CRISPR technology in ovarian cancer research: advances from gene editing to precision diagnosis and therapy

Introduction

Ovarian cancer (OC) represents a malignancy characterized by significant molecular heterogeneity and clinical intractability, posing a substantial challenge in the field of gynecologic oncology. According to epidemiological data from the Global Cancer Observatory (GLOBOCAN) 2020, this disease accounts for approximately 4.7% of cancer-related mortality among women. The prognosis of OC is heavily dependent on the stage at diagnosis, with 5-year survival rates exceeding 90% for localized tumors [International Federation of Gynecology and Obstetrics (FIGO) stages I–II] but declining to below 30% for advanced-stage disease (FIGO stages III–IV) (1,2). This marked disparity in clinical outcomes is primarily attributable to two interrelated biological and clinical factors. First, the disease’s insidious onset and largely nonspecific symptomatology often result in delayed diagnosis, with only 15–20% of cases identified at early stages (3). Second, there is a near-universal development of therapeutic resistance, most notably reflected by the fact that approximately 80% of patients experience platinum-resistant recurrence within 24–36 months following initial treatment (4). Collectively, these challenges underscore the urgent need for innovative diagnostic paradigms, the identification of novel therapeutic targets to overcome resistance, and the implementation of precision medicine strategies capable of effectively addressing the genomic complexity of the disease.

The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing technology has catalyzed a significant paradigm shift in addressing these previously unmet needs. Originating from the adaptive immune system of Streptococcus pyogenes, the CRISPR-Cas9 platform was initially repurposed as a programmable genome-editing tool through the seminal work of Doudna and Charpentier (5,6). Compared with earlier genome-editing technologies, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), this system represents a substantial advancement by offering enhanced modularity, scalable multiplexed targeting, and cost-effectiveness (7,8). These advantages have enabled the systematic deconstruction of OC biology, allowing researchers to map driver mutations in tumor suppressor genes and to delineate complex molecular networks underlying platinum resistance, including drug efflux transporters and DNA damage response pathways (9,10).

In light of this rapidly evolving landscape, a timely synthesis of recent advancements is essential for monitoring progress and delineating future research directions. Accordingly, this review aims to provide a comprehensive framework for CRISPR applications in OC, encompassing the identification of therapeutic targets, the construction of advanced disease models, and the development of precision diagnostic and therapeutic strategies. By consolidating current knowledge and emerging evidence, this review seeks to contribute to the advancement of more personalized and effective management strategies for patients with OC.

Development of CRISPR-Cas9 gene editing technology

The intricate analysis of the significant molecular heterogeneity in OC and the identification of its genetic vulnerabilities have historically posed substantial challenges. Before the advent of CRISPR technology, genome editing tools such as ZFNs and TALENs facilitated targeted genome modification (11). However, these tools were constrained by the necessity for complex protein engineering for each specific target, low throughput, and high costs, which significantly limited their scalability for systematic studies in OC (12,13). The discovery and adaptation of the prokaryotic CRISPR-Cas9 immune system into a programmable genome-editing platform fundamentally addressed these limitations (14). This paradigm shift enabled OC researchers to transition from merely correlative observations to causal validation, facilitating high-throughput functional screens, precise disease modeling, and the development of cellular therapies. This evolution can be broadly categorized into three critical phases, each defined by conceptual and technical breakthroughs that have successively redefined the scope and potential of genomic manipulation (Figure 1).

Figure 1 Timeline of key milestones in the development of CRISPR technology. This schematic summarizes the major conceptual and technological advances in the CRISPR field, from the initial discovery of clustered repetitive sequences to the establishment of CRISPR-Cas9 genome editing, and the subsequent emergence of base editing, prime editing, and AI-assisted guide RNA design. AI, artificial intelligence; CRISPR, clustered regularly interspaced short palindromic repeats; sgRNA, single-guide RNA.

The foundational phase of CRISPR research began with the discovery and mechanistic elucidation of the CRISPR system. This trajectory dates back to 1987, when Ishino et al. first identified unusual clustered repeats interspersed with spacer sequences within the Escherichia coli genome (7). However, the biological function of these sequences remained unclear until 2002, when Jansen et al. formally introduced the term “CRISPR” and identified associated cas genes, thereby suggesting a role in microbial defense (15). A pivotal conceptual advancement occurred in 2005, when independent bioinformatic analyses conducted by Mojica and Pourcel demonstrated that CRISPR spacer sequences shared homology with bacteriophage and plasmid DNA, providing compelling evidence that CRISPR constitutes a component of the adaptive immune system in prokaryotes (8,16).

The subsequent translational phase was marked by the repurposing of this endogenous defense mechanism into a programmable genome-editing platform. A major breakthrough occurred in 2011, when Gasiunas et al. successfully reconstituted the Streptococcus pyogenes CRISPR-Cas9 system in vitro, elucidating its RNA-guided DNA cleavage mechanism and establishing the essential targeting role of the protospacer adjacent motif (PAM) (17). In 2012, the collaborative work of Doudna and Charpentier led to the engineering of a single-guide RNA (sgRNA), thereby simplifying the system into a versatile two-component platform composed of the Cas9 endonuclease and sgRNA, enabling sequence-specific DNA cleavage (9). The translational impact of this innovation was rapidly demonstrated in eukaryotic systems when, in 2013, research teams led by Zhang and Mali independently achieved highly efficient gene knockout in mammalian cells, an advance that dramatically accelerated the widespread adoption of CRISPR-Cas9 technology (18). In parallel, Mali et al. independently confirmed the feasibility of CRISPR-Cas9-mediated genome editing in human cells, further validating its broad applicability (19).

The most recent phase of CRISPR technology has focused on improving editing precision and expanding functional versatility. Early efforts to mitigate off-target effects associated with wild-type SpCas9 prompted the development of high-fidelity variants, including the Cas9 nickase (Cas9n, D10A) introduced in 2013 (20), followed by the rationally engineered SpCas9-HF1 in 2016 (21). Beyond canonical double-strand break (DSB)-mediated editing, innovative platforms such as base editing [2016] and prime editing [2019] have been developed to enable precise single-nucleotide substitutions and small insertions or deletions (indels) without inducing DNA DSBs (22,23). More recently, advances in CRISPR methodology have driven functional genomics toward single-cell resolution and combinatorial perturbation analysis. Technologies such as Direct-capture Perturb-seq [2019] and Cas13-based RNA Perturb-seq (CaRPool-seq, 2023) now facilitate high-throughput, combinatorial genetic screening integrated with single-cell transcriptomic profiling (24,25). The integration of artificial intelligence and machine-learning approaches has substantially improved sgRNA design, off-target prediction, and editing efficiency, thereby accelerating the development of intelligent and scalable genome-engineering strategies (26).

Technical process and diversity of CRISPR-Cas9 library screening

Technical process of CRISPR-Cas9 library screening

A significant challenge in OC research has been the unbiased and systematic identification of genes that are crucial for malignant phenotypes. Traditional loss-of-function screens utilizing RNA interference (RNAi) have been constrained by issues such as incomplete knockdown and off-target effects, which frequently result in ambiguous outcomes (27). In contrast, CRISPR-Cas9 library screening addresses these limitations by facilitating permanent, biallelic gene knockout with high specificity, thereby transforming functional genomics into a discovery-driven field. This methodology enables the simultaneous examination of every gene in the genome under selective pressures pertinent to OC, directly associating genotype with phenotypes such as drug resistance or metastatic capability. The optimized workflow described below has become essential for delineating the genetic vulnerabilities associated with OC.

CRISPR-Cas9 library screening constitutes a systematic, genome-scale interrogation strategy that enables the simultaneous perturbation of thousands of genes within a single experiment. This approach relies on the design and synthesis of extensive libraries comprising tens to hundreds of thousands of sgRNAs. These sgRNAs direct the Cas9 nuclease to induce knockout, activation, or inhibition of specific target genes across a cellular population. By integrating this systematic genetic perturbation with subsequent phenotypic selection, including but not limited to the evaluation of cell survival under drug treatment or the assessment of metastatic potential, researchers can identify genes that are critical for distinct biological processes or disease states. The feasibility of genome-wide loss-of-function screening using CRISPR-Cas9 was first demonstrated by the pioneering studies of Shalem et al. and Gilbert et al. in 2013, thereby ushering in a transformative era of functional genetic discovery (28,29).

The effective execution of a CRISPR screen requires a meticulously optimized and highly coordinated workflow encompassing library design, delivery, phenotypic selection, and data analysis (Figure 2). The process typically begins with the computational design and synthesis of an sgRNA library, tailored to the specific objectives of the screen, such as genome-wide gene knockout or transcriptional activation. These sgRNA sequences are subsequently cloned into lentiviral vectors, among which commonly used systems include lentiCRISPRv2 for gene knockout, lentiSAMv2 for transcriptional activation, and lentiGuide-Puro, all of which facilitate efficient delivery and stable genomic integration in target cells (30). Following viral transduction, the sgRNA library is introduced into target cells to generate a pooled cell population harboring stable genetic perturbations, after which defined screening conditions are applied to impose phenotypic selection. Finally, enriched or depleted sgRNAs are quantified by high-throughput sequencing, allowing for the identification of candidate genes associated with the selected phenotype.

Figure 2 Schematic workflow of CRISPR library screening. The diagram illustrates the standard pipeline for pooled CRISPR screening. A genome-scale sgRNA library is first designed, synthesized, and cloned to generate a lentiviral sgRNA library. Following viral production, target cells are transduced to achieve stable library integration. Phenotypic screening is then performed to identify genetic perturbations associated with specific cellular outcomes, such as proliferation, metastasis, or drug resistance. Genomic DNA is subsequently extracted, and sgRNA sequences are PCR-amplified and quantified by high-throughput sequencing. Bioinformatic analysis determines sgRNA enrichment or depletion, enabling the identification of candidate genes, which are further validated through multi-omics integration, in vitro and in vivo functional assays, and clinical sample analysis. CRISPR, clustered regularly interspaced short palindromic repeats; gDNA, genomic DNA; PCR, polymerase chain reaction; sgRNA, single-guide RNA.

