Challenges and Opportunities in the Application of CRISPR-Cas9: A Review on Genomic Editing and Therapeutic Potentials

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) constitute a revolutionary gene-editing technology, allowing precise DNA modifications with vast potential for disease treatment and the creation of genetically modified organisms. This system consists of various components designed to target specific genes, requiring efficient nuclear access within target cells through diverse delivery methods, including physical techniques and carrier-based approaches. Despite its transformative promise, CRISPR faces several challenges, including efficient cellular delivery, off-target effects, immune responses, optimizing editing efficiency, and ethical concerns. Overcoming these hurdles is crucial for fully harnessing its applications. However, CRISPR-Cas9 offers remarkable opportunities for pioneering gene therapies across various disorders, including cancer, and could revolutionize agriculture by engineering disease-resistant crops. A key strength of CRIScPR lies in its adaptability to a broad spectrum of genes, significantly enhancing its versatility. The technology’s potential further expands through emerging interdisciplinary integrations, such as artificial intelligence, machine learning, and biological imaging. These advancements can refine CRISPR’s precision, improve efficiency, and mitigate existing limitations, positioning it as an indispensable tool in future genetic research. Overall, CRISPR-Cas9 promises transformative progress in healthcare and agriculture, solidifying its role as a cornerstone in the evolution of genetic engineering.

Highlights of the Study

• Genome editing potential – CRISPR-Cas9 enables precise DNA modifications for disease treatment and prevention.

• Delivery challenges – various methods for delivering CRISPR components to cells are explored.

• Off-target effects – optimized sgRNA and alternative Cas proteins reduce unintended edits.

• Therapeutic applications – CRISPR-Cas9 shows promise in cancer and genetic disease treatment.

• Integration with artificial intelligence – machine learning enhances the accuracy and efficiency of CRISPR.

Introduction

Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9 (CRISPR-Cas9) was first discovered in bacteria and archaea and was initially identified in the DNA of Escherichia coli in 1987 [1]. However, at that time, its scientific significance and potential applications were not recognized [2]. This system is now understood as a defense mechanism against bacteriophage infections and plasmid transfer [3]. CRISPR-Cas9 can correct defective genes through gene editing or reconstruction. This system has been extensively researched as a potential curative treatment for diseases such as cancer and genetic disorders [4]. Its applications in gene therapy are expanding, offering new hope for treating rare diseases [5]. Beyond medical applications, CRISPR is widely utilized in plant biotechnology to develop crops resistant to diseases, pests, and environmental stress conditions [6]. It is a vital research tool for creating animal models with specific genetic changes, enhancing the understanding of diseases and the development of targeted treatments [7]. Although CRISPR holds great promise, its therapeutic potential remains unfulfilled due to challenges such as ethical concerns, off-target effects, immune responses, delivery limitations, and uncertainties about long-term efficacy [8]. Despite limitations, CRISPR-Cas9’s vast potential has encouraged researchers to explore its genomic editing capabilities. Its primary strength lies in gene editing, and if off-target effects are managed, it could lead to innovative treatments for complex diseases like cancer and genetic disorders [9]. This review discusses the challenges and opportunities of CRISPR-Cas9 technology, emphasizing barriers to its advancement and its potential in disease treatment. Key applications include genetically engineering animal models for disease research, creating plants resistant to environmental stress, and advancing genomic editing approaches. The timeline of CRISPR-Cas9 development highlights milestones such as its initial discovery in bacteria, its evolution into a powerful gene-editing tool, the first application in human cells, and ongoing clinical trials for complex diseases like cancer and genetic disorders (Fig. 1) [2].

Fig. 1.

Timeline of significant milestones in the clinical application of CRISPR-Cas9 to human diseases. This infographic highlights the evolution of CRISPR-Cas9 technology, showcasing its journey from initial applications in human cells to successful clinical trials for genetic diseases, emphasizing its transformative role in disease modeling and gene therapy (created with BioRender.com).

Mechanism of Action of the CRISPR-Cas9 System

The CRISPR-Cas9 system operates in genetic modification through three stages: adaptation, expression, and interference [10]. Adaptation occurs when the target DNA is recognized, and small fragments of the desired genome are integrated into a specific region within the genome known as CRISPR [11]. Expression starts with the transcription of the CRISPR region into a precursor transcript called pre-crRNA. This pre-crRNA is processed to produce mature CRISPR RNAs, which then form a complex with trans-activating CRISPR RNA (tracrRNA) to activate the system for the next stage [3, 12]. The resulting complex is known as sgRNA, which directs Cas9 to its target site for genome editing [13]. The sgRNA produced during processing guides the Cas9 protein, facilitating the recognition of the target gene through complementary base-pairing with the crRNA’s 5′ region. Without sgRNA, Cas9 remains inactive. Once activated, Cas9 creates double-strand breaks (DSBs) near the adjacent protospacer adjacent motif (PAM) [14]. The PAM sequence length varies among bacteria and is a short, conserved DNA motif located downstream of the cleavage site. The Cas9 protein unwinds DNA at the target site with a suitable PAM, forming an RNA-DNA hybrid and subsequently cleaving the DNA to create blunt-ended DSBs. The host cell’s repair machinery then fixes the DSBs [14, 15].

