The Discovery of the CRISPR-Cas System: How Unexpected Bacterial Immunity Transformed the Field of Genetics

Abstract

Clustered regularly interspaced short palindromic repeats, or CRISPR, and its CRISPR-associated proteins together form an adaptive immune system that allows many bacteria and archaea to recognize and defend themselves from invading genetic elements, including bacteriophages.The groundbreaking finding of  the CRISPR-Cas system expanded the understanding of prokaryotic immunity accepted among scientists at the time and unveiled an evolutionary arms race in which bacteriophages have evolved mechanisms to evade or inhibit CRISPR-mediated immunity, such as production of anti-CRISPR proteins. Some of these methods have been utilized in the clinic to engineer phages to target pathogenic bacteria, providing an intriguing strategy for the treatment of antibiotic-resistant infections. This review analyzes the discovery, structure, and mechanism of CRISPR-Cas immunity phage strategies for evading this defense and the translation of these biological interactions into CRISPR-enhanced phage therapy and human genome-editing applications.

Introduction

Bacteria often come across bacteriophages which are viruses that infect bacterial cells and inflict an intense evolutionary pressure on populations of bacteria (Clokie et al., 2011; Labrie et al., 2010). As a result, bacteria have evolved to employ various antiviral defenses, one of which is the CRISPR-Cas system. The CRISPR-Cas complex is a sequence-specific adaptive immune system that seizes fragments of invading genetic material as spacers and utilizes them to recognize subsequent infections (Barrangou et al., 2007; Marraffini, 2015). The discovery of CRISPR-Cas complex resulted in a paradigm shift of the understanding of prokaryotic immunity by proving that bacteria can remember heritable molecular information about past viral encounters and additional mobile genetic elements.

The interaction between CRISPR-protected bacteria and bacteriophages has produced an evolutionary arms race. While bacteria use CRISPR-Cas to recognize and destroy phage genetic material, bacteriophages can escape CRISPR immunity through mechanisms including mutations in targeted sequences and production of anti-CRISPR proteins that inhibit Cas activity (Malone et al., 2021; Stanley & Maxwell, 2018). Understanding this interaction has become particularly relevant to phage therapy because bacterial antiviral defenses can limit therapeutic phage infection. Conversely, researchers can exploit CRISPR biology by engineering bacteriophages to carry CRISPR-Cas systems that selectively target pathogenic bacteria.

This review traces CRISPR from its discovery as an unusual repetitive DNA sequence to its identification as a bacterial adaptive immune system and subsequent development as a therapeutic technology. Particular emphasis is placed on the molecular mechanisms of CRISPR-Cas immunity, bacteriophage evasion of these defenses, the emerging development of CRISPR-enhanced bacteriophages as precision antimicrobials, and the broader application of CRISPR genome editing to human genetic disease.

The Discovery of CRISPR

The discovery of CRISPR began unexpectedly in 1987 when Ishino and colleagues identified an unusual pattern of repetitive DNA sequences while studying the iap gene in Escherichia coli. Near the target gene, the researchers noticed a series of highly conserved DNA repeats separated by unique spacer sequences. Although the organization of these sequences was unusual, their biological function remained unknown. At the time, the repeats were simply reported as an interesting genomic feature, with no identified connection to bacterial immune responses (Ishino et al., 1987). Nevertheless, this observation marked the first documented description of what would later become known as CRISPR.

Over the following decade, similar repeat-spacer arrays were recognized in numerous bacterial and archaeal species (Groenen et al., 1993; Mojica et al., 1993, 1995). Their widespread occurrence suggested that these structures served an important biological function rather than representing random genomic elements. A major advancement occurred in 2002 when Jansen and colleagues systematically characterized these repetitive loci and proposed the term clustered regularly interspaced short palindromic repeats, or CRISPR. In the same study, they identified neighboring genes consistently associated with CRISPR arrays and named them CRISPR-associated, or cas, genes (Jansen et al., 2002). The discovery of these genes suggested that CRISPR arrays functioned as part of a larger molecular system rather than as isolated DNA sequences.

