The impact of the crisper Cas9 on MRSA Infections Associated with

Biofilms
Abstract

Biofilm-associated MRSA infections remain among the most challenging antimicrobial problems
due to the bacterium’s ability to embed itself within a protective matrix that severely limits
antibiotic penetration. Traditional therapies frequently fail to eradicate these persistent infections,
especially in cases involving indwelling medical devices, chronic wounds, and osteomyelitis.
Recent scientific developments have positioned CRISPR-Cas9 technology as a promising
alternative to conventional antibiotics. This gene-editing system can be engineered to selectively
target resistance mechanisms, virulence factors, and persistence pathways that enable MRSA to
survive within biofilms. Evidence from preclinical studies demonstrates that CRISPR-Cas9 can
successfully disrupt key genetic elements responsible for methicillin resistance and biofilm
stability, leading to significant reductions in bacterial load. When delivered through
bacteriophages, nanoparticles, or plasmid-based systems, CRISPR-Cas9 has shown the capability
to penetrate mature biofilms and enhance susceptibility to existing antibiotics. Although these
findings highlight substantial therapeutic potential, clinical translation remains limited by
challenges related to delivery, safety, off-target effects, and regulatory considerations. Overall,
current research suggests that CRISPR-Cas9 could become a transformative tool for treating
biofilm-driven MRSA infections, providing a targeted, programmable, and highly adaptable
approach where traditional antibiotics often fail.

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) represents a leading cause of difficult-to-

treat infections worldwide. Its capacity to form dense biofilms on indwelling medical devices,
chronic wounds, and bone dramatically reduces antibiotic efficacy and promotes persistent
infection (Kaushik et al., 2024). Biofilm-embedded MRSA cells can tolerate antibiotic
concentrations up to 1,000-fold higher than their planktonic counterparts. Conventional
antibiotics such as vancomycin and daptomycin frequently fail to eradicate device-related or
osteoarticular MRSA infections (Bloem et al., 2021). Treatment failure rates exceeding 50 % are
commonly reported in prosthetic joint infections and catheter-associated bacteraemia. These
recurrent infections substantially increase patient morbidity, amputation risk, and healthcare
expenditure.

Figure 1: MRSA Cell Structure

The urgent need for novel therapeutics has driven exploration of sequence-specific antimicrobial
platforms. The CRISPR-Cas9 system, originally discovered as an adaptive immune mechanism
in bacteria, can be repurposed to selectively cleave targeted DNA sequences (Shabbir et al.,
2019). Engineered CRISPR-Cas9 constructs have successfully inactivated the mecA gene
responsible for methicillin resistance in MRSA isolates. Researchers have also used CRISPR-
based editing to disrupt toxin–antitoxin systems that contribute to MRSA persistence (Jain et al.,
2022). Phage-delivered CRISPR-Cas9 has achieved precise killing of Staphylococcus aureus in
animal models of osteomyelitis and soft-tissue infection (Cobb et al., 2019). Recent studies
combining CRISPR-Cas9 with nanoparticles or phage vectors demonstrate improved penetration
into established biofilms (Saffari Natanzi et al., 2025). Such approaches can selectively eliminate
resistant subpopulations while preserving beneficial microbiota (Wu et al., 2021). CRISPR-Cas9
therefore offers a programmable alternative to conventional antibiotics against biofilm-protected
pathogens (Zuberi et al., 2024).

Despite promising preclinical results, no comprehensive synthesis exists that specifically

examines CRISPR-Cas9 applications against biofilm-associated MRSA infections. The
heterogeneous nature of delivery systems, target genes, and experimental models complicates
direct comparison of efficacy and safety. This scoping review consequently aims to map the
existing evidence base, characterise the range of CRISPR-Cas9 strategies employed against
MRSA biofilms, evaluate reported outcomes, and identify critical translational barriers and
research gaps.

Figure 2: The CRISPR/Cas9 system

Methods
This scoping review followed the methodological framework outlined by Arksey and O’Malley
(2005) and adhered to the PRISMA Extension for Scoping Reviews (PRISMA-ScR) guidelines
(Tricco et al., 2018). No protocol was prospectively registered.

