Using CRISPR-Cas to fight carbapenem antibiotic resistance: from initial tests to clinical use

This review was based on peer-reviewed publications indexed in PubMed and Scopus (2015–2025) using the keywords CRISPR, Cas13a, Cas9 and carbapenem resistance. Priority was given to original experimental studies, translational models, and clinical trials.

The Cas9 nuclease (Type II) recognizes target DNA adjacent to a protospacer-adjacent motif (PAM) and introduces double-strand breaks through its RuvC and HNH domains. When guided to a plasmid-borne resistance gene such as bla_KPC-2, bla_NDM-1 or bla_OXA-48, Cas9 can disrupt gene function or trigger plasmid loss. Its major strength lies in its simplicity and high cleavage efficiency (Citorik et al., 2014; Yosef et al., 2015; Hsu et al., 2013); however, potential off-target cleavage and limited in vivo delivery remain obstacles to therapeutic use. Cas9 is best suited for ex vivo plasmid curing or ex vivo plasmid curing, or for engineered probiotic applications rather than direct in-patient therapy.

Cas13a (Type VI) is unique among CRISPR systems in that it targets RNA rather than DNA (Kiga et al., 2020). Upon binding its target transcript—such as an mRNA encoding a carbapenemase enzyme such as KPC-2—Cas13a becomes activated and nonspecifically degrades both the target and nearby RNAs, leading to bacterial cell death. Because it does not introduce double-stranded DNA breaks, Cas13a minimizes genomic integration risks and is suitable for delivery via bacteriophage capsids or nanoparticles. These features make it a highly promising candidate for clinical antimicrobial applications, capable of selectively eliminating carbapenemase-expressing bacteria while sparing nonresistant strains.

Kiga et al. (2020) pioneered the use of Cas13a as an antimicrobial tool against carbapenem-resistant Escherichia coli. By packaging Cas13a with customized crRNAs into bacteriophage capsids (M13 or Φ80/PICI), the authors achieved sequence-specific killing of strains harboring bla_IMP-1, bla_NDM-1,bla_KPC-2,bla_VIM-2, and bla_OXA-48. In a Galleria mellonella infection model, treatment with Cas13a phage targeting bla_IMP-1 significantly improved larval survival. Because Cas13a acts on RNA, it eradicated cells expressing the resistance gene regardless of whether it was chromosomal or plasmid-borne. Hart et al. (2025) demonstrated that CRISPR–Cas13d enables highly precise RNA targeting with minimal off-target activity. Their work expands CRISPR applications beyond DNA editing, offering a programmable tool for post-transcriptional regulation. This level of specificity strengthens the potential of RNA-targeted antimicrobials and complements plasmid-curing strategies that act at the DNA level.

Hao et al. (2020) used a Cas9–sgRNA plasmid (pCasCure) delivered by electroporation to Serratia marcescens, successfully restoring carbapenem susceptibility this study demonstrated that Cas9 can eliminate carbapenemase genes (bla_KPC, bla_NDM, bla_OXA-48) or their associated plasmids in multiple Enterobacterales species. After transformation with inducible Cas9–sgRNA constructs, resistant clinical isolates of Klebsiella pneumoniae, E. coli, Enterobacter spp., and Serratia marcescens lost the targeted genes and exhibited an eight-fold reduction in carbapenem minimum inhibitory concentrations. Although delivery required laboratory transformation, the study confirmed that precise DNA cleavage could restore carbapenem susceptibility in clinical strains.

A recent study by Yen et al. (2024) introduced a Cas9-based conjugative vector designed to cure common plasmids in Gram-negative bacteria. The system leverages conjugation to deliver a programmable CRISPR–Cas9 module directly into recipient cells, enabling targeted plasmid cleavage without the need for electroporation or other non-physiological delivery methods. Importantly, the conjugative vector achieved efficient plasmid elimination across multiple hosts, demonstrating broad applicability. This work highlights the clinical potential of conjugation-mediated Cas9 delivery, addressing a major limitation of earlier approaches that relied on laboratory-only transformation techniques. However, as with all conjugative systems, host range and transfer efficiency remain important considerations for in vivo translation.

Calvo-Villamañán et al. (2025) employed CRISPR interference (CRISPRi) with dCas9 to perform plasmid-wide knockdowns on the bla_OXA-48 plasmid across clinical Enterobacterales isolates. The work identified bla_OXA-48 itself and plasmid-stability modules as key fitness burdens, providing molecular targets for future multiplexed CRISPR antimicrobials.