Diversity of CRISPR-Cas9 library screening

The intricate nature of OC necessitates the use of advanced functional genomics tools that extend beyond mere gene knockout approaches. Although CRISPR knockout (CRISPRko) techniques are effective in identifying essential genes, they may overlook vulnerabilities arising from haploinsufficiency or gene overexpression. The expansion of CRISPR library methodologies addresses this limitation. At present, three primary methodologies dominate this field: CRISPRko for gene disruption, CRISPR activation (CRISPRa) for transcriptional upregulation, and CRISPR interference (CRISPRi) for targeted gene repression. In this section, we provide a critical analysis of their distinct molecular mechanisms, comparative advantages, and inherent limitations. In addition, we discuss the emerging class of base-editing libraries, which enable precise single-nucleotide conversions without inducing DSBs (Figure 3).

Figure 3 Classification of CRISPR-based technologies and their core features. (A) CRISPRko uses Cas9 and sgRNA to generate double-strand DNA breaks, resulting in permanent gene disruption. It enables efficient loss-of-function screening, including in non-coding regions, but is limited by variable editing efficiency, off-target DNA damage, and the need for multiple sgRNAs. (B) CRISPRa employs dCas9 fused to transcriptional activators (e.g., VP64) to induce endogenous gene expression without genomic alteration. This approach supports reversible gain-of-function studies but is sensitive to target accessibility and often requires cell-type-specific optimization. (C) CRISPRi utilizes dCas9 fused to repressors (e.g., KRAB) to silence gene transcription without inducing DNA breaks. It is effective for non-coding RNAs but may exhibit sequence-dependent and transient repression. (D) Base editing combines nCas9 with deaminases to achieve precise single-nucleotide conversions without double-strand breaks. While suitable for single-nucleotide variant interrogation, its application is constrained by PAM dependency, target accessibility, and potential off-target editing. CRISPRa, clustered regularly interspaced short palindromic repeats activation; CRISPRi, clustered regularly interspaced short palindromic repeats interference; CRISPRko, clustered regularly interspaced short palindromic repeats knockout; DSB, double-strand break; IncRNA, long non-coding RNA; PAM, protospacer adjacent motif; sgRNA, single-guide RNA; SNP, single nucleotide polymorphism.

CRISPRko library

The CRISPRko library technology represents a robust high-throughput screening platform for genome-wide functional gene deletion studies. This system leverages the CRISPR-Cas9 mechanism to induce targeted DNA cleavage and frameshift mutations through the use of precisely designed sgRNAs. Typically comprising tens of thousands of sgRNAs, these libraries guide the Cas9 nuclease to coding regions of target genes, thereby inducing DSBs. Subsequent DNA repair, predominantly mediated by error-prone non-homologous end joining (NHEJ), frequently results in indels that disrupt gene function via frameshift mutations (10). To enhance screening efficiency, lentiviral delivery systems are widely adopted to ensure stable sgRNA integration into host genomes, while pooled infection strategies facilitate comprehensive gene coverage (28). A landmark advancement in this area is the Genome-scale CRISPR Knock-Out (GeCKO) library, which employs a dual-vector system to separately express Cas9 and sgRNAs, thereby improving knockout efficiency and specificity (31). In OC research, CRISPRko libraries have been extensively applied to the systematic identification of genes governing tumor growth, survival, and metastatic behavior.

Compared with traditional RNAi methodologies, CRISPRko libraries demonstrate substantially superior performance in functional genomics screens (Figure 3A). The DNA-level gene disruption achieved by CRISPRko circumvents key limitations of RNAi, including incomplete gene silencing and pronounced off-target effects (32). Moreover, the ability of CRISPRko to target specific protein-coding regions enables higher-resolution functional interrogation. For instance, CRISPRko screening has revealed that genetic ablation of components of the poly [adenosine diphosphate (ADP)-ribose] polymerase (PARP) pathway, particularly BRCA1 and BRCA2, significantly enhances the sensitivity of OC cells to PARP inhibitors (33). Recent technological refinements, exemplified by the Brunello library, have incorporated advanced sgRNA design algorithms and increased guide density (4–6 sgRNAs per gene), thereby improving screening robustness and reproducibility (34).

Despite its widespread application, CRISPRko technology remains subject to several limitations. First, functional redundancy among gene families may result in compensatory mechanisms following gene knockout, thereby masking essential phenotypes (35). To mitigate this issue, inducible Cas9 systems, such as doxycycline-regulated platforms, have been developed to enable temporal control of gene inactivation and capture immediate phenotypic consequences (36). Second, although off-target effects are markedly reduced relative to RNAi, additional safeguards, including the use of high-fidelity Cas9 variants (e.g., SpCas9-HF1) and paired sgRNA strategies, have been implemented to further minimize nonspecific editing (21). Furthermore, whereas conventional CRISPRko libraries primarily target coding regions, next-generation whole-genome libraries now encompass noncoding elements, enabling functional interrogation of regulatory regions and noncoding RNAs (37). In OC research, the integration of single-cell sequencing with CRISPRko screening is emerging as a powerful strategy to address tumor heterogeneity and enhance the resolution of functional genomic analyses (38).

CRISPRa library

In contrast to CRISPRko libraries, which rely on Cas9-mediated DNA cleavage, CRISPRa libraries employ catalytically inactive dCas9 proteins fused to transcriptional activation domains, such as VP64, p65, or SunTag systems (39). These complexes are directed to gene promoters or enhancer regions, where they stimulate transcription by relieving epigenetic repression or recruiting endogenous transcriptional machinery, including the Mediator complex or synthetic activators (40). In OC research, CRISPRa libraries enable systematic gene overexpression, facilitating functional analyses of tumor suppressors, oncogenes, and chemotherapy resistance-associated genes. This gain-of-function approach complements CRISPRko-based loss-of-function screens, providing insight into gene dosage effects and pathway-level synergies.

The translational relevance of CRISPRa in OC lies in its ability to recapitulate endogenous gene activation, thereby uncovering subtle or redundant gene functions often overlooked in knockout screens. A seminal study by Ye et al. employed genome-wide CRISPRa screening to identify alternative activation pathways, including TGF-β and WNT signaling, that contribute to chemotherapy resistance in high-grade serous ovarian cancer (HGSOC) (41). Moreover, CRISPRa libraries facilitate the systematic mapping of gene interaction networks through single- or multi-gene co-activation strategies, as demonstrated by the identification of compensatory DNA repair pathways in BRCA1/2-deficient contexts (42). Compared with conventional cDNA overexpression libraries, CRISPRa minimizes artifacts associated with ectopic expression by modulating endogenous regulatory elements, thereby achieving more physiologically relevant gene regulation (43). Clinically, CRISPRa screens have identified candidate targets for immunotherapy sensitization, such as TAP1 activation, which contributes to remodeling the OC immune microenvironment (44).

Despite these advantages, CRISPRa technology faces several technical challenges (Figure 3B). First, activation efficiency is strongly influenced by chromatin accessibility, with heterochromatic regions exhibiting resistance to transcriptional upregulation (45). Optimization strategies include sgRNA targeting of DNase-hypersensitive sites and the use of multiplexed activation platforms, such as the SAM system combined with MS2-p65-HSF1 modules (40). Second, excessive or non-physiological gene activation may introduce experimental artifacts; for example, overactivation of pro-apoptotic genes may obscure biologically relevant phenotypes. To address this, inducible CRISPRa systems, such as Tet-On-regulated dCas9-VPR platforms, enable temporal control of gene activation (46). Finally, delivery challenges in complex models are being addressed through adeno-associated virus (AAV) vectors and lipid nanoparticle (LNP) formulations, which enhance CRISPRa library performance in OC patient-derived xenograft (PDX) models (47).

CRISPRi library

CRISPRi libraries utilize a modified CRISPR-Cas9 system to achieve targeted transcriptional repression. This approach employs a nuclease-inactivated dCas9 protein fused to transcriptional repressor domains, most commonly KRAB, forming a dCas9-repressor complex43. Guided by sgRNAs, this complex is recruited to promoter regions or transcription start sites (Figure 3C) (48). where it represses transcription by sterically blocking RNA polymerase II or by recruiting chromatin-modifying enzymes, including histone deacetylases. Unlike CRISPRko, CRISPRi induces reversible gene silencing without generating DSBs, thereby reducing genomic instability and enabling functional interrogation of essential genes in OC models.

CRISPRi offers several advantages in loss-of-function screening. First, its high specificity minimizes confounding DNA damage responses, such as inappropriate p53 activation, that can accompany CRISPRko-based approaches (35). For example, CRISPRi-mediated repression of the MYCN oncogene promotes differentiation in OC cells without inducing cytotoxic DNA damage (49). Second, CRISPRi supports combinatorial gene repression, facilitating systematic exploration of genetic interaction networks. A genome-wide CRISPRi screen conducted by Gilbert et al. identified transcriptional regulators, including NF-κB and STAT3, as mediators of chemotherapy resistance in OC through modulation of anti-apoptotic BCL2 family proteins (37). Additionally, CRISPRi has proven valuable for functional studies of noncoding RNAs, exemplified by targeted repression of the oncogenic long noncoding RNA HOTAIR (50).

Nonetheless, CRISPRi is not without limitations. Similar to CRISPRa, its efficacy depends on chromatin accessibility, with heterochromatic regions exhibiting reduced repression efficiency (51). Sustained expression of dCas9-KRAB may also induce cellular toxicity, particularly in primary cells or in vivo settings (43). Furthermore, constraints in sgRNA design, such as high GC content, can compromise dCas9-DNA binding stability (52). Emerging strategies to overcome these challenges include chromatin-informed sgRNA design and transient expression systems to enhance specificity and biocompatibility.

Base editing library

Base editing libraries constitute a class of precision CRISPR tools that enable single-nucleotide substitutions without inducing DSBs (Figure 3D). This technology was pioneered by David Liu’s group in 2016 through the fusion of engineered deaminases with Cas9n. Cytosine base editors (CBEs) were initially developed to mediate C•G-to-T•A conversions, followed by adenine base editors (ABEs) in 2017, which enable A•T-to-G•C transitions (22,53). Mechanistically, sgRNA-directed localization positions the editor at the target locus, where the deaminase catalyzes base conversion, while Cas9n introduces a nick in the non-edited strand to bias DNA repair toward the desired substitution (54). By avoiding DSB formation, base editing eliminates uncontrolled indel generation associated with NHEJ, thereby substantially improving editing precision.

