Applications of CRISPR-Cas9 in mitigating cellular senescence and age-related disease progression

Abstract

Aging is a multifaceted process influenced by many elements. During cell division, the repetitive DNA sequences at the ends of chromosomes called telomeres protect them from degradation. Telomeres shorten alongside each cell division, eventually contributing to cellular senescence and aging. Telomerase as an enzyme has a role in the maintenance of telomere length. Reduced function of telomerase is linked to acceleration of aging and age-related diseases. By affecting cellular function, mutations in particular genes can cause aging. Genes involved in DNA repair, cellular metabolism, and inflammation play the key roles in this process. Accumulated mutations result in cellular dysfunction and age-related diseases over time. Epigenetic changes are the modifications that impact gene expression without altering the DNA sequence. Lifestyle factors (diet, exercise, stress) and environmental influences (toxins, trauma) can cause epigenetic alterations. DNA methylation as well as histone modifications are examples of epigenetic alterations. They influence how cells work and are essential to the aging process. Understanding these molecular mechanisms is essential for developing interventions to promote healthy aging and prevent age-related diseases. This paper explores the potential of CRISPR/Cas9 as a gene-editing tool to target these mechanisms and mitigate age-related conditions, ultimately enhancing longevity and quality of life.

Introduction

Aging refers to the natural biological process characterized by the gradual decline in physiological functions and heightened vulnerability to age-related ailments. Ample research has focused on the molecular and cellular mechanisms associated with aging, uncovering the involved factors like telomere shortening, reduced telomerase activity, gene mutations, and epigenetic alterations [1]. Telomeres act as the protective caps at the ends of chromosomes, and when they shorten with each cell division, cellular senescence and aging occur. Telomerase is an enzyme that aids in maintaining the telomere length, and its diminished function has been associated with accelerated aging and age-related illnesses [2–4].

Mutations in genes involved in DNA repair, cellular metabolism, and inflammation can also contribute to the aging process via impairing cellular function and promoting the accumulation of damage over time. Furthermore, the aging process can be affected by epigenetic changes like alterations in DNA methylation and histone modifications through changing the gene expression patterns and cellular function [5]. Understanding how aging works at the molecular and cellular levels, such as telomere shortening, decreased telomerase function, gene mutations, and epigenetic changes, is important for creating ways to support healthy aging and avoid diseases related to aging. The present paper strives to unravel the complex interplay of these factors and identify potential targets for therapeutic interventions to enhance longevity and quality of life in aging populations [5]. Today, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 (CRISPR/Cas9) seems to be a powerful gene-editing tool that has garnered significant attention in scientific investigation. The current study tries to explore how telomerase can be leveraged to mitigate age-related diseases.

CRISPR/Cas9 and aging

Scientists have made significant progress in comprehending the molecular mechanisms of aging by CRISPR/Cas9 application, which has changed genetics and molecular biology [6]. The process of aging involves the deterioration of cellular functions, leading to health issues such as heart disease, cancer, and Alzheimer’s disease. In one notable study, mice suffered from Hutchinson–Gilford progeria syndrome, a rare genetic disorder that accelerates aging. The mice exhibited signs such as DNA damage, cardiac dysfunction, and a dramatically shortened lifespan [7, 8]. The researchers developed a novel CRISPR/Cas9 genome-editing therapy to conceal accelerated aging in these mice. Specifically, they targeted the LMNA gene, which produces two similar proteins: lamin A and lamin C. Progeria shifts the production toward a toxic form called progerin. Using CRISPR/Cas9, researchers were able to reduce the harmful effects and better understand the aging process by focusing on both lamin A and progerin [8]. Another study on aging used genome-wide CRISPR/Cas9 screening to identify several new senescence-promoting genes in humans [9]. Of note, cellular senescence, characterized by permanent growth arrest, plays a critical role in aging. [10]. It should be mentioned that this discovery opened up fresh therapeutic avenues for addressing aging-related pathologies (Fig. 1) [9].

Fig. 1.

The Evolution of Discoveries in Aging Biology and Epigenetics. From the earliest discoveries on cell division limits and the role of telomerase to cutting-edge technologies like deep learning and epigenome restoration, this image provides a comprehensive overview of key advancements in understanding the aging process. Looking ahead, the coming years could bring a revolution in biology and therapies related to longevity

CRISPR-Cas9: overview, mechanism, and significance

CRISPR-Cas9 is a revolutionary gene-editing tool that allows scientists to accurately change the DNA of living things, opening up many possibilities for treating genetic diseases, enhancing crop yields, and possibly changing the genes of an embryo [11]. CRISPR-Cas9 has several benefits compared to older gene-editing methods like TALENs and ZFNs, mainly because it is easier to use, more flexible, and more accurate. It is due to its simplicity, adaptability, and enhanced precision [12].

Mechanism

The CRISPR-Cas9 method is derived from a naturally occurring defense mechanism employed by bacteria to detect and remove viral DNA infiltrating their cells [11]. The mechanism of CRISPR-Cas9 genome editing involves several factors and steps (Fig. 2), including 1. Guide RNA (gRNA) synthesis: The initial step involves creating an RNA molecule with a short ‘guide’ sequence that binds to a specific DNA sequence in a genome. The RNA molecule also creates a binding connection with the Cas9 enzyme [13]. 2. Complex formation: The gRNA combines with the Cas9 enzyme to create a complex and facilitates the targeted binding of the Cas9 enzyme to the specific genomic locus that corresponds to its nucleotide sequence [14]. 3. DNA cleavage: Upon binding to the target DNA sequence, the Cas9-sgRNA complex starts a cleavage event by the Cas9 nuclease, leading to the simultaneous cutting of both strands of the DNA at a precise location within the target sequence. This event leads to the creation of a double-strand break (DSB) in the DNA molecule.[15] 4. DNA repair: The Cas9 nuclease repairs the double-stranded break (DSB), which is carried out by the cellular machinery of the host organism. The two primary repair mechanisms consist of non-homologous end joining (NHEJ) and homology-directed repair (HDR). Non-homologous end joining (NHEJ) is the most common and effective cellular method for DNA damage repair. This mechanism shows increased activity in cells; nonetheless, it is characterized by a tendency to introduce errors. Explicitly, NHEJ may result in small, stochastic insertions or deletions (indels) at the site of DNA cleavage. As a result, such changes in DNA can contribute to the occurrence of frame-shift mutations or premature stop codons. However, HDR is a more accurate mechanism in which a homologous DNA template is utilized to repair the DSB, resulting in a more precise restoration of the target DNA sequence [16].

Fig. 2.

Mechanism of CRISPR/Cas9

Significance

CRISPR-Cas9 has significant applications in many sectors, including medicine, agriculture, and basic biological investigation.

Medicine

The utilization of CRISPR-Cas9 in the medical field has promise for treating genetic disorders through correcting DNA mutations. This approach can be applied to treat various medical conditions, including genetic disorders such as cystic fibrosis and sickle cell disease, as well as complex illnesses like cancer and HIV [14, 17].

Agriculture

CRISPR-Cas9 can be employed in agriculture so as to increase crop yields, improve nutritional composition, and boost resistance against pests and diseases. This technology may address the urgent need for sustainable and resilient food systems in light of the growing world population and climate change effects [18].

