CRISPR/Cas9 gene editing in gastric cancer: Mechanisms, advances, and therapeutic potential

Abstract

Gastric cancer (GC) remains one of the leading causes of cancer-related mortality worldwide, necessitating innovative approaches for its diagnosis and treatment. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a revolutionary gene-editing technology, has emerged as a powerful tool for unraveling the molecular mechanisms underlying GC and for advancing precision medicine strategies. This review explores the current applications of CRISPR/Cas9 in GC research, including the identification of oncogenes and tumor suppressors, modeling tumor microenvironment interactions, and developing gene-based therapies. We highlight recent breakthroughs in genome editing that have enhanced our understanding of GC pathogenesis and resistance mechanisms to conventional therapies. Additionally, we discuss the potential of CRISPR/Cas9 for therapeutic gene editing in GC, addressing challenges such as off-target effects, delivery methods, and ethical considerations. By summarizing the progress and limitations of CRISPR/Cas9 in GC, this review aims to provide a comprehensive perspective on how this transformative technology could shape future strategies for the prevention, diagnosis, and treatment of GC.

Key Words: Gastric cancer; Gene-editing technologies; Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9; Helicobacter pylori; Precision genome editing

Core Tip: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing has emerged as a transformative tool in gastric cancer research, offering precise manipulation of oncogenes and tumor suppressor genes. This technology facilitates the dissection of gastric tumorigenesis mechanisms and the development of targeted therapies. Recent advances highlight its potential in overcoming chemotherapy resistance and enhancing immunotherapeutic strategies. Despite challenges such as off-target effects and delivery mechanisms, ongoing research continues to refine CRISPR/Cas9 applications, underscoring its promise in improving outcomes for gastric cancer patients.

INTRODUCTION

Gastric cancer (GC) is a leading cause of cancer-related morbidity and mortality worldwide, exhibiting significant regional variations in incidence and survival outcomes. GC is a mosaic of molecular alterations. At the crossroads of genetic instability, environmental factors, and cellular memory, epigenetic changes, microsatellite instability (MSI), and Helicobacter pylori (H. pylori) infection emerge as pivotal forces that shape the very foundation of this malignancy. The complex interplay of genetic mutations, epigenetic modifications, and environmental factors in GC underscores how subtle molecular alterations can lead to significant changes in the gastric epithelium, contributing to tumor initiation and progression. With the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing technology, there is new hope for precision oncology. CRISPR-Cas9 allows for precise alterations to the genome, offering a promising tool to address genetic mutations in GC. This review explores the molecular mechanisms of GC, focusing on key genetic mutations, and discusses the potential of CRISPR-Cas9 technology in combating this disease. Despite a global decline in incidence, it continues to pose significant health challenges, particularly in East Asia, Eastern Europe, and parts of South America, where the incidence rates remain high[1]. GC is often diagnosed at an advanced stage, contributing to its poor prognosis and high mortality rate[2]. The 5-year survival rate for patients with advanced GC remains low, underscoring the importance of early detection and effective therapeutic strategies[3]. The development of GC is influenced by various risk factors, including environmental, dietary, and genetic factors. A major contributor to GC is chronic infection with H. pylori, which induces a state of persistent inflammation in the gastric mucosa and is considered the most significant risk factor for GC[4]. Dietary habits, such as high salt consumption, low intake of fruits and vegetables, and consumption of smoked or preserved foods, also contribute to disease development[5]. Additionally, genetic factors, including mutations in tumor suppressor genes (e.g., TP53 and CDKN2A) and the presence of hereditary syndromes like hereditary diffuse GC (linked to mutations in CDH1), play a critical role in gastric carcinogenesis[6]. At the molecular level, GC is a heterogeneous disease characterized by genetic mutations, epigenetic alterations, and disruptions in critical signaling pathways. Alterations in the a key tumor suppressor gene TP53 are commonly found in GC[7]. Furthermore, the activation of oncogenes such as KRAS and HER2 and mutations in genes involved in cellular adhesion and epithelial-to-mesenchymal transition (EMT), like CDH1 (E-cadherin), are frequently observed in gastric tumors[8]. Dysregulated signaling pathways, such as the phosphatidylinositol 3-kinase/protein kinase B, Wnt/β-catenin, and mitogen-activated protein kinase pathways, contribute to unchecked cellular proliferation, survival, and migration, facilitating tumor growth and metastasis[9]. These molecular mechanisms highlight the complexity of GC and emphasize the need for targeted therapies to improve patient outcomes.

CRISPR-CAS9 AND ITS POTENTIAL IN ONCOLOGY

In recent years, CRISPR-Cas9 technology has revolutionized genetic research and holds immense promise for oncology. The CRISPR-Cas9 system allows for precise, targeted modifications to the genome, enabling researchers to investigate the roles of specific genes in cancer development and progression. By inducing double-strand breaks (DSBs) at specific genomic loci, CRISPR-Cas9 facilitates gene editing through either error-prone repair mechanisms or homology-directed repair (HDR), providing an opportunity to correct mutations or alter gene expression in cancer cells[10]. In the context of GC, CRISPR-Cas9 offers numerous applications, from understanding the functional impact of mutations to developing potential therapeutic interventions. One of the promising uses of CRISPR-Cas9 is the creation of genetically engineered animal models that mimic human GC, allowing for more accurate preclinical testing of novel therapeutic agents[11]. Additionally, CRISPR-Cas9 enables the manipulation of genes involved in the GC microenvironment, such as those that regulate immune evasion or tumor-stroma interactions, offering avenues for immunotherapy and combination therapies[12]. The potential of CRISPR-Cas9 extends to the development of personalized medicine in oncology. By editing tumor cells or immune cells like T-cells, researchers aim to enhance the immune system’s ability to recognize and attack cancer cells with greater specificity. Moreover, CRISPR-Cas9 has shown promise in the development of chimeric antigen receptor T-cell (CAR-T) therapies, which are engineered to target tumor-specific antigens in GC and other malignancies[13]. As technology advances, CRISPR-Cas9 holds the potential to provide novel therapeutic strategies that could address the underlying genetic causes of GC, ultimately improving patient prognosis and treatment options.