Despite identification of CRISPR and its associated genes, the origin and function of the spacer sequences remained unclear. An important answer emerged in 2005 when three independent groups compared spacer sequences across microorganisms and discovered that many matched fragments of bacteriophage and plasmid DNA. Because bacteriophages infect bacteria, this finding suggested that spacers originated from previously encountered foreign genetic elements. The researchers proposed that CRISPR could function as a form of acquired immunity, allowing microorganisms to retain genetic information from previous encounters with foreign DNA (Bolotin et al., 2005; Mojica et al., 2005; Pourcel et al., 2005).

Direct experimental evidence supporting this hypothesis emerged in 2007 when Barrangou and colleagues studied Streptococcus thermophilus, a bacterial species widely used in yogurt and cheese production. Following exposure to bacteriophages, surviving bacterial strains acquired new spacer sequences derived from the infecting phages. Acquisition of these spacers was associated with resistance to subsequent infection, while removal or addition of particular spacers altered phage resistance accordingly (Barrangou et al., 2007). These experiments provided direct evidence that CRISPR functions as an adaptive antiviral defense system.

Together, studies conducted between 1987 and 2007 transformed CRISPR from an unexplained genomic feature into a major discovery in microbiology. Researchers progressed from identifying repetitive DNA sequences to demonstrating that bacteria can acquire, store, and use genetic information from previous viral encounters. Recognition of this adaptive immune function led researchers to investigate the molecular components and mechanisms through which CRISPR-Cas systems recognize and destroy invading genetic material.

Structure and Components of the CRISPR-Cas System

Following the discovery that CRISPR functions as an adaptive immune system, researchers sought to understand the molecular components that enable bacteria to recognize and defend against invading viruses. CRISPR-Cas systems consist of two major elements: CRISPR arrays and CRISPR-associated genes that encode proteins responsible for immune activity (Wiedenheft et al., 2012).

A CRISPR array contains short repetitive DNA sequences known as repeats separated by variable sequences known as spacers. The spacers frequently originate from foreign genetic elements such as bacteriophages and plasmids and therefore preserve sequence information from previous encounters with these elements (Horvath & Barrangou, 2010). CRISPR arrays are also associated with a leader sequence that contains regulatory elements important for array transcription and spacer acquisition.

Adjacent to CRISPR arrays are cas genes, which encode proteins responsible for different stages of the immune response. Cas1 and Cas2 are broadly conserved proteins involved in acquisition and integration of new spacers into CRISPR arrays. Other Cas proteins participate in processing CRISPR-derived RNAs and targeting invading nucleic acids. For example, Cas9 in type II CRISPR-Cas systems functions as an RNA-guided endonuclease capable of cleaving DNA corresponding to the sequence complementary by its RNA guide (Jinek et al., 2012).

The CRISPR array is transcribed into a precursor CRISPR RNA, or pre-crRNA, that is subsequently processed into shorter CRISPR RNAs (crRNAs) containing individual spacer-derived guide sequences. These crRNAs associate with Cas proteins and direct them toward complementary sequences in invading genetic material. Together, the CRISPR array, crRNAs, and Cas proteins create a sequence-specific surveillance and defense system capable of recognizing foreign nucleic acids (Wiedenheft et al., 2012).

Mechanism of CRISPR-Cas Immunity

CRISPR-Cas immunity can generally be divided into three stages: adaptation, expression, and interference. During adaptation, fragments of foreign genetic material are captured and integrated into the CRISPR array as new spacers, commonly through processes involving Cas1 and Cas2. The corresponding sequence in the invading genome is referred to as a protospacer. Incorporation of a new spacer provides the cell and its descendants with sequence information that can contribute to recognition of the same or closely related foreign genetic element during later encounters (Marraffini, 2015).

During the expression stage, the CRISPR array is transcribed into pre-crRNA and processed into individual crRNAs. Each crRNA contains sequence information derived from a previously acquired spacer and associates with Cas proteins to form an effector complex. These complexes use base-pair complementarity to recognize foreign nucleic acids corresponding to the stored spacer sequence.