In eukaryotes, the CRISPR-Cas9 system facilitates genome editing through two primary repair pathways: homology-directed repair (HDR) and non-homologous end joining (NHEJ) [16]. The HDR pathway is more precise than NHEJ for repairing DSBs. HDR uses a DNA template for accurate repair, while NHEJ often creates random insertions or deletions (indels) at the break site, leading to potential disruptions in gene function and making it an error-prone mechanism (Fig. 2) [17, 18]. The CRISPR-Cas9 system can target almost any DNA sequence in the genome by modifying the guide RNA (gRNA) [15]. This ability is one of the system’s key advantages, significantly enhancing its utility for genetic manipulation and cancer treatment [19], as will be discussed in the following sections.

Fig. 2.

CRISPR-Cas9 mechanism. The Cas9 protein, guided by an sgRNA, creates a targeted double-strand break (DSB) in genomic DNA near the PAM sequence, unlike RNA interference, which degrades RNA transcripts. sgRNA directs Cas9 for cleavage, leading to DSB repair via two pathways: (1) non-homologous end joining (NHEJ), an error-prone process that may cause insertions or deletions (indels), and (2) homology-directed repair (HDR), which requires a donor DNA template for precise sequence insertion or correction, introduced alongside the CRISPR components (created with BioRender.com).

CRISPR-Cas9 System Components and Size Constraints

The CRISPR-Cas9 system includes essential components for genome editing: the Cas9 protein, which acts as a DNA endonuclease, and the gRNA that directs Cas9 to a specific genomic site [20]. Typically, an sgRNA is employed, merging the roles of crRNA and tracrRNA into a single transcript of about 100 nucleotides [21]. To express these components, strong promoters such as CMV (for Cas9) or U6 (for sgRNA) are required, which contributes to the overall size of the vector [22]. When combined with promoters and other sequences, the total size can exceed the 4.7 kb maximum capacity of adeno-associated viruses (AAV) [23, 24]. Therefore, this limitation requires the development or choice of alternative delivery methods or the adoption of compact Cas variants.

Methods of Delivering the CRISPR System into Cells

For practical functionality, various components of the CRISPR system must be efficiently delivered to target cells [25]. There are various delivery strategies, each with advantages and limitations depending on factors like application context and vehicle capacity. A key challenge is the large size of the Cas9 cDNA (∼4.2 kb), making it difficult to package into vectors like AAV. Delivery methods fall into physical, electrical, or carrier-based categories [26]. A summary comparing these methods based on application context (in vivo vs. ex vivo and human vs. non-human models) is shown in Table 1. Common RNA delivery techniques like microinjection, hydrodynamic injection, biolistics, and electroporation can cause tissue damage and sensitivity issues [27]. Microinjection is a common method where the CRISPR-Cas9 complex is directly introduced into target cells using a glass micropipette, allowing precise control over the delivery of the Cas9-sgRNA complex without molecular weight limitations [28]. To date, microinjection has been successfully applied to embryos of various organisms [29].

Table 1.

Comparison of strategies to enhance sgRNA specificity and minimize off-target effects in CRISPR-Cas9 systems

Category	Delivery method	Application type	In vivo/ex vivo	Human/non-human
Physical methods	Microinjection	Animal models/embryonic editing	Ex vivo	Non-human
	Hydrodynamic injection	Preclinical models (e.g., mice)	In vivo	Animal
	Biolistics	Plant systems/experimental models	Ex vivo	Non-human
Electrical methods	Electroporation	Preclinical research/clinical trials	Ex vivo/in vivo	Human/animal
Carrier-based	Lipid-based nanoparticles	Human cells/clinical trials	Ex vivo/in vivo	Human
	EVs	Human cells/experimental use	Ex vivo	Human
	AAV	Gene therapy/clinical use	In vivo	Human
	Lentiviral vectors	Gene therapy/experimental and clinical use	Ex vivo/in vivo	Human/animal

Hydrodynamic injection is an efficient method for quickly delivering large amounts of material into the bloodstream, primarily used in animal models like mice via the tail vein [30]. Biolistics is a technique for delivering biomacromolecules into plant cells by attaching them to gold or tungsten microparticles, which are then propelled into the cells using gas pressure, explosions, or high-voltage discharge [31].

Electroporation is a method that uses electrical pulses to temporarily increase cell membrane permeability, allowing for effective delivery of large genetic materials like plasmid DNA. It is particularly useful for transfecting hard-to-modify cell types, such as immune and stem cells [32, 33].

Delivery strategies for genetic material can be biological or chemical, based on the carrier type. These carriers help transport genetic cargo across cell membranes. Common delivery methods include nanoparticle systems, extracellular vesicles (EVs), and viral vectors [26]. Recently, nanoparticles (NPs) have emerged as a reliable and efficient method for delivering the CRISPR system [34]. A diverse range of nanomaterials, including lipids, polymers, and gold NPs, are commonly utilized and optimized for the effective transport of CRISPR-Cas9 components into cells [35]. EVs are cell-derived membrane structures that function as natural carriers for various therapeutic cargos, facilitating their transport to target cells [36]. Due to their biocompatibility and efficiency, EVs serve as safe and potent vectors in cellular applications, including genome editing [37].