Molecular mechanisms of GC and the potential of CRISPR-Cas9 for genetic editing

GC is a heterogeneous disease characterized by a broad range of genetic alterations. These mutations disrupt essential cellular functions such as apoptosis, cell cycle regulation, DNA repair, and cellular signaling. Among the most common genetic mutations associated with GC are those involving tumor suppressor genes, oncogenes, and genes involved in DNA repair mechanisms.

TP53 mutation: The guardian of the genome

One of the most critical mutations in GC is the alteration of the TP53 gene, which encodes the p53 protein. p53 is known as the “guardian of the genome” due to its crucial role in maintaining cellular integrity. Under normal conditions, p53 responds to DNA damage by initiating DNA repair or triggering apoptosis if the damage is irreparable. However, mutations in TP53 results in the inability to induce cell cycle arrest or apoptosis. This allows cells with DNA damage to proliferate, contributing to tumorigenesis. Studies have shown that TP53 mutations are present in a significant proportion of GCs, particularly in advanced stages, underscoring its role in disease progression[14].

CDKN2A mutation: Disruption of cell cycle regulation

The CDKN2A gene encodes the p16INK4a protein, a crucial inhibitor of cyclin-dependent kinases involved in cell cycle regulation. Loss of CDKN2A function leads to unchecked cell cycle progression, promoting tumor growth. The loss of p16INK4a is often observed in GC, particularly in tumors associated with hypermethylation of the CDKN2A promoter region. This disruption accelerates the cell cycle, rendering cells more prone to malignant transformation. Research has demonstrated that silencing of CDKN2A correlates with poor prognosis and chemotherapy resistance in GC patients[15].

KRAS mutation: Oncogenic activation in GC

Mutations in the KRAS gene, which encodes a small GTPase involved in the mitogen-activated protein kinase/extracellular signal-regulated kinases signaling pathway, are common in various cancers, including GC. In its active form, KRAS drives the activation of downstream pathways that promote cell survival, proliferation, and invasion. Mutations in KRAS lead to constitutive activation of these pathways, even in the absence of growth factor stimulation, resulting in unchecked tumor growth. Although less frequent than TP53 mutations, KRAS mutations have been associated with a poor prognosis and chemotherapy resistance in GC patients. Targeting KRAS mutations with specific inhibitors remains a challenge, but this area of research is gaining momentum[16].

Human epidermal growth factor receptor 2 amplification: A target for therapeutic intervention

Human epidermal growth factor receptor 2 (HER2) amplification is found in approximately 10%-20% of GCs. HER2 is a receptor tyrosine kinase that plays a crucial role in regulating cell growth and differentiation. In GC, HER2 overexpression promotes tumorigenesis by enhancing cell survival, proliferation, and angiogenesis. The anti-HER2 monoclonal antibody Trastuzumab has been shown to improve survival in patients with HER2-positive GC, highlighting the importance of targeting HER2 in therapeutic strategies. The amplification of HER2 and its role in GC has made it a prime candidate for targeted therapies, offering a personalized treatment approach for patients with this genetic alteration[17].

Epigenetic modifications as critical drivers in gastric carcinogenesis

In addition to genetic mutations, epigenetic modifications play an indispensable role in GC. DNA methylation, histone modifications, and microRNA dysregulation contribute to the silencing of tumor suppressor genes and activation of oncogenes. For instance, hypermethylation of the CDH1 gene, which encodes E-cadherin, is frequently observed in GC and is associated with EMT, a process that enhances cancer cell invasiveness. Loss of E-cadherin function disrupts cell adhesion, promoting metastasis. Furthermore, the dysregulation of microRNAs, such as miR-21, whose targets include tumor suppressor genes like PTEN, plays a crucial role in promoting GC cell proliferation and survival. The molecular interplay between DNA methylation and histone modifications constitutes a fundamental mechanism in the regulation of gene expression. It is the foundation upon which cancer cells stand[18].

The role of CRISPR-Cas9 in targeting GC mutations

CRISPR-Cas9, a revolutionary genome-editing tool, has opened new doors for targeted therapies in cancer treatment, including GC. This technology allows for precise genome modifications, including the introduction of mutations, deletions, or repairs at specific loci. The potential applications of CRISPR-Cas9 in GC are vast, ranging from gene correction to targeting of specific genetic mutations responsible for tumorigenesis.

Gene editing of TP53 mutations

Restoring the function of TP53 in GC cells using CRISPR-Cas9 could reverse the malignant phenotype induced by its mutation. Recent studies have demonstrated that CRISPR-mediated correction of TP53 mutations restores tumor suppressor function of p53 in various cancer cell lines, including GC (Table 1). This approach has been shown to induce apoptosis and inhibit cell proliferation in p53-deficient GC cells, suggesting that gene editing could offer a promising therapeutic avenue[19]. Given the pivotal role of KRAS mutations in GC progression, CRISPR-Cas9 could be employed to knock out or edit KRAS mutations, potentially halting tumor growth. CRISPR has been used to target KRAS mutations in preclinical models, leading to a decrease in tumor size and metastasis. This offers an exciting prospect for overcoming the challenges of KRAS-targeted therapies, which have historically been difficult to develop due to the gene’s complex structure and function[20]. CRISPR interference (CRISPRi) and CRISPR activation techniques allow for the repression or activation of specific genes without altering the underlying DNA sequence. These techniques could be utilized to silence oncogenes such as HER2 or reactivate silenced tumor suppressor genes like CDKN2A. For example, CRISPRi has been employed to reduce HER2 expression in HER2-positive GC cells, leading to decreased cell proliferation and enhanced sensitivity to trastuzumab[21].

Table 1 Overview of clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 applications in gastric cancer: Gene targets, delivery strategies, and clinical relevance.