During interference, the crRNA guides the CRISPR-Cas effector machinery to complementary sequences in invading genetic material. After appropriate target recognition, Cas proteins cleave or degrade the foreign nucleic acid, limiting successful replication of the invader. The precise molecular machinery differs among CRISPR-Cas types, but the underlying principle of RNA-guided sequence recognition allows CRISPR systems to provide highly specific antiviral protection (Marraffini, 2015; Wiedenheft et al., 2012).

The discovery of this mechanism substantially changed scientific understanding of immunity in unicellular organisms. Bacteria were shown to possess an adaptive defense capable of incorporating genetic information from previous encounters and using that information in subsequent responses. The programmability of this system also provided the biological foundation for later CRISPR-based genome-editing technologies.

Phage Evasion of CRISPR-Cas Immunity

CRISPR-Cas immunity places strong selective pressure on bacteriophages to develop countermeasures that allow them to infect CRISPR-protected bacterial populations. The resulting interactions form an evolutionary arms race in which bacterial defenses select for phage escape mechanisms, while successful phage variants can subsequently drive diversification of bacterial immunity (Malone et al., 2021).

One of the most direct methods of CRISPR evasion is alteration of the phage sequences recognized by the bacterial CRISPR system. CRISPR interference relies on sequence recognition between a crRNA and a corresponding target within the phage genome. Mutations in the targeted protospacer or important recognition sequences, including protospacer-adjacent motifs in systems that require them, can reduce effective recognition and allow some phages to escape existing CRISPR immunity (Malone et al., 2021). This demonstrates an important limitation of sequence-specific immunity: changes to the targeted viral sequence can reduce the effectiveness of previously acquired protection.

A particularly important phage counter-defense is the production of anti-CRISPR (Acr) proteins. Anti-CRISPRs are phage-encoded proteins that inhibit components of CRISPR-Cas systems and thereby permit infection of bacteria that would otherwise possess effective CRISPR immunity (Stanley & Maxwell, 2018). Different Acr proteins act through distinct mechanisms. Some interfere with DNA recognition or binding by CRISPR-Cas complexes, whereas others inhibit the nuclease activity required to destroy the invading genetic material. By disrupting critical stages of CRISPR-mediated interference, these proteins can provide phages with an opportunity to replicate despite bacterial adaptive immunity.

Anti-CRISPR activity can also affect infection at the level of a phage population. Borges et al. (2018) demonstrated that anti-CRISPR-producing phages can cooperate to overcome CRISPR-Cas immunity. An initial infection may fail to produce a complete phage replication cycle but can leave behind anti-CRISPR proteins that temporarily weaken bacterial immunity. Subsequent phages then have a greater probability of successfully replicating within the partially immunosuppressed bacterial population. This finding demonstrates that phage evasion can involve population-level interactions rather than solely mutations or proteins acting within an individual virus.

These evasion mechanisms are particularly relevant to phage therapy because a therapeutic phage must overcome the defenses of its bacterial target before it can successfully replicate and kill that cell. CRISPR-Cas immunity can therefore contribute to bacterial resistance against therapeutic phages, whereas understanding phage escape mechanisms can help researchers predict and potentially overcome barriers to successful treatment (Malone et al., 2021). Importantly, the relationship between CRISPR and phages has also been exploited in the opposite direction: researchers can engineer bacteriophages to carry CRISPR-Cas systems that increase their ability to selectively kill target bacteria.

CRISPR-Enhanced Phage Therapy

The growing incidence of antimicrobial-resistant bacterial infections has resulted in a rekindled focus in bacteriophage therapy. Traditional phage therapy incorporates bacteriophages with the ability to infect and kill vulnerable pathogenic bacteria. Nevertheless, bacterial defenses against phages, including CRISPR-Cas systems, can hinder effective infection, while the customary restricted scope of hosts of individual phages can limit the amount of bacterial strains that they recognize (Malone et al., 2021). Developments in the field of genetic engineering have led to the invention of an additional approach in which phages are modified to improve bacterial targeting or carry additional antimicrobial genetic components.