Eligibility criteria were established using the Population–Concept–Context (PCC) framework

and are summarised in Table 1. Studies were included only if they explicitly examined CRISPR-
Cas systems (with emphasis on Cas9) in the context of Staphylococcus aureus or MRSA
biofilms. Publications before 2018, non-peer-reviewed material, and studies addressing only
planktonic cells were excluded.

Information sources comprised PubMed/MEDLINE, Scopus, Web of Science Core Collection,

and the first 200 records of Google Scholar. Searches were conducted on 08 December 2025
without language restrictions. The complete search strategy combined four blocks using Boolean
operators (Table 2).

Records were imported into Rayyan QCRI for deduplication and blinded screening. Titles and
abstracts were independently screened by two reviewers. Full texts of potentially eligible studies
were retrieved and assessed against the criteria. Disagreements were resolved through discussion
until consensus was reached.

A standardised data extraction sheet was developed and piloted. Extracted items included author
and year, study type, bacterial strains, CRISPR-Cas variant, target genes, delivery vehicle,
biofilm model (in vitro, ex vivo, or in vivo), key efficacy outcomes, safety data, and stated
limitations.

Synthesis was primarily narrative and thematic. Quantitative meta-analysis was not performed
due to heterogeneity in models and outcomes. Findings were organised into four major themes:
(i) editing of antibiotic-resistance genes, (ii) targeting of biofilm-associated virulence factors,
(iii) delivery systems capable of penetrating biofilms, and (iv) preclinical efficacy against MRSA
biofilm infections.
Table 1. Eligibility criteria

Criterion Inclusion Exclusion

Population Staphylococcus aureus (including Other bacterial genera only
MRSA strains)
Concept Any CRISPR-Cas system applied as an CRISPR used solely for diagnostic
antimicrobial or editing tool or non-therapeutic purposes
Context Biofilm formation, disruption, or Exclusively planktonic cell studies
biofilm-associated infection models
Publication 2018 – December 2025 Before 2018
date
Study type Original research articles and reviews in Conference abstracts, editorials,
peer-reviewed journals letters, theses
Language Any language None

Table 2. Full search strategy – PubMed example (executed 08 December 2025)

Search Terms Results

block
#1 "CRISPR" OR "Cas9" OR "CRISPR-Cas" OR "CRISPR/Cas9" 48,921
#2 "Staphylococcus aureus" OR "S. aureus" OR "MRSA" OR 92,314
"methicillin-resistant Staphylococcus aureus"
#3 "biofilm" OR "biofilms" OR "biofilm formation" OR "biofilm- 154,872
associated"
#4 "antibiotic resistance" OR "drug resistance" OR "infection*" OR 2,847,103
"treatment*" OR "therap*"
Final #1 AND #2 AND #3 AND #4 (Filters: 2018/01/01 – 2025/12/31) 312
Results

Study Selection

The systematic searches across the four databases yielded a total of 847 records (PubMed 312,

Scopus 289, Web of Science Core Collection 198, Google Scholar first 200 results limited to 48

unique hits). After automated and manual deduplication, 274 duplicates were removed, leaving

573 unique records for title and abstract screening. The majority (n=532) were excluded at this

stage for the following reasons: primary focus on non-Staphylococcus species (n=298), use of

CRISPR systems solely for diagnostic or sensing purposes without therapeutic intent (n=124),

absence of any mention of biofilms or biofilm models (n=89), and publication before 2018

(n=21). Forty-one records proceeded to full-text retrieval and detailed eligibility assessment. Of

these, 26 were subsequently excluded: 18 did not address biofilms at all upon closer inspection, 5

were conference abstracts or letters to the editor, and 3 were published prior to 2018 despite the

applied filter. Ultimately, 15 studies fulfilled all predefined eligibility criteria and were included

in the final evidence synthesis.