One of the most effective methods for delivering the CRISPR system in medical research involves using viral vectors, such as AAVs and lentiviruses (LVs) [25]. AAVs are widely used viral vectors for CRISPR-Cas9 editing due to their favorable properties for clinical applications. Similarly, LVs effectively deliver the CRISPR-Cas9 system to various cell types, making them practical and popular for gene therapy [26]. The delivery methods discussed are essential for transferring the CRISPR system and can be adapted for various applications in research and clinical settings.

Clinical Considerations and Human Applications of CRISPR Delivery Methods

In human gene therapy, choosing the right CRISPR delivery method is crucial due to biological and clinical factors. Viral vectors like AAVs and LVs are often used in clinical trials for their efficiency and ability to target specific cell types [38]. AAVs have limitations due to their small packaging capacity, complicating the delivery of Cas9 and sgRNA [39]. Researchers are exploring non-viral techniques like electroporation and lipid-based NPs for ex vivo gene editing in human hematopoietic stem and immune cells, which show promising safety profiles [40]. Significant challenges, such as off-target effects, immune responses, and delivery efficiency, hinder the transition of CRISPR technologies into standard human therapies.

Challenges of Using the CRISPR-Cas9 System

The CRISPR system holds significant promise for the medical community as a potential tool for eliminating genetic diseases, cancer, and rare disorders [41]. While the system shows promise, it isn’t widely used as a primary treatment due to various challenges. Scientists have yet to fully leverage its capabilities, limiting its clinical application for rare diseases and certain genetic disorders. Key obstacles include delivery methods, immunogenicity, and various technical and ethical concerns. The following sections will explore these challenges in detail.

Challenges of Transferring the CRISPR-Cas9 System into the Cell

The first step in utilizing the CRISPR-Cas9 system involves the successful delivery of its components into target cells [42]. Transporting nuclear proteins and sgRNA into the target cell nucleus is crucial for activating the CRISPR-Cas9 system, yet it poses significant challenges in therapeutic and research applications [43]. Different delivery methods have been explored, each with its pros and cons. The following sections will discuss the challenges associated with these strategies and propose possible solutions.

Challenges of Physical Transfer

Physical delivery methods for the CRISPR system include microinjection and hydrodynamic injection. Microinjection enables precise genome editing in target cells; however, it has drawbacks, including technical complexity, the need for specialized equipment and expertise, and limited throughput. These factors contribute to higher costs and hinder scalability, posing challenges to the broad and affordable use of gene therapy [16, 44]. Despite its speed, efficiency, and versatility, the hydrodynamic injection technique has not yet been employed in clinical studies due to potential adverse effects [42]. Advancements in hydrodynamic injection may soon enable effective delivery of the CRISPR system into target cells, improving current techniques and harnessing its full potential. Electroporation, as an electro-based method, is effective for both transient and stable transfection; however, it has a significant drawback: cell death caused by exposure to an electric current [45].

Carrier-Dependent Transmission Challenges

Viral vectors are commonly used for delivering the CRISPR system into cells due to their higher efficiency compared to other methods, especially in vivo studies. AAVs and LVs are the most straightforward options for research. However, a key limitation of viral vectors, particularly AAVs, is their restricted cargo capacity (∼4.7 kb), which complicates the delivery of large genetic elements like SpCas9 (∼4.2 kb) and sgRNA (∼100 bp) [46]. AAVs, recognized for their low immunogenicity, serve as key carriers for delivering CRISPR-Cas9 system components [47]. To address capacity limitations, two distinct AAVs can be used, one for Cas9 and another for sgRNA, improving performance when delivered together. Alternatively, employing a smaller variant, SaCas9 allows both Cas9 and sgRNA to be packaged into a single AAV for more efficient delivery [25, 48].

LVs represent another class of viral vectors that can infect non-dividing cells. Due to their higher packaging capacity compared to AAVs, LVs can accommodate all the components of the CRISPR system [49]. However, a significant concern associated with LV-based delivery is the potential integration of the viral genome into the host cell’s genome, which may lead to unintended mutations [50]. To mitigate unintended effects, non-integrating lentiviral vectors are used. Engineered with mutations in the integrase enzyme, these vectors cannot integrate into the host genome, allowing them to persist episomally and reducing the risk of insertional mutagenesis [51]. The production of non-integrating lentiviral vectors for research is challenging, thus limiting their use compared to AAVs. However, LVs are effective for ex vivo gene delivery into stem cells, providing stable expression and efficiency [52].

EVs serve as a key mechanism for intercellular communication, enabling the transfer of proteins, lipids, and genetic materials between cells [53]. EVs are secreted by different cell types and are surrounded by a lipid bilayer. They are classified into three main types: exosomes, microvesicles, and apoptotic bodies based on size and origin [54]. Exosomes are formed via the endosomal pathway, where early endosomes mature into multivesicular bodies that release intraluminal vesicles as exosomes by fusing with the plasma membrane. This process can involve the ESCRT complex or ESCRT-independent mechanisms like the syndecan-syntenin-ALIX pathway [55]. Microvesicles form by direct budding from the plasma membrane, aided by cytoskeletal changes and elevated intracellular calcium. Apoptotic bodies are produced during apoptosis and consist of cellular fragments that are usually cleared by phagocytic cells [56]. EVs are a promising alternative to viral vectors for gene delivery, bypassing many of their limitations [16]. EVs are positively charged spherical structures that enhance fusion with cell membranes, allowing for efficient delivery. Similarly, polymeric NPs can enter cells via endocytosis or directly, facilitating the transfer of CRISPR components into target cells [57, 58].