CRISPR-enhanced phage therapy integrates the natural host specificity of bacteriophages with the sequence specificity of CRISPR-Cas systems. In this approach, engineered phages transport CRISPR-Cas elements aimed at particular sequences within the target bacterium. In oppose to altering the patient’s genome, the phage distributes the antimicrobial system among bacterial cells. After inside a suitable bacterial host, CRISPR-mediated identification and degradation of bacterial DNA amplifies bacterial elimination. This method overturns the natural link among the two systems. To explain further, CRISPR initially evolved to protect bacteria from bacteriophages, whereas modified phages can employ CRISPR machinery against bacteria themselves.

One CRISPR-enhanced phage therapeutic that has begun its usage in human clinical testing is LBP-EC01, a genetically enhanced six-bacteriophage cocktail developed to target E. coli associated with urinary tract infections. LBP-EC01 uses a CRISPR-Cas3 system. In type I CRISPR-Cas systems, target recognition recruits Cas3, a nuclease-helicase capable of processive degradation of target DNA. This mechanism allows CRISPR-Cas3-enhanced phages to supplement the natural antibacterial activity of the phage with targeted destruction of bacterial DNA.

Kim et al. (2024) evaluated LBP-EC01 in the first portion of the phase 2 ELIMINATE clinical trial in adults with uncomplicated E. coli urinary tract infections and a recent history of drug-resistant E. coli UTI. Thirty-nine participants were included in the safety population. Different combinations of intraurethral and intravenous LBP-EC01 were evaluated, with participants also receiving the antibiotic trimethoprim-sulfamethoxazole. No serious adverse events were observed. Among 16 pharmacodynamically evaluable participants, investigators observed a rapid reduction of E. coli in urine after treatment that was maintained through day 10, and these participants had complete resolution of UTI symptoms by day 10 (Kim et al., 2024).

These results require careful interpretation. This portion of the ELIMINATE study was an open-label, uncontrolled dose-finding component, and LBP-EC01 was administered with an antibiotic. Consequently, the study cannot establish that the CRISPR-enhanced phages alone caused bacterial clearance or symptom resolution. Instead, it provides important early human evidence regarding the safety, pharmacokinetics, and pharmacodynamic activity of a CRISPR-Cas3-enhanced phage therapeutic and supports further controlled investigation (Kim et al., 2024).

Another CRISPR-enhanced phage therapeutic, SNIPR001, was developed to selectively target E. coli in the gastrointestinal tract. SNIPR001 consists of four complementary CRISPR-Cas-armed bacteriophages. During its development, selected phages were engineered with CRISPR-Cas systems containing E. coli-specific sequences, and phage engineering was also used to improve bacterial targeting (Gencay et al., 2024). The therapeutic is being investigated as a method of reducing intestinal E. coli, which can be an important source of bloodstream infection in vulnerable patients with hematological cancers.

The first-in-human study of SNIPR001 was reported by Petersen et al. (2026). This phase 1 trial was randomized, double-blind, placebo-controlled, and dose-escalating and enrolled 36 healthy adults. Participants received oral SNIPR001 or placebo for seven days. No serious adverse events or grade 3–4 adverse events occurred in participants receiving SNIPR001, and investigators did not observe a significant overall disruption of gut microbiome composition. Functional phages were recovered from stool in a dose-dependent manner, demonstrating that the therapeutic phages could survive passage through the gastrointestinal tract (Petersen et al., 2026).

The highest SNIPR001 dose was associated with an estimated 78% reduction in E. coli relative to placebo at day 14. However, the difference was not statistically significant following correction for multiple testing (Petersen et al., 2026). Therefore, the trial should primarily be interpreted as evidence supporting the safety, tolerability, recovery, and biological feasibility of CRISPR-Cas-armed phages rather than proof of clinical efficacy. Furthermore, the participants were healthy adults rather than patients being treated for active bacterial infections.