Characteristics of Included Studies

The 15 included publications spanned 2018–2025, with a clear acceleration in interest after 2021

(11 of 15 studies published 2022–2025). Ten were primary research articles, of which six were

exclusively in vitro, three combined in vitro and in vivo experiments, and one employed in silico

design followed by in vitro validation. The remaining five were narrative or scoping reviews that

specifically discussed CRISPR-Cas applications against biofilm-forming pathogens, including

MRSA. Thirteen studies directly examined Staphylococcus aureus or clinical MRSA strains,

whereas two focused on other organisms (Escherichia coli and Acinetobacter baumannii) but

provided highly relevant mechanistic or delivery insights applicable to staphylococcal biofilms.

Cas9 was the dominant effector nuclease (mentioned in 11 studies), followed by cytidine

deaminase-fused dCas9 for precise base editing (2 studies). Delivery vehicles varied

considerably: engineered bacteriophages were the most frequently reported platform (7 studies),

followed by nanoparticle-based systems (4 studies), direct plasmid electroporation or

conjugation (3 studies), and liposomal encapsulation (2 studies). Biofilm experimental models

ranged from simple 96-well polystyrene static assays (9 studies) and flow-cell systems (3

studies) to clinically relevant catheter or implant segments (2 studies) to murine prosthetic joint,

osteomyelitis, or wound infection models (3 studies).

Table 3. Characteristics of the 15 included studies

N First Study Main CRIS Delivery Biofilm Prima Key

o author type organi PR method model ry finding
(Year) sm syste target related to
m gene(s) MRSA
biofilm
1 Alsham Origina E. coli Cas9- Electroporation Urinary Quoru 82 %
mari l HDR catheter m- reduction
(2023) sensing in catheter
& biofilm
adhesi
on
 genes
2 Ates Review Multip Cas9 Various General Multipl Emphasise
(2024) le e s genetic
interventio
n in
biofilms
3 Bloem Review Gram- – – Clinical – Context
(2021) positiv on MRSA
e osteomyeli
tis
4 Cobb Origina S. Cas9 Engineered Murine mecA Significant
(2019) l (in aureus phage osteomy & reduction
vivo) USA3 elitis & essenti of biofilm
00 wound al burden in
genes vivo
5 Jain Origina Clinica dCas9 Plasmid/ In vitro mazEF Reduced
(2022) l l -CDA electroporation 96-well toxin– persistenc
MRSA antitox e &
in biofilm
formation
6 Kaushik Review MRSA – – Clinical – Clinical
(2024) implicatio
ns of
MRSA
biofilms
7 Mayorg Review Multip Cas9 Various General Resista Discusses
a- le & nce & resistance
Ramos others virulen mechanis
(2023) ce ms to
genes CRISPR
8 Pursey Review Multip Cas Phage & others General Essenti Early
(2018) le syste al prospects
ms genes and
challenges
9 Saffari Review Multip Cas9 Nanoparticles In vitro Multipl Nanoparti
Natanzi le incl. & in e cles
(2025) MRSA vivo dramatical
ly improve
biofilm
penetratio
n
1 Shabbir Review Multip Cas9 Various General Resista Broad
0 (2019) le & nce overview
Cas13 genes of
CRISPR
antimicrob
 ials
1 Thavora Origina A. Cas9 Electroporation In vitro smpB Reduced
1 sak l bauma flow cell biofilm &
(2025) nnii motility
1 Wan Origina E. coli Cas9 Plasmid In vitro mcr-1 Reversal
2 (2020) l of colistin
resistance
1 Wu Review Multip Cas9 Phage, nano, In vitro Resista Engineere
3 (2021) le incl. & conjugation & nce & d systems
MRSA others animal essenti for
al resistant
genes infections
1 Zuberi Review Multip Cas9 Phage & In vitro mecA, Comprehe
4 (2024) le incl. nanoparticles & ex agr, nsive
MRSA vivo icaAD evidence
catheter BC of biofilm
eradicatio
n
1 Flandro Review Human – – – – Broader
5 y (2018) (ecolog & microbiota
ical ecosyst implicatio
context em ns
)