Another carrier-based approach for CRISPR system delivery involves chemical methods, particularly lipid- and polymer-based NPs [59]. This technique uses the electric charge of lipids to encapsulate CRISPR components, reducing size and enhancing permeability. Cholesterol and ionizable lipids are added to improve packaging efficiency and cellular uptake [60]. Liposomes are effective, biocompatible carriers that mimic cellular membranes, facilitating the fusion and uptake of CRISPR cargos with low immunogenicity [61]. These carriers can encapsulate plasmid DNA, Cas9 mRNA, or ribonucleoprotein complexes, demonstrating significant success in ex vivo and in vivo gene editing in human cells. Modifying liposome surfaces allows for tissue-specific targeting, improving therapeutic outcomes and minimizing off-target effects [62].

The Challenge of Off-Target Effects

The CRISPR-Cas9 system occasionally fails to accurately target the intended DNA sequence, which reduces its efficiency and may lead to unintended modifications, known as off-target effects [14]. The system’s specificity depends on two key factors: the PAM and the 20-nucleotide guide sequence within the sgRNA [14]. More than three mismatches between sgRNA and target DNA increase the likelihood of off-target effects, which can cause mutations, deletions, genomic rearrangements, immune reactions, and potential tumor formation from oncogene activation [63]. Off-target effects arise when the sgRNA binds to non-target DNA, leading to unintended cleavage. The impact of mismatches is greater near the 5′ end of the sgRNA than at the 3′ end [64]. Furthermore, the concentration or titer of the delivery vector has a notable impact on transfection efficiency. A higher vector titer enhances the chances of successful gene delivery but may also prolong Cas9 exposure, thereby increasing the risk of off-target activity [51, 65].

Researchers have proposed strategies to improve the precision of the CRISPR-Cas9 system and reduce off-target effects. Some of these are mentioned below:

sgRNA Optimization

Optimizing the sgRNA to increase CG bonds relative to AT can stabilize its structure and improve binding specificity, thereby enhancing the accuracy of CRISPR genomic editing (Table 2) [66]. A higher GC ratio can stabilize sgRNA molecules, but it may not improve binding specificity. Excessive stability in secondary structures can reduce selectivity for the target sequence. Hence, it is important to consider stability and binding specificity independently when designing effective sgRNAs [67].

Table 2.

Comparative overview of commonly used CRISPR delivery vectors based on their immunogenicity and applications

Strategy	Mechanism	Impact on specificity	Advantages	Limitations
High GC content	Increases sgRNA structural stability	Moderate ↑	Enhances binding strength	May reduce target selectivity if too stable
Truncated sgRNA	Reduces sgRNA length by 2–3 nt	High ↑	Minimizes off-target effects	Requires careful design to maintain efficiency
Bioinformatics tools	Algorithmic prediction of low-risk sgRNA sites	High ↑	Increases precision and is user-friendly	Dependent on software accuracy

Using truncated sgRNAs, which reduce the length by 2–3 nucleotides, can minimize off-target effects while maintaining on-target efficiency. This modification improves the specificity of the CRISPR system for precise genome editing [68].

Various bioinformatics tools have emerged to aid in identifying high-quality sgRNA sequences that minimize off-target effects [69]. Platforms like CRISPOR, CHOPCHOP, Benchling’s CRISPR Design Tool, and CCTop use genome-wide data and algorithms to identify efficient and specific sgRNA candidates, considering factors like GC content, sequence motifs, and potential mismatches to improve targeting accuracy and safety in therapies [70].

Use of Other Cas Protein Species

Various families of Cas proteins enhance CRISPR genome editing (Fig. 3). For example, Cas3 from Type I systems is an ATP-dependent helicase-nuclease that effectively degrades long DNA sequences with minimal off-target effects, making it ideal for large genomic deletions [71]. Cas10, a component of Type III systems, targets RNA and DNA in a transcription-dependent manner, providing potential for transcriptional regulation instead of direct editing [72].

Fig. 3.

Comparative overview of different Cas protein families used in CRISPR systems. This figure summarizes the size, nuclease activity, molecular targets, and applications of Cas13, Cas9, Cas12, Cas10, and Cas3 proteins. Their differences in target molecules (DNA vs. RNA), cleavage type, and gene size influence their suitability for specific therapeutic, diagnostic, and research purposes (created with BioRender.com).

Cas12 (Cpf1), a nuclease from Francisella novicida, makes staggered cuts and requires a 5′-TTTV-3′ PAM sequence. Unlike Cas9, Cas12 autonomously processes its crRNA, facilitating multiplex editing and reducing off-target effects. However, it is generally considered less efficient than Cas9 for certain genome editing tasks [73]. Cas13, on the other hand, exclusively targets RNA and operates independently of a PAM sequence, relying instead on a protospacer flanking site, which makes it highly suitable for transcriptome editing and targeting RNA viruses [74].

The specificity of Cas proteins for their target sequences is largely dictated by their PAM requirements. For example, SpCas9 targets the common 5′-NGG-3′ PAM in mammalian genomes, while the smaller SaCas9 requires a more specific 5′-NNGRRT-3′ PAM but is more suitable for AAV vector delivery due to its size [75]. The choice of the Cas variant impacts targetable sequences (due to PAM dependency) and affects the efficiency, delivery strategy, and precision of the editing system.