Compared with conventional CRISPR libraries, base editing platforms offer two principal advantages: single-nucleotide resolution and compatibility with non-dividing cells, such as ovarian stromal cells, enabling functional interrogation of the tumor microenvironment (55). However, several limitations remain. The editable window is typically restricted to 4–8 nucleotides within the sgRNA target region, constraining correction of certain pathogenic mutations (56). Additionally, deaminase-mediated off-target edits have been detected at both DNA and RNA levels, particularly due to the promiscuous activity of APOBEC1 (57). Moreover, the large size of base editors, such as the ~5.2 kb ABE, exceeds the packaging capacity of AAV vectors, posing challenges for in vivo delivery (58).

Future efforts should focus on improving editor specificity through the development of high-fidelity deaminase variants and on advancing delivery technologies to facilitate the clinical translation of base-editing strategies for precision OC therapy.

Application of the CRISPR system in OC research

The utilization of the CRISPR system in OC research has evolved from interrogating fundamental molecular mechanisms to driving advances in precision diagnosis and targeted therapy. By enabling precise editing of oncogenic driver genes, tumor suppressor genes, and key regulators involved in chemotherapy resistance and immune evasion, this technology allows comprehensive dissection of the molecular networks underlying OC initiation, progression, and therapeutic response (Figure 4). CRISPR-based disease models faithfully recapitulate tumor heterogeneity and the metastatic microenvironment, while their high-precision gene-editing capabilities provide innovative avenues for the identification of early diagnostic biomarkers. An overview of representative CRISPR systems, key gene hits, functional categories, and underlying mechanisms discussed in this section is provided (Table 1).

Figure 4 Applications of CRISPR-Cas systems in ovarian cancer research and therapy. This schematic summarizes the multifaceted applications of CRISPR technology in ovarian cancer, encompassing functional library screening, disease model construction, metastasis research, drug resistance analysis, and molecular diagnosis. CRISPR-based screening enables systematic identification of genes driving tumor initiation, metastatic progression, and therapeutic resistance. Precise genome editing facilitates the generation of genetically defined cell lines, organoids, and animal models that recapitulate tumor heterogeneity and microenvironmental interactions. In addition, CRISPR approaches are applied to interrogate mechanisms of resistance to chemotherapy, targeted therapy, immunotherapy, and radiotherapy, as well as to develop DNA- and RNA-based diagnostic strategies. Diverse delivery platforms, including viral vectors, nanoparticles, physical methods, and exosomes, further expand the translational potential of CRISPR-based interventions in ovarian cancer. CRISPR, clustered regularly interspaced short palindromic repeats; sgRNA, single-guide RNA.

Study of OC driver genes using the CRISPR system

The accurate differentiation between driver mutations and passenger alterations presents a fundamental challenge in the study of OC, due to its intricate and heterogeneous genomic landscape. Although correlative sequencing studies have cataloged numerous genetic alterations, historically, establishing direct causal evidence has depended on low-throughput functional assays. The emergence of CRISPR-Cas9 technology has transformed this paradigm by offering a comprehensive framework for both the unbiased discovery and direct functional validation of genetic dependencies. This is accomplished through two complementary strategies: high-throughput library screening for genome-wide interrogation and precise gene editing for mechanistic dissection. This integrated approach has not only reinforced the significance of canonical oncogenic pathways but has also identified novel drivers, including those originating from endogenous viral elements and metabolic regulators (59,60), thereby broadening the therapeutic landscape for OC.

Precise gene editing has served as the foundational approach for validating candidate drivers, particularly those arising from conventional protein-coding genes. By directly manipulating genomic sequences, researchers can establish causal links between specific genetic alterations and downstream phenotypic consequences. Li et al. demonstrated that knockout of SKP1 and CUL1 results in cyclin E1 accumulation and chromosomal instability, thereby identifying these genes as critical early drivers in the pathogenesis of high-grade serous ovarian carcinoma (61). Beyond these core cell cycle regulators, similar editing strategies have uncovered drivers in less expected contexts. In ovarian clear cell carcinoma (OCCC), ablation of APELA revealed its role in promoting cellular proliferation and migration through a noncanonical, APLNR-independent activation of p53 signaling, highlighting a previously unrecognized therapeutic vulnerability (62). Similarly, CRISPR-mediated knockout of ATAD2 confirmed that its genomic amplification drives tumor proliferation via activation of the JNK-MAPK pathway, and that elevated ATAD2 expression correlates with poor clinical prognosis, supporting its potential utility as both a biomarker and therapeutic target in aggressive OC subtypes (63).

Remarkably, CRISPR-based editing has also uncovered driver genes originating from genomic elements traditionally considered non-coding or even non-human. Notably, knockout of the human endogenous retrovirus HERV-K env gene significantly suppressed proliferation, migration, and invasion in OC cell lines. This effect was mediated through differential regulation of RB and cyclin B1, implicating a viral-derived genomic element as a functional dependency and a potential therapeutic target (59). Parallel investigations have revealed that metabolic regulators represent another emerging class of unconventional drivers. Deletion of the lipogenic enzyme PLA2G3 resulted in reduced tumor growth and metastatic capacity, increased sensitivity to platinum-based chemotherapy, and restoration of the tumor-suppressive primary cilium. These findings position PLA2G3 as a key mediator of metastasis and chemoresistance in OC (60).

While targeted editing provides mechanistic depth, high-throughput CRISPR library screening offers complementary breadth by enabling systematic, unbiased discovery of genetic vulnerabilities. Genome-wide knockout libraries, such as GeCKO and Brunello, together with activation libraries like SAM, enable unbiased discovery of genes essential for cancer cell survival. Applying these screening platforms to genetically defined OC subtypes has revealed context-specific dependencies. In ARID1A/PIK3CA-mutant OCCC, these screens identified the epigenetic regulator KDM2A and the translation initiation factor PAIP1 as selectively essential; their loss disrupts chromatin homeostasis and oncoprotein translation, thereby nominating them as precision therapeutic targets (64). Similarly, independent screening efforts identified the phosphate transporter XPR1 (SLC53A1) as indispensable for OCCC cell survival. It’s knockout reprograms phosphate metabolism, induces cell-cycle arrest and apoptosis in vitro, and suppresses tumor growth in vivo, suggesting a novel metabolism-based therapeutic strategy (65).

Beyond identifying essential genes directly, CRISPR screening has proven particularly powerful for uncovering synthetic lethal interactions, which provide a conceptual framework for targeted therapy development. A comprehensive genome-wide knockout screen identified approximately 140 genes whose loss confers a specific dependency on DNA polymerase theta (Pol θ, encoded by POLQ), a phenomenon termed “Pol θ addiction”. This dependency is observed in cells harboring defects across multiple DNA damage response pathways, extending beyond homologous recombination deficiency. Given that a substantial proportion of OCs harbor alterations in these pathways, pharmacological inhibition of Pol θ emerges as a promising therapeutic strategy applicable to a broader patient population. This paradigm exemplifies the capacity of CRISPR screens to uncover context-specific therapeutic vulnerabilities (66).

Collectively, the integration of precision genome editing and high-throughput CRISPR screening is systematically mapping the landscape of OC driver genes. This dual approach has not only validated canonical oncogenic pathways but has expanded the driver concept to include viral elements, metabolic regulators, and context-dependent synthetic lethal partners, thereby elucidating the molecular basis of tumor heterogeneity and uncovering novel targets for personalized therapeutic intervention.

Study of OC metastatic mediators using the CRISPR system

The CRISPR system has emerged as a pivotal instrument for elucidating the intricate mechanisms underlying OC metastasis. By enabling both precise functional validation and comprehensive genome-wide interrogation, this technology provides unprecedented insights into the molecular networks governing tumor invasion, metastatic colonization, and adaptation to diverse microenvironmental contexts.

Targeted CRISPR editing has been instrumental in establishing causal relationships between specific genes and metastatic phenotypes, particularly within core signaling pathways. By selectively manipulating specific genetic targets, this approach allows researchers to establish direct causal relationships between gene function and metastatic phenotypes. For example, disruption of the LKB1-STRAD signaling axis through a combination of CRISPR-mediated knockout and RNAi demonstrated its essential role in peritoneal dissemination. Mechanistically, LKB1 regulates matrix metalloproteinase expression and fibronectin remodeling, and therapeutic targeting of this pathway resulted in a significant reduction in tumor burden in preclinical models (67). Extending this logic to other pro-metastatic regulators, dual CRISPR strategies involving gene knockout in SKOV3 and OVCAR8 cells and gene overexpression in OVAR3 cells identified EIF5A2 as a potent promoter of metastasis through activation of TGFβ-mediated epithelial-mesenchymal transition (EMT) (68). Together, these findings exemplify how targeted genome editing can validate key regulators of the metastatic microenvironment and core EMT signaling cascades.

While targeted approaches provide mechanistic depth, genome-wide CRISPR screening has substantially expanded the landscape of metastasis-associated genes by enabling unbiased discovery across diverse biological processes. Through unbiased functional genomics, these screens have uncovered previously unrecognized genetic dependencies spanning diverse biological processes. A landmark study in HGSOC integrated genome-wide CRISPRko screening with multi-omics profiling and identified ACADVL, ECHDC2, and ULK1 as essential mediators of anoikis resistance. Loss of these genes compromised the survival of detached tumor cells, with metabolic analyses implicating fatty acid reprogramming as a critical driver of peritoneal dissemination (69).

Beyond core survival mechanisms, CRISPR screens have revealed that metastatic vulnerability is profoundly shaped by context-specific factors, including the tumor microenvironment and systemic host conditions. In vivo CRISPR screening performed under psychological stress conditions demonstrated that depletion of the serotonin receptor HTR1E exacerbates EMT and peritoneal metastasis via activation of the proto-oncogene tyrosine-protein kinase SRC signaling pathway. Pharmacological modulation using either HTR1E agonists or SRC inhibitors effectively attenuated stress-induced tumor progression (70). Parallel screening efforts have identified key mediators of metabolic reprogramming that confer metastatic competence while simultaneously creating therapeutic vulnerabilities. The lipid-metabolizing enzyme ACSL4 enhances membrane fluidity through increased synthesis of polyunsaturated fatty acids, while concurrently sensitizing cells to ferroptosis-a paradoxical effect counterbalanced by the homeostatic functions of ABHD6, ECI1, and ECH1 (71). The importance of the immune microenvironment has further emerged from screens identifying FCGR1A, which promotes EMT through regulation of LSP1, and the metastasis suppressor PTGES3, which inhibits metastatic progression via modulation of glycolysis through PFKL (72,73).