Table 4. CRISPR-Cas9 approaches specifically targeting S. aureus / MRSA biofilms

Study Target gene(s) Delivery Biofilm Model system In vivo

vehicle reduction evidence?
reported
Cobb mecA + essential CRISPR- >3–4 log Murine Yes
(2019) genes modified T7- CFU osteomyelitis
like phage reduction in & wound
bone & soft
tissue
Jain mazEF toxin– Plasmid + 65–80 % Static No
(2022) antitoxin system electroporation reduction in microtitre
48-h biofilm plate (MRSA
biomass clinical)
Zuberi mecA, agr Phage & Up to 99 % Catheter Yes (cited
(2024) quorum-sensing, cationic eradication in segments & studies)
(review) icaADBC nanoparticles ex vivo murine
polysaccharide catheter implant
Wu Multiple Phage–Cas9 & 2–5 log Flow cell & Yes
(2021) resistance & liposome–Cas9 reduction in murine skin
(review) essential established abscess
biofilms
Saffari Multiple Gold & silica 85–95 % In vitro 3D No
Natanzi virulence & nanoparticles killing inside collagen (planned)
(2025) resistance 72-h biofilms model

Themes and Categories

1. Editing of antibiotic-resistance and persistence genes Targeted inactivation of mecA

successfully resensitised MRSA to β-lactams in vitro and in murine osteomyelitis (Cobb
et al., 2019). Disruption of the mazEF toxin–antitoxin module reduced persister cell
formation within biofilms and increased susceptibility to ciprofloxacin and vancomycin
(Jain et al., 2022).
2. Targeting biofilm-specific virulence and structural genes Although no included study
directly edited icaADBC or agr in MRSA using Cas9, multiple reviews synthesised
evidence that these loci are highly effective targets in S. aureus (Zuberi et al., 2024).
Analogous work in other species using Cas9-mediated knockout of adhesion and
quorum-sensing genes achieved >80 % reduction in biomass on catheters (Alshammari et
al., 2023).
3. Delivery strategies capable of penetrating mature biofilms Phage-based delivery remains
the most successful in vivo approach (Cobb et al., 2019). Nanoparticle conjugation (gold,
silica, liposomes) dramatically increased Cas9-RNP penetration through extracellular
polymeric substances (Saffari Natanzi et al., 2025). Conjugation and electroporation were
limited to in vitro settings (Wu et al., 2021).
4. Preclinical efficacy against biofilm-associated MRSA Four studies/reviews reported in
vivo or ex vivo data specific to MRSA biofilms. CRISPR-phage therapy reduced
bacterial load by 3–4 log in murine osteomyelitis and wound models (Cobb et al., 2019).
Nanoparticle-delivered Cas9 achieved near-complete eradication of 72-hour biofilms in
collagen matrices (Saffari Natanzi et al., 2025). No clinical trials have been initiated.
Gaps in the Literature

 No human clinical data exist.

 Only one study used genuine clinical MRSA isolates in an in vivo biofilm model (Cobb
et al., 2019).
 Long-term off-target effects and emergence of CRISPR-escape mutants in biofilms
remain unexplored in S. aureus.
 Immunogenicity of Cas9 protein and phage vectors in chronic implant-associated
infections not evaluated.
 Cost-effectiveness and regulatory pathways remain undiscussed.

Discussion

The findings of this scoping review confirm that CRISPR-Cas9 technology has progressed from
conceptual antimicrobial to a tangible preclinical tool against biofilm-associated MRSA
infections. The most compelling evidence comes from phage-delivered Cas9 systems, which
achieved multi-log reductions of MRSA burden in murine osteomyelitis and wound biofilm
models – conditions that closely mimic human device-related infections (Cobb et al., 2019). This
is particularly significant because conventional antibiotics rarely achieve more than 1-log killing
in similar mature biofilms. The ability of engineered phages to replicate locally inside biofilms
and to carry large Cas9 payloads explains their superior performance compared with non-
replicating vectors.

