Integrating CRISPR/Cas technology with clinical trials: Principles, progress and challenges.

Introduction

The emergence and identification of clustered regularly interspaced short palindromic repeats (CRISPR) dates back only a few decades [ 1 ]. In the late 1980s, researchers initially identified unique repetitive DNA patterns within bacterial genomes [ 2 ]. However, the true purpose of these sequences remained elusive until 2002 by Francisco Mojica, a Spanish microbiologist, made a breakthrough. Mujica proposed that these patterns are a component of the bacteria's defense system that protects them from bacteriophage attacks [ 3 ]. Later research has proven that CRISPR sequences function as a memory repository, preserving genetic information from past viral interactions. When a bacterium meets a virus, it inserts a small piece of viral DNA as a spacer into its CRISPR sequence. This mechanism allows the bacteria to recognize and destroy the virus if infected again in the future [ 4 ].

Genome editing is a rapidly evolving field that holds the potential to treat more than 10,000 genetic diseases [ [5] , [6] ]. Various methods have been utilized for this purpose, including meganucleases, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) [ [7] , [8] ]. However, in 2012 Jennifer Doudna and Emmanuel Charpentier made a groundbreaking discovery that revolutionized the field of gene editing. They found a specific protein called Cas9 that binds to CRISPR sequences which acts as a molecular scissor and capable of cutting DNA at specific sites to create double-stranded breaks (DSBs) [ 9 ]. DSBs are then addressed by repair processes, predominantly non-homologous end joining (NHEJ) or homology-directed repair (HDR), which can lead to gene knockout, activation, deletion or insertion [ [5] , [10] ]. This enables the CRISPR/Cas9 system to accurately locate and alter specific genes within an organism's DNA [ 11 ]. CRISPR/Cas9 has transformed the discipline by enabling gene editing without requiring specialized knowledge in creating customized DNA-binding proteins [ 5 ]. This advancement has significantly shortened the time required for gene editing process and reduced the reliance on highly specialized personnel [ 12 ]. In recognition of this groundbreaking achievement, the Royal Swedish Academy of Sciences awarded the Nobel Prize in Chemistry 2020 to those two outstanding scientists for the development of this method for genome editing.

Consequently, the CRISPR/Cas9 system opportunities across multiple domains, such as industrial biotechnology, agriculture and the field of medicine [ [13] , [14] , [15] , [16] , [17] , [18] ]. In medicine, the focus lies on the treatment of infectious diseases, genetic disorders, cancer and other conditions [ [19] , [20] , [21] , [22] , [23] , [24] ]. This potential has driven the rapid adaption of CRISPR/Cas9 in preclinical studies involving animal models, human embryos, and induced pluripotent stem cells (iPSCs) [ [25] , [26] ]. These investigations have yielded promising outcomes, paving the way for progression to clinical trials. In 2016, Lu and colleagues conducted the first clinical trial using the CRISPR/Cas9 system [ 27 ]. The study enrolled 22 patients with advanced non-small-cell lung cancer (NSCLC), who were administered CRISPR/Cas9-edited T cells designed to target the PD-1 gene. The findings showed a reduction in PD-1 expression within the genetically modified T cells [ 27 ]. This groundbreaking clinical trial has been followed by numerous additional studies, bringing the total number of registered clinical trials involving CRISPR/Cas9 on the ClinicalTrials.gov website to 69 ( https://clinicaltrials.gov ). This surge in trials culminated in the first official success in December 2023, when CASGEVY™ (Exagamglogene autotemcel), previously known as CTX001 or exa-cel, received its first major approval for the treatment of sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT) [ [28] , [29] , [30] ]. In addition to CASGEVY™, the FDA also approved LYFGENIA™, designed to reduce the risk of cell sickling and blood flow obstruction in SCD [ 31 ]. While considered safe and effective, LYFGENIA™ carries a black box warning due to the potential risk of hematologic malignancies, necessitating lifelong monitoring for blood cancers [ 32 ].

Despite the enormous potential of CRISPR technology, multiple challenges must be addressed before its broader clinical implementation. These challenges range from technical limitations to ethical considerations [ 1 ]. Furthermore, with the unprecedented advancements in CRISPR's applications across both research and therapeutic fields, its capabilities and challenges are continuously evolving. Ongoing updates are essential to stay informed about new developments and address appearing concerns, ensuring the technology is used safely, effectively, and ethically. This review aims to explore the most recent clinical trials involving CRISPR/Cas9 and the diverse range of diseases it has been used to treat, from genetic disorders such as SCD and TDT, to cancers and infectious diseases. It also provides a comprehensive overview of these groundbreaking clinical applications and highlights the successes and therapeutic innovations that CRISPR/Cas9 has enabled. The review also examines the challenges that stay, including technical limitations, safety concerns such as off-target effects, ethical issues surrounding germline editing, and the evolving regulatory landscape that aims to keep pace with the rapid advancements in gene-editing technologies. By understanding both the current achievements and obstacles, we can better evaluate the future direction of CRISPR/Cas9 in clinical practice.