Collectively, the integration of precise gene manipulation with systematic genome-wide screening has enabled CRISPR technology to delineate the complex and heterogeneous landscape of OC metastasis across metabolic, signaling, and microenvironmental dimensions. This integrative framework reveals that metastatic progression is not governed by a single pathway but rather by a network of context-dependent adaptations, including anoikis resistance via metabolic reprogramming, stress-induced signaling cascades, and immune modulation. Such multidimensional insights are essential for the identification of novel, context-specific therapeutic targets aimed at disrupting metastatic progression.

Study of OC drug-resistance genes using the CRISPR system

Therapeutic resistance to chemotherapy, PARP inhibitors, and immunotherapy represents a primary obstacle to successful treatment outcomes in OC. Traditional methodologies have constrained our understanding of this complex adaptive process by providing only a fragmented perspective on the underlying mechanisms. The advent of CRISPR-Cas9 library screening offers a solution by facilitating the unbiased, genome-wide identification of genes whose loss influences cell survival under therapeutic pressure. This systems-level approach elucidates the integrated networks that drive resistance. As elaborated below, such screenings have identified novel genetic determinants of chemoresistance and PARP inhibitor resistance, while also revealing previously unrecognized mechanisms of immune evasion, collectively offering a strategic framework for overcoming therapeutic failure.

CRISPR-based loss-of-function screens have been particularly impactful in dissecting resistance mechanisms to PARP inhibitors, revealing both established and previously unrecognized genetic dependencies. Through genome-wide knockout screening, the ubiquitin-activating enzyme UBA1 was identified as a critical genetic dependency. Pharmacological inhibition of UBA1 disrupts DNA repair pathways and exhibits synergistic antitumor activity when combined with PARP inhibitor in homologous recombination-proficient models, supporting a promising combinatorial therapeutic strategy (74). Beyond identifying individual resistance genes, these screens have illuminated complex genetic interaction networks that govern PARP inhibitor sensitivity. Additional screening efforts have revealed complex genetic interaction networks, identifying ATM, MUS81, and BRCA2 as predictive biomarkers of olaparib sensitivity, while also implicating CDK12 as an essential regulator of global cellular viability (75). The synthetic lethality paradigm has been further expanded through screens revealing collateral dependencies in DNA repair-deficient backgrounds. Findings that BReast CAncer (BRCA)-deficient tumors harbor vulnerabilities in genes such as APEX2 and FEN1, indicating collateral dependencies within base excision repair and microhomology-mediated end-joining pathways and thereby revealing new therapeutic opportunities (76).

Extending beyond PARP inhibitor resistance, CRISPR screening has elucidated the multifactorial nature of resistance to platinum-based chemotherapy, revealing both core regulators and unconventional survival strategies. Unbiased genome-wide investigations have identified previously unrecognized regulators, including ZNF587B and SULF1, as contributors to platinum resistance (77). Perhaps more strikingly, these screens uncovered adaptive metabolic reprogramming as a key resistance mechanism. Tumor evasion of ferroptotic cell death via PAX8-mediated reprogramming of glutathione metabolism exemplifies how cancer cells exploit non-canonical pathways to survive chemotherapy (78). Taken together, these findings contribute to a detailed molecular landscape of chemoresistance encompassing dysregulated DNA damage responses, metabolic plasticity, and altered cell death pathways.

Parallel CRISPR screening efforts have increasingly focused on delineating immune evasion mechanisms, revealing how tumor cells actively sculpt their immunological environment to escape detection. Genome-wide approaches have identified GPAA1 as a novel regulator of an innate immune checkpoint, enhancing the CD24 Siglec-10 “don’t eat me” signal and thereby suppressing macrophage-mediated phagocytosis (79). In vivo CRISPR screening models have further revealed how immune modulation operates in a context-dependent manner within the tumor microenvironment. Loss of the cytokine receptor IL20RA was shown to inhibit metastatic dissemination by skewing macrophage polarization toward an antitumor M1 phenotype, a finding corroborated by clinical progression data (80). Moreover, screens have uncovered unexpected links between innate immune signaling and adaptive antitumor immunity. Deletion of TRAF3 remodeled the tumor microenvironment by enhancing major histocompatibility complex (MHC) class I antigen presentation and promoting a B cell-activating, IgA-enriched ascitic milieu, revealing a previously unappreciated connection between intracellular signaling pathways and broader immunological reprogramming (81).

By integrating these complementary screening strategies across chemotherapy, PARP inhibition, and immunotherapy contexts, CRISPR technology has moved beyond cataloging discrete resistance-associated genes to reconstructing the interconnected molecular circuits that underlie therapeutic failure. This systems-level perspective reveals that resistance is not a monogenic trait but emerges from coordinated adaptations involving DNA repair plasticity, metabolic reprogramming, and immune microenvironment remodeling. Such integrated understanding is essential for the rational development of next-generation therapeutic strategies capable of preempting or reversing resistance, thereby advancing toward more durable and effective management of OC.

Construction of OC models using CRISPR system

A significant challenge in OC research has been the absence of experimental models that accurately reflect the disease’s intricate genetics and tissue microenvironment. Traditional models, such as immortalized cell lines and PDXs, often suffer from limitations related to genetic relevance, physiological context, and throughput capacity for systematic analysis. The CRISPR-Cas9 technology effectively addresses these limitations by allowing for the precise introduction of single or combinatorial driver mutations into specific cellular contexts. This capability enables the creation of genetically accurate models, including organoids and advanced in vivo systems, which mimic disease initiation, progression, and treatment response. As elaborated below, these CRISPR-engineered models are providing crucial insights into the effects of cell origin, the dynamics of metastatic dissemination, and the functional interactions between tumor genetics and the immune microenvironment. Consequently, they are establishing a new generation of manageable and physiologically relevant platforms for therapeutic discovery.

CRISPR-engineered in vitro models, particularly organoid systems, have revolutionized our ability to study tumor initiation by enabling the de novo generation of malignancies from normal epithelial cells while preserving tissue-specific contexts. A landmark study by Lohmussaar et al. employed CRISPR-mediated editing to induce high-frequency mutations in Trp53 and Brca1 within murine fallopian tube and ovarian surface epithelial organoids, thereby establishing a dual-cell-of-origin model of HGSOC. This work demonstrated that the cellular origin of transformation critically influences malignant potential and lineage-specific chemotherapeutic responses (82). Building upon this foundational insight, subsequent organoid models have proven valuable for recapitulating subtype-specific treatment responses. A tubal organoid model combining Trp53 knockout with Ccne1 and Akt2 overexpression successfully recapitulated subtype-specific chemoresistance and enabled the identification of effective immunotherapeutic combinations for CCNE1-amplified tumors, highlighting its predictive value for personalized treatment strategies (83). Complementing organoid systems, CRISPR-modified cell lines have provided scalable platforms for dissecting genotype-phenotype relationships. In parallel, CRISPR-engineered ID8 cell lines have elucidated genotype-phenotype relationships, demonstrating that Trp53 deletion promotes an immunosuppressive microenvironment through CCL2-mediated recruitment of myeloid cells. Moreover, the simultaneous deletion of Brca1 and Brca2 establishes a homologous recombination-deficient state, markedly enhancing sensitivity to PARP inhibitor (84,85).

To interrogate the dynamic interplay between tumor-intrinsic genetics and the host microenvironment, CRISPR technology has enabled the development of increasingly physiologically relevant in vivo models that capture the complexity of tumor-stroma interactions. These advanced systems include both genetically engineered mouse models and refined transplantation-based platforms, each providing complementary insights into tumor evolution and immune remodeling. In genetically engineered mice, CRISPR-mediated knockout of Ankrd22 revealed that its loss accelerates tumor progression by promoting polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC)-driven immunosuppression via upregulation of WDFY1, thereby identifying a novel regulator of the immunosuppressive niche (86). Beyond conventional GEMMs, innovative delivery strategies have expanded the repertoire of manipulable in vivo systems. In the context of HGSOC, an innovative in vivo electroporation strategy was used to introduce Brca1 and Pten mutations, generating a model that faithfully recapitulates human disease pathology. Importantly, integration of Cre-based lineage tracing provided unprecedented insights into the early events of peritoneal micrometastatic dissemination (87).

Beyond fully engineered genetic models, transplantation systems utilizing CRISPR-modified cells offer a flexible and scalable framework for dissecting the combinatorial effects of defined oncogenic alterations within an intact immune microenvironment. Enhanced ID8 transplantation models, particularly those incorporating dual Trp53 and Brca2 knockout, have demonstrated substantial translational relevance. These models not only reproduce clinically meaningful homologous recombination deficiency but also actively reshape the immune microenvironment, fostering the formation of tertiary lymphoid structures (84,85). The modularity of these systems represents a particular advantage for systematic investigation. The modularity of these systems, which allows for the incorporation of additional driver mutations such as Nf1 and Pten, facilitates systematic investigation of chemoresistance mechanisms and immunotherapy responsiveness within an integrated physiological setting (83,84).

In conclusion, the strategic implementation of CRISPR-Cas9 technology in the construction of OC models is significantly advancing our understanding of the relationship between specific genetic alterations and complex disease phenotypes. These advanced platforms, which include organoids, genetically engineered mice, and transplantable systems, have collectively revolutionized our capacity to investigate the role of mutations in tumorigenesis, metastatic behavior, and immune response modulation. By facilitating precise genetic manipulation within physiologically relevant settings, these models are essential for the validation of therapeutic targets, the mechanistic exploration of resistance pathways, and the informed development of personalized treatment strategies for OC.

Application of CRISPR system in OC diagnosis

The early detection of OC is crucial for enhancing patient survival rates; however, it remains a significant challenge due to the absence of sensitive and specific biomarkers. Traditional diagnostic techniques, including serum CA-125 testing and imaging, frequently fail to detect the disease at its early, more treatable stages, underscoring the urgent need for more sophisticated molecular tools (88). CRISPR-based diagnostic platforms, which utilize the programmable accuracy of Cas enzymes, directly address this necessity. These platforms facilitate the direct, amplification-free detection of tumor-derived nucleic acids with single-molecule sensitivity through minimally invasive liquid biopsies. This innovative approach not only holds the potential to surpass the efficacy of conventional assays but also enables the multiplexed profiling of biomarkers such as extracellular vesicles (89). As elaborated below, the application of CRISPR diagnostics in OC is progressing beyond early detection to include real-time molecular subtyping and monitoring of therapeutic responses, thereby establishing a new paradigm for the precision management of the disease.