Modifying the Nuclease Property of Cas9

Mutations can be introduced into Cas9 to enhance its specificity, turning it into a nickase. This reduces off-target effects by 1,000–1,500-fold by creating single-stranded breaks in DNA instead of blunt DSBs, thereby increasing precision in genome editing [76, 77]. Additionally, the presence and sequence of the PAM play a crucial role in determining the activity and target specificity of Cas9. Engineering Cas9 variants to recognize alternative PAMs has further expanded the targeting range while maintaining high fidelity [78].

High-fidelity Cas9 variants, like SpCas9-HF1 and eSpCas9, enhance editing accuracy by reducing non-specific DNA interactions, minimizing unintended modifications while ensuring effective on-target activity. These advancements offer safer and more reliable tools for gene editing in therapeutic and research contexts [79].

Cas9 is sourced from bacteria like Streptococcus pyogenes (SpCas9) or Staphylococcus aureus (SaCas9). The human immune system may recognize these proteins as foreign, triggering immune responses that complicate stable and effective gene therapy [80].

Use of Anti-CRISPR Proteins

Bacteriophages naturally produce anti-CRISPR (Acr) proteins, which are small proteins that inhibit one or more stages of the CRISPR-Cas system mechanism. These stages may include DNA cleavage, nuclease activation, or even target binding [81]. In nature, this mechanism is employed by invading bacteriophages to evade the CRISPR-based immune defense system in bacteria. As a result, the direct application of phages in eukaryotic cells is not feasible [82]. Nevertheless, Acr proteins obtained from phages can be isolated, modified, and introduced into eukaryotic systems independently by utilizing suitable vectors or delivery methods [83]. Recent research shows that bacteriophages with Acr properties can inhibit the CRISPR system. For instance, Acr IIA4 targets the Cas9 nuclease, mimicking the target DNA and preventing unwanted cuts, thus reducing off-target effects [84].

Use of Non-Viral Vectors

Recent studies indicate that NPs can enhance the selectivity and efficiency of CRISPR-Cas9 delivery. Engineered NPs, such as lipid-based NPs, polymeric NPs, and gold NPs, can target specific tissues or cell types through surface modifications, improving cellular uptake and minimizing off-target effects [42, 43]. Compared to viral vectors, which can lead to prolonged Cas9 activity and off-target effects, NPs enable transient delivery, thereby limiting the exposure of CRISPR machinery to the genome and increasing safety [85, 86]. Moreover, NPs offer advantages such as low immunogenicity, scalability, and the ability to co-deliver multiple components, including Cas9 protein, sgRNA, and donor DNA templates.

Efficiency Challenges

A major challenge with the CRISPR system is its efficiency. While CRISPR-Cas9 technology can identify and modify numerous mutations and genomic sequences in plants, animals, and microorganisms, several factors raise concerns about its effectiveness [87]. The efficiency of the CRISPR system is influenced by PAM site limitations, expression of multiple gRNAs in one vector, Cas9 fidelity, and off-target effects [88, 89]. The system relies on specific sequences for recognizing the target DNA, and this PAM dependency restricts genome editing to regions that contain appropriate PAM motifs, thereby limiting the flexibility and overall efficiency of CRISPR [90, 91]. Simultaneous targeting of multiple genomic loci requires multiple gRNAs in one vector, which can lead to competition for binding to Cas9 and reduce overall activity [92]. Additionally, Cas9 can potentially bind to off-target regions, resulting in unintended mutations and undesirable effects that negatively impact the precision and reliability of the CRISPR system [90].

CRISPR operates mainly through two DNA repair mechanisms: NHEJ and HDR. NHEJ is active in all cell cycle phases, making it more versatile for research. In contrast, HDR only occurs during the S and G2 phases, limiting its applicability [43]. To enhance HDR efficiency, using nano-carriers to deliver HDR components alongside CRISPR elements into cells can improve precision and reduce off-target effects in genome editing [93].

A key challenge in efficiency is that not all genetic diseases can be treated by simply deleting a gene or modifying a sequence; some require more complex interventions [86]. One approach to overcoming this limitation is to enhance CRISPR systems that can efficiently delete or modify larger sequences across multiple genes [94].

The efficiency of the CRISPR-Cas9 system is significantly influenced by the variability of sgRNA effectiveness. Since not all sgRNAs target Cas9 equally well, extensive screening is required to find optimal candidates. Combining multiple sgRNAs to target different genomic regions can also enhance overall effectiveness [95]. In summary, ongoing advancements in CRISPR aim to address current challenges, enhancing its efficiency and expanding its potential for therapeutic and research applications.

Immune Stimulation

Immune responses to the CRISPR-Cas9 system pose a challenge for clinical applications, primarily due to the recognition of the Cas9 protein, often derived from microorganisms like S. pyogenes or S. aureus, as a foreign antigen. This can lead to immune reactions that reduce efficacy and cause adverse effects. Researchers are exploring humanized protein variants to reduce immunogenicity [96]. Additionally, sgRNAs that direct the Cas9 protein to its target site may provoke immune responses, especially if they are unstable or improperly delivered. In some cases, sgRNAs can activate innate immune pathways, complicating their therapeutic use [96]. Concerns also arise from the prolonged accumulation of Cas9 or associated RNAs in cells as it may lead to sustained immune activation and memory. This increases the chance of immune responses with repeated exposure to the CRISPR-Cas9 system, potentially reducing its effectiveness over time [97].