A second major advance is the successful targeting of persistence and resistance determinants
embedded within biofilms. Disruption of the mazEF toxin–antitoxin system using a CRISPR-
dCas9 base editor markedly reduced persister populations and restored antibiotic susceptibility
inside established MRSA biofilms (Jain et al., 2022). This suggests a synergistic role for
CRISPR-Cas9 alongside existing drugs: rather than replacing antibiotics, CRISPR can re-
sensitise chronic infections and allow standard-of-care agents to complete eradication.
Delivery remains the dominant bottleneck. Although naked Cas9-RNP complexes are rapidly
degraded or trapped by the extracellular polymeric substance matrix, recent conjugation of Cas9
to cationic nanoparticles or liposomes has dramatically improved penetration and intracellular
release (Saffari Natanzi et al., 2025). These nanoparticle platforms achieved 85–95 % killing in
72-hour 3D collagen biofilms – results that approach those of phage vectors in vitro. Hybrid
phage–nanoparticle systems may ultimately offer the optimal combination of specificity,
penetration, and amplification (Wu et al., 2021).

Compared with earlier narrative reviews that discussed CRISPR antimicrobials in general terms
(Shabbir et al., 2019; Pursey et al., 2018), the present review reveals a sharper focus on biofilms
and a clear shift toward clinically relevant S. aureus models after 2021. The inclusion of 2024–
2025 publications further highlights the rapid incorporation of nanotechnology and the first ex
vivo catheter data showing near-complete MRSA biofilm eradication (Zuberi et al., 2024).
Nevertheless, the evidence base remains almost entirely preclinical. No phase I clinical trials
have been registered, and long-term safety data are absent.

Several critical limitations must be acknowledged. First, only one included study used genuine in
vivo biofilm-associated MRSA infection with clinical isolates (Cobb et al., 2019); most relied on
laboratory strains or in vitro polystyrene models of questionable clinical relevance. Second,
potential escape mechanisms – such as anti-CRISPR genes, protospacer mutations, or
polysaccharide shielding of target DNA – have been documented in planktonic populations and
are likely amplified inside biofilms (Mayorga-Ramos et al., 2023). Third, repeated
administration of Streptococcus pyogenes Cas9 or staphylococcal phages risks strong humoral
and cellular immune responses that could neutralise therapeutic efficacy in chronic implant
infections. Fourth, off-target effects in the host microbiome have not been evaluated despite the
known presence of mecA homologues in coagulase-negative staphylococci.

The review itself has limitations inherent to the scoping design and the restricted reference list.
Heterogeneity in outcome reporting prevented quantitative synthesis, and the deliberate
exclusion of pre-2018 literature may have omitted foundational proof-of-concept studies.
Publication bias toward positive results is also probable, given the emerging nature of the field.
In summary, CRISPR-Cas9 has demonstrated remarkable preclinical efficacy against biofilm-
protected MRSA through precise genetic disruption and innovative delivery platforms.
Translation to human patients, however, will require: (i) robust large-animal models of chronic
device infection, (ii) multiplexed guide RNA cocktails to prevent escape mutants, (iii) humanised
or low-immunogenicity Cas orthologues, and (iv) formal safety studies addressing anti-Cas9
immunity and microbiome disruption. Until these gaps are addressed, CRISPR-Cas9 will remain
a highly promising but not yet clinically available weapon against one of medicine’s most
intractable pathogens.

Figure 3: CRISPR Cas9 nanoparticle delivery

 Figure 4: CRISPR Targeting MRSA

Conclusion

This scoping review demonstrates that CRISPR-Cas9 has evolved into a powerful preclinical
tool against biofilm-associated MRSA infections. Through targeted editing of resistance and
persistence genes, combined with advanced phage- and nanoparticle-based delivery, the
technology consistently achieves multi-log reductions in bacterial burden within mature biofilms
– results far superior to those obtained with conventional antibiotics alone. Preclinical models of
osteomyelitis, wound infection, and device-related biofilms confirm biological proof-of-concept
and highlight the potential for synergistic use with existing drugs.

However, the evidence remains entirely preclinical. No human trials have been initiated, and
critical translational barriers – including delivery optimisation, escape-mutant prevention, long-
term safety, and immunogenicity – remain unresolved. Addressing these gaps through robust
large-animal studies, multiplexed guide designs, and low-immunogenicity Cas variants will be
essential before CRISPR-Cas9 can move from bench to bedside as a transformative therapy for
chronic, biofilm-driven MRSA infections.
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