A seminal demonstration of this diagnostic paradigm employed a CRISPR-Cas13a-based system for profiling microRNAs within plasma-derived extracellular vesicles, establishing the foundational framework for amplification-free liquid biopsy in OC. The CRISPR-Cas13a platform selectively recognizes and cleaves target RNA sequences, enabling highly sensitive quantification of miR-21-5p at the level of individual EVs. Notably, this strategy eliminates the need for RNA extraction and enzymatic amplification, thereby substantially simplifying the diagnostic workflow. The utilization of EVs as diagnostic substrates is particularly advantageous, as these vesicles encapsulate tumor-derived nucleic acids and faithfully reflect the molecular features of the tumor microenvironment, offering a minimally invasive liquid biopsy approach (90-92). Clinical validation of this platform demonstrated that patients with OC exhibit markedly elevated proportions of miR-21-5p-positive EVs in plasma, ranging from 2% to 10%, compared with benign controls, in whom levels typically remain below 0.65%. These findings exhibited strong concordance with RT-qPCR measurements, highlighting the sensitivity, specificity, and analytical robustness of the assay (90). The ability to detect tumor-derived EVs with such high precision underscores the potential of this method for early OC diagnosis, a context in which timely intervention can dramatically improve patient outcomes.

Building upon single-biomarker detection, further refinement of this diagnostic paradigm has been achieved through the integration of surface protein labeling with miRNA detection, substantially enhancing multiplexing capacity and diagnostic specificity. This innovation allows for the high-resolution characterization of distinct EV subpopulations originating from ovarian tumors (93). By facilitating the simultaneous detection of multiple molecular features on single EVs, this multiplexed approach significantly improves diagnostic specificity while increasing overall sensitivity and accuracy. The combined profiling of EV surface proteins and intravesicular miRNA signatures thus establishes a novel liquid biopsy framework, offering a non-invasive, high-throughput strategy for molecular-level OC screening (94).

Beyond initial detection and characterization, CRISPR-based diagnostic platforms hold considerable promise for longitudinal disease monitoring, creating opportunities for real-time assessment of therapeutic response and early identification of recurrence. Dynamic tracking of tumor-derived EVs during treatment may provide real-time insights into disease evolution and therapeutic efficacy, thereby informing adaptive treatment strategies (95). As these technologies advance, CRISPR-enabled molecular diagnostics are set to revolutionize the clinical management paradigm for OC. This transformation involves a shift from static, snapshot-based detection to continuous, dynamic monitoring. Such an approach integrates early diagnosis, molecular subtyping, treatment guidance, and recurrence surveillance into a cohesive precision oncology framework (96).

Delivery methods for CRISPR technology for OC

CRISPR-Cas technology holds substantial promise for transforming the therapeutic landscape of OC; however, its successful clinical translation is fundamentally dependent on the development of delivery systems that are both safe and efficient, while also enabling tumor-selective targeting. The unique anatomical and pathological features of OC, including widespread peritoneal dissemination and a profoundly immunosuppressive tumor microenvironment, impose stringent requirements on delivery platforms capable of traversing multiple biological barriers. Accordingly, the optimization of CRISPR delivery remains a central challenge in the field. This section provides a systematic overview of the principal strategies currently employed for the delivery of CRISPR components into OC cells, encompassing viral vectors, non-viral nanoparticle systems, physical delivery approaches, and exosome-based platforms (Figure 4). Each strategy is discussed with respect to its underlying delivery mechanisms, translational potential, and the technical, safety, and ethical challenges associated with its application.

Viral vector systems for CRISPR delivery in OC

Viral vectors represent one of the most effective platforms for the delivery of CRISPR machinery, providing high transduction efficiencies across a broad spectrum of cell types. This characteristic is particularly advantageous for targeting the heterogeneous cellular populations present within ovarian tumors and their surrounding microenvironment. To fully exploit this potential in the context of OC’s distinctive pattern of peritoneal dissemination, research is progressing towards advanced engineering strategies. These strategies encompass the modification of viral tropism through capsid engineering to enhance tumor selectivity, the use of intraperitoneal administration to optimize local biodistribution, and the development of conditionally replicative vectors to increase therapeutic payloads at disease sites. The subsequent discussion elucidates how these innovations are being integrated into lentiviral, adenoviral, and AAV platforms, thereby paving the way for more precise and efficacious in vivo gene editing therapies targeting OC.

Lentiviral vectors engineered with modified envelopes have demonstrated enhanced stability and tumor selectivity in preclinical models, offering valuable insights for OC applications. Although the following study was conducted in liver cancer, its design principles directly inform strategies to overcome ovarian-cancer-specific barriers. A preclinical study utilizing lentiviruses pseudotyped with hepatitis C virus (HCV) E1E2 envelope glycoproteins achieved systemic and tumor-selective gene delivery in orthotopic liver cancer models. This approach enabled efficient disruption of the kinesin spindle protein (KSP) gene, resulting in pronounced tumor growth inhibition. Compared with conventional vesicular stomatitis virus (VSV)-pseudotyped lentiviral vectors, the E1E2-modified system exhibited enhanced serum stability and reduced off-target immune activation, thereby underscoring its potential applicability for targeted CRISPR delivery in solid tumors (97). Such pseudotyping strategies could be adapted to improve lentiviral vector performance in the ascitic environment, where stability and immune evasion are critical for reaching disseminated peritoneal lesions.

Adenoviral vectors, particularly high-capacity variants, offer high transduction efficiency and reduced genotoxicity, features that are attractive for OC therapy where repeated or high-dose administration may be required. Adenoviral vectors constitute another powerful delivery modality, distinguished by their high transduction efficiency and episomal expression, which minimizes the risk of insertional mutagenesis. High-capacity adenoviral vectors (HCAdVs) engineered to deliver human papillomavirus (HPV)-specific CRISPR-Cas9 constructs targeting the E6 oncogene have demonstrated selective cytotoxicity in HPV-positive cervical cancer models, markedly reducing tumor cell viability while sparing HPV-negative controls (98). This tumor-selective killing strategy could be repurposed for OC by targeting prevalent mutations such as TP53 or BRCA1/2, and the large cargo capacity of HCAdVs allows co-delivery of multiple guide RNAs or therapeutic genes to address the heterogeneity of ovarian tumors.

AAVs bring the advantage of a well-established clinical safety profile and are being actively explored for OC applications, particularly for local administration routes. In parallel, AAVs offer a well-established clinical safety profile and are currently under investigation in multiple gene therapy trials, with potential adaptations for OC applications (99). Intraperitoneal delivery of AAVs could exploit their low immunogenicity and ability to transduce quiescent cells, making them suitable for targeting disseminated tumor cells within the peritoneal cavity.

Collectively, lentiviral, adenoviral, and AAV vectors provide a complementary arsenal for CRISPR delivery in OC, each offering distinct advantages that can be harnessed to address the unique challenges posed by peritoneal dissemination, ascitic fluid, and the need for tumor-selective targeting.

Non-viral nanoparticle delivery systems for OC

Non-viral nanoparticle systems constitute a highly adaptable and synthetically customizable platform for CRISPR delivery, characterized by their advantageous safety profiles and substantial cargo loading capacity. A significant benefit of these systems is their adjustable design parameters, including size, surface charge, and functionalization with targeting ligands such as folate or hyaluronic acid (HA). This programmable nature facilitates the development of precision carriers capable of targeting disseminated peritoneal metastases and supporting combination therapies in OC. As illustrated in the following examples, these systems are being advanced not only for efficient CRISPR delivery but also to address critical clinical challenges, such as multidrug resistance (MDR).

A key challenge in OC therapy is achieving selective delivery to tumor cells scattered throughout the peritoneal cavity while sparing healthy tissues. Folate-functionalized nanoparticles address this by exploiting the overexpression of folate receptors on OC cells. A representative example is provided by folate-functionalized nanoparticles specifically engineered to target OC peritoneal metastases. These multifunctional nanostructures enable the co-delivery of chemotherapeutic agents and radiopharmaceuticals within a monodisperse architecture, while folate ligands facilitate receptor-mediated tumor cell uptake. Comparative analyses have demonstrated superior therapeutic efficacy of these targeted nanoparticles relative to non-targeted formulations in both in vitro and in vivo models (100). This active targeting strategy could be similarly applied to CRISPR payloads, enabling selective gene editing in disseminated peritoneal lesions while minimizing off-target effects in normal tissues.

Beyond targeting, overcoming MDR represents another critical hurdle in OC treatment, where conventional chemotherapy frequently fails. HA-based nanocomplexes have been engineered to simultaneously address both delivery efficiency and chemoresistance reversal. Parallel advances in nanotechnology have focused on overcoming MDR, a major impediment to effective OC treatment. HA-based nanocomplexes have been developed to deliver MDR1-targeting siRNA into chemoresistant OC cells. These HA-conjugated hybrid nanoparticles achieved concomitant suppression of MDR1 and P-glycoprotein expression, thereby restoring sensitivity to paclitaxel. This work highlights the potential of nanocarrier-mediated genetic modulation to reverse chemoresistance through precision molecular intervention (101,102). Importantly, HA functionalization serves a dual purpose: it targets CD44 receptors overexpressed on OC cells while also enhancing nanoparticle stability in ascitic fluid, demonstrating how rational design can simultaneously address multiple ovarian-cancer-specific barriers.

Physical methods for CRISPR delivery in OC

Physical and hybrid delivery methods facilitate the direct cytosolic delivery of CRISPR ribonucleoproteins (RNPs) through transient membrane disruption, offering rapid action and avoiding risks from genomic integration. This approach is particularly suited for the peritoneal cavity, where engineered platforms such as stimulus-responsive nanocarriers and injectable hydrogels can enhance local retention and controlled release. These systems enable not only efficient gene editing but also potent synergistic combinations with conventional therapies, as demonstrated by the following advanced material designs.

A major obstacle in peritoneal delivery is the rapid clearance of therapeutic agents from the abdominal cavity, which limits their exposure to disseminated tumor nodules. Liposome-templated hydrogel nanoparticles (LHNPs) address this challenge by combining the stability of hydrogels with the delivery efficiency of liposomal formulations. A notable example is the development of LHNPs, which are designed for the simultaneous delivery of Cas9 protein and nucleic acids. By incorporating minicircle DNA technology, these nanostructures demonstrated superior delivery efficiency in vitro compared with commercially available transfection reagents. Their modular and tunable design enables adaptation to specific molecular targets, as exemplified by PLK1-directed LHNP-CRISPR constructs that significantly suppressed tumor proliferation and prolonged survival in murine OC models (103). The hydrogel component provides sustained release within the peritoneal cavity, overcoming the dilution and clearance effects of ascitic fluid, while the liposomal shell facilitates cellular uptake by tumor cells.

