, and the spe-
cific barriers can vary widely even between related
bacteria. Some of these barriers can be overcome
through common techniques that have been developed
through efforts to improve DNA transformation [40].
For example, mimicry of the target’s DNA methylation
and codon optimization of any delivered DNA may help
to overcome the target bacterium’s restriction-
modification systems and improve translation effi-
ciency, respectively [41,42]. However, more work is
needed to understand which major barriers are posed in
each strain and how to systematically overcome each
barrier.
Aside from barriers to foreign DNA, there are multiple
scenarios in which a fully functional CRISPR antimi-
crobial may fail to kill the target bacterium (Figure 3).
Multiple modes of heritable resistance have been sug-
gested or demonstrated, such as loss of the target site, or
mutation of the surface receptors for phage adsorption
[19]. Other potential mechanisms include the expres-
sion of compatible anti-CRISPR proteins in a lysoge-
nized phage that could inhibit the activity of delivered
CRISPR antimicrobials [43], or the presence of a
lysogenized phage that blocks infections by similar
B Heritable resistance to killing C Off-target killing
Superinfection Anti-CRISPR Mutated Guide Activation of Cas9 toxicity
exclusion genomic target competition endogenous
guides
Endogenous
CRISPR
guides
Unknown
mechanism
Processing of
endogenous
guides
Cell survival Cell death
Current Opinion in Biomedical Engineering
Current Opinion in Biomedical Engineering 2017, 4:57–64
62 Synthetic Biology and Biomedical Engineering
phage through superinfection exclusion [44]. Despite
these potential mechanisms of resistance, current
studies have found that deleterious mutations in the
CRISPR antimicrobial constructs are the primary driver
of escapes rather than innate bacterial resistance. For
instance, Bikard et al. found that all S. aureus cells that
survived phage-delivered Cas9 antimicrobials remained
susceptible to reinfection by the phage [19], while
Gomaa et al. found that E. coli cells expressing the I-E
CRISPR-Cas system and transformed with genome-
targeting CRISPR plasmid survived through recombi-
nation within the guide RNAs prior to plasmid isolation.
Therefore, resistance to CRISPR antimicrobials may be
less prominent, although future research efforts should
aim to elucidate and counteract the most common
mechanisms of bacterial escape.
Finally, CRISPR antimicrobials could also adversely
impact non-targeted bacteria (Figure 3C). Aside from
the potential for off-target cleavage, undesired killing
could result from nuclease cytotoxicity or promiscuous
cleavage activity at elevated expression levels
[23,24,45]. The introduction of Cas9 has also been
shown to interact with orphaned Type II CRISPR arrays,
the natural form of guide RNAs, in Enterococcus faecalis
that could result in incidental self-targeting [46]. These
challenges could be overcome in part by using a rare
CRISPR nuclease that is unlikely to be compatible with
an endogenous array. Rare nucleases would also have the
added benefit of being unlikely to encounter inhibitory
anti-CRISPR proteins. Overall, CRISPR antimicrobials
face multiple potential hurdles to ensure efficient de-
livery and on-target killing, although the versatility and
tractability of CRISPR-Cas systems and phage would
suggest that each barrier can be overcome through
further engineering efforts.
Conclusions and future perspectives
CRISPR antimicrobials represent one of many uses of
CRISPR-derived technologies. The CRISPR antimi-
crobial field is still in its infancy and faces numerous
challenges for the design and delivery of these antimi-
crobials. We also envision future hurdles as CRISPR
antimicrobials move from the lab to animal models and
finally to human trials. Specifically, further investigation
and engineering is needed to ensure that the phage
delivery vehicles reach the site of infection and effi-
ciently deliver the CRISPR payload to pathogens in the
affected tissue. Depending on the pathogen type and
infection site, CRISPR antimicrobials may be more or
less amenable as therapeutic agents; for instance, topical
infections offering potentially simpler targets than gut
infections while planktonic pathogens may be simpler
targets than intracellular pathogens or those that form
biofilms. Aside from the challenge of targeting patho-
gens in vivo, additional barriers are anticipated as this
technology begins to navigate a regulatory approval
Current Opinion in Biomedical Engineering 2017, 4:57–64
process with little precedent for phage-based thera-
peutic strategies. Despite these envisioned challenges,
these efforts should continue given the increasing need
to combat multidrug-resistant infections and an
emerging emphasis on preserving a healthy human
microbiome. There is also the potential to use CRISPR
antimicrobials to selectively perturb complex microbial
communities or clear niches for introduced bacteria.
Overall, there remains incredible potential for CRISPR
as an antimicrobial agent that could greatly expand the
umbrella of CRISPR technologies and address existing
challenges in the prevention and treatment of human
infectious diseases.
Acknowledgements
This work was supported by the Bill and Melinda Gates Foundation [grant
number OPP1140021], the North Carolina Biotechnology Center [Tech-
nology Enhancement Grant], and the Bay Area Lyme Foundation
[Emerging Leader Award]. C.L.B. is a co-founder of Locus Biosciences and
is an inventor on patent applications related to CRISPR technologies.
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