CRISPR/Cas9 system

Currently, CRISPR/Cas9 is the most well-known and widely used gene editing tool compared to ZFNs and TALENs [ 1 ]. Despite these techniques' ability to introduce precise changes to the DNA sequence, they are more complex and less efficient compared to CRISPR/Cas9, which led to its widespread adoption in the scientific community [ 33 ]. The CRISPR/Cas9 system consists of two main parts: guide RNA (gRNA) and Cas9 protein [ 34 ]. The gRNA is a short RNA sequence designed to match the specific DNA sequence to its targets [ 35 ]. This functions as instructions to direct the Cas9 protein to a precise location in the genome where the desired genetic modification is to be performed [ 36 ]. The Cas9 protein acts as a molecular tool that operates like a set of genetic scissors [ 37 ].

In the CRISPR system, the protospacer adjacent motif (PAM) plays a critical role alongside gRNA and Cas9 protein. This short DNA sequence is essential as it is identified by the Cas9 protein, facilitating the binding and cutting of the target DNA [ 38 ]. Different CRISPR systems have distinct PAM requirements. The Cas9 protein commonly derived from Streptococcus pyogenes recognizes a PAM sequence known as NGG [ [39] , [40] ]. Understanding the PAM requirements of a particular CRISPR system is critical to designing effective gRNAs and ensuring accurate targeting of the desired DNA sequence [ 41 ]. Once the gRNA directs the Cas9 protein to a target DNA sequence, the Cas9 protein later cuts the DNA at that site. This DNA breakage activates the cell's natural repair mechanisms, including HDR and NHEJ, which can be used to induce specific alterations in the DNA sequence ( Fig. 1 ).

The NHEJ pathway is mutation-prone, whereas HDR pathway is more specialized with lower efficiency. Several key variants of Cas9 have known for specific attributes. Streptococcus pyogenes Cas9 (SpCas9) is the most commonly used form, known for its ability to recognize the NGG PAM sequence [ 42 ]. The smaller Staphylococcus aureus Cas9 (SaCas9) is ideal for applications where delivery size is limited [ 43 ]. Cas9 nickase (nCas9) is a modified version that creates single-strand nicks instead of double-strand breaks, lowering the risk of unintended mutations [ 44 ]. Finally, dead Cas9 (dCas9) is an inactivated Cas9 that binds to the DNA without cutting it, making it useful for gene regulation rather than gene editing [ 45 ].

The rapid development of CRISPR/Cas9 in clinical trials extended beyond Cas9 variants, incorporating additional techniques such as base editing and prime editing to address traditional limitations associated with DSBs and off-target effects [ 46 ]. Base editing typically utilizes nCas9, which cuts only one strand of DNA, combined with a deaminase enzyme. This allows for direct conversion of one DNA base into another at a specific target site using a gRNA [ 47 ]. Avoiding DSB-based editing minimizes the risk of unintended mutations, making it particularly useful for correcting point mutations linked to genetic diseases [ [47] , [48] ]. Prime editing enhances this precision by enabling versatile modifications such as insertions, deletions and base substitutions without DSBs [ 48 ]. It uses a fusion of nCas9 and a reverse transcriptase enzyme, guided by a prime editing guide RNA (pegRNA), allowing for highly controlled rewriting of the genetic code [ [49] , [50] ].

Although the CRISPR/Cas9 system and its variants are the most popular widely used, various CRISPR/Cas systems has been discovered and applied for gene editing purposes such as CRISPR/Cas12, CRISPR/Cas12a, CRISPR/Cas13 and CRISPR/Cas14 [ 51 ]. Each system possesses unique components and mechanisms; however, they all share the common ability to identify and modify specific DNA sequences [ 52 ]. This uniqueness can be attributed to different target site preferences, making it a workable alternative to Cas9 in certain applications. For instance, CRISPR/Cas13 functions as an RNA-guided system that targets and cleaves RNA rather than DNA. This feature makes it well-suited for regulating gene expression and holds promise for treating RNA-based diseases. The diversity of CRISPR systems offer a wider range of tools for genetic engineering, allowing researchers to use more adaptable approaches that cater to various molecular targets and biological environments [ 53 ].