The heterogeneous and adaptive nature of OC cells often requires combination strategies that simultaneously deliver genetic modifiers and chemotherapeutic agents. Reduction-responsive nanocarriers have been engineered to exploit the distinct redox environment of the tumor microenvironment for coordinated drug and gene delivery. An alternative physical delivery strategy employs reduction-responsive fluorinated platinum (IV) nanocarriers (PtUTP-F) to achieve combinatorial therapeutic effects. This multifunctional platform co-delivers a CRISPRa system targeting the cancer-testis antigen CT45 alongside platinum-based chemotherapeutics. By inducing CT45 overexpression, the system suppresses DNA repair pathways while concurrently sensitizing OC cells to platinum treatment, thereby demonstrating a sophisticated approach to synergistic gene-drug therapy (104). The reduction-responsive design ensures preferential release within the intracellular environment of tumor cells, where glutathione levels are elevated, providing an additional layer of specificity for targeting peritoneal metastases.

Exosomes

Exosomes, as naturally occurring extracellular vesicles, present a biocompatible and targeted platform for the delivery of CRISPR technology. Their intrinsic properties, including low immunogenicity, circulatory stability, and tissue-specific homing capabilities, render them promising “stealth” carriers. By engineering these vesicles to encapsulate CRISPR components, researchers can exploit their natural tumor tropism, facilitating precise gene editing and potentially reversing therapy resistance in OC, as demonstrated in the following study.

The peritoneal cavity presents a uniquely challenging environment for synthetic delivery vehicles, which often face rapid clearance and protein corona formation in ascitic fluid. Exosomes offer a distinct advantage in this context due to their endogenous origin and inherent stability in biological fluids. Experimental studies have shown that tumor-derived exosomes preferentially accumulate in OC xenografts. When engineered to encapsulate CRISPR-Cas9 plasmids targeting PARP1, these exosomes effectively suppressed PARP1 expression, induced apoptotic cell death, and restored cisplatin sensitivity in OC cells. This strategy capitalizes on the natural tropism of exosomes to achieve targeted gene editing within the ovarian tumor microenvironment, while their lipid bilayer membrane protects CRISPR payloads from degradation in ascitic fluids. This strategy capitalizes on the natural tropism of exosomes to achieve targeted gene editing and combinatorial therapeutic effects (105,106). Future engineering efforts may focus on enhancing exosome loading efficiency and incorporating surface ligands for even greater specificity toward OC cell subtypes.

Current challenges in CRISPR delivery for OC

Despite substantial technological progress, several critical challenges continue to hinder the clinical translation of CRISPR-based therapies for OC. Off-target genome editing remains a principal safety concern, as unintended DNA modifications may disrupt essential gene functions and potentially induce oncogenic or other deleterious effects (107). Moreover, delivery vehicles themselves introduce additional risks; viral vectors may provoke immune responses, whereas the long-term biodistribution, persistence, and potential toxicity of nanoparticle-based systems require comprehensive evaluation.

Beyond technical considerations, the application of CRISPR technology in oncology raises significant ethical and regulatory issues. Although current therapeutic strategies are largely confined to somatic genome editing, the theoretical possibility of germline modification, together with precedents established by related research, necessitates rigorous ethical oversight. Issues surrounding informed consent, equitable access to advanced therapies, and the broader societal implications of human genome editing warrant sustained discussion within both the scientific community and the public sphere (108). Addressing these intertwined scientific, safety, and ethical challenges is essential for the responsible and effective clinical implementation of CRISPR-based interventions in OC.

Discussion

CRISPR-Cas9 technology has fundamentally reshaped OC research by enabling a transition from correlative genomic analyses to direct functional validation. By permitting precise genome interrogation at scale, CRISPR approaches have systematically deconstructed the molecular architecture underlying OC initiation, metastasis, and therapeutic resistance. Functional studies have identified key regulators across these processes, including drivers of tumorigenesis, mediators of metastatic spread, and determinants of drug resistance and immune evasion, highlighting the central roles of DNA repair, metabolic reprogramming, and tumor-immune interactions.

A major conceptual advance arising from CRISPR-based studies is the redefinition of cancer drivers as context-dependent functional dependencies rather than static genetic alterations. Genes such as ATAD2, EIF5A2, LKB1, ACSL4, UBA1, and GPAA1 exemplify vulnerabilities that emerge only within specific genetic or microenvironmental contexts. While these findings extend beyond earlier association-based studies by establishing causality, they also reveal important limitations. Functional dependencies are not universally conserved across OC subtypes, experimental models, or selective pressures, underscoring the need to interpret CRISPR screens within defined biological contexts. Collectively, these observations challenge linear oncogenic models and instead support a network-based framework in which tumor fitness is sustained by adaptive and compensatory pathways.

CRISPR functional genomics contributes to the refinement of existing cancer models by emphasizing systems-level organization and plasticity. The identification of synthetic lethal interactions and noncoding regulatory dependencies suggests that OC progression is governed by dynamic regulatory networks rather than isolated driver events. Integrating CRISPR-derived functional maps with multi-omics, single-cell, and spatial profiling is therefore essential for constructing more generalizable models that capture intratumoral heterogeneity and therapy-induced evolution (109,110).

A central promise of CRISPR technology lies in its potential to integrate diagnosis and treatment into a unified precision medicine framework for OC. As highlighted throughout this review, CRISPR-based diagnostic platforms are advancing early detection through amplification-free, ultrasensitive profiling of tumor-derived nucleic acids in liquid biopsies. These approaches enable not only initial diagnosis but also real-time monitoring of therapeutic response and early identification of recurrence. Concurrently, CRISPR functional genomics is systematically expanding the repertoire of targetable vulnerabilities, while advances in delivery systems are progressively overcoming barriers to in vivo therapeutic editing. The convergence of these capabilities creates the opportunity for a closed-loop paradigm: molecular diagnosis informs treatment selection; engineered models derived from patient samples enable functional validation and personalized drug testing; and dynamic monitoring of circulating biomarkers guides adaptive therapy and early intervention upon resistance emergence. Achieving this vision will require continued innovation in multiplexed diagnostic sensors, tumor-selective delivery vehicles, and clinically predictive model systems, alongside the development of integrated clinical workflows that bridge molecular diagnostics with therapeutic decision-making. By uniting the diagnostic and therapeutic dimensions of CRISPR, this integrated approach holds the potential to transform OC from a late detected, uniformly treated disease toward a continuously monitored, molecularly guided precision medicine.

Beyond mechanistic insight, CRISPR technologies are beginning to exert translational impact. CRISPR-based molecular diagnostics, exemplified by Cas13a-mediated extracellular vesicle miRNA detection, offer highly sensitive, amplification-free approaches for early OC detection, addressing the persistent challenge of late-stage diagnosis (90). In parallel, CRISPR-engineered models, including patient-derived organoids and genetically refined in vivo systems, more accurately recapitulate tumor heterogeneity and treatment response, thereby strengthening the translational bridge between experimental discovery and clinical application (83).

Despite recent advancements, numerous interconnected obstacles continue to hinder the clinical translation of CRISPR-based interventions for OC, many of which are exacerbated by the disease’s distinct biological characteristics. Firstly, achieving efficient and tumor-specific delivery poses a significant challenge within the context of peritoneal dissemination. Therapeutic vectors must navigate ascitic fluid, which can dilute and degrade therapeutic payloads, penetrate the dense fibrotic stroma surrounding tumor nodules, and evade sequestration by phagocytic cells within the immunosuppressive tumor microenvironment. Secondly, the pronounced genomic instability that typifies OC—particularly mutations in TP53—raises concerns regarding the safety of nuclease-induced DNA DSBs, as these may aggravate chromosomal aberrations or inadvertently select for malignant clones. Third, intratumoral heterogeneity guarantees that even effective editing of dominant clones may leave resistant subpopulations unaffected, thereby necessitating combination strategies that concurrently target multiple vulnerabilities. Fourth, although current preclinical models are advancing, they frequently do not accurately replicate the intricate spatial architecture and immune composition of human OC, which constrains the predictive validity of efficacy and toxicity evaluations. Finally, the expected complexity and cost associated with personalized CRISPR therapies introduce ethical and accessibility concerns, especially for patient populations already experiencing disparities in cancer care. To address these challenges, a coordinated and multidisciplinary approach is essential. Progress in delivery science must prioritize formulations specifically designed for the peritoneal environment, such as mucopenetrating particles or matrix-degrading enzyme-conjugated carriers. Safety assessment frameworks should integrate assays that evaluate genomic instability risks, particularly in TP53-mutant contexts. Combination screening platforms, including arrayed CRISPR libraries in patient-derived organoids, are crucial for identifying synergistic targets that can circumvent heterogeneity-related escape mechanisms. Additionally, the development of clinically predictive model systems, such as peritoneal organoid co-cultures and humanized mouse models, should be prioritized to enhance the prediction of human responses. Equally important is the proactive engagement with ethical and regulatory bodies to establish guidelines for patient selection, informed consent, and equitable access as these technologies progress toward clinical application (111,112).

Looking ahead, progress in OC research will depend on the convergence of improved editing precision, intelligent delivery strategies, and integrative analytical frameworks. Advances in base and prime editing, tumor-targeted delivery platforms, and microenvironment-responsive carriers are expected to expand therapeutic feasibility. Coupling CRISPR functional genomics with single-cell and spatial technologies will further elucidate tumor-microenvironment co-evolution and enable proactive therapeutic strategies, including rational combination therapies designed to prevent resistance.

In summary, CRISPR technology has evolved into a transformative framework for OC research, linking molecular precision with functional causality and translational relevance. Its continued integration into experimental and clinical pipelines holds substantial promise for redefining OC management and improving long-term patient outcomes.

Acknowledgments

None.