Gene target	Function	Delivery strategy	Preclinical/clinical relevance	Ref.
TP53	Tumor suppressor gene regulating cell cycle and apoptosis	Lentiviral vector, lipid nanoparticles	Restoration of p53 function suppresses tumor growth in GC models	[62,63]
CDH1	Encodes E-cadherin; involved in cell adhesion and EMT	CRISPR/Cas9 ribonucleoprotein complexes	CDH1 knockout mimics hereditary diffuse gastric cancer; used in disease modeling	[64,65]
KRAS	Oncogene involved in MAPK signaling pathway	AAV vectors	Editing KRAS mutations reduces cell proliferation and tumorigenesis	[66]
PIK3CA	Encodes PI3K catalytic subunit; promotes growth signaling	Liposomal CRISPR delivery	Disruption impairs PI3K/AKT signaling, reducing tumor progression	[67]
HER2 (ERBB2)	Oncogene, overexpressed in about 15%-20% of GC cases	Nanoparticle-based CRISPR delivery	Knockout increases response to chemotherapy in HER2 + GC models	[30]
MET	Proto-oncogene encoding hepatocyte growth factor receptor	Electroporation-based plasmid delivery	Targeting MET suppresses invasion and metastasis in GC cells	[68]
PD-L1 (CD274)	Immune checkpoint protein; mediates immune evasion	Dual CRISPR-Cas9/anti-PD-L1 delivery systems	Enhances antitumor immunity, improves immunotherapy response	[68]

CRISPR: Clustered regularly interspaced short palindromic repeats; Cas9: Clustered regularly interspaced short palindromic repeats-associated protein 9; RNP: Ribonucleoprotein; AAV: Adeno-associated virus; EMT: Epithelial-mesenchymal transition; CDH1: Cadherin 1; MAPK: Mitogen-activated protein kinase; KRAS: Kirsten rat sarcoma viral oncogene homolog; PI3K: Phosphoinositide 3-kinase; AKT: Protein kinase B; GC: Gastric cancer; HER2: Human epidermal growth factor receptor 2; MET: Mesenchymal-epithelial transition factor; PD-L1: Programmed death-ligand 1.

Exploring CRISPR for epigenetic modifications

CRISPR-based epigenome editing tools, such as CRISPR-dead Cas9 (dCas9) fused to epigenetic regulators, offer an innovative approach to reversing epigenetic alterations in GC. For instance, CRISPR can be used to demethylate the CDH1 promoter region, restoring E-cadherin expression and inhibiting EMT. Similarly, CRISPR-based strategies could modulate the expression of microRNAs involved in GC progression, providing a therapeutic approach for targeting epigenetic disease drivers[22]. In the intricate world of GC, where genetic chaos intertwines with bacterial cunning, two forces emerge as significant players: H. pylori, the relentless invader, and MSI, the echo of genetic turmoil. Together, they form a powerful duo that drives the malignant transformation of gastric epithelial cells. Yet, in the arsenal of modern science, CRISPR-Cas9, a revolutionary gene-editing technology, offers a gleam of hope - a precise scalpel poised to target these molecular culprits and potentially reverse the cascade of events that lead to GC. But can this elegant tool, with its ability to alter DNA with unparalleled accuracy, rewrite the fate of gastric tumors?

MSI: A marker of genetic turbulence

Alongside H. pylori infection, MSI, a hallmark of mismatch repair (MMR) deficiency, represents a critical driver in GC development. The failure of MMR proteins, such as DNA mismatch repair protein Mlh1 (MLH1) and DNA mismatch repair protein Msh2, to correct replication errors leads to the accumulation of mutations in microsatellite regions, which are short, repetitive genomic sequences. This genetic chaos gives rise to instability at these loci, which, when left unchecked, promotes tumorigenesis. MSI-positive GCs are often associated with better prognosis, as they tend to respond more favorably to immunotherapy[23]. However, the genetic instability of these cancers hints at an underlying turmoil that is a double-edged sword in the war against cancer. Here again, CRISPR-Cas9 emerges as a potential key to unlocking the door to targeted intervention. The tool's precision offers the ability to correct MMR deficiencies at the genetic level, potentially restoring normal function to genes like MLH1 or DNA mismatch repair protein Msh2. In vitro models have already demonstrated the ability to repair these defective genes using CRISPR, leading to a reduction in mutation rate and a restoration of genetic stability[24]. By reintroducing functional MMR proteins, CRISPR could halt mutation accumulation, preventing the progression from precancerous lesions to fully invasive GC. Furthermore, CRISPR-Cas9 could target the pathways that enable the persistence of MSI-positive tumors. By correcting the mutations in tumor suppressor genes or oncogenes caused by MSI, this technology could remove the genetic fuel that drives tumor progression. In this sense, CRISPR does not merely cut away at the symptoms - it offers the potential to correct the molecular foundations of the disease.

H. pylori: The chronic instigator

The role of H. pylori, the spiral bacterium nestled in the gastric mucosa, cannot be overstated. Its persistent infection triggers chronic inflammation, which, over decades, fosters an environment ripe for genetic alterations. H. pylori induces changes to the gastric mucosa, leading to atrophic gastritis, intestinal metaplasia, and dysplasia, all precursors to GC. The bacterium's ability to manipulate host immune responses and induce DNA damage further drives the development of cancerous lesions[25]. Through the regulation of host inflammatory responses and modulation of the gastric microenvironment, H. pylori sets the stage for carcinogenesis, illustrating how an external pathogen can insidiously alter the fabric of cellular processes. For decades, H. pylori has been a known provocateur in GC, lurking within the folds of the stomach lining, inflaming and damaging cells with its acidic touch. However, CRISPR-Cas9 offers a potential answer to this microbial menace. By harnessing the specificity of this genome-editing tool, scientists can potentially target and silence H. pylori genes responsible for its virulence. For example, CRISPRi could be employed to downregulate H. pylori’s key virulence factors such as cytotoxin-associated gene A, a protein that manipulates host cell signaling to promote inflammation and survival[26]. Such precise interventions might reduce H. pylori’s ability to induce DNA damage and chronic inflammation, halting the early stages of gastric carcinogenesis. Moreover, CRISPR-based strategies could extend beyond bacterial manipulation to enhance host resistance. By editing host immune response pathways or promoting more efficient bacterial clearance, CRISPR could act as both a sword and shield, weakening the pathogen’s grip on the gastric mucosa while reinforcing the body’s defenses[27].