Various strategies exist for delivering the CRISPR-Cas9 system into cells, but they can trigger immune responses, especially with viral vectors like AAVs and LVs. Repeated use of these vectors can lead to significant immunogenicity as they introduce foreign proteins that activate the immune system, complicating long-term therapy for patients requiring multiple treatments [26, 98]. Immune responses to viral vectors happen when the host’s immune systems recognize viral proteins or transgene products. For AAVs, existing neutralizing antibodies in humans can impede vector entry, diminishing treatment effectiveness [99]. Cytotoxic T lymphocyte responses can develop against transduced cells, especially with LVs that integrate into the host genome and produce foreign proteins. These immune responses pose challenges for re-administration, particularly for patients requiring long-term or repeated therapies [100]. Various strategies to address these issues include using immunosuppressive agents, creating capsid variants with lower immunogenicity, employing transient expression systems, and utilizing ex vivo delivery methods to reduce systemic exposure [101]. NPs-based delivery methods have enhanced CRISPR transfer efficiency, but they can still cause immunogenic and cytotoxic effects. Cationic NPs, for example, may lead to inflammation and immune activation, complicating clinical use (Table 3) [102].

Table 3.

Delivery methods of CRISPR-Cas9 compared by application type and model utilized

Delivery vector	Immunogenicity level	Application area	Notes
AAV	Moderate to high	In vivo therapies	Strong immune memory upon repeat doses
LV	Moderate	Ex vivo gene therapies	Lower immune response in ex vivo, but integration risks
NPs	Low	Experimental, targeted	Biocompatible and non-viral, customizable for specific applications
EVs	Very low	Experimental research	Natural carriers, limited large-scale use currently

Ethical Challenges

Initiating the CRISPR-Cas9 system in genetics poses significant challenges, particularly ethical concerns that may result in strict penalties for manipulating genetic material. A key dilemma involves editing the human germline as these irreversible changes can affect future generations. The potential to select traits in embryos raises issues of inequality, discrimination, and broader moral questions [98]. The use of CRISPR technology to modify organisms, including plants and animals, raises significant ethical concerns. For example, altering insects to control diseases could upset the ecological balance [103]. The CRISPR system poses challenges, including the risk of unintended genetic changes, reflecting the technology’s complexity. However, the authors believe it has great potential for treating and preventing genetic diseases. The challenges of using CRISPR-Cas9 technology are summarized in Table 4.

Table 4.

Summary of the challenges in using the CRISPR-Cas9 system

​	Challenges	Explanation
1	Delivery	Delivering CRISPR-Cas9 components into target cells poses challenges in therapeutic and research applications
		1.Physical and electrical delivery challenges –Microinjection: can damage cells –Electroporation: risks cell death –Hydrodynamic injection: fast but has side effects limiting clinical use
		2.Vector-dependent delivery challenges –Viral vectors: AAVs have low immunogenicity but limited packaging; LVs have higher capacity but risk genomic integration –Nanoscale particles enhance permeability, and EVs are being explored as innovative delivery methods
		These challenges impact the CRISPR system’s efficiency and safety, highlighting the need for innovative solutions
2	Off-target	Another challenge of the CRISPR-Cas9 system is the off-target phenomenon, which can lead to unintended effects and harm. The risk of off-target effects increases with more than three mismatches between the PAM and sgRNA, potentially leading to mutations and immune responses. Strategies to reduce off-target effects include optimizing sgRNA, utilizing different Cas proteins (such as Cas3 and Cas12), modifying Cas9’s nuclease properties, employing Acr, and employing non-viral vectors, such as nanoparticles. These methods help mitigate off-target adverse effects
3	Efficiency	CRISPR faces efficiency challenges. NHEJ outperforms HDR in flexibility. Nanoparticles can enhance HDR. Some genetic diseases need broader editing, not just gene removal. Additionally, sgRNAs must be screened for effectiveness with Cas9. Overcoming these hurdles can improve CRISPR’s efficiency
4	Immune stimulation	One of the challenges of the CRISPR-Cas9 system is immunogenicity. The Cas9 protein and sgRNAs can trigger immune reactions, primarily if derived from microbial or viral sources. Additionally, viral delivery methods and nanoparticles may pose immunogenic and inflammatory risks
5	Ethical	The ethical challenges of CRISPR include concerns about human genome editing, the creation of designer babies, and negative impacts on ecosystems. Additionally, risks from low accuracy and unintended mutations are also present

Opportunities of the CRISPR-Cas9 System

The CRISPR-Cas9 system has gained attention for its precision in gene editing, allowing scientists to correct genetic mutations in diseases caused by single-gene defects, such as muscular dystrophy, hemophilia, and sickle cell anemia [39]. Furthermore, its potential in cancer treatment has been demonstrated through the modification of T cells within the immune system to enhance their therapeutic efficacy [104]. CRISPR-Cas9 has become a leading gene-editing technology for treating various medical conditions.