Basic biological research

The application of CRISPR-Cas9 has considerably enhanced our capacity to engage in fundamental biological investigations. Researchers now have easier access to manipulate model organism genomes to explore gene function, provide disease models, and investigate genetic interactions. The use of this technology has significantly sped up our understanding of genetics and cellular biology [19].

Telomeres and telomerase editing

Telomeres are the protective structures considered as the heterochromatin repetitive sequences, consisting of TTAGGG units at the end of chromosomes, which are covered by six protein complexes known as Shelterin. Shelterin, along with telomeres, plays a role in both binding and telomere length protection. In eukaryotic cells, the special structures at the ends of the chromosomes that prevent the separation of terminal bases and the shortening of chromosomes and end joining of chromosomes are called telomerase, a ribonucleoprotein consisting of a reverse transcriptase (TERT) and an RNA subunit (TERC), playing a role as a template to increase telomere length. Telomerase is responsible for maintaining the telomere length in some cells, such as stem cells, lymphocytes, and proliferating cells; however, the telomere length is different owing to the different activity of telomerase [20, 21]. It is well-established that alterations in telomere length, resulting in telomere damage, lead to cellular disorders, including aging and various diseases [22]. CRISPR/Cas9 is now a valuable tool for investigating the impact of mutations in living cancer cells and for modeling cellular aging. Researchers can employ the CRISPR/Cas9 method to study telomere damage, specifically at the DNA double-strand break and single-nucleotide levels (Fig. 3). For instance, researchers apply it to induce DNA double-strand breaks (DSBs), activate the repair system, and completely remove telomeres [23, 24]. Besides, a new class of CRISPR-based tools efficiently corrects single-nucleotide changes in cell lines, animal models, and perhaps the clinic [25]. Research shows that when telomeres shorten and DNA gets damaged in key tissues, it leads to problems in those areas and can predict the development of age-related diseases, such as cancer, in other tissues [26–28].

Fig. 3.

Telomeres and Telomerase Editing by CRISPR/Cas9

The most common TERT promoter mutation in malignant melanoma, which is associated with increased mRNA and protein expression, telomerase activity, telomere length, and very poor prognosis, highlights the critical role of telomerase in cancer progression. Investigation of this malignancy has shown that the frequency of mutations in the TERT promoter is higher than the occurrence of all known mutations in melanoma [29]. By manipulating the hTERT promoter, expression can be amplified or reduced, a phenomenon likely occurring in cancer cells or cells undergoing aging and death [30, 31]. Using CRISPR and dual CKM and TERT promoters, inhibition of malignant osteosarcoma cells and induction of apoptosis in cancer cells were observed [32]. These findings suggest that with structural modifications to telomerase, it is possible to design control mechanisms for this enzyme, some of which may be implemented within living organisms.

Adding a halo protein tag to the N-terminal end of TERT or introducing a single DNA substitution mutation in the hTERT promoter can impact telomerase expression, as observed in urothelial cancer cells where telomerase levels are reduced [33, 34]. Epigenetic modification, such as m6A, is a key regulator of telomerase activity and can be modulated using CRISPR genome editing. For instance, if the m6A regulator hnRNP affects hTERC, it could activate telomerase and delay stem cell aging. Similarly, if ALKBH5 acts as a methylation regulator influencing telomerase RNA (hTER), it could be pivotal in controlling telomerase activity and cellular function [35–37].

Alternative lengthening of telomeres (ALT) is a cellular mechanism that allows some cancers to extend their telomeres. Deletion of the TERT or TERC subunits of the telomerase enzyme using CRISPR can activate the ALT mechanism, leading to increased telomere length. Similarly, deleting hTERT also results in telomere elongation [30, 38–40]. Reprogramming fibroblast cells with hTERT is being used to create new models for studying Werner syndrome [30, 41]. Mutations in the TERT gene promoter are the most common clonal oncogenic mutations in glioblastoma, one of the most lethal cancers, making telomerase a promising therapeutic target. When TERT is inactivated, glioblastoma cells lose their ability to replicate due to telomere shortening and chromatin bridge formation over time. CRISPR technology can be employed to eliminate TERT expression in glioblastoma cell lines with TERT promoter mutations [42].

Epigenetics—editing

During genomic editing, there is a comprehensive alteration in the nucleotide sequence configuration, specifically targeting the encoded regions [43]. This procedure may result in the formation of new antigens due to genetic mutations [30]. However, epigenetic editing is a form of manipulation through a non-cutting process, and it has a persistent and reversible nature [43]. Instead of altering the fundamental DNA codes, it targets the sections of DNA responsible for controlling gene transcription and the way that DNA sequences interact with proteins [43]. This process can change the molecular makeup of the chromatin structure, and the way DNA is packaged [43]. Epigenetic editing can also reactivate genes that are generally only expressed in particular organs with distinct immune traits [30].

Epigenetic editing can be performed via four main mechanisms, including DNA methylation, post-histone translational modification, chromatin remodeling regulation, and regulation of noncoding RNA [30, 44]. DNA methylation is a regulatory mechanism that plays a key role in cell differentiation, tissue development, and homeostasis [44]. It is associated with physiological and pathological processes like gene expression, chromosomal inactivation, aging, and disease in tissues [44]. During the methylation process, DNA covalent modifications are made in the cytosine of the nucleotide sequence of CPG within the promoter region, which suppresses transcription [43, 45, 46]. The DNA methyltransferases (DNMTs) are catalyzed into 5-methyl cytosine (5MC) by S-adenosyl methionine (SAM) as a methyl donor. Of note, a group of proteins known as epigenetic writers, epigenetic readers, and epigenetic erasers regulates the methylation process. Alterations across the genome are made by the ‘writer’ that is identified by the ‘reader,’ and if these changes are not needed, the ‘eraser’ removes them [45].

There are four important types of DNMTs proteins: NMT3A, DNMT3B, DNMT1, and DNMT3L. NMT3A and DNMT3B belong to the group of de novo methyltransferases [44, 45]. They are involved in the methylation of CPG dinucleotide islands during the embryonic period [44]. DNMT1 preserves the methylation pattern by adding methyl groups to hemimethylated CPG during chromosome replication [44, 45]. Unlike other types, DNMT3L lacks enzymatic function [43]. The main function of DNA methyltransferases in epigenetic processes is to act as fusion proteins in the methylation of off-target DNA. By targeting it, the target gene can be repressed by inducing de novo 5mc [46]. If a mutation occurs in DNMT, it impacts both catalytic and multimerization activity through the activation of protein kinase and reduces the amount of off-target 5MC and constrains methylation near the target sites [46]. The post-translational modification of the histone is one of the mechanisms of epigenetic editing and is considered a covalent modification that mainly includes methylation, acetylation, ADP-ribosylation, phosphorylation, ubiquitylation, SUMOylation, and glycosylation [47].