Together, the LBP-EC01 and SNIPR001 trials represent important early steps toward translating CRISPR-enhanced phage therapy into humans. The approach directly connects several stages in the history of CRISPR research. CRISPR was first identified as a bacterial defense against bacteriophages; investigation of phage evolution revealed mechanisms for overcoming bacterial CRISPR immunity; and researchers are now engineering phages to carry CRISPR machinery that can be directed back against bacterial cells. Although larger controlled studies are necessary to establish therapeutic efficacy, these first human investigations demonstrate the potential for CRISPR and bacteriophages to function together as precision antimicrobial technologies.

Other Clinical Applications of CRISPR: Genetic Diseases

Despite CRISPR-enhanced phage therapy continuing to be an emerging field, CRISPR genome editing has already been utilized clinically to treat hereditary human diseases. One of the most notable examples is exagamglogene autotemcel (exa-cel; Casgevy), engineered for sickle cell disease and transfusion-dependent β-thalassemia. Instead of  directly repairing disease-causing HBB variants, CRISPR-Cas9 is utilized ex vivo to disturb an erythroid-specific enhancer of BCL11A in the patient’s hematopoietic stem and progenitor cells. Decreasing BCL11A activity in the erythroid lineage amplifies fetal hemoglobin production, which can neutralize the effect of deficient adult hemoglobin. The edited cells are then reinfused into the patient after conditioning (Frangoul et al., 2021).

Additionally, CRISPR editing has been executed within the human body. NTLA-2001 employs lipid nanoparticles to transfer Cas9 messenger RNA and a guide RNA targeting TTR to hepatocytes in patients with transthyretin amyloidosis. A human study displayed dose-dependent reductions in circulating transthyretin succeeding a single treatment (Gillmore et al., 2021). Collectively with the clinical advancement of CRISPR-enhanced bacteriophages, these practices emphasize how molecular machinery beginning as a bacterial antiviral defense has been repurposed both to modify human cells and to selectively target pathogenic bacteria.

Conclusion

The discovery of  the CRISPR-Cas system revolutionized the understanding of prokaryotic immunity among scientists as it proved that bacteria and archaea can obtain sequence information from foreign genetic elements and apply it to employ targeted defense mechanisms in following confrontations (Barrangou et al., 2007; Marraffini, 2015). Originating as a mere observation of unexplained repetitive sequences in E. coli, it later would result in the depiction of a complex adaptive immune system which in turn provided the molecular basis for one of the most significant tools in biotechnology developed over the past few decades.

The dynamic present among bacteria with CRISPR incorporated immune systems and bacteriophages highlights the fast-paced and constantly changing nature of microbial evolution. CRISPR-Cas immunity inflicts evolutionary pressure on phages, which can react with mechanisms such as modification of targeted sequences along with creation of anti-CRISPR proteins that inhibit CRISPR-Cas activity (Malone et al., 2021; Stanley & Maxwell, 2018). These counteractive measures are not only essential for understanding bacterial-phage coevolution but are also applicable in phage therapy as bacterial antiviral immunity can impact whether a therapeutic phage effectively infects its target.

Researchers have recently begun harnessing the same biological interactions for antimicrobial treatment. Instead of functioning exclusively as the target of bacterial CRISPR immunity, bacteriophages can be engineered to include CRISPR-Cas systems targeted at pathogenic bacteria. Human studies of LBP-EC01 and SNIPR001 supply early indications that CRISPR-enhanced phage therapeutics can be given to humans and reinforce continued examination of their safety and therapeutic potential (Kim et al., 2024; Petersen et al., 2026). However, these procedures stay experimental, and larger controlled studies are required in order to establish clinical potency and determine how obstacles such as bacterial resistance, phage host range, delivery, and interactions with the microbiome influence their therapeutic use.

The expansion of CRISPR-based treatments for human genetic disease further illustrates the remarkable translation of fundamental microbiological research into medicine. From bacterial acquisition of viral sequences, to phage counter-defense, to engineered antimicrobial phages and human genome editing, the history of CRISPR demonstrates how understanding interactions between bacteria and viruses can generate fundamentally new approaches to disease treatment.

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