A concise summary of representative studies is presented in Table 1.

These early human trials directly tested several uncertainties that had persisted since the preclinical phase. In animal and in vitro studies, questions remained about whether CRISPR elements could persist or propagate within commensal microbiota, whether host immunity would neutralize engineered phages, and whether phage replication might trigger systemic inflammation (Hart et al., 2025). The LBP-EC01 and SNIPR001 studies began to resolve these issues: both demonstrated that CRISPR-armed phages were transient, largely confined to target bacterial populations, and did not elicit strong inflammatory or adaptive immune responses (Kim et al., 2024; SNIPR Biome, 2023).

This evidence not only supports the biosafety of CRISPR delivery but also validates the phage capsid as a scalable and immunologically tolerated vector for subsequent carbapenem-targeted constructs. By closing this feedback loop between bench and bedside, these trials shift CRISPR-based therapy from theoretical feasibility to a regulated, monitorable intervention. The challenge now lies less in if delivery works and more in how precisely it can be adapted to target carbapenemase-producing Enterobacterales.

For combating carbapenem resistance specifically, Cas13a-based RNA-targeting systems could logically follow this model. Phage-encapsidated Cas13a constructs capable of degrading bla_NDM-1 or bla_KPC-2 transcripts may offer sequence-specific killing with minimal risk of horizontal gene transfer. Compact RNA-targeting variants such as Cas13d (Hart et al., 2025) are also emerging as safer payloads for human application due to their smaller size and reduced collateral activity.

Beyond phage-based vectors, several alternative delivery technologies are emerging for CRISPR antimicrobial applications and may overcome some of the intrinsic limitations of bacteriophages. Nanoparticle-based platforms—including lipid nanoparticles, polymeric nanocarriers, and charge-modified inorganic particles—can encapsulate CRISPR–Cas ribonucleoproteins or guide RNAs and deliver them directly to bacterial cells (Gupta et al., 2021; Rui et al., 2020; Chowdhry et al., 2023). These systems offer advantages such as protection of the CRISPR payload from degradation, reduced dependence on species-specific receptors, and potential for targeted surface functionalization. However, nanoparticle delivery faces significant challenges, including limited penetration in biofilms and rapid clearance by host immune mechanisms. Additional non-phage strategies include outer membrane vesicles engineered to package CRISPR cargo, conjugative plasmid systems equipped with containment circuits to prevent uncontrolled horizontal transfer, and live biotherapeutics designed to transiently release CRISPR constructs within the gut (Rahmati et al., 2025). Although these approaches remain in early development, integrating them into the translational landscape provides a more realistic and comprehensive view of future CRISPR delivery pathways beyond bacteriophage-mediated systems (see Table 2).

Although CRISPR–Cas antimicrobials have shown promising proof-of-concept activity, several fundamental limitations restrict their current translational potential. The most significant constraints relate to delivery efficiency. Bacteriophage-based vectors, despite being the most advanced platforms, suffer from an inherently narrow host range, dependence on specific surface receptors, and variable stability within infected tissues. Host immune responses can neutralize incoming phage particles, while phage replication dynamics remain unpredictable in vivo. These limitations restrict the reliable targeting of diverse carbapenemase-producing Enterobacterales populations (Guo et al., 2024). Beyond phages, alternative platforms such as nanoparticles, outer membrane vesicles, and conjugative plasmids face their own challenges. Nanoparticle formulations often struggle with limited biofilm penetration, rapid clearance, and potential cytotoxicity, while their surface functionalization for bacterial targeting is still technically demanding. Conjugative plasmid systems, although effective in controlled settings, rely on successful donor–recipient contact, are influenced by competing plasmids in the microbial community, and carry the theoretical risk of horizontal transfer beyond the intended host range. Engineered live biotherapeutics also present uncertainties in colonization stability and biosafety.

Finally, all CRISPR antimicrobials must contend with bacterial escape mechanisms (Mayorga-Ramos et al., 2023). Mutations within protospacer or PAM regions, acquisition of anti-CRISPR proteins (Pinilla-Redondo et al., 2020), and structural rearrangements of resistance plasmids can impair nuclease targeting. Biofilm-associated phenotypes and heterogeneous metabolic states further reduce the uniformity of CRISPR-mediated killing. Collectively, these limitations highlight the need for continued optimization of both CRISPR design and delivery modalities to ensure robust and predictable therapeutic performance. Addressing off-target activity remains a critical safety requirement for CRISPR/Cas9-based antimicrobials (Guo et al., 2023). Unintended cleavage at partially homologous genomic sites can trigger genotoxic lesions, stress-induced mutagenesis, or unpredicted transcriptional changes in bacterial populations, particularly under infection-associated physiological states. High-fidelity Cas9 variants, rational gRNA design, and regulated nuclease expression minimize—but do not eliminate—these risks. Consequently, off-target assessment using orthogonal verification methods and stringent biosafety thresholds is essential before Cas9-based constructs can advance toward human therapeutic application.