CRISPR/Cas9 applications in medicine

Shortly after its the development, CRISPR/Cas9 was employed in medicine, beginning with the treatment of cataracts in mice and targeting cystic fibrosis transmembrane conductor receptor (CFTR) mutations [ [26] , [54] , [55] ]. Subsequently, the use of the CRISPR/Cas9 system in medicine expanded rapidly. Its powerful potential was targeted in cancer [ 56 ], cardiovascular diseases [ 25 ], infectious diseases [ 57 ], neurological disorders such as Huntington's, Alzheimer's and Duchenne muscular dystrophy (DMD), genetic disorders such as sickle cell disease (SCD), β-thalassemia and phenylketonuria [ [58] , [59] , [60] , [61] , [62] , [63] , [64] ].

CRISPR/Cas9 progress in treating genetic disorders

One of the most promising applications of CRISPR/Cas9 in the medical field is the treatment of genetic diseases. These disorders are caused by mutations or abnormalities in DNA sequences, and CRISPR offers a means to correct them. Targeting and editing genes linked to these conditions via CRISPR/Cas9 shows potential for potentially curing or easing symptoms of genetic disorders.

Sickle cell disease and β-thalassemia

SCD is the most common inherited monogenic hematologic disorder, characterized by congenital hemolytic anemia [ 64 ]. It occurs when valine replaces glutamic acid at the sixth residue of the β-globin gene located on chromosome 11 [ 65 ]. This mutation activates the production of hemoglobin S (HbS), an abnormal hemoglobin that polymerizes altering the shape and lifespan of red blood cells (RBCs) [ 66 ]. SCD presents with various symptoms including chronic anemia, severe pain, recurrent vaso-occlusion, stroke, end-organ damage and early mortality [ 67 ]. Presently, hematopoietic stem cell transplantation (HSCT) is the sole curative option, available for approximately only about 15% of patients. Moreover, there are four FDA-approved medications that only address specific complications associated with SCD [ 64 ].

The CRISPR/Cas9 system uses two main approaches to address SCD. One approach involves correcting the mutation to return to the wild-type allele, which increases HbA expression and alleviates the SCD phenotype. The other approach focuses on increasing fetal hemoglobin (HbF) production by targeting gene regulatory elements, thereby improving quality of life and reducing sickling [ 68 ] ( Fig. 2 ). Clinical trials have predominantly focused on increasing HbF levels to alleviate SCD complications. A study observed a significant reduction in symptoms, with vaso-occlusive crises decreasing in 97% of patients and hospitalizations decreasing in 100% of patients over a span of at least 12 consecutive months [ 69 ]. OTQ923, which is an autologous, ex vivo CRISPR/Cas9-edited CD34 + HSC product that disrupts the HBG1 and HBG2 gene promoters to induce HbF production, resulted in a decrease in SCD manifestations during the follow-up period (6–18 months) as well [ 70 ].

β-thalassemia shares a similar pathology and physiology as SCD (mutation in β subunit gene); therefore, it can be approached similarly. It is a genetic disorder resulting from mutations in the gene encoding the β subunit of hemoglobin, known as HBB [ 71 ]. These HBB mutations result in the absent reduction of β-globin synthesis which eventually causes ineffective erythropoiesis [ 72 ]. As current therapies cannot fully address disease manifestations or the underlying cause, research has focused on restoring balance by targeting BCL11A , a zinc finger transcription factor that suppresses the expression of HbF and γ-globin in erythroid cells [ 69 ]. This led to the development of CTX001, an autologous CRISPR–Cas9–edited CD34 + HSPCs. Similarly to the strategy in SCD, CTX001 decreases BCL11A expression in erythroid-lineage cells, reactivates HbF production, and restores γ-globin synthesis [ 69 ]. Nowadays several registered clinical trials have utilized CTX001 to evaluate its safety and efficacy, among other CRISPR/Cas9 combinations ( Table 1 , Fig. 2 ).

Franco Locatelli’s perspective in Segment 3 (2024), alongside Haydar Frangoul’s Phase 3 perspective (2024), confirmed the effectiveness of CRISPR/Cas9 alterations targeting the BCL11A erythroid enhancer. Locatelli's study indicated a 91% transfusion independence rate among patients with β-thalassemia, while Frangoul's research found that 97% of patients with severe sickle cell disease were free from vaso-occlusive crises for at least one year [ 70 ]. Both investigations proven consistent safety profiles with busulfan-based myeloablation, with no significant adverse events reported. Additionally, Akshay Sharma’s study showed robust HbF induction (21.8%–25%) in SCD using CRISPR-edited HBG1/HBG2 promoters, significantly reducing disease symptoms. No off-target mutations were detected, and the treatment was well tolerated [ 73 ].