It should be mentioned that stone acetylation is a process of epigenetic modification, affecting the function of proteins. The neutralization of unmodified lysine residual loads is carried out through the actions of two enzymes, histone acetyl transferase (HAT), and histone deacetylases (HDAC) [45]. Through doing so, they reduce the electrostatic interaction between negatively charged DNA and histone [48]. Subsequently, the structure of chromatin can be alternately opened and closed, leading to the relaxation of the chromatin [49]. As a result, the transcription rate increases [45]. All aspects of this process potentially can affect the composition of proteins and their interactions with cofactors, substrates, and macromolecules [45, 50]. Besides, it should be noted that mutations in the HAT enzyme contribute to its diminished function, which has been reported in numerous ailments like cancer [45]. In the histone methylation process, methyl groups are added to specific amino acids in histone proteins [51]. This process acts as a transcription suppressor and gene silencer [46, 51]. The residues of lysine and arginine in the tail of the histone can be the target of histone methylation [45, 46]. In contrast to histone acetylation, in histone methylation, the DNA and histone physical interaction does not result in neutralizing the histone load [45]. Subsequently, histone methylation provides a more complex chromatin structure compared with histone acetylation [45, 46]. Nonetheless, it can generate a more durable and stable post-translational correction. Histone methylation is catalyzed by histone methyltransferases (HMTs); however, it can be removed by histone demethylase (HDMs) enzymes, allowing for dynamic regulation of gene expression and chromatin remodeling [51]. It is worth mentioning that zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9-based RNA-guided DNA endonuclease as the targeted genome-editing tools are effective for modeling human disease through the creation of isogenic cells and transgenic animals [52] (Fig. 4) (Table 1). These efficient programmable chimeric nucleases are much faster and more cost-effective in comparison to the traditional homologous recombination-based mutagenic approach [53]. These tools investigate the role of epigenetic modulators in gene activity in native tissues and cell-type-specific chromatin states [54]. Among the mentioned tools, CRISPR-based genome-editing tools are a more usable and popular system for gene-editing and gene transcription adjustment. This is due to its high versatility with chemical modification, efficiency, multiplexity, simplicity in goal design, and cost-effectiveness [53].

Fig. 4.

Genome-editing tools are based on RNA-guided endonuclease, DNA based on zinc finger nuclease (ZFN), transcription-like activating nuclease (TALEN)

Table 1.

Overview of Targeted Gene-Editing Approaches in Various Age-related Disorders

Disorders	Target Sites	Model (cell line)	Model of action	Delivery Method	Advantages	Disadvantage	Refs
Amyotrophic Lateral Sclerosis (ALS)	SOD1 gene	Patient-derived induced pluripotent stem cells (iPSCs)	Suppress the expression	Adeno-associated virus (AAV) vectors or lipid nanoparticles	Enhancements in motor function and an extended lifespan	May require complex delivery methods, and off-target effects could be a concern	[95, 96]
	C9orf72 gene	iPSCs	Mutation	Viral vectors, typically AAV or lentivirus	Reinstatement of typical gene expression and enhanced the viability of motor neurons in a cellular environment	Potential challenges in achieving efficient and precise gene editing in clinical settings	[97]
	scAAV9-hIGF1	Mice	Inhibition of the NF-κB signaling pathway	Adeno-associated virus serum type 9 (scAAV9-hIGF1)	Delay the initiation and decelerate the advancement of the ailment	Risks associated with viral vector delivery, including immune response and off-target effects	[98]
Colorectal Cancer (CRC)	KRAS	HT29, WIDR, HCT116, LS174T, and HEK293T; SW480 and A549; and CFPAC‐1	Knock out	Two‐vector lentivirus system	GRB7‐PLK1 has a critical axis for RTK tolerance. PLK1 and thus a suitable target for synergizing MEK inhibitors in CRC patients with KRAS mutations	Potential resistance development and off-target effects, leading to unintended gene alterations	[99]
	Klotho	Caco‐2	Knock out	Lentiviral vectors for delivering CRISPR/Cas9 to target cells	By causing apoptosis, Klotho gene overexpression in Caco‐2 cells by CRISPR/Cas9 inhibits cell growth	Limited to specific cancer types; potential off-target effects and challenges in gene delivery	[100]
	uPAR	CRL1619, CCCL247	Knock out	Okayama‐Berg vector	Knockout of the uPAR gene Leads to tumor growth inhibition, EGFR downregulation, and an increase in stemness markers	May require complex delivery methods, and the long-term effects of gene knockout are still unknown	[101]
Breast Cancer	MYC gene	Animal models	Target and suppress oncogenes	Lentiviruses, adeno-associated viruses, non-viruses like nanoparticles and lipids	Beneficial effects of CRISPR/Cas9 in cancer treatment	Off-target	[102, 103]
Ovarian Cancer	EGFL6 exons	SKOV3	Delete	Lentiviral vectors	The removal of EGFL6 significantly reduced the migration, invasion, and growth of SKOV3 cells, while also promoting the death of tumor cells	Potential off-target effects and challenges in achieving precise gene editing in a clinical setting	[104]
	BMI1, CXCR2, MTF1, miR-21, and BIRC5	ovarian cancer cells	Knock out	Lentiviral CRISPR/Cas9 nickase vector	targets multiple genes associated with cancer progression, offering a comprehensive approach to treatment	Complexities in delivery and potential off-target effects could limit its effectiveness	[105–113]
Prostate Cancer	Androgen receptor (AR) gene	LNCaP, VCaP, and PC-3 prostate cancer cell lines	Disruption of AR gene expression	AAV or lentiviral vectors	Slow down prostate cancer growth	Potential development of resistance and off-target effects could pose challenges in treatment	[114]
	ER-β gene	Mice	Knock out	Lentiviral vectors	Regulated the prostate tumor size and acted as a tumor-suppressor gene	Delivery challenges and off-target effects may limit its clinical application	[114, 115]
	Erythropoietin-producing hepatocyte receptor A2 (EphA2)	DU145 and PC-3 tumor cells	CRISPR/Cas9 targeting of EphA2 to inhibit cell migration, invasion, and proliferation, potentially reducing metastatic potential	Calcium phosphate-based nanomedicine	Uses a novel nanomedicine approach for targeted delivery, potentially increasing treatment efficacy	Risks associated with nanomedicine delivery, including toxicity and off-target effects, are still under investigation	[116]

The CRISPR/Cas9 system consists of two important components [55]: the guide component, responsible for specificity, which includes a single-stranded RNA molecule called sgRNA, and an effective component. sgRNA targets a specific DNA sequence in a genomic area and binds to the effective component via a scaffold RNA welding component. Cas protein is considered the most effective component, and the most common is Cas9 [55].

Promoters and insulators serve as barriers to inhibit interactions between enhancers and promoters, and they also hinder the spread of heterochromatin by neutralizing its effects [56]. In the process of gene activation using the CRISPR/Cas9 strategy, an effector domain (ED) can be fused to dCas9 (dCas9-ED) and precisely targeted to prevent insulator and promoter activity. This approach enables targeted DNA methylation, histone methylation or demethylation, and histone acetylation or deacetylation in mammalian cells, allowing for precise control of gene expression [53].

The efficiency of the CRISPR editing system depends on sgRNA and directs Cas9 protein for genome cutting [57]. dCas9-ED with sgRNA-mediated can transcribe local epigenetic localized states such as the histone sequence 5mC and modify transcription activity.