The precision CRISPR-Cas9 gene editing in GC: Mechanisms, applications, and delivery strategies

CRISPR-Cas9 technology has emerged as a precise and efficient tool for targeted gene editing, enabling the modification of specific genomic sequences. Its application in GC research holds significant promise for elucidating gene functions and developing potential therapeutic strategies. CRISPR-Cas9 targets specific DNA sequences, introduces DSBs, and engages the cell's repair machinery to edit or correct genetic mutations. Yet the journey from concept to clinic is fraught with the challenges of delivery - how to precisely introduce this molecular scalpel into cancer cells. This CRISPR-Cas9 action, the repair pathways and delivery systems offer a new frontier in the battle against GC. At the core of the CRISPR-Cas9 system lies its remarkable ability to precisely target specific DNA sequences. Guided by a small RNA molecule, Cas9 protein acts as a molecular scissors, directed to a predetermined genetic locus. Once there, Cas9 introduces a DSB in the DNA. This break, though destructive in the short term, provides a fertile ground for genetic modifications - be it gene disruption, insertion, or correction. In the context of GC, CRISPR-Cas9 targets genes whose mutations fuel the malignant transformation of gastric epithelial cells. For instance, targeting oncogenes such as KRAS or tumor suppressor genes like TP53 could disrupt the signaling pathways driving disease progression[28]. The DSBs are repaired by one of two key DNA repair pathways: Non-Homologous End Joining (NHEJ) or HDR. The fate of a CRISPR-induced DSB lies in the hands of the cell's repair machinery. The two predominant pathways, NHEJ and HDR, are critical in determining the outcome of gene editing. NHEJ is the more error-prone repair mechanism, often resulting in insertions or deletions (indels) at the break site. These indels can cause frameshift mutations, leading to target gene inactivation - an effective means of gene knock-out. In GC, NHEJ could be harnessed to disrupt oncogenes, thereby blocking the signals that drive tumorigenesis[29]. In contrast, HDR offers a more precise form of genetic editing, utilizing a donor template to guide the repair process. HDR allows for the insertion of new genetic material, enabling the correction of mutations that may contribute to cancer progression. In the context of GC, HDR could be employed to restore tumor suppressor gene function, such as MLH1, whose inactivation contributes to MSI and drives carcinogenesis[30]. However, HDR is a more intricate process and is less efficient than NHEJ in many cell types, particularly in the context of cancer cells with dysregulated repair machinery. Together, these two pathways offer a toolkit for CRISPR-Cas9 in GC therapy. By guiding the system to utilize one pathway over the other, scientists can either knock out deleterious genes or correct mutations that underlie disease.

Delivery systems: The Journey of CRISPR to the tumor

While CRISPR-Cas9 holds immense therapeutic potential, its successful application in GC depends on overcoming the challenge of delivering this powerful tool to the right cells. The stomach is a complex organ; its thick mucosal lining and hostile acidic environment makes gene delivery particularly difficult. However, a range of delivery systems have been developed to ferry CRISPR-Cas9 into target cells, each with its own strengths and challenges. Viral vectors have long been employed to deliver genetic material into cells. Adenoviral and lentiviral vectors are particularly useful for their ability to efficiently deliver CRISPR-Cas9 components into host cells. These vectors can carry the necessary genetic material - both the Cas9 protein and the guide RNA - to GC cells, where the editing can begin. However, viral vectors carry a risk of immune responses, and their use in clinical settings must balance efficiency with safety concerns[31]. Nanoparticles, on the other hand, offer a less invasive and potentially safer alternative to viral delivery. Lipid-based nanoparticles or polymeric nanoparticles can encapsulate the CRISPR-Cas9 machinery and protect it from degradation, allowing for more controlled release at the target site. Their use in GC holds promise due to their ability to cross cellular barriers and target specific tissues through modifications to their surface. Once nanocarriers have moved into the fluid portion of the cell, a substantial amount of their cargo must reach the nucleus if the genetic material requires processing to achieve its intended function, or if the contents need direct access to a specific gene for immediate action. Various strategies have enhanced the movement of these carriers into the nucleus. One such approach involves the use of a specialized amino acid sequence that serves as a molecular tag, helping large biological molecules more effectively navigate toward the nucleus[32]. These nanoparticles offer a precision-guided system for directly delivering CRISPR-Cas9 to the tumor cells, reducing the risk of off-target effects and enhancing therapeutic efficacy. Electroporation, a technique that applies an electric field to cells, temporarily disrupts the cell membrane, creating pores through which CRISPR-Cas9 components can enter. This technique is often used in ex vivo gene editing, where cells are removed from the body, edited in the laboratory, and then reintroduced. While electroporation is effective in introducing CRISPR-Cas9 into cells, its application in vivo is still under exploration, particularly in GC, due to the challenges of accessing target tissues through non-invasive means[33].