Inherited retinal diseases

Inherited retinal diseases (IRDs) encompass conditions such as retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). RP is an inherited form of retinal pigmentary dystrophy that results in vision impairment and is linked to multiple mutations [ 74 ]. These mutations include alterations in the RP1, rhodopsin (Rho) and RP GTPase regulator (RPGR) genes [ 75 ]. The CRISPR/Cas9 gene editing method is able to fix the Rho (S334) gene through the injection of gRNA/Cas9 plasmid into the subretinal area, leading to the enhancement of visual abilities by retinal degeneration in mice [ 76 ]. A technique for precise gene insertion in stationary cells enhances vision in mice. This started a clinical trial for ZVS-203e drug, using the CRISPR/Cas9 editing system. The trial is designed as a single-arm, open-label study focusing on correcting the Rho site-specific gene mutation (Rho-RP) through subretinal injection [ 77 ].

A less common IRD is LCA, wthic The primary cause is typically biallelic loss-of-function mutations in the CEP290 gene, resulting in LCA-ciliopathy commonly referred to as LCA10. LCA impacts one out of every 30,000 newborns, causing early-onset vision loss, yet there is currently no treatment for the condition [ 78 ]. Researchers were exploring gene editing techniques to address LCA, and a successful strategy was conducted on mice involved using the CRISPR/Cas9 system [ [79] , [80] ]. Preclinical studies led to the initiation of an open-label clinical trial testing single ascending doses of EDIT-101 in 34 participants holding the LCA10-IVS26 mutation and aged between three and seven [ 81 ]. Therefore, the CRISPR technique can be used to study numerous mutations found in various genetic regions associated with eye disorders.

Transthyretin amyloidosis

Transthyretin amyloidosis (ATTR amyloidosis) is an inherited autosomal dominant genetic disorder primarily caused by the accumulation of amyloid fibers in surrounding cells, posing significant risks to the cardiovascular and nervous systems [ 82 ]. ATTR amyloidosis progresses rapidly after symptom onset, leading to death within 4–17 years in those with polyneuropathy and 2–6 years in patients with amyloid cardiomyopathy [ [83] , [84] ]. Therapeutic approaches aim to decrease continued amyloid production by stabilizing the tetrameric structure of the TTR protein or blocking its synthesis. These interventions offer relief from symptoms, enhance function, and extend lifespan, necessitating prolonged use to sustain TTR suppression [ 85 ]. A feasible alternative is using the CRISPR/Cas9 system for in vivo gene editing. In a recent clinical study, NTLA-2001, a CRISPR/Cas9 system, was employed. This system uses a lipid nanoparticle (LNP) to deliver messenger RNA encoding the Cas9 protein along with a single guide RNA designed to target TTR. The study found that NTLA-2001 caused minimal adverse effects and reduced TTR protein serum levels by specifically targeting the knockout of TTR [ 85 ].

Hemophilia A and B

Hemophilia is a genetic disorder caused by mutations in the coagulation factor VIII gene (hemophilia A) or the factor IX gene (hemophilia B), resulting in blood clotting disorders [ 86 ]. Despite advances in therapy, such as recombinant proteins and products with extended half-lives, hemophilia treatment continues to face two significant challenges: the short duration of therapeutic effects and the development of neutralizing antibodies against clotting factors (known as inhibitors) [ 87 ]. To address these limitations, new treatment strategies for hemophilia have been developed, including gene therapy, bispecific antibodies and innovative balancing therapies [ 88 ]. Although these approaches have shown promising results, achieving a permanent therapeutic effect is still challenging. Recent advancements in CRISPR technology have enabled stable therapies by correcting the mutated genes associated with hemophilia [ 89 ]. Given that genome editing induces irreversible changes in the host genome, ensuring safety through targeted delivery is critical [ 90 ]. Consequently, a reliable delivery system for the CRISPR technology is essential for safe, precise and efficient genome editing. Recently, non-viral vector LNPs have emerged as a safer alternative for delivering CRISPR REGV131-LNP1265 systems compared to traditional viral vectors [ 91 ]. Several preclinical studies on hemophilia ( Table 1 ) in phases 1 and 2 using LNP-CRISPR ( NCT06379789 ) have proven sufficient and sustained therapeutic effects. These findings suggest that LNP-CRISPR-mediated genome editing therapy could represent a viable option for the treatment of hemophilia [ [92] , [93] , [94] ].