Due to reversible and flexible nature of epigenetic modification, it has the potential to be utilized as an anticancer treatment and enables the readjustments of the cancer epigenome [44]. Based on several studies on the genomic sequence of cancer cells, it has been clearly revealed that a high rate of changes in epigenetic regulatory genes occurs [30, 52]. These alterations affect all cancer hallmarks, such as the interaction between the immune system and tumor cells [58]. Hence, epigenetic editing mechanisms, in particular targeting epigenetic regulators, importantly provide an interaction between immune cells and tumor cells and enhance immune responses against tumors [54]. Of note, epigenetic regulatory mechanisms play a critical role in the interactions between cancer cells and immune cells in the tumor microenvironment through a number of specific proteins, including 1. ‘Writers’ add epigenetic marks to DNA. 2. ‘Erasers’ remove epigenetic marks from DNA. 3. ‘Readers’ are to identify specific epigenetic marks that act as mediators in DNA. 4. ‘Remodelers’ to moderate chromatin status [30].

In the cancer-immunity cycle, epigenetic mechanisms can influence cytokine production, leading to a dysregulated antigen presentation machinery in tumor cells. This dysregulation may prevent tumor cells from becoming invisible to T cells, thereby affecting immune recognition [30]. In addition, dysregulation results in gene reactivation with limited development and the creation of tumor differentiation antigens [58]. By regulating programmed death-ligand 1 (PD-L1) expression and binding to T cells, the proliferation of T cells, and the production and activity of cytokines can be suppressed, leading to T cell exhaustion [30].

The epigenetic machinery in immune cells plays a role in determining the fate of lymphocyte cells during lymphocyte development. Furthermore, it impacts the occurrence of functional and phenotypic changes when the adaptive immune system is activated. Alterations in epigenetic regulators play a direct role in the development of hematologic malignancies [58]. Chromatin organization is involved in T cell exhaustion. T cell exhaustion is defined via altered transcriptional and epigenetic states, reduced proliferative capacity and production of effector cytokines, sustained expression of inhibitory receptors, and impaired metabolic activity [59]. Exhausted T cells originated from infection have many open chromatin regions, providing a very different structure compared to effective lymphocytes [30]. Chromatin changes caused by T cell exhaustion can be reversible at the first stage; however, at the next stage they enter a fixed state, which cannot be reversed, leading to resisting changes and reprogramming [30].

During tumor development, the epigenome undergoes several changes, such as a decrease in DNA methylation across the genome and an increase in methylation, especially in the CpG islands of tumor-suppressor genes (TSGs). Epigenetic alterations can be considered as the drivers of tumorigenesis onset, especially in children’s brain tumors, which are defined as an aberrant epigenetic pattern [44, 54]. Epigenetic alterations can be considered as the drivers of tumorigenesis onset, especially in children’s brain tumors, which are defined as an aberrant epigenetic pattern [44].

The role of epigenetic disorders in human disease etiology and the aging process has been revealed. Epigenetic regulation disorder is recognized as a significant biological characteristic of aging and lifespan [56]. Aberrant transcription regulation caused by incorrect epigenetic patterns can accelerate aging and reduce life expectancy. Environmental factors can trigger heritable changes in the epigenome, leading to faster aging and the development of age-related diseases [53]. Genome and epigenomic engineering mechanisms involved in aging and age-associated diseases in humans reduce hemostatic processes over time and increase degenerative conditions, leading to an enhanced risk of many chronic diseases [44]. The contribution of genetic and epigenetic components in animal model lifespan has been recognized by applying the CRISPR-Cas9 technology. Thus, it is possible to quickly explore their roles in health and longevity with the functional investigation of genome-mapped genetic variants of the human genome [11]. Genome and epigenome-editing technology seem to be promising in age-related disease treatment. Therefore, targeted editing of genes associated with aging offers a new therapy for multiple diseases [53].

General mechanisms of aging in mammals include loss of histone associated with chromatin regeneration, imbalance in activation and suppression of histone changes, localized or general alterations in DNA methylation, change of transcription, nuclear reorganization, and subtractive or gradual changes in heterochromatin [44, 53]. It should be mentioned that the depletion of histone methyltransferase SUV39H1, which specifically methylates lysine nine in histone H3 and performs as a transcriptional suppressant, has been shown to improve DNA repair capacity, expand life expectancy, and delay the aging process in animal models [53].

Based on numerous lines of evidence, several afflictions have been shown to be related to epigenetic changes. Those alterations are linked to aging, cancer, and neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) [60]. In other words, mechanisms involving aberrant DNA methylation could be relevant to human ALS pathobiology and therapeutic targeting. Abnormal DNA methylation patterns that originate from environmental factors play a role in the beginning or evolution of neurodegenerative diseases like ALS [61]. DNA methylation is involved in proliferation and differentiation of neural stem cells, synaptic plasticity, neuronal reparation, learning, and memory [62]. According to pathway analysis, genes with different methylation in sporadic ALS were particularly involved in calcium homeostasis, neurotransmission, and oxidative stress [63].

The etiology of colorectal cancer (CRC) is primarily associated with a number of genetic and epigenetic abnormalities within normal colonic epithelial cells, coupled with the reshaping of the tumor microenvironment in the surrounding stroma [60]. Based on prior investigations, epigenetic regulation is a key factor in acquiring underlying inherent drug resistance in CRC patients. It has been shown that unusual expression of the CRC epigenome contributes to resistance against conventional drugs, including cetuximab, 5-fluorouracil, and oxaliplatin. Epigenetic alterations often occur in the early progression of diseases and have a key role in nearly all cancer-associated pathways. Therefore, these alterations are prime candidates for cancer detection, diagnosis, and prognostic biomarkers. It is worth noting that to acquire the early CRC screening, classic CRC biomarkers, including mutations in KRAS and BRAF, methylation modifications of BMP3, NDRG4, and SEPT9, high expression micro-RNAs like miR-92a and miR-144, and the presence of F. nucleatum, are used in liquid biopsies [64–67].