CRISPR-CAS9 APPLICATIONS IN GC RESEARCH: PRECISION IN THE WAR AGAINST ONCOGENESIS

One of the most profound applications of CRISPR-Cas9 in GC research lies in gene knockout studies, where specific genes are deactivated to uncover their roles in tumorigenesis. Among the many oncogenes implicated in GC, HER2 and MET have garnered significant attention. HER2, a receptor tyrosine kinase, is overexpressed in approximately 10%-20% of GCs, where it drives aggressive tumor growth, metastasis, and poor prognosis. In many cancers, HER2 amplification and overexpression trigger downstream signaling cascades that promote cell proliferation, survival, and invasiveness. However, the advent of targeted therapies such as trastuzumab has only underscored the need for deeper understanding and refined therapeutic strategies. CRISPR-Cas9 enables the precise knockout of HER2, allowing researchers to study its exact contribution to tumorigenesis. Through such studies, scientists have found that HER2 not only drives the malignant phenotype but also alters the tumor microenvironment, facilitating angiogenesis and immune evasion[34]. Knockout models using CRISPR-Cas9 have shown that silencing HER2 leads to a significant reduction in tumor growth, thereby affirming its role as a key therapeutic target. Similarly, MET, the receptor for hepatocyte growth factor, is another oncogene frequently implicated in GC. MET amplification leads to enhanced tumorigenic properties, such as increased cell motility, invasiveness, and resistance to apoptosis. By using CRISPR-Cas9 to disrupt the MET gene, researchers have demonstrated decreased metastasis and tumor progression in both in vitro and in vivo models. These findings confirm that MET plays a crucial role in GC’s ability to invade surrounding tissues, making it a potential target for future therapies[35]. Through these knockout studies, CRISPR-Cas9 not only deepens our understanding of individual oncogenes but also provides insights into the dynamic molecular networks that underlie cancer progression. Beyond studying individual oncogenes, CRISPR-Cas9 opens the door to more expansive inquiries, such as genome-wide screening for novel therapeutic targets. Traditional methods for identifying cancer-driving genes have been labor-intensive and often limited to a small subset of genes. In contrast, CRISPR-based screens allow for systematic, large-scale identification of genes that influence GC cell survival and proliferation. By introducing CRISPR-Cas9 libraries into GC cell lines, researchers can simultaneously disrupt thousands of genes to determine which knockouts lead to decreased cancer cell viability. This approach has illuminated several novel targets for therapeutic intervention, many of which had previously been overlooked due to their subtle roles in cellular function. For example, SMAD4, a gene involved in the transforming growth factor β signaling pathway, has been identified as a critical gene for GC cell survival. Inactivation of SMAD4 using CRISPR not only diminishes cell proliferation but also sensitizes cells to chemotherapeutic agents[36]. These genome-wide screens are not only transforming our ability to uncover previously unrecognized cancer-driving genes but are also paving the way for the development of more effective combination therapies. Moreover, CRISPR-based screens can be adapted to identify genes that mediate resistance to existing treatments, such as chemotherapy or targeted therapies. This capability is crucial in overcoming one of the greatest hurdles in cancer treatment: Drug resistance. By disrupting individual genes involved in resistance mechanisms, CRISPR-Cas9 may reveal new avenues for overcoming the challenges posed by therapeutic resistance, offering hope for more durable treatments. While gene knockout studies and genome-wide screens can define the genetic foundation of GC, the role of the epigenome is equally profound. The epigenome, which consists of chemical modifications to DNA and histones, governs gene expression without altering the underlying genetic code. In cancer, these epigenetic modifications often silence tumor suppressor genes or activate oncogenes, contributing to uncontrolled cell division and cancer progression. CRISPR-Cas9 technology has evolved to extend its reach beyond simple gene editing, enabling the targeted modification of the epigenome itself. By using CRISPR-based epigenetic editing tools, researchers can selectively modify the epigenetic landscape of GC cells. One promising approach involves the use of CRISPR-dCas9, a dead version of Cas9 that no longer cuts DNA but can be fused with epigenetic modifiers such as DNA methyltransferases or histone acetyltransferases. This system allows for precise epigenetic modifications that can either silence oncogenes or activate tumor suppressor genes, effectively reprogramming the epigenome of cancer cells. For instance, dCas9-DNMT3A, a fusion of dCas9 with a DNA methyltransferase, can induce DNA methylation at the promoter regions of tumor suppressor genes, effectively silencing them in GC cells[37]. Conversely, dCas9-p300, which fuses dCas9 with a histone acetyltransferase, can be employed to activate silenced tumor suppressor genes, reestablishing normal growth control. These epigenetic modifications could help restore normal gene expression patterns, counteracting the malignant phenotype of GC cells and offering a potential therapeutic strategy that does not rely on direct genetic modifications. Epigenetic editing with CRISPR holds a unique advantage: Unlike permanent genetic mutations, epigenetic changes can be reversible, offering a more flexible and dynamic approach to cancer therapy. This reversibility is particularly significant in the context of GC, where environmental factors such as H. pylori infection and chronic inflammation contribute to the epigenetic reprogramming of the gastric mucosa. By targeting these reversible epigenetic changes, CRISPR-mediated epigenetic modifications could lead to more personalized and adaptive therapies[38].

TARGETING TUMOR SUPPRESSOR GENE MUTATIONS

At the heart of GC’s pathogenesis lies a complex interplay of genetic mutations, many of which occur in tumor suppressor genes. These genes normally function to control cellular growth, repair DNA, and initiate apoptosis when cells are damaged. However, in GC, mutations or silencing of these tumor suppressor genes allow for uncontrolled cell proliferation, evasion of cell death, and the promotion of metastasis. The ability of CRISPR-Cas9 to precisely target and modify these genes holds immense potential for therapeutic strategies aimed at restoring normal cellular functions and halting the progression of cancer. One of the most frequently mutated tumor suppressor genes in GC is p53, a gene known for its pivotal role in DNA damage response and apoptosis. p53 mutations are present in approximately 50% of GC cases and are associated with poor prognosis. Restoration of p53 function could provide an effective therapeutic approach. CRISPR-Cas9 has been utilized to restore p53 function by correcting point mutations or reactivating its expression. In one study, researchers used CRISPR-Cas9 to repair p53 mutations in GC cells, leading to restored tumor suppression and induction of cell cycle arrest and apoptosis[39]. Another key tumor suppressor gene involved in GC is Adenomatous polyposis coli (APC). Mutations in APC disrupt the Wnt signaling pathway, a critical pathway for regulating cell growth and differentiation. This disruption contributes to GC initiation and progression. CRISPR-Cas9 has been applied to edit and restore APC function in GC models, demonstrating its potential as a therapeutic target for restoring proper cell growth regulation. By using CRISPR to restore APC activity, researchers were able to inhibit GC cell proliferation and promote cell differentiation, offering promising therapeutic possibilities[40].