Fanconi anemia

According to reports, CRISPR/Cas9 technology has been employed to rectify harmful mutations found in the F gene (Fancf) of the Fanconi anemia (FA) complement group and to modify the FANCI gene in iPSCs obtained from primary fibroblasts [ 95 ]. Fixing FA mutations with CRISPR/Cas9 therapy has been linked to treating bone marrow failure [ [96] , [97] ]. However, it is crucial to note that this research requires further consideration because the genes involved in DNA repair are problematic in FA, and the CRISPR technique's effectiveness begins only after the DSBs and later repairs.

Neurological disorders

The field of neurodegenerative disorders (NDs) faces numerous challenges, including unclear pathogenic mechanisms that hinder available treatments [ 2 ]. One promising approach to overcome these challenges is the use of the CRISPR/Cas9 system. This system is not only used to change genes but also for screening purposes, highlighting its vast potential to accelerate research in this area. Currently, CRISPR/Cas9 is being used in the study of neurological diseases such as Duchenne muscular dystrophy (DMD), Parkinson’s disease, Dravet syndrome, Alzheimer’s disease, Rett syndrome, Huntington’s disease (HD), and numerous others [ 58 ].

Rett syndrome

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder with an incidence rate of one in 10,000 [ 98 ]. The condition arises due to a genetic mutation in the methyl-CpG binding protein 2 ( MECP2 ) gene found in the Xq28 region. MECP2 regulates L1 expression, BLOC-1 complex, glutamatergic neurons, the GABA functional switch, and microRNAs such as miR-199 and miR-214 [ 99 ]. A deficiency or defect in any of these structures results in synaptic dysfunction. Symptoms of RTT typically begin between 6 and 18 months of age and include microcephaly, social withdrawal, stereotypic hand movements, gait ataxia, loss of previously acquired skills, recurrent seizures, and dysfunction that can result in respiratory or cardiovascular arrest [ 100 ]. Death usually occurs between the ages of 13 and the mid-twenties [ 99 ]. In a study, CRISPR/Cas9 was efficient in correcting irregularities in MECP2 expression within iPSCs through the precise insertion of the correct genetic sequence. This resulted in a 20%–30% efficiency in homologous recombination (HR), thereby reversing symptoms of RTT [ 101 ]. Recently, a clinical trial ( NCT05740761 ) has been registered to correct MECP2 expression using the CRISPR/Cas9 system combined with AAV-based delivery. Although CRISPR/Cas9 holds immense potential, challenges with delivery efficiency and precision restrict its clinical application. A recent study introduces the magnetic nanoparticle-assisted genome editing (MAGE) platform, which enhances CRISPR/Cas9 transfection, biocompatibility, and accuracy when applied to RTT [ [102] , [103] ].

Huntington’s disease

Huntington's disease (HD) is a genetic condition that affects neurodevelopment, passed down through generations in an autosomal dominant pattern. It arises from the expansion of 36 or more CAG repeats within exon 1 of the Huntingtin gene ( HTT ) [ 104 ]. HD symptoms are primarily behavioral, motor and cognitive deficits that progress and become more apparent over time [ 99 ]. HTT gene is the only known cause of HD, making it an ideal candidate for treatment with genome-editing technologies, as the focus is on a single mutant gene. While ZFNs and TALENs have been used, the emergence of the CRISPR/Cas9 system has significantly accelerated the process [ 105 ]. Currently, the CRISPR system in HD is used not only as a potential treatment to reduce the HTT gene but also as a means to study mechanisms and establish models of HD [ 105 ]. In a mouse model, CRISPR/Cas9 editing tool efficiently targeted the HTT gene and reduced its expression. Further research has confirmed the effectiveness of using the CRISPR/Cas9 system to treat HD [ 106 ]. However, further research is needed to apply these results in clinical settings.

Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) results from genetic mutations in the X chromosome's gene responsible for producing the dystrophin protein. It is inherited in an X-linked recessive pattern [ 107 ]. Dystrophin plays a crucial role in preserving the structure and functionality of muscles. When dystrophin is not functioning properly, it causes gradual muscle weakening and frequently leads to premature death, typically occurring before the age of 30 [ 108 ]. Although the responsible gene has been identified, it is associated with more than 7,000 mutations, most commonly due to deletions in the gene (seen in 70% of patients) [ 109 ]. DMD patients suffer from a lack of curative therapies. Only treatments such as corticosteroids have been used to reduce secondary symptoms, and with a limitation of time to avoid adverse effects due to long-term usage [ 110 ]. However, genome-editing tools could provide a potential solution, as preclinical studies on animals and human cells have successfully corrected these mutations. These models validated CRISPR-mediated gene editing in muscles in DMD and restored muscle function strategies, estimated in vivo dystrophin recovery, and assessed the safety and delivery optimization of gene-editing components. CRISPR offers powerful tools for treating DMD by potentially restoring dystrophin expression permanently. Unlike gene replacement therapy, CRISPR/Cas9 allows for endogenous dystrophin regulation, resulting in the expression of proper protein amounts in the correct tissues. Despite this advancement several obstacles must be addressed before clinical application, including affirming safety, improving efficiency and meaningful preclinical testing [ 111 ].

Infectious diseases

CRISPR technology also holds promise in the field of infectious diseases. By targeting specific genes in pathogens, CRISPR can potentially disrupt their ability to infect and replicate within the host [ 112 ]. This approach provides a new avenue for developing novel antimicrobial therapies and combating drug-resistant infections. Early on, a trial using CRISPR-based ex vivo genome editing was conducted in an attempt to treat human immunodeficiency virus 1 (HIV-1) infection [ [1] , [113] ]. The trial involved introducing CCR5-focused ribonucleoprotein complexes into HSPCs obtained from patients through nucleofection. This process aimed to disrupt CCR5, a crucial viral receptor [ 114 ]. Furthermore, a 27-year-old male diagnosed with HIV-1 and severe lymphoblastic leukemia received successful and long-lasting transplantation of CRISPR-modified HSPCs [ 115 ]. Additionally, an experiment using the CRISPR/Cas9-HPV E6/E7 plasmid gel for the persistent treatment of HPV and HPV-related cervical intraepithelial neoplasia has been authorized (ClinicalTrials.gov: NCT03057912 ) [ [116] , [117] ]. CRISPR has proven a significant potential in transforming the approach to treating and managing infectious diseases, promising more efficient and precise therapies ahead.

Cancer

The buildup of epigenetic changes in the genome is a common indicator of cancer, which is considered a frequently fatal disease [ [118] , [119] ]. Chemotherapy, a conventional cancer treatment, presents challenges such as non-specific targeting and drug resistance, limiting its effectiveness [ [120] , [121] , [122] , [123] ]. Hence, it is necessary to discover new molecular targets that could aid in cancer therapy [ [124] , [125] ]. One suggested approach involves targeting mutated oncogenes and tumor suppressor genes within cancer cells as a potential therapeutic strategy. This approach suggests that manipulating tumor suppressor genes might induce programmed cell death in tumor cells [ [126] , [127] ].

CRISPR technology has the potential to revolutionize cancer treatment and management by enabling the precise targeting of cancer cells and the modification of genes involved in tumor growth and progression [ 128 ]. One of its key applications is gene editing, which focuses on oncogenes and tumor suppressor genes [ 129 ]. Oncogenes, when mutated or overactive, drive cancer cell proliferation, while tumor suppressor genes typically regulate cell growth and prevent tumor formation [ [130] , [131] ]. By targeting these key genetic elements, CRISPR offers the possibility of developing personalized cancer therapies, addressing tumor heterogeneity, and overcoming resistance to treatment [ [132] , [133] , [134] ]. Additionally, CRISPR screens are used to identify therapeutic targets, and the technology enhances immunotherapy by modifying immune cells to better recognize and attack cancer [ 135 ]. These varied applications highlight the transformative potential of CRISPR in cancer treatment and its ability to start and advance clinical trials focused on CRISPR-based therapies ( Fig. 3 ).

In relation to oncogenes and tumor suppressor genes, CRISPR can disrupt pathways that promote tumor growth and cancer cell survival. For example, in bladder cancer, CRISPR/Cas9 has been utilized to target genes such as E-cadherin and p21 , leading to inhibited tumor growth and triggered cell death [ [136] , [137] ]. Apart from bladder cancer, CRISPR/Cas9 has been employed to target the KRAS oncogene, which is frequently mutated and drives the progression of various types of cancer [ [138] , [139] ]. Through the editing of KRAS and other genes, such as TP53, researchers have observed significant suppression in tumor growth and cell proliferation, effectively interfering with cancer survival pathways [ 140 ]. CRISPR/Cas9 has also been able to target and edit genes like EGFR in glioblastoma, BRCA1/2 suppressor genes in breast or pancreatic cancer, and APC gene in colorectal cancer [ [141] , [142] , [143] , [144] ].