The progression of breast cancer (BC) is a complicated and multistep procedure. This process includes interactions between genetic and epigenetic changes, influenced by various internal and external factors. Factors such as the cell’s internal microenvironment, availability of nutrients, cellular stress, and exposure to endocrine disruptors or carcinogens in the external environment can all play a role in BC pathogenesis. The two main players in epigenetic alterations in BC are the methylation of DNA and the modification of histone proteins. Overall, important genes that control cell growth, death, movement, and invasion are affected by the epigenetic changes associated with the progression of breast cancer [68]. Several BC genes show CpG island hypermethylation or aberrant promoter methylation, leading to silencing of tumor-suppressor genes involved in estrogen signaling, apoptosis, cell cycle, and DNA repair, such as HOXA5, TMS1, p16, RASSF1A, BRCA, E-cadherin, GSTP1, and p21/CIP1/WAF1 [69–75]. Histone methylation occurs on lysine and arginine side chains, adding methyl groups without changing protein charge. Methyltransferases and demethylases dynamically control this process [76]. Methylation affects gene regulation, activation, and repression. Various BC subtypes show differences in histone modifications, correlating with tumor characteristics and metastasis. Histone marks like H3K4 and H4K20 play key roles in cancer progression. Epigenetic changes impact gene expression and therapeutic targets in BC treatment [77, 78]. The enhancer of zeste homolog 2 (EZH2) is a key histone methyltransferase in BC, upregulated and promoting EMT [79]. Lysine methyltransferase 2 (KMT2) family members facilitate growth by activating estrogen-dependent genes [68]. The histone methylase disruptor silencing 1-like (DOT1L) is a potential therapeutic target for invasive BC [80]. Lysine-specific histone demethylase 1 (LSD1) negatively regulates cell growth genes [81]. Several demethylases (KDMs) are involved in BC progression and metastasis [82]. Plant homeodomain finger proteins 8 (PHF8) and PHF20L1 have oncogenic roles in BC metastasis [83, 84]. Noncoding RNA, like long noncoding RNAs (lncRNAs) and micro-RNAs (miRs), plays a crucial role in regulating gene expression in breast cancer. Micro-RNAs, especially the let-7 family, miR-148a & miR-152, miR-125b, miR-126, miR-31, miR-663, miR-148, miR-9-1, miR-152, and miR-124a3, have been studied a lot for how they affect gene activity in breast cancer [85–89].

Ovarian cancer (OC) also utilizes epigenetic modifications, such as DNA methylation and histone deacetylation, to manipulate gene expression for cell division and metastasis. One significant example of hypermethylation is reported the silencing of BRCA genes [90]. In addition, two gene sets have been proposed to be related to OC. First gene set have been proposed to be associated with frequency of histotype-specific DNA methylation including BRCA1, PTEN, CDKN2A, MLH1, RASSF1A, and CDH1. Second gene set focused on chromatin remodeling such as ARID1A, SPOP, and KMT2D. Targeting these processes with pharmaceutical agents may reverse unusual changes in cancer cells, inhibiting growth, invasion, metastasis, and chemotherapy resistance [91].

Epigenetic abnormalities are intricately linked to the development and progression of prostate cancer (PC). Similar to cancers mentioned above, aberrant DNA methylation, histone modifications, and noncoding ribonucleic acids play a role in the initiation and progression of PC [92]. The hypermethylated genes are involved in DNA repair, cell cycle, growth suppression, apoptosis, and cell adhesion, such as GSTP1, MGMT, CDKN2, RASSF1, APC, RARβ, DCR1, DCR2, XAF1, TMS1, CDH1, and CD44 [93]. It should be mentioned that PC also harbors genomic hypomethylation. However, the late phase of PC tumorigenesis, such as metastasis, often reports DNA hypomethylation more frequently than the early stage [94]. Hypomethylated genes in PC include PLAU, HPSE, and CYP1B1. Furthermore, changes in histone modifications, another form of epigenetic modification, significantly influence PC. Researchers have highlighted methylation, acetylation, phosphorylation, and ubiquitination among these modifications [93]. Hypomethylated genes in PC include PLAU, HPSE, and CYP1B1. Furthermore, changes in histone modifications, another form of epigenetic modification, significantly influence PC. Among these modifications, methylation, acetylation, phosphorylation, and ubiquitination have been highlighted [90]. Furthermore, researchers have proposed a relationship between two gene sets and OC. The first gene set has been proposed to be associated with the frequency of histotype-specific DNA methylation, including BRCA1, PTEN, CDKN2A, MLH1, RASSF1A, and CDH1. The second gene set focused on chromatin remodeling, such as ARID1A, SPOP, and KMT2D. Targeting these processes with pharmaceutical agents may reverse unusual changes in cancer cells, inhibiting growth, invasion, metastasis, and chemotherapy resistance [91].

Epigenetic abnormalities are intricately linked to the development and progression of prostate cancer (PC). Similar to cancers mentioned above, aberrant DNA methylation, histone modifications, and noncoding ribonucleic acids play a role in the initiation and progression of PC [92]. The hypermethylated genes are involved in DNA repair, cell cycle, growth suppression, apoptosis, and cell adhesion, such as GSTP1, MGMT, CDKN2, RASSF1, APC, RARβ, DCR1, DCR2, XAF1, TMS1, CDH1, and CD44 [93]. It should be mentioned that PC also harbors genomic hypomethylation. Nonetheless, DNA hypomethylation is more frequently reported in the late phase, such as metastasis, rather than the early stage of tumorigenesis in PC [94]. Hypomethylated genes in PC include PLAU, HPSE, and CYP1B1. Moreover, alterations in histone modifications as another epigenetic change play a key role in PC. Among these modifications, methylation, acetylation, phosphorylation, and ubiquitination have been highlighted [93]. Understanding how epigenetic regulatory mechanisms work will help us gain a better insight into PC and identify new epigenetic biomarkers that can indicate the risk of developing cancer, predict how patients will respond to drugs, or determine their likelihood of a poor prognosis.

CRISPR-Cas9 therapeutic role in age-related diseases

CRISPR-Cas9 gene therapy in amyotrophic lateral sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS), colloquially referred to as Lou Gehrig’s disease, is a degenerative neurological condition characterized by the progressive impairment of motor neurons located in the brain and spinal cord [117]. At present, ALS lacks a definitive cure, and the available treatment modalities remain constrained [118]. Nevertheless, recent advancements in gene therapy applying the CRISPR-Cas9 approaches have exhibited potential in the treatment of this debilitating ailmen[119]. As the CRISPR-Cas9 system is a highly effective mechanism to manipulate the DNA sequence of cells, this method is able to modify patient-derived induced pluripotent stem cells (iPSCs). IPSCs can be further developed into motor neurons for the purposes of disease modeling and drug screening [120]. Superoxide dismutase 1 (SOD1) is the gene being studied in relation to ALS, as it has been connected to the development of this disease. A particular subset of familial ALS instances exhibit changes in the SOD1 gene. Furthermore, the abnormal accumulation of SOD1 protein has been identified in both familial and sporadic cases of ALS [121].

Several investigations have shown that the effective application of CRISPR-Cas9 technology in rectifying a mutation found in the SOD1 gene leads to notable enhancements in motor function and an extended lifespan [117, 120, 122]. Another potential approach includes using CRISPR-Cas9 technology to target and suppress the production of harmful proteins, such as TDP-43 and FUS, known to play a role in causing ALS. Several studies have revealed that this approach is practical and effective in cellular and animal models of ALS. Currently, clinical trials are being carried out to evaluate how safe and effective this method is for human subjects [120]. Another potential approach includes using CRISPR-Cas9 technology to target and suppress the production of harmful proteins, such as TDP-43 and FUS, known to play a role in causing ALS. Several studies have revealed that this approach is practical and effective in cellular and animal models of ALS. Currently, clinical trials are being carried out to evaluate how safe and effective this method is for human subject [123]. In other studies, the delivery of self-complementary adeno-associated virus serum type 9 encoding the human insulin-like growth factor 1 (scAAV9-hIGF1) was administered to the subarachnoid space of mice with ALS utilizing the CRISPR-Cas9 system. The results showed that using scAAV9-hIGF1 significantly helped to slow down the start and progression of the disease by blocking the NF-κB signaling pathway [124].