Immunotherapy has revolutionized cancer treatment, particularly with the advent of immune checkpoint inhibitors. GC, however, often develops mechanisms to evade immune detection, making it resistant to immune checkpoint blockade. One of the most critical immune evasion strategies involves the expression of the immune checkpoint molecule programmed death-ligand 1 (PD-L1), which binds to the programmed death 1 receptor on T cells, dampening immune responses against the tumor. CRISPR-Cas9 offers a promising method for enhancing the immune response in GC by knocking out PD-L1 expression in tumor cells or immune cells, thus overcoming immune evasion mechanisms. Studies have shown that PD-L1 knockout using CRISPR-Cas9 in GC cell lines can sensitize tumors to immune cell-mediated killing. By disrupting the PD-L1/programmed death 1 interaction, T cells can more effectively recognize and attack the cancer cells. In one notable study, CRISPR-Cas9 was used to delete PD-L1 in GC cell lines, resulting in increased T cell cytotoxicity and enhanced anti-tumor immunity[41]. This suggests that CRISPR-Cas9-mediated PD-L1 knockout could serve as a potential strategy to enhance the efficacy of immune checkpoint inhibitors in GC treatment. Another exciting application of CRISPR in GC immunotherapy is CAR-T cell engineering. CAR-T cell therapy has shown remarkable success in hematologic cancers, but its application in solid tumors like GC remains limited due to the inability of CAR-T cells to effectively target cancer cells in the tumor microenvironment. CRISPR-Cas9 can be used to genetically modify T cells, equipping them with receptors that specifically recognize GC-associated antigens. In preclinical studies, CRISPR-engineered CAR-T cells have been shown to effectively target GC cells in vitro and in vivo, improving anti-tumor responses. This approach holds great promise for overcoming the challenges faced by conventional CAR-T therapy in solid tumors[42]. Despite advances in chemotherapy and targeted therapy, drug resistance remains a major challenge in the treatment of GC. The heterogeneity of GC and the ability of cancer cells to rapidly adapt to therapeutic pressures lead to the emergence of resistant clones. Understanding the molecular mechanisms underlying drug resistance is crucial for developing strategies to overcome it. Here, CRISPR-Cas9-based genome-wide screening offers an invaluable tool to dissect the complex biology of drug resistance and identify novel therapeutic targets. Using CRISPR-Cas9, researchers can perform high-throughput screens to identify genes that are critical for drug-resistant GC cell survival. In one study, a CRISPR-Cas9 screen was conducted on GC cell lines treated with chemotherapy, revealing that genes involved in the phosphatidylinositol 3-kinase/protein kinase B signaling pathway were essential for the survival of drug-resistant cells. Inhibiting this pathway with specific inhibitors led to the sensitization of resistant cells to chemotherapy, suggesting that targeting this pathway could help overcome drug resistance[43]. Another key mechanism of drug resistance in GC is ATP-binding cassette transporter overexpression, which pump chemotherapy drugs out of cancer cells and reduce their efficacy. CRISPR-Cas9-based screening has been employed to identify the role of specific ATP-binding cassette transporters in mediating drug resistance. By knocking out these transporters, researchers were able to sensitize GC cells to chemotherapy, providing a potential strategy for overcoming multidrug resistance[44]. Moreover, CRISPR-Cas9 can be used to identify and validate novel drug resistance-associated genes in GC. By performing genome-wide CRISPR screens, researchers have discovered new genes involved in the development of resistance to targeted therapies such as HER2 inhibitors. These findings open the door for new therapeutic strategies that target these resistance mechanisms, potentially improving the efficacy of existing treatments[45].

CHALLENGES AND LIMITATIONS OF CRISPR-CAS9 IN GC: OFF-TARGET EFFECTS AND GENOME INSTABILITY

The advent of CRISPR-Cas9 technology has undeniably heralded a new era in gene-editing and molecular medicine, offering unprecedented precision in targeting specific genes and pathways. In GC research, its potential to rewrite the genetic landscape, repair mutations, and create more effective therapies has fueled optimism for the future of treatment. However, like all innovations, CRISPR-Cas9 is not without its challenges. While its precision has made it a revolutionary tool, the very nature of genome editing presents a unique set of limitations, notably the risk of off-target effects and genome instability. These challenges are particularly pronounced in GC, where the complexity of genetic mutations, the intricacy of tumor heterogeneity, and the dynamic tumor microenvironment further complicate therapeutic outcomes.

Off-target effects refer to unintended edits made at sites in the genome other than the intended target. While CRISPR-Cas9’s precision is its hallmark, these off-target effects remain one of the most significant hurdles to its clinical application. The risk of unanticipated mutations at loci outside of the targeted region could potentially lead to harmful, unpredictable outcomes, including oncogene activation, tumor suppressor gene silencing, or the induction of harmful genetic rearrangements. In the context of GC, where mutations in critical genes such as TP53, APC, and CDH1 are common, the inadvertent modification of non-target regions may aggravate the very molecular pathways that drive tumor progression rather than offering therapeutic benefit.

Several studies have highlighted the prevalence of off-target effects in CRISPR-Cas9-mediated genome editing, especially when using the wild-type Cas9 enzyme. For example, a study by Fu et al[46] explored the genome-wide off-target activity of CRISPR-Cas9 in human cells and found that despite its high precision, Cas9 can introduce unintended cuts in hundreds of other genomic sites, some of which are associated with critical cellular processes, including apoptosis and DNA repair. These unwanted changes can contribute to tumorigenesis or genomic instability, thereby complicating the therapeutic application of CRISPR in GC[46]. To mitigate these risks, researchers have focused on optimizing CRISPR systems to improve specificity. Modifications of the Cas9 enzyme, such as high-fidelity Cas9 variants and the development of CRISPR/Cas9 base editors, have been introduced to reduce off-target cutting. These engineered versions exhibit a higher degree of accuracy in gene editing, minimizing the unintended changes that could lead to detrimental outcomes in GC therapy[47]. However, even with these improvements, the complete elimination of off-target effects remains an unresolved challenge.