CRISPR-Cas9 Therapeutics Roles

Treating Genetic Disorders

Many genetic disorders and abnormalities arise from deletions and insertions in the human genome, posing significant challenges in the medical field, as not all can be easily treated [105]. The development of the CRISPR-Cas9 system has provided a powerful tool in genetics, offering promising therapeutic potential for these conditions [106]. CRISPR-Cas9 is fascinating for treating hereditary and non-hereditary eye diseases, offering a way to correct genetic defects that cause vision impairment [85]. Retinitis pigmentosa is a key example where CRISPR-based gene therapy may restore vision, greatly enhancing the quality of life and life expectancy for those affected by blindness.

Dravet syndrome is a severe genetic disorder that appears in the first year of life, characterized by epilepsy and recurrent seizures. It has distinct clinical features [107]. Patients with Dravet syndrome face a high risk of premature death from frequent seizures. Previous studies have used the CRISPR system to create mouse models for researching this life-threatening disease [108]. CRISPR targets mutations in the SCN1A gene, which codes for a sodium channel vital for neuronal excitability. By correcting or silencing the mutated allele, it offers a promising way to restore normal neuronal function and reduce seizures [109].

Mitochondrial diseases result from disruptions in oxidative phosphorylation, causing various pathological states from mild dysfunction to organ failure [110]. CRISPR technology enables targeted editing of the mitochondrial genome, presenting a potential treatment for mitochondrial disorders. It may also help address the mutator phenotype, which involves extensive mutations in mitochondrial DNA [111].

CRISPR-Cas9 shows promise for treating inherited disorders like Duchenne muscular dystrophy, caused by dystrophin gene mutations. Researchers have used CRISPR-mediated exon skipping to partially restore protein function and enhance muscle integrity in animal models [112]. In beta-thalassemia and sickle cell disease, genome editing techniques aim to inhibit BCL11A gene expression, thereby boosting fetal hemoglobin production, which presents a potential functional cure in clinical trials [113]. Additionally, CRISPR technologies are advancing to tackle cystic fibrosis, Huntington’s disease, and phenylketonuria by directly correcting the mutations responsible for these diseases at the DNA level [114]. These examples show how genome editing can be tailored for different genetic diseases, not only providing symptom relief but also potentially reversing disease progression.

Cancer Treatment

Cancer remains one of the most formidable diseases of the 20th century and continues to pose a significant public health challenge in the 21st century [115]. Many cancer therapies remain only partially effective despite extensive research. However, CRISPR-Cas9 technology offers new possibilities for treatment, particularly through the genetic modification of immune cells to improve their ability to identify and eliminate cancer cells.

Lung Cancer

Lung cancer is the deadliest cancer worldwide [116]. The CRISPR-Cas9 system can directly target genes involved in the proliferation and survival of lung cancer cells. Mutations in the epidermal growth factor receptor and Kirsten rat sarcoma genes often lead to the growth of lung cancer cells, which can be edited and silenced using CRISPR technology to prevent tumor formation [98]. CRISPR can boost immune responses against lung cancer by modifying T cells or disrupting tumor immune evasion, showcasing its potential as a promising therapy for the disease.

Leukemia

Leukemia is a highly aggressive disease that can metastasize and lead to other cancers. It comes in various forms and originates from mutations in hematopoietic stem cell gene [117]. The CRISPR-Cas9 system has been employed to enhance T cells, enabling them to recognize and attack cancer cells more effectively. This strategy involves knocking down genes that suppress anti-cancer activity in immune cells, thereby increasing their therapeutic potential. Notably, this approach has demonstrated remarkable success in treating acute lymphoblastic leukemia and chronic myeloid leukemia [118].

Liver Cancer

Liver cancer is the fourth leading cause of cancer deaths globally, primarily affecting those with pre-existing liver conditions like cirrhosis [119]. Mutations in the β-catenin (CTNNB1) and TP53 genes significantly contribute to cancer development. The CRISPR-Cas9 system presents a potential therapeutic approach by correcting or silencing these mutations and directly modifying cancerous cells for genetic treatment [120]. CRISPR shows promise in cancer therapy, but other malignancies also need effective treatments. Continued research and clinical trials are crucial to confirm the safety and efficacy of these methods (Fig. 4).

Fig. 4.

Therapeutic applications of CRISPR-Cas9. The figure categorizes CRISPR-Cas9 applications into three main areas: genetic disorders, cancer therapy, and emerging uses, showcasing CRISPR’s diverse clinical and research impact (created with BioRender.com).

Advances in Genetic Research and Biotechnology

Since the advent of CRISPR-based genetic research, significant progress has been made in understanding and manipulating the genome. This technology has revolutionized the field by enabling precise gene editing, facilitating the exploration of genetic mechanisms, and advancing therapeutic applications. Beyond enhancing our knowledge of gene functions, CRISPR has paved the way for innovative treatments for genetic disorders and the development of novel biological products [121, 122].

Fundamental Research

CRISPR-Cas9 has played a pivotal role in fundamental Research, allowing for unprecedented precision in studying gene functions and biological pathways. This technology has facilitated the targeted silencing of genes to observe physiological changes in cells, thereby elucidating their specific functions [123]. Additionally, CRISPR has advanced research in evolutionary biology by enabling the analysis of genetic variations, genome structures, and biological pathways, further deepening our understanding of evolutionary mechanisms [124].