CRISPR-Cas9 sys gene therapy in colorectal cancer

Targeting the CRISPR-associated protein-9 nuclease (Cas9) within a living cell enables the highly efficient genome editing of CRISPR/Cas9 technology [126, 127]. The CRISPR/Cas9 method has been applied to in vitro CRC by inhibiting some molecular pathway [100, 101, 128]. The estimation showed that CRC is the second leading cause of deaths among cancers and the third most common malignancy worldwide [129–132]. Telomerase reverse transcriptase (TERT and hTERT in humans) is the catalytic portion of telomerase which is known as the limiting factor of this enzyme [133]. Classified as a ribonucleoprotein, telomerase consists of both RNA and proteins. Specifically, it consists of two molecules of telomerase reverse transcriptase, telomere RNA, and dyskerin [134–136]. Each of these subunits plays an important role in the biological function of telomerase. Nevertheless, it is critical to note that the catalytic component, known as telomerase reverse transcriptase (TERT or hTERT in humans), is typically the limiting factor when it comes to telomerase activity and the elongation of telomeres [133].

The transforming growth factor b (TGF- β) pathways are context-dependent signal transduction cascades that can act as both a tumor promoter and tumor suppressor [137]. TGF-β1 acts as a controller of proliferation and differentiation in many tissues [138]. Besides, higher TGF-b1 protein expression is related to advanced tumor stages and metastasis in colorectal carcinoma [139]. By using CRISPR/Cas9 technology, previous studies indicated that TGF-β1 gene expression has a direct correlation with tumor progression in non-small cell carcinoma (NSCLC), gastric cancer, prostate cancer, and also colorectal cancer [140–143].

Vaults are barrel-shaped ribonucleoprotein (RNP) particles in many eukaryotic cells, with their role being unclear still [144]. These structures contain MVP (major vault protein), TEP1, vPARP, and noncoding RNAs [145, 146]. It has been demonstrated that they participate in developing drug resistance in malignant cells [147]. By using CRISPR/Cas9, current studies have demonstrated that MVPs are involved in regulating migration and proliferation of cancer cells and are highly expressed in tumor metastasis in colorectal adenocarcinoma; they are also essential components for HAP1 cells to survive [148]. CST, which stands for the ssDNA-binding trimetric complex, is composed of the CTC1, STN1, and TEN1 subunits [149, 150]. This complex plays a crucial role in various aspects of telomere replication [150, 151]. Via applying CRISPR/Cas9 technology, recent studies utilized have demonstrated that CTC1-STN1 actively terminates telomerase activity to prevent excessive extension of the G-overhang [152]. In addition, it has been described that STN1-TEN1 is essential for C-strand synthesis during telomere replication [152].

CRISPR-Cas9 gene therapy in breast cancer

BC is the world’s second most prevalent cancer diagnosis. Its heterogenic nature is due to the involvement of various gene mutations [102, 153]. While traditional BC therapies like radiation and chemotherapy can be beneficial, they often cause adverse effects and fail to target-specific mutations. In contrast, CRISPR-Cas9 gene therapy can help target-specific gene mutations in BC [154]. CRISPR-Cas9 offers exceptional accuracy, efficiency, affordability, and reduced risk, which makes it a better option than previous gene therapies such as ZFN and TALEN [153–156]. CRISPR-Cas9 technology could repair cancer-causing mutations in animal models as a first step toward translational applications. This method enables scientists to determine the responsible gene in various cancers, including BC. This is achieved by creating and deploying guide RNAs (gRNAs) that specifically target-specific sections of the gene [157]. Targeting and suppressing oncogenes with CRISPR-Cas9 can slow down the progression and metastasis of BC [158]. The majority of clinical trials that use CRISPR-Cas9 rely on genetically engineered T cells and cancer immunotherapy [159]. Moreover, viruses such as lentiviruses and adeno-associated viruses and non-viruses like nanoparticles and lipids can deliver CRISPR/Cas9 components to target tissues in vivo. Gold nanomaterials offer several benefits that make them useful in theranostics, delivery vehicles, imaging, and photothermal agents [102, 160, 161]. BC CRISPR-Cas9 gene therapy may target the MYC gene (a type of oncogene). Schuijers et al. used CRISPR-Cas9 to downregulate the MYC gene, which increases 30–50% in high-grade BC. Although there are several beneficial effects of CRISPR/Cas9 in cancer treatment, it has also been associated with higher carcinogenesis [162]. CRISPR-Cas9 stimulates both genotoxicity and immunotoxicity, with genotoxicity linked to off-target outcomes. These issues can lead to unintended mutations that may harm humans [163]. Furthermore, tumor heterogeneity is considered an issue in treatment because tumors are typically made up of distinct subclones [45].

CRISPR-Cas9 gene therapy in ovarian cancer

The seventh most prevalent disease among women worldwide is OC. This variation increases the genetic susceptibility to OC and can be inherited as a result of rare, moderately to highly penetrant mutations. A woman’s lifetime risk of acquiring OC is 1–2%, and there is a 1 in 100 probabilities of dying from it. Every year, OC causes 239,000 new cases and 152,000 fatalities worldwide [164, 165]. Additionally, ovarian malignancies typically spread to distal organs such as the liver, colon, and omentum through peritoneal dissemination from the main tumor site [166]. CRISPR-Cas9, a promising gene-editing technique, offers the potential to treat OC through gene-editing procedures [167]. CRISPR knockouts of various oncogenes implicated in the OC etiology, including BIRC5, BMI1, MTF1, CXCR2, and miR-21, have been described in studies, demonstrating the promise of the CRISPR-Cas9 approach to efficiently treat OC [168].

OC progresses and metastasizes due to tumor angiogenesis [169] and exhibits high levels of epidermal growth factor-like domain multiple (EGFL6) protein expression, suggesting that this protein is crucial for encouraging tumor angiogenesis [170]. EGFL6 is a member of the EGFL (epidermal growth factor-like) family of proteins [171]. Embryos and most tumor tissues express EGFL6 extensively. EGFL6 enhanced the OC stem cells’ asymmetric division, phosphorylated ERK, and supported the proliferation and spread of OC. Additionally, Noh et al. found that the TWIST transcription factor regulated EGFL6 and helped the growth of new blood vessels in OC by managing integrin/Tie2/AKT [172]. In one study, EGFL6 was knocked out using CRISPR-Cas9 by transfecting a guide RNA (gRNA) that was specifically engineered to target EGFL6 exons into an OC cell line. The EGFL6 gene was removed from the ovarian cancer cell line SKOV3 using the CRISPR/Cas9 system after a guide RNA was designed to specifically target the EGFL6 exons. The elimination of EGFL6 dramatically decreased the migration, invasion, and proliferation of SKOV3 cells and facilitated the death of tumor cells. This study suggests that EGFL6 might regulate the FGF-2/PDGFB signaling pathway, which could influence blood vessel formation, movement, and growth of ovarian cancer cells [170].