GENOME INSTABILITY: THE DOUBLE-EDGED SWORD

Another critical challenge is the risk of inducing genome instability during CRISPR-Cas9-mediated gene editing. The CRISPR system works by creating DSBs in the DNA, which, if not properly repaired, can lead to chromosomal rearrangements, mutations, or large deletions. While the cell’s natural repair mechanisms, namely NHEJ and HDR, are responsible for fixing these breaks, their efficiency and accuracy can vary. The NHEJ pathway is error-prone and often results in indels, which can disrupt genes or regulatory regions critical for cellular function. In cancer cells, this heightened instability can fuel the very process of carcinogenesis that researchers aim to control. In GC, where genetic instability is already a hallmark of tumor progression, introducing additional mutations via CRISPR-Cas9 could exacerbate the tumor’s aggressive behavior. For instance, p53 mutations, which are common in GC, can already lead to dysfunctional DNA damage response and increased genomic instability. Editing this pathway via CRISPR could inadvertently enhance the very characteristics that make GC so difficult to treat[48]. Furthermore, recent studies have shown that DSBs induced by CRISPR can result in chromosomal translocations or oncogene amplification, such as myelocytomatosis oncogene or HER2, which are both involved in gastric carcinogenesis. These alterations could lead to the emergence of new clones with enhanced malignancy, undermining the therapeutic potential of CRISPR-based interventions[49]. To address genome instability, researchers have developed strategies to enhance the repair process by promoting HDR, which is more accurate than NHEJ. One promising approach involves the use of repair templates to guide the repair process towards a more precise edit, reducing the likelihood of error-prone repair outcomes. However, HDR is often inefficient, particularly in dividing cells, which limits its utility in certain cancer types. Finding a balance between precision and the risk of inducing instability remains a formidable challenge in CRISPR-based therapies for GC.

ETHICAL AND TECHNICAL CHALLENGES IN CLINICAL APPLICATION

Beyond the biological limitations of CRISPR-Cas9, there are also significant ethical and technical challenges to consider, especially in the context of GC therapy. The potential for off-target effects and genome instability raises concerns about the long-term consequences of CRISPR-based interventions in human patients. Germline modification, for instance, introduces additional ethical dilemmas about the transmission of unintended mutations to future generations. While germline editing is not a current focus in GC therapy, the long-term impact of somatic edits on the patient’s genome remains a subject of intense scrutiny. Moreover, the delivery of CRISPR-Cas9 components to tumor cells poses another significant hurdle. Despite significant advances in delivery technologies, such as viral vectors and nanoparticles, efficiently delivering CRISPR-Cas9 to GC cells remains a difficult task. With its dense extracellular matrix and heterogeneous cell populations, the tumor microenvironment often impedes the effective delivery of gene-editing tools. Thus, improving the specificity and efficiency of CRISPR delivery to GC cells is essential to ensure successful therapeutic outcomes[50]. To overcome the delivery barrier, researchers have turned to various vectors for CRISPR-Cas9 delivery. These include viral vectors (such as lentiviruses and adenoviruses), non-viral vectors (such as liposomes and polymers), and nanoparticle-based systems. Each has its own advantages and drawbacks in GC therapy. Viral vectors, particularly adenoviruses and lentiviruses, have been widely studied for gene delivery in cancer therapy. Adenoviruses can efficiently transduce a variety of tumor cells and provide high transfection rates. However, their use in GC is limited by immune responses that can lead to rapid clearance from the body and inadequate delivery to deep tumor tissues[51]. Moreover, concerns over insertional mutagenesis, where the viral genome integrates into the host cell’s DNA, pose a risk of further genomic instability. In contrast, non-viral delivery systems such as lipid nanoparticles or polymeric carriers offer greater safety profiles by avoiding the immune response triggered by viral vectors. These systems can also be designed to carry larger genetic payloads. However, their efficiency in delivering CRISPR-Cas9 complexes to solid tumors, particularly in the case of GC, remains suboptimal. These systems can also struggle with overcoming the dense tumor stroma and achieving precise delivery to targeted cancer cells. Nanoparticle-based delivery systems have emerged as a promising solution to many of these issues. Gold nanoparticles and lipid nanoparticles are being investigated for their ability to penetrate the tumor microenvironment and deliver genetic payloads directly to cancer cells. Nanoparticles can also be engineered for targeted delivery by attaching specific ligands that bind to overexpressed receptors on cancer cells. These innovations hold promise for improving targeting to GC cells but still require further refinement to increase their precision and efficiency in vivo[52].

TUMOR HETEROGENEITY: A COMPOUNDING FACTOR

Like many solid tumors, GC is characterized by significant tumor heterogeneity, which further complicates CRISPR-Cas9-based therapies. The diverse genetic and epigenetic landscape of a gastric tumor means that different cancer cells may require different genetic modifications to effectively halt tumor growth. This heterogeneity not only affects the success of CRISPR-based gene editing but also hampers the delivery of the CRISPR machinery. For instance, certain tumor cells may exhibit higher expression of drug transporters that pump the CRISPR components out of the cell before they can be fully delivered. Other cells may have altered surface markers that make them less susceptible to certain delivery methods. As a result, even with precise delivery systems, not all cancer cells will receive treatment, leading to partial responses and potentially the development of resistance. Cancer cells can sometimes be “addicted” to a certain molecule. Aberrations in genes such as EGFR, ALK, HER2, MET may lead a normal cell to become a cancerous cell. The use of tyrosine kinase inhibitors, including gefitinib, afatinib, and crizotinib, offers a promising strategy for suppressing tumor growth by targeting specific molecular pathways. However, over time, cancer cells often develop resistance to these treatments, driven by the activation of alternative escape mechanisms. This resistance may arise through newly acquired genomic alterations, allowing the tumor to circumvent the effects of tyrosine kinase inhibitors and continue proliferating[53].

Overcoming delivery challenges: Towards optimized CRISPR-Cas9

As the limitations of CRISPR-Cas9 delivery in GC become clearer, researchers are actively seeking ways to enhance the specificity and efficiency of gene-editing therapies. Optimizing delivery vectors, such as tunable nanoparticles and CRISPR-Cas9 base editing, could offer safer and more efficient methods of gene editing in GC. Additionally, combining CRISPR-Cas9 with other therapies such as immunotherapy might help overcome barriers by simultaneously attacking the tumor on multiple fronts. For example, engineering CAR-T cells to carry CRISPR-Cas9 could create a therapeutic synergy, allowing for both genetic editing and immune cell targeting of the tumor[54]. Despite the challenges, the future of CRISPR-Cas9 in GC holds promise. By improving delivery strategies, refining CRISPR tools for enhanced precision, and understanding the tumor microenvironment in greater detail, researchers will continue to push the boundaries of what is possible in cancer therapy.