Footnote

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2820/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2820/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021;71:209-49. [Crossref] [PubMed]
2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin 2020;70:7-30. [Crossref] [PubMed]
3. Menon U, Gentry-Maharaj A, Hallett R, et al. Sensitivity and specificity of multimodal and ultrasound screening for ovarian cancer, and stage distribution of detected cancers: results of the prevalence screen of the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS). Lancet Oncol 2009;10:327-40. [Crossref] [PubMed]
4. Bowtell DD, Böhm S, Ahmed AA, et al. Rethinking ovarian cancer II: reducing mortality from high-grade serous ovarian cancer. Nat Rev Cancer 2015;15:668-79. [Crossref] [PubMed]
5. Patch AM, Christie EL, Etemadmoghadam D, et al. Whole-genome characterization of chemoresistant ovarian cancer. Nature 2015;521:489-94. [Crossref] [PubMed]
6. Li X, Heyer WD. Homologous recombination in DNA repair and DNA damage tolerance. Cell Res 2008;18:99-113. [Crossref] [PubMed]
7. Ishino Y, Shinagawa H, Makino K, et al. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 1987;169:5429-33. [Crossref] [PubMed]
8. Mojica FJ, Díez-Villaseñor C, García-Martínez J, et al. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 2005;60:174-82. [Crossref] [PubMed]
9. Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337:816-21. [Crossref] [PubMed]
10. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014;157:1262-78. [Crossref] [PubMed]
11. Joung JK, Sander JD. TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 2013;14:49-55. [Crossref] [PubMed]
12. Lei Y, Guo X, Liu Y, et al. Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs). Proc Natl Acad Sci U S A 2012;109:17484-9. [Crossref] [PubMed]
13. Mussolino C, Cathomen T. TALE nucleases: tailored genome engineering made easy. Curr Opin Biotechnol 2012;23:644-50. [Crossref] [PubMed]
14. Tian X, Gu T, Patel S, et al. CRISPR/Cas9 - An evolving biological tool kit for cancer biology and oncology. NPJ Precis Oncol 2019;3:8. [Crossref] [PubMed]
15. Jansen R, Embden JD, Gaastra W, et al. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 2002;43:1565-75. [Crossref] [PubMed]
16. Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology (Reading) 2005;151:653-63. [Crossref] [PubMed]
17. Gasiunas G, Barrangou R, Horvath P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A 2012;109:E2579-86. [Crossref] [PubMed]
18. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339:819-23. [Crossref] [PubMed]
19. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science 2013;339:823-6. [Crossref] [PubMed]
20. Ran FA, Hsu PD, Lin CY, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 2013;154:1380-9. [Crossref] [PubMed]
21. Kleinstiver BP, Pattanayak V, Prew MS, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 2016;529:490-5. [Crossref] [PubMed]
22. Komor AC, Kim YB, Packer MS, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 2016;533:420-4. [Crossref] [PubMed]
23. Anzalone AV, Randolph PB, Davis JR, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 2019;576:149-57. [Crossref] [PubMed]
24. Replogle JM, Norman TM, Xu A, et al. Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing. Nat Biotechnol 2020;38:954-61. [Crossref] [PubMed]
25. Wessels HH, Méndez-Mancilla A, Hao Y, et al. Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq. Nat Methods 2023;20:86-94. [Crossref] [PubMed]
26. Gong J, Zhao Z, Niu X, et al. AI reshaping life sciences: intelligent transformation, application challenges, and future convergence in neuroscience, biology, and medicine. Front Digit Health 2025;7:1666415. [Crossref] [PubMed]
27. Schmich F, Szczurek E, Kreibich S, et al. gespeR: a statistical model for deconvoluting off-target-confounded RNA interference screens. Genome Biol 2015;16:220. [Crossref] [PubMed]
28. Shalem O, Sanjana NE, Hartenian E, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 2014;343:84-7. [Crossref] [PubMed]
29. Gilbert LA, Horlbeck MA, Adamson B, et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell 2014;159:647-61. [Crossref] [PubMed]
30. Shan Q, Wang Y, Li J, et al. Genome editing in rice and wheat using the CRISPR/Cas system. Nat Protoc 2014;9:2395-410. [Crossref] [PubMed]
31. Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 2014;11:783-4. [Crossref] [PubMed]
32. Kim D, Kim J, Hur JK, et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol 2016;34:863-8. [Crossref] [PubMed]
33. Konstantinopoulos PA, Ceccaldi R, Shapiro GI, et al. Homologous Recombination Deficiency: Exploiting the Fundamental Vulnerability of Ovarian Cancer. Cancer Discov 2015;5:1137-54. [Crossref] [PubMed]
34. Doench JG, Fusi N, Sullender M, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 2016;34:184-91. [Crossref] [PubMed]
35. Munoz DM, Cassiani PJ, Li L, et al. CRISPR Screens Provide a Comprehensive Assessment of Cancer Vulnerabilities but Generate False-Positive Hits for Highly Amplified Genomic Regions. Cancer Discov 2016;6:900-13. [Crossref] [PubMed]
36. Adamson B, Norman TM, Jost M, et al. A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response. Cell 2016;167:1867-1882.e21. [Crossref] [PubMed]
37. Horlbeck MA, Gilbert LA, Villalta JE, et al. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. Elife 2016;5:e19760. [Crossref] [PubMed]
38. Dixit A, Parnas O, Li B, et al. Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens. Cell 2016;167:1853-1866.e17. [Crossref] [PubMed]
39. Gilbert LA, Larson MH, Morsut L, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 2013;154:442-51. [Crossref] [PubMed]
40. Konermann S, Brigham MD, Trevino AE, et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 2015;517:583-8. [Crossref] [PubMed]
41. Joung J, Kirchgatterer PC, Singh A, et al. CRISPR activation screen identifies BCL-2 proteins and B3GNT2 as drivers of cancer resistance to T cell-mediated cytotoxicity. Nat Commun 2022;13:1606. [Crossref] [PubMed]
42. Wang C, Wang G, Feng X, et al. Genome-wide CRISPR screens reveal synthetic lethality of RNASEH2 deficiency and ATR inhibition. Oncogene 2019;38:2451-63. [Crossref] [PubMed]
43. Chavez A, Tuttle M, Pruitt BW, et al. Comparison of Cas9 activators in multiple species. Nat Methods 2016;13:563-7. [Crossref] [PubMed]
44. Garcia-Diaz A, Shin DS, Moreno BH, et al. Interferon Receptor Signaling Pathways Regulating PD-L1 and PD-L2 Expression. Cell Rep 2019;29:3766. [Crossref] [PubMed]
45. Thakore PI, D'Ippolito AM, Song L, et al. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat Methods 2015;12:1143-9. [Crossref] [PubMed]
46. Tian R, Gachechiladze MA, Ludwig CH, et al. CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons. Neuron 2019;104:239-255.e12. [Crossref] [PubMed]
47. Matharu N, Rattanasopha S, Tamura S, et al. CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency. Science 2019;363:eaau0629. [Crossref] [PubMed]
48. Qi LS, Larson MH, Gilbert LA, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013;152:1173-83. [Crossref] [PubMed]
49. Kessler JD, Kahle KT, Sun T, et al. A SUMOylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis. Science 2012;335:348-53. [Crossref] [PubMed]
50. Liu SJ, Horlbeck MA, Cho SW, et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 2017;355:aah7111. [Crossref] [PubMed]
51. Horlbeck MA, Witkowsky LB, Guglielmi B, et al. Nucleosomes impede Cas9 access to DNA in vivo and in vitro. Elife 2016;5:e12677. [Crossref] [PubMed]
52. Koh CM, Bezzi M, Low DH, et al. MYC regulates the core pre-mRNA splicing machinery as an essential step in lymphomagenesis. Nature 2015;523:96-100. [Crossref] [PubMed]
53. Gaudelli NM, Komor AC, Rees HA, et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 2017;551:464-71. [Crossref] [PubMed]
54. Anzalone AV, Koblan LW, Liu DR. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol 2020;38:824-44. [Crossref] [PubMed]
55. Koblan LW, Doman JL, Wilson C, et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol 2018;36:843-6. [Crossref] [PubMed]
56. Komor AC, Zhao KT, Packer MS, et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv 2017;3:eaao4774. [Crossref] [PubMed]
57. Grünewald J, Zhou R, Garcia SP, et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 2019;569:433-7. [Crossref] [PubMed]
58. Richter MF, Zhao KT, Eton E, et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol 2020;38:883-91. [Crossref] [PubMed]
59. Ko EJ, Kim ET, Kim H, et al. Effect of human endogenous retrovirus-K env gene knockout on proliferation of ovarian cancer cells. Genes Genomics 2022;44:1091-7. [Crossref] [PubMed]
60. Ray U, Roy D, Jin L, et al. Group III phospholipase A2 downregulation attenuated survival and metastasis in ovarian cancer and promotes chemo-sensitization. J Exp Clin Cancer Res 2021;40:182. [Crossref] [PubMed]
61. Lepage CC, Palmer MCL, Farrell AC, et al. Reduced SKP1 and CUL1 expression underlies increases in Cyclin E1 and chromosome instability in cellular precursors of high-grade serous ovarian cancer. Br J Cancer 2021;124:1699-710. [Crossref] [PubMed]
62. Yi Y, Tsai SH, Cheng JC, et al. APELA promotes tumour growth and cell migration in ovarian cancer in a p53-dependent manner. Gynecol Oncol 2017;147:663-71. [Crossref] [PubMed]
63. Liu Q, Liu H, Li L, et al. ATAD2 predicts poor outcomes in patients with ovarian cancer and is a marker of proliferation. Int J Oncol 2020;56:219-31. [PubMed]