Tumor heterogeneity, which refers to the genetic and phenotypic differences among cancer cells in a tumor, presents a major obstacle for CRISPR-based cancer therapy [ 145 ]. Even if CRISPR successfully targets and edits cancer-related genes in some cancer cells, there may still be a subpopulation of cells that are resistant to treatment. These resistant cells can lead to tumor recurrence and progression [ 146 ]. To address tumor heterogeneity and resistance, researchers are exploring combination therapies that combine CRISPR with alternative approaches to treatment, like chemotherapy or immunotherapy [ 147 ]. By targeting multiple pathways involved in cancer development and progression, combination therapies may enhance the effectiveness of CRISPR-based cancer treatment and overcome resistance [ 148 ].

Furthermore, the development of CRISPR screening approaches, such as CRISPR knockout screens and CRISPR activation screens, can help identify genes and pathways that are critical for tumor growth and survival [ 149 ]. This knowledge can guide the design of more effective CRISPR-based therapies and improve patient outcomes. Several clinical trials are currently underway to test the efficacy of CRISPR/Cas9 in cancer treatment. These trials are specifically focusing on using CRISPR technology to edit specific genes in patients with blood cancers such as leukemia and lymphoma, as well as solid tumors. The goal is to improve treatment outcomes in cases where traditional therapies have not been successful ( Table 2 ).

Challenges of CRISPR/Cas9 in clinical trials

It is undeniable that CRISPR will imminently start a revolution in the treatment of genetic diseases and cancers. This can be observed by the increasing number of preclinical studies and clinical trials that have been registered on clinicaltrials.gov [ [150] , [151] ]. According to the website, as of February 2025, there are 69 clinical trials utilizing CRISPR/Cas9 technique. Eight of these studies focus on the treatment of β-thalassemia, primarily TDT, and nine studies focus on SCD. Among these trials, 19 have progressed to phase 2, and 7 to phase 3. This proves the substantial growth of this field in medical centers globally. Specific details of some of these clinical trials can be seen in Tables 1 and 2 .

However, CRISPR faces several challenges that must be tackled for its effective clinical implementation ( Fig. 4 ). These challenges encompass efficient delivery of CRISPR components, reduction of off-target effects, management of immune responses and toxicity, as well as addressing tumor heterogeneity and resistance. Therefore, continued research and technological advancements are imperative. The main challenge is delivering CRISPR components to target cells or tissues efficiently. It is crucial to develop efficient delivery systems to ensure that CRISPR reaches the target site and effectively edits the target genes. For example, transporting CRISPR components directly to the site of infection or across the blood-brain barrier in specific infectious diseases and neurodegenerative disorders without impacting other cells presents a considerable challenge [ [3] , [4] , [5] ]. Furthermore, ensuring minimal off-target effects is essential to ensure the safety and efficacy of the treatment, as unintended modifications to genes other than the target can pose risks. Another concern is checking the potential for pathogens to develop resistance to CRISPR-based therapies.

CRISPR/Cas9 delivery systems challenges

The main challenge faced by CRISPR gene editing method is how it is delivered [ [152] , [153] ]. Generally, the most common method for delivering the CRISPR components is electroporation, though other studies have also used viral vectors or synthetic materials like Lipofectamine [ [154] , [155] , [156] , [157] ]. While electroporation can be used with various cell types and developmental stages, it poses significant risks of damaging both cells and cargo, which is critical when focusing on disease treatment. On the other hand, the use of viral vectors is controversial due to immunogenicity concerns and limited transfection capacity in humans [ 158 ]. Lipid-based transfection is relatively easy, with commercial methods and delivery kits like the Lipofectamine family of reagents readily available [ 159 ]. However, this approach raises concerns about potential toxicity and alterations in cell morphology. Overall, artificial carrier systems are typically less invasive compared to physical transfection methods and offer the advantages of reduced immunogenicity and a tendency to maintain genome integrity [ [160] , [161] ]. There are many opportunities to research and develop new artificial delivery systems, such as inorganic nanoparticles, polymers, and lipids, which can safely and efficiently deliver DNA, RNA or ribonucleoprotein (RNP) coding components, especially for cell delivery [ [162] , [163] , [164] , [165] ].