FUTURE DIRECTIONS

Various engineering strategies have been developed to enhance CRISPR/Cas9 delivery for tumor treatment. One approach involves improving passive tumor targeting by adjusting nanocarrier sizes, such as using polyethyleneimine-β-cyclodextrin for efficient genome editing in HeLa cells. Chemical modifications, like zwitterion-like polyethyleneimine derivatives, reduce polymer-serum protein interactions, prolong circulation, and enhance transfection efficiency. Angiopep-functionalized nanocarriers have also demonstrated effective brain tumor targeting and gene editing. Active tumor targeting strategies, including lipid nanoparticles for selective lung, spleen, and liver targeting, and ligand-mediated systems like tetrahedral DNA nanostructure–engineered extracellular vesicle for hepatocellular carcinoma, have further improved precision in gene delivery[55].

To enhance cellular uptake and endosomal escape, methods such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine–polyethylene glycol lipid-chondroitin-protamine-Cas9/single guide RNA formulations have been employed to increase transfection rates and inhibit tumor growth. Multi-component lipid nanoparticles have also shown superiority in inducing endosomal escape and improving delivery efficiency. Responsive drug delivery systems, including pH-responsive lipid-polymer hybrids, effectively inhibit tumor migration and metastasis, while enzyme-responsive DNA nano-frameworks targeting polo-like kinase 1 have achieved high cancer inhibition rates. Additionally, temperature and light-responsive delivery systems have shown promising results in controlling tumor growth and metastasis[55,56].

Innovative nanoparticle-based gene-editing approaches have also been explored. Mout et al[57] developed an alternative strategy using gold nanoparticles to form nanoassemblies containing a modified Cas9 protein and guide RNA. By introducing a glutamate peptide tag for self-assembly with cationic arginine gold nanoparticles and adding a nuclear localization signal to enhance nuclear transport, this system improved protein and nucleic acid delivery to the nucleus, highlighting the therapeutic potential of gold nanoparticles. Meanwhile, Chen et al[58] designed liposome-templated hydrogel nanoparticles with a polyethylenimine hydrogel core for Cas9 encapsulation and a cationic 1,2-dioleoyl-3-trimethylammonium-propane lipid shell for genetic material delivery. These hybrid nanoparticles leverage an autocatalytic tumor-targeting poly (amine-co-ester) terpolymer to selectively reach the brain and efficiently penetrate the blood-brain barrier, offering a promising avenue for targeted gene editing in neurological disorders.

Furthermore, natural polymer-based delivery systems, such as aptamer-hyaluronic acid-chitosan-CRISPR/Cas9 nanoparticles, have exhibited excellent biosafety and prolonged survival in tumor-bearing mice. Organic biomimetic nanomaterials and CRISPR/Cas9-loaded exosomes have demonstrated low immunogenicity and the ability to trigger anti-tumor immune responses, further expanding the potential of nanotechnology-driven gene-editing therapies in oncology[55,58-60]. Last but not least, CRISPR/Cas12, which is an RNA-guided endonuclease similar to Cas9, could be used to develop targeted cancer therapies, such as to introduce the CAR gene into T cells enhancing efficacy against cancer cells. Cas9 and Cas12 target DNA, whereas Cas13 binds and cleaves RNA molecules. Cas13 could be used in knocking-down genes across the genome, identifying genes that play significant role in cancer cell survival[61].

CONCLUSION

As CRISPR-Cas9 continues to evolve, so too does its potential in the fight against GC. With its precision targeting and ability to engage repair mechanisms, this technology offers a new dimension in molecular medicine. The challenge now lies in optimizing the delivery systems - whether through viral vectors, nanoparticles, or electroporation - to ensure that CRISPR-Cas9 safely and effectively reaches its destination. In this molecular symphony, where each note is a carefully orchestrated step in DNA repair, CRISPR-Cas9 stands as a promising conductor, offering hope for the future of GC therapy. In GC, where the roles of H. pylori infection and MSI are both well-established and insidiously destructive, the promise of CRISPR-Cas9 lies not just in treating the symptoms but in addressing the root causes of the disease. Through precise genetic interventions, it is possible to disrupt H. pylori’s ability to initiate carcinogenesis while simultaneously stabilizing the genetic landscape of the host gastric epithelium. CRISPR’s potential to manipulate the host genome, to correct genetic defects, and to downregulate pathogens opens the door to a new era in GC treatment - one where precision is paramount, and where the molecular warfare against cancer can be fought at its most fundamental level. While the road from laboratory to clinic remains fraught with challenges, particularly in the aspect of delivery mechanisms and off-target effects, the intersection of CRISPR-Cas9 technology with GC holds promise. If harnessed effectively, CRISPR could rewrite the story of GC. The convergence of epigenetic changes, MSI, and H. pylori infection within the gastric milieu forms the intricate landscape of GC. Each factor, whether it is the silent power of epigenetic modifications, the chaotic instability of microsatellites, or the chronic inflammation induced by H. pylori, plays a crucial role in tipping the balance toward malignancy. Understanding these forces offers hope for more precise diagnostics and targeted therapies, transforming the fight against GC. GC is a genetically complex disease, driven by a multitude of mutations and epigenetic alterations. Key genetic mutations in TP53, CDKN2A, KRAS, and HER2 play central roles in its pathogenesis and progression. The advent of CRISPR-Cas9 technology offers a transformative approach to combating these genetic mutations, providing a tool for gene correction, targeted therapy, and personalized medicine. While challenges remain in translating CRISPR-Cas9 into clinical practice, its potential to address the genetic underpinnings of GC represents a promising frontier in cancer therapy. The future of CRISPR in GC treatment will depend on striking a delicate balance between harnessing its gene-editing power and mitigating the risks that come with altering the genome. Only through continued exploration and innovation can CRISPR-Cas9 evolve into a safer, more effective therapeutic tool for combating this devastating disease.