While delivery systems such as bacteriophages currently dominate translational efforts, several complementary strategies are emerging to overcome the inherent constraints of phage-based vectors. Compact CRISPR variants and RNA-guided effectors with reduced payload size may broaden compatibility across delivery platforms, including nanoparticles, outer membrane vesicles, and engineered live biotherapeutics (Rahmati et al., 2025). These approaches aim to address limitations such as narrow host range, receptor dependence, and variable phage stability in vivo.

A parallel challenge involves fine-tuning the in vivo activity of CRISPR systems. Heterogeneous bacterial physiology, stress responses, and biofilm architecture can reduce the efficiency of nuclease targeting. Controlled-expression circuits, self-limiting designs, and multiplexed guide strategies may enhance precision while reducing the likelihood of bacterial escape. Integrating delivery innovations with real-time CRISPR-based diagnostics could ultimately enable a more adaptive and targeted therapeutic framework.

Unlike chemical antibiotics, CRISPR-based agents act on the genetic fabric of microbial populations, which means their ecological footprint may extend beyond the immediate infection site. Microbial communities evolve under constant selective pressure; any intervention that introduces a new genetic weapon can, over time, select for phage resistance, anti-CRISPR proteins, or CRISPR-tolerant variants (Hillary and Ceasar, 2023). Such adaptive countermeasures could reshape resistance reservoirs rather than eliminate them.

Anticipating these evolutionary responses is therefore as critical as ensuring biosafety. Modeling population-level dynamics and incorporating "evolutionary containment"—through self-limiting phages, inducible circuits, or temporally restricted expression of CRISPR payloads—may prevent long-term ecological distortion. These considerations place CRISPR antimicrobials within the broader framework of evolutionary stewardship, where success is measured not only by immediate pathogen eradication but by maintaining the stability of microbial ecosystems.

From a clinical-ethical perspective, informed consent and risk communication will require special attention, as patients may perceive CRISPR treatment as "gene therapy," even when directed exclusively at microbes. Transparency about off-target effects, phage persistence, and potential microbiome alterations will be essential to maintain public trust.

A parallel discussion is unfolding at the regulatory level. Agencies differ in how they classify CRISPR-enhanced biologics: the U.S. FDA currently treats bacteriophage products delivering CRISPR payloads as live biotherapeutic agents or biologic antibacterials, evaluated under frameworks similar to phage therapy; the European Medicines Agency (EMA), meanwhile, has considered such constructs under the gene therapy umbrella when the CRISPR component exerts heritable genetic modification within microbial populations. This divergence matters for trial design, biosafety thresholds, and long-term surveillance obligations. Clarifying whether CRISPR antimicrobials fall under the rules for conventional biologics or gene-therapy medicinal products will shape how—and how quickly—they move from experimental use to regulated therapy. Among regulatory authorities, the EMA maintains a stricter classification framework than the FDA, treating CRISPR-enhanced bacteriophage products as gene therapy medicinal products when heritable genetic modification is involved, whereas the FDA applies a more functional, biologics-based assessment (European Medicines Agency Amsterdam, The Netherlands, 2018; U.S. Department of Health and Human Services, Food and Drug Administration, Center For Biologics Evaluation and Research Silver Spring, MD, 2022, 2023; Science and regulation of bacteriophage therapy, Silver Spring, MD, 2023).

CRISPR antimicrobials are unlikely to replace conventional antibiotics, yet they are redefining what "therapy" means in microbiology—from chemical inhibition to genetic correction. Among the current systems, Cas13a stands closest to clinical readiness: its RNA-guided activity avoids double-strand DNA breaks and has been shown to mediate sequence-specific killing of carbapenemase-producing E. coli in vivo (Shi and Wu, 2024; Li et al., 2022). Cas13a payloads are small enough for packaging into bacteriophage capsids or nanoparticles, and their biosafety has indirect human validation through early CRISPR-phage trials, notably the Cas3-armed LBP-EC01 program (Kim et al., 2024).