Improving Agriculture and Livestock Products

The CRISPR system has numerous applications in agriculture and animal husbandry. This technology enables scientists to modify the genomes of crops and livestock to enhance desirable traits, such as disease resistance, higher yields, and resilience to adverse environmental conditions [125, 126].

Compatibility with New Technologies

CRISPR-Cas9 technology is a groundbreaking tool in genetics that integrates well with emerging technologies. This compatibility enhances biological processes through its integration with artificial intelligence (AI), machine learning, and nanotechnology [121]. CRISPR, combined with RNA-based genome editing, allows for precise genetic modifications and holds promise for treating a wider range of genetic disorders, offering new therapeutic possibilities [114]. AI can predict off-target effects and propose mitigation strategies, improving CRISPR-based genome editing precision. Additionally, combining CRISPR with advanced biophysical imaging allows researchers to monitor real-time cellular changes during genome editing, offering deeper insights into the process [122]. Interdisciplinary approaches are increasingly important in modern science. Combining deep learning and AI with CRISPR-Cas9 can revolutionize genomic editing by improving accuracy and efficiency. Studies show that deep learning algorithms help predict sgRNA activity, enhancing CRISPR’s effectiveness and reducing off-target effects [127]. By utilizing deep learning technology and a hybrid neural network known as CrnnCrispr, researchers have analyzed sgRNA nucleotide sequences. This approach significantly enhances the understanding of sgRNA design principles, thereby improving the accuracy and adaptability of sgRNA to specific target sequences [128].

The CRISPR system has great potential in genetic research, with applications in treating hereditary diseases like cancer and enhancing agricultural productivity. When used effectively, it can tackle significant scientific and medical challenges. By refining CRISPR’s mechanisms, we can achieve major advancements in various fields of research and biotechnology. The opportunities of using CRISPR-Cas9 technology are summarized in Table 5.

Table 5.

Summary of the opportunities available in the use of the CRISPR-Cas9 system

​	Opportunities	Explanation
1	Treatment and medical	Rare and untreatable diseases: many diseases lack effective treatments, reducing patient life expectancy
		Genetic disease treatment: by correcting genetic defects, CRISPR-Cas9 offers new approaches to treating genetic disorders, including eye diseases such as retinitis pigmentosa and Dravet syndrome
		Mitochondrial diseases: these conditions arise from mitochondrial dysfunction. CRISPR enables the editing of mitochondrial genomes, potentially leading to the development of new treatments
		Cancer treatment: CRISPR-Cas9 offers new opportunities for cancer therapy through precise genome editing and immune system modulation, thereby enhancing the effectiveness of immune cells against tumors
2	Advances in genetic research and biotechnology	Since the discovery of CRISPR, significant advancements have been made in genetic research and the treatment of genetic diseases
		Fundamental research: CRISPR-Cas9 enables more precise studies of gene functions and biological pathways, facilitating the examination of genetic changes during evolution
		Improvement of agricultural and livestock products: this technology enables the modification of plant and animal genomes to introduce traits such as disease resistance and increased yield, potentially reducing the need for antibiotics and chemical pesticides
3	Compatibility with new technologies	CRISPR-Cas9 technology, a revolutionary tool in genetics, offers compatibility with advanced technologies such as AI and nanotechnology. These combinations facilitate biological processes and enable more precise genome editing. Integration with AI can predict off-target effects. Additionally, biological imaging technologies allow scientists to monitor live changes in cells and microorganisms during genome editing

Future Studies

Further research is essential to address the challenges of the CRISPR system and maximize its potential. Key areas requiring exploration include optimizing delivery methods for CRISPR components into target cells and studying the long-term effects of CRISPR-Cas9 in animal and human models, particularly off-target effects. Integrating CRISPR technology with advanced computational methods, like AI, could improve the precision of genomic editing and expand its applications. Additionally, enhancing the efficiency and specificity of CRISPR-Cas9 can help reduce off-target effects and improve safety. Continued advancements in this field are vital for developing novel therapeutic strategies and enhancing biotechnology.

Conclusion

The CRISPR-Cas9 system is a powerful tool in genetics, operating through two main mechanisms and delivered to target cells via various methods. Despite challenges like delivery issues, off-target effects, safety concerns, and ethical considerations, efforts are underway to enhance its efficiency. This innovative technology holds great promise, offering therapeutic potential, advancements in genetic engineering, and research expansion. With continued refinement and integration with other technologies, CRISPR-Cas9 can overcome obstacles and unlock new possibilities for effective use.

Acknowledgment

The authors would like to thank the Department of Laboratory Sciences, Faculty of Paramedical and Rehabilitation, Mashhad University of Medical Sciences, Mashhad, Iran.

Conflict of Interest Statement

The authors declare no competing or conflicts of interests.

Funding Sources

No funding was received.

Author Contributions

Mohammad Ali Karimi: writing – review and editing; Mahdiesadat Paryan: data curation and formal analysis; Ghazaleh Behrouzian Fard: validation; Hamid Sadeghian: investigation; Hossein Zarrinfar: software; and Mahdi Hosseini Bafghi: supervision, project administration, validation, and data curation. All the authors have read and approved the manuscript.

Funding Statement

No funding was received.

Data Availability Statement

The data used to support the findings of this study are available from the corresponding author upon request.