DNMT1 is a key biological target for cancer epigenetic treatment [173]. It is an oncogene that inhibits tumor-suppressor genes when it is overexpressed and is essential for cancer stem cell maintenance. It is associated with carcinogenesis and OC relapse, tumorigenesis, and resistance. Frequently, it has been shown that patients with poor prognoses had elevated levels of DNMT1. Thus, DNMT1 has been considered a viable molecular target for OC therapy [106]. Targeting DNMT1 with CRISPR-Cas9 was found to suppress DNMT1 expression and slow tumor growth, indicating the promise of DNMT1 as an OC therapeutic target. In a recent study, for the delivery of CRISPR plasmid DNA co-expressing Cas9 and single-guide RNA addressing the OC-related DNMT1 gene, a folate receptor-targeted liposome (F-LP) was employed. Cas9 plasmids were effectively delivered to cancer cells using cationic liposomes to edit the targeted genome in vivo. Nonetheless, additional research on non-specific anticancer effects is required [174].

Patients with OC typically have a poor prognosis or low survival rate because BIRC5 is highly expressed in these cells. The gene BIRC5 encodes the survivin protein, which is an inhibitor of apoptosis family members [175]. Its expression is frequently linked to tumor spread and chemoresistance. In contrast to expression in normal adult tissues, survivin is highly expressed in a wide range of malignancies, including OC [176]. It has also been demonstrated that survivin has a role in tumor spread and chemoresistance in OC [177]. To modify BRIC5 expression, one may use the lentiviral CRISPR/Cas9 nickase vector or the tiny pharmacological inhibitor YM155. This approach would prevent EMT, significantly reduce cell growth and invasion, and initiate apoptosis. Loss of BIRIC5 expression decreased TGF-pathway activity [178].

In a study, the efficiency of the CRISPR-Cas9 genome-editing technique in knocking down the expression of the ABCB1 gene in OC cells resistant to adriamycin was examined [179]. Multidrug resistance (MDR) is an important challenge to effective OC chemotherapy [180]. The current study examined how well CRISPR-Cas9 can help overcome multiple drug resistance (MDR) in adriamycin-resistant ovarian cancer cells. A typical phenomenon in cancer cells is the overexpression of ATP-binding cassette (ABC) drug transporters, which is linked to chemo-resistant phenotypes and multiple drug resistance (MDR) in solid tumors [179]. Up-regulation of ABCB1/P-gp in OC is highly correlated with a poor response to chemotherapy [113]. The ABCB1 gene was downregulated using the CRISPR-Cas9 gene-editing technology, which led to a sharp decrease in ABCB1 gene expression and a rise in the doxorubicin sensitivity of cells transfected with sgRNAs [179].

CRISPR-Cas9 vectors are effective methods for altering noncoding RNAs, such as miRNA genes, in addition to gene expression [181]. According to one study, lentiviral CRISPR-Cas9 vectors are extremely successful at disrupting miRNA expression [182]. miR-21 had oncogenic properties in OC [183]. Pre-miR-21 sequence alteration decreased cell invasion, migration, and proliferation while increasing the sensitivity to chemotherapeutic drugs. E-cadherin, a hallmark of epithelial cells, was upregulated, and mesenchymal markers were downregulated in both cells when the epithelial-to-mesenchymal transition (EMT) was blocked by miR-21 disruption [182].

PARP inhibitors have significantly improved epithelial OC treatment [184]. Particularly for those with recombination repair abnormalities or BRCA1/2 mutations [185]. Adult patients with advanced OC who have a BRCA mutation can take olaparib as maintenance therapy. It is a PARP inhibitor, meaning that it prevents the DNA repair enzyme poly ADP-ribose polymerase (PARP) from working [186]. It works against breast, ovarian, and prostate cancers as well as malignancies in those with inherited BRCA1 or BRCA2 mutations. After the discovery of 93 genes, the CRISPR-Cas9 mutagenesis test was used to predict the olaparib response. The study identified the genes NBN, MUS81, BRCA2, RAD51B, and ATM as predictive markers. Along with three PARPi and carboplatin, the study looked at nine important candidate genes, and the results showed similar dropout rates that were not affected by the genes or the drugs used. Across EOC cell lines, CDK12 is frequently vulnerable to olaparib-induced survival and proliferation, underscoring its potential as a therapeutic target [187, 188].

A different investigation was conducted on poly (ADP-ribose) polymerase-1 (PARP-1) using the CRISPR-Cas9 technology, which had an important impact on controlling the cell cycle, responding to DNA damage, and apoptosis, as a possible therapeutic target. Cancer cells die as a result of PARP-1 inhibition. Inhibiting PARP-1 using CRISP-Cas9 resulted in SKOV3 cell (OC cell line) apoptosis and limited cancer growth [189]. These investigations demonstrate the CRISPR-Cas9 system’s therapeutic promise for treating OC. However, there are restrictions that limit CRISPR-Cas9 biomedical applications and the deployment of ovarian cancer gene therapy. Examples include CRISPR-Cas9’s effect on nontargeting normal cells and off-target DNA damage [190].

CRISPR-Cas9 gene therapy in prostate cancer

PC is the second most common cancer diagnosis made in men and the fifth leading cause of death globally [191]. PC is initially treated by inhibiting androgen receptor signaling, but eventually progresses to castration-resistant PC (CRPC). Next-generation androgen receptor (AR) signaling inhibitors have improved survival for patients with CRPC, but resistance remains an issue. Scientists can use CRISPR/Cas9 to target the AR gene, which plays a major role in the growth and survival of PC cells. By editing this gene, the growth and metastasis of cancer cells can be reduced. Inhibiting the AR function could help slow down PC growth [115, 192]. The CRISPR/Cas system disrupted the AR at specific sites and inhibited the growth of androgen-sensitive PC cells by inducing cellular apoptosis [193].

The use of CRISPR/Cas technology to target the AR gene and the combination of AR inhibition with PRMT1 inhibition may provide promising therapeutic strategies for the treatment of advanced PC [194]. Also, by using CRISPR/Cas9, knockout of the ER-β gene in the mouse genome effectively regulated the prostate tumor size and acted as a tumor-suppressor gene [115]. In another research, researchers found that a CRISPR-Cas9-based genome-editing calcium phosphate-based nanomedicine was developed to target erythropoietin-producing hepatocyte receptor A2 (EphA2), a receptor presented in tumor cells, for PC therapy [116]. The results show that removing IL30 led to a decrease in cancer-causing genes and an increase in genes that help prevent tumors, which slowed down tumor growth and spread in pancreatic cancer models. The researchers suggest that CRISPR/Cas9-mediated targeting of Interleukin-30 (IL30) could be a promising clinical approach for curbing PC progression [195].

An additional prospective strategy showed that CRISPR-Cas9 can be effectively delivered to cancer cells using graphene quantum dots as carriers. This approach targets the TP53 gene mutation, which is commonly overexpressed in many types of cancer. The delivery of CRISPR RNP and a gene repair template leads to successful restoration of the healthy TP53 gene, promoting cellular repair pathways and causing cancer cell apoptosis [196]. They found that the use of nanocarriers for CRISPR/Cas9 delivery holds great potential for targeted gene therapy in PC treatment. The aptamer-liposome-CRISPR/Cas9 chimera provides promising evidence for the clinical development of this technology [197]. Findings suggest that the aptamer-liposome-CRISPR/Cas9 chimera has enormous potential as a targeted gene therapy for PC. The use of an RNA aptamer as a ligand allows for specific binding to cancer cells, while the cationic liposomes enable efficient delivery of therapeutic CRISPR/Cas9 [198]. These findings suggest that metal–organic frameworks (MOFs) could be a promising platform for targeted gene therapy in PC, and the use of ZIF-C through RNAi and CRISPR/Cas9 technologies could provide more precise and effective treatment options. The use of the addition of a green tea phytochemical coating may also enhance the delivery of therapeutic agents to cancer cells [199].