64. Kawabata A, Hayashi T, Akasu-Nagayoshi Y, et al. CRISPR/Cas9 Screening for Identification of Genes Required for the Growth of Ovarian Clear Cell Carcinoma Cells. Curr Issues Mol Biol 2022;44:1587-96. [Crossref] [PubMed]
65. Akasu-Nagayoshi Y, Hayashi T, Kawabata A, et al. PHOSPHATE exporter XPR1/SLC53A1 is required for the tumorigenicity of epithelial ovarian cancer. Cancer Sci 2022;113:2034-43. [Crossref] [PubMed]
66. Feng W, Simpson DA, Carvajal-Garcia J, et al. Genetic determinants of cellular addiction to DNA polymerase theta. Nat Commun 2019;10:4286. [Crossref] [PubMed]
67. Trelford CB, Buensuceso A, Tomas E, et al. LKB1 and STRADα Promote Epithelial Ovarian Cancer Spheroid Cell Invasion. Cancers (Basel) 2024;16:3726. [Crossref] [PubMed]
68. Zhao G, Zhang W, Dong P, et al. EIF5A2 controls ovarian tumor growth and metastasis by promoting epithelial to mesenchymal transition via the TGFβ pathway. Cell Biosci 2021;11:70. [Crossref] [PubMed]
69. Wheeler LJ, Watson ZL, Qamar L, et al. Multi-Omic Approaches Identify Metabolic and Autophagy Regulators Important in Ovarian Cancer Dissemination. iScience 2019;19:474-91. [Crossref] [PubMed]
70. Qin X, Li J, Wang S, et al. Serotonin/HTR1E signaling blocks chronic stress-promoted progression of ovarian cancer. Theranostics 2021;11:6950-65. [Crossref] [PubMed]
71. Wang Y, Hu M, Cao J, et al. ACSL4 and polyunsaturated lipids support metastatic extravasation and colonization. Cell 2025;188:412-429.e27. [Crossref] [PubMed]
72. Qi Y, Zhu W, Mo K, et al. CRISPR/Cas9-based genome-wide screening for metastasis ability identifies FCGR1A regulating the metastatic process of ovarian cancer by targeting LSP1. J Cancer Res Clin Oncol 2024;150:306. [Crossref] [PubMed]
73. Chen S, Wu Y, Gao Y, et al. Allosterically inhibited PFKL via prostaglandin E2 withholds glucose metabolism and ovarian cancer invasiveness. Cell Rep 2023;42:113246. [Crossref] [PubMed]
74. Awasthi S, Dobrolecki LE, Sallas C, et al. UBA1 inhibition sensitizes cancer cells to PARP inhibitors. Cell Rep Med 2024;5:101834. [Crossref] [PubMed]
75. Coelho R, Tozzi A, Disler M, et al. Overlapping gene dependencies for PARP inhibitors and carboplatin response identified by functional CRISPR-Cas9 screening in ovarian cancer. Cell Death Dis 2022;13:909. [Crossref] [PubMed]
76. Mengwasser KE, Adeyemi RO, Leng Y, et al. Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets. Mol Cell 2019;73:885-899.e6. [Crossref] [PubMed]
77. Ouyang Q, Liu Y, Tan J, et al. Loss of ZNF587B and SULF1 contributed to cisplatin resistance in ovarian cancer cell lines based on Genome-scale CRISPR/Cas9 screening. Am J Cancer Res 2019;9:988-98. [PubMed]
78. Luo Y, Liu X, Chen Y, et al. Targeting PAX8 sensitizes ovarian cancer cells to ferroptosis by inhibiting glutathione synthesis. Apoptosis 2024;29:1499-514. [Crossref] [PubMed]
79. Mishra AK, Ye T, Banday S, et al. Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer. Cell Rep 2024;43:114041. [Crossref] [PubMed]
80. Li J, Qin X, Shi J, et al. A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis. Elife 2021;10:e66222. [Crossref] [PubMed]
81. Zorea J, Motro Y, Mazor RD, et al. TRAF3 suppression encourages B cell recruitment and prolongs survival of microbiome-intact mice with ovarian cancer. J Exp Clin Cancer Res 2023;42:107. [Crossref] [PubMed]
82. Lõhmussaar K, Kopper O, Korving J, et al. Assessing the origin of high-grade serous ovarian cancer using CRISPR-modification of mouse organoids. Nat Commun 2020;11:2660. [Crossref] [PubMed]
83. Zhang S, Iyer S, Ran H, et al. Genetically Defined, Syngeneic Organoid Platform for Developing Combination Therapies for Ovarian Cancer. Cancer Discov 2021;11:362-83. [Crossref] [PubMed]
84. Walton J, Blagih J, Ennis D, et al. CRISPR/Cas9-Mediated Trp53 and Brca2 Knockout to Generate Improved Murine Models of Ovarian High-Grade Serous Carcinoma. Cancer Res 2016;76:6118-29. [Crossref] [PubMed]
85. Walton JB, Farquharson M, Mason S, et al. CRISPR/Cas9-derived models of ovarian high grade serous carcinoma targeting Brca1, Pten and Nf1, and correlation with platinum sensitivity. Sci Rep 2017;7:16827. [Crossref] [PubMed]
86. Chen H, Yang K, Pang L, et al. ANKRD22 is a potential novel target for reversing the immunosuppressive effects of PMN-MDSCs in ovarian cancer. J Immunother Cancer 2023;11:e005527. [Crossref] [PubMed]
87. Teng K, Ford MJ, Harwalkar K, et al. Modeling High-Grade Serous Ovarian Carcinoma Using a Combination of In Vivo Fallopian Tube Electroporation and CRISPR-Cas9-Mediated Genome Editing. Cancer Res 2021;81:5147-60. [Crossref] [PubMed]
88. Jacobs IJ, Menon U. Progress and challenges in screening for early detection of ovarian cancer. Mol Cell Proteomics 2004;3:355-66. [Crossref] [PubMed]
89. Guo Y, Guo L, Su Y, et al. CRISPR-Cas system manipulating nanoparticles signal transduction for cancer diagnosis. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2023;15:e1851. [Crossref] [PubMed]
90. Hong JS, Son T, Castro CM, et al. CRISPR/Cas13a-Based MicroRNA Detection in Tumor-Derived Extracellular Vesicles. Adv Sci (Weinh) 2023;10:e2301766. [Crossref] [PubMed]
91. Chen Q, Shi J, Ruan D, et al. The diagnostic and therapeutic prospects of exosomes in ovarian cancer. BJOG 2023;130:999-1006. [Crossref] [PubMed]
92. Zhang C, Qin M. Extracellular vesicles targeting tumor microenvironment in ovarian cancer. Int J Biol Macromol 2023;252:126300. [Crossref] [PubMed]
93. Chen M, Choi HK, Goldston LL, et al. Advanced Cancer Liquid Biopsy Platform for miRNA Detection in Extracellular Vesicles Using CRISPR/Cas13a and Gold Nanoarrays. ACS Nano 2025;19:31438-56. [Crossref] [PubMed]
94. Su X, Shan Z, Duan S. Harnessing extracellular vesicles using liquid biopsy for cancer diagnosis and monitoring: highlights from AACR Annual Meeting 2024. J Hematol Oncol 2024;17:55. [Crossref] [PubMed]
95. Kumar MA, Baba SK, Sadida HQ, et al. Extracellular vesicles as tools and targets in therapy for diseases. Signal Transduct Target Ther 2024;9:27. [Crossref] [PubMed]
96. Di Carlo E, Sorrentino C. State of the art CRISPR-based strategies for cancer diagnostics and treatment. Biomark Res 2024;12:156. [Crossref] [PubMed]
97. Lee S, Kim YY, Ahn HJ. Systemic delivery of CRISPR/Cas9 to hepatic tumors for cancer treatment using altered tropism of lentiviral vector. Biomaterials 2021;272:120793. [Crossref] [PubMed]
98. Ehrke-Schulz E, Heinemann S, Schulte L, et al. Adenoviral Vectors Armed with PAPILLOMAVIRUs Oncogene Specific CRISPR/Cas9 Kill Human-Papillomavirus-Induced Cervical Cancer Cells. Cancers (Basel) 2020;12:1934. [Crossref] [PubMed]
99. Song Z, Tao Y, Liu Y, et al. Advances in delivery systems for CRISPR/Cas-mediated cancer treatment: a focus on viral vectors and extracellular vesicles. Front Immunol 2024;15:1444437. [Crossref] [PubMed]
100. Werner ME, Karve S, Sukumar R, et al. Folate-targeted nanoparticle delivery of chemo- and radiotherapeutics for the treatment of ovarian cancer peritoneal metastasis. Biomaterials 2011;32:8548-54. [Crossref] [PubMed]
101. Fathabadi EG, Shelling AN, Al-Kassas R. Nanocarrier systems for delivery of siRNA to ovarian cancer tissues. Expert Opin Drug Deliv 2012;9:743-54. [Crossref] [PubMed]
102. Yang X, Iyer AK, Singh A, et al. MDR1 siRNA loaded hyaluronic acid-based CD44 targeted nanoparticle systems circumvent paclitaxel resistance in ovarian cancer. Sci Rep 2015;5:8509. [Crossref] [PubMed]
103. Chen Z, Liu F, Chen Y, et al. Targeted Delivery of CRISPR/Cas9-Mediated Cancer Gene Therapy via Liposome-Templated Hydrogel Nanoparticles. Adv Funct Mater 2017;27:1703036. [Crossref] [PubMed]
104. Lu H, Zhang Q, He S, et al. Reduction-Sensitive Fluorinated-Pt(IV) Universal Transfection Nanoplatform Facilitating CT45-Targeted CRISPR/dCas9 Activation for Synergistic and Individualized Treatment of Ovarian Cancer. Small 2021;17:e2102494. [Crossref] [PubMed]
105. Godbole N, Quinn A, Carrion F, et al. Extracellular vesicles as a potential delivery platform for CRISPR-Cas based therapy in epithelial ovarian cancer. Semin Cancer Biol 2023;96:64-81. [Crossref] [PubMed]
106. Kim SM, Yang Y, Oh SJ, et al. Cancer-derived exosomes as a delivery platform of CRISPR/Cas9 confer cancer cell tropism-dependent targeting. J Control Release 2017;266:8-16. [Crossref] [PubMed]
107. Stone RL, Sood AK, Coleman RL. Collateral damage: toxic effects of targeted antiangiogenic therapies in ovarian cancer. Lancet Oncol 2010;11:465-75. [Crossref] [PubMed]
108. Turocy J, Adashi EY, Egli D. Heritable human genome editing: Research progress, ethical considerations, and hurdles to clinical practice. Cell 2021;184:1561-74. [Crossref] [PubMed]
109. Cheng J, Lin G, Wang T, et al. Massively Parallel CRISPR-Based Genetic Perturbation Screening at Single-Cell Resolution. Adv Sci (Weinh) 2023;10:e2204484. [Crossref] [PubMed]
110. Dhainaut M, Rose SA, Akturk G, et al. Spatial CRISPR genomics identifies regulators of the tumor microenvironment. Cell 2022;185:1223-1239.e20. [Crossref] [PubMed]
111. Xu X, Chemparathy A, Zeng L, et al. Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Mol Cell 2021;81:4333-4345.e4. [Crossref] [PubMed]
112. Paranthaman S, Uthaiah CA, Md S, et al. Comprehensive strategies for constructing efficient CRISPR/Cas based cancer therapy: Target gene selection, sgRNA optimization, delivery methods and evaluation. Adv Colloid Interface Sci 2025;341:103497. [Crossref] [PubMed]