The activation of the activating protein-1 (AP-1) transcription factor has been associated with the onset and progression of cancer, particularly in prostate cancer, where the proto-oncogenes JUN and FOS play a pivotal role. CRISPR/Cas9-mediated knockout of nuclear paraspeckle assembly transcript 1 (NEAT1) in AGS cells caused S phase cell cycle arrest and apoptosis and upregulated FAS level [200]. Besides focusing on TP53 mutations, CRISPR/Cas9 can also help find new genes that influence how PC cells respond to PARP inhibitors [201]. These studies demonstrate the power of CRISPR/Cas9 technology in identifying genes involved in the response to PARP inhibitors in PC cells. By targeting both known and unknown genes, researchers have identified potential biomarkers for PARP inhibition and new therapeutic targets for overcoming resistance [202]. PC modeling has led to the development of tools to examine therapies for this type of cancer. Cultured somatic rat models for prostate cancer have been produced, and nanomedicine can target cancer cells with reduced side effects and increased efficacy. PEG-targeted liposomal loaded nanomedicine is being investigated for potential treatment of PC, along with CRISPR/Cas9 gene editing [203]. The application of CRISPR/Cas9 technology in genetic screening has helped uncover the mechanisms behind cancer progression and drug resistance. This process has led to the discovery of new potential genes that can be targeted for therapy [204].

Concluding remarks and future perspectives

Aging is an intricate biological phenomenon marked by a progressive decline in cellular and physiological functions, rendering individuals increasingly susceptible to a spectrum of age-related diseases. This manuscript has meticulously explored the molecular underpinnings of aging, spotlighting key contributors such as telomere shortening, diminished telomerase activity, genetic mutations, and epigenetic alterations. These elements collectively precipitate cellular senescence—a state of permanent growth arrest that serves as a cornerstone for age-related pathologies. Within this context, the advent of CRISPR-Cas9 gene-editing technology emerges as a transformative tool, offering unprecedented precision in targeting and modulating these molecular drivers to mitigate cellular senescence and its associated diseases.

At the heart of aging lies the erosion of telomeres, the protective caps at chromosome ends, which shorten with each cell division. This process, exacerbated by reduced telomerase activity, accelerates cellular aging and heightens disease risk. Concurrently, mutations in genes governing DNA repair, metabolism, and inflammation accumulate over time, impairing cellular integrity. Epigenetic changes, including DNA methylation and histone modifications, further compound this decline by altering gene expression without modifying the DNA sequence itself. CRISPR-Cas9 addresses these challenges head-on, enabling precise interventions at both genetic and epigenetic levels. For instance, studies highlighted in this manuscript demonstrate its efficacy in targeting the LMNA gene in progeria mouse models, reducing toxic progerin production, and shedding light on aging pathways. Similarly, genome-wide CRISPR-Cas9 screens have unveiled novel senescence-promoting genes, broadening the therapeutic landscape.

The versatility of CRISPR-Cas9 extends beyond genetic correction to epigenetic reprogramming. By editing telomere-related genes, such as the TERT promoter in cancer cells, or modulating telomerase activity, CRISPR-Cas9 offers insights into telomere dynamics and their role in aging and malignancy. Its application in epigenetic editing—targeting DNA methylation or histone modifications—provides a reversible means to restore youthful gene expression patterns, holding promise for reversing age-related epigenetic drift. This dual capability underscores CRISPR-Cas9’s potential as a multifaceted tool in aging research and therapy. The manuscript further delineates CRISPR-Cas9’s therapeutic applications across a range of age-related diseases. In ALS, it targets genes like SOD1 and C9orf72, correcting mutations or suppressing toxic protein production, resulting in improved motor function and survival in preclinical models. In colorectal cancer, it disrupts pathways like KRAS or enhances tumor-suppressor genes, such as Klotho, inhibiting tumor progression. Breast cancer benefits from CRISPR-Cas9’s ability to silence oncogenes like MYC, while in ovarian cancer, it knocks out angiogenesis-promoting genes like EGFL6 or multidrug resistance genes like ABCB1, enhancing chemotherapy sensitivity. Prostate cancer research leverages CRISPR-Cas9 to target the androgen receptor (AR) gene, slowing cancer growth and offering novel therapeutic avenues. These examples collectively illustrate CRISPR-Cas9’s capacity to address both the hallmarks of aging and the specific molecular aberrations driving age-related diseases.

Despite its transformative potential, CRISPR-Cas9 is not without challenges. Off-target effects remain a critical concern, as unintended genetic modifications could compromise safety in therapeutic settings. Delivery mechanisms—whether viral vectors, nanoparticles, or liposomes—require optimization to ensure efficient and tissue-specific targeting. Ethical considerations, particularly regarding germline editing, demand rigorous scrutiny to balance innovation with societal implications. These hurdles necessitate ongoing refinement to translate CRISPR-Cas9 from bench to bedside effectively. Looking ahead, the future of CRISPR-Cas9 in aging and disease mitigation is bright yet contingent on continued innovation. Integrating this technology with AI could enhance precision by predicting off-target effects and optimizing guide RNA design. Advances in delivery systems, such as next-generation nanoparticles or bioengineered vectors, promise to improve efficacy and safety. Also, using CRISPR-Cas9 for more age-related issues—like other brain diseases besides ALS or heart problems—could make it more useful. Collaborative efforts to address ethical frameworks will further ensure its responsible deployment.

In summary, CRISPR-Cas9 stands at the forefront of a paradigm shift in combating cellular senescence and age-related diseases. By targeting the molecular roots of aging—telomere attrition, genetic mutations, and epigenetic dysregulation—it offers a pathway to extend lifespan and enhance health span. As research progresses, the synergy of CRISPR-Cas9 with cutting-edge technologies and a more profound understanding of aging biology holds the potential to revolutionize therapeutic strategies, fostering healthier aging and improving quality of life for aging populations worldwide. This manuscript underscores the promise and complexity of this endeavor, paving the way for a future where aging is not merely endured but actively managed.

Authors’ contributions

A.A., M.SH., R.D., and S.A. contributed to the hypothesis, investigating, gathering data, and writing the main text of the manuscript. P.L., A.HK., Z.Z., and H.GH. contributed to investigating, writing, and reviewing the final draft of the manuscript. H.GH., H.F., and, H.R. contributed to data gathering and designing figures and tables. as well as content and grammatical editing. S.T. and Q.B. contributed to the hypothesis, scientific, and structural editing, supervision, and verifying the manuscript before submission.

Funding

This research received no grant from any funding agency, commercial or not-for-profit sectors.

Availability of data and material

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare no competing interests.