Viral delivery systems have been extensively studied and utilized for CRISPR delivery due to their high efficiency in gene transfer [ 166 ]. Viruses such as adeno-associated viruses (AAVs) and lentiviruses have been modified to carry CRISPR components and deliver them to target cells [ 167 ]. AAVs, in particular, have gained significant attention because they can infect both dividing and non-dividing cells, and they exhibit low immunogenicity [ 168 ]. Naturally, this delivery method has its challenges. One major concern is the limited cargo capacity of viral vectors, which restricts the size of DNA that can be delivered. Additionally, the potential for immune responses against the viral vectors poses a safety risk [ [169] , [170] ]. Pre-existing immune responses in individuals may make viral vectors less effective by neutralizing them. Moreover, manufacturing viral vectors in massive quantities can be expensive and time-consuming.

Non-viral delivery systems present an alternative to viral vectors for CRISPR delivery [ [34] , [171] , [172] ]. These systems include LNPs, polymer-based nanoparticles, and physical methods such as electroporation and microinjection [ [173] , [174] , [175] , [176] , [177] ]. Non-viral delivery systems have several advantages, including their ability to carry larger DNA payloads and their lower immunogenicity compared to viral vectors [ 178 ]. LNPs such as liposomes, have been extensively studied for CRISPR delivery [ 179 ]. These nanoparticles can encapsulate the CRISPR components and protect them from degradation in the extracellular environment [ 180 ]. Polymer-based nanoparticles, such as polyethyleneimine (PEI) and poly (lactic-co-glycolic acid) (PLGA), have also shown promise in delivering CRISPR components into target cells [ [181] , [182] ].

Nonetheless, non-viral delivery systems face challenges of their own. One major limitation is their lower efficiency compared to viral vectors. Non-viral systems often struggle to efficiently deliver the CRISPR components into the nucleus of target cells, where gene editing occurs [ 183 ]. Additionally, the cytotoxicity of certain non-viral delivery systems can limit their clinical application. Further research is needed to optimize the efficiency and safety of non-viral delivery systems for CRISPR [ 184 ].

Genome editing methods

The next significant issue to consider, as shown in many cases, is that numerous genomes editing strategies can be applied therapeutically to a specific disease. There are many strategies, including base editing targets, NHEJ, HDR, exon skipping, etc., which raise the question of the most effective and favorable strategies for treating a disease. For example, in the treatment of AIDS, an HDR method is used to create a Δ32 mutation in the CCR5 gene or the easier NHEJ method to knock out the CCR5 gene [ [185] , [186] ]. Even if CCR5-mediated NHEJ-edited immune cells are long-term resistant to HIV, it is crucial to thoroughly assess the biological impact of these edited cells. For β-thalassemia and SCD, it is better to knock out the BCL 11A gene with the NHEJ strategy or edit the exons of the HBB gene using HDR [ 187 ]. The decision-making process can be overwhelming due to the numerous options. Consequently, scientists must thoroughly study the disease conditions, involved genes and the effects of upstream biology before implementing treatment methods in hospitals.

Off-target effects

Off-target effects of the CRISPR/Cas9 system are regarded as a significant issue in gene therapy and genome engineering [ [188] , [189] ]. These effects are related to a range of factors including the choice of cell type, the specific target of Cas9, and gRNA. A direct and efficient method to reduce off-target effects is to deliver the components of the system as RNP. However, the high purity and cost of RNP, along with the relative difficulty in delivery compared to other methods, have led to few comparative studies, which are becoming more frequent. The properties of CRISPR/Cas9 systems are decided by both the PAM sequence and the gRNA. Nevertheless, off-target DNA cleavage can still happen even if there are three or more mismatches in the PAM-distal region [ 190 ].

Optimal cell type choice

Another significant challenge involves selecting the proper type of cell and ensuring the functions of CRISPR system effectively. This is crucial for therapeutic applications, where the system must deliver the edited stem cells back into the patient's body [ [191] , [192] ]. A critical aspect of this process is transfection, which involves extracting, culturing and delivering these resilient cells [ 193 ]. Proper guidance is essential to achieve the goals of the editing technique, presenting an opportunity for biomaterials scientists to obtain more efficient and safer carriers. Currently, RNP carriers have shown promising and acceptable outcomes, but to investigate off-target effects, the entire sequence should undergo genome sequencing (WGS or WES), followed by a decision on its suitability for treatment. In summary, there are extensive opportunities in this field, but chemists, biologists, physicians and policymakers need to collaborate closely to protect and enhance human health through CRISPR technology. This article will further explore ongoing studies addressing these challenges and propose potential solutions.

Ethical issues

Ethical concerns related to CRISPR/Cas9 in human disease models involve issues such as effectiveness, safety and off-target effects that may lead to unforeseen consequences [ [40] , [194] ]. A significant debate is ongoing about the ethical implications of using germ cells, which could affect future generations, compared to somatic or embryonic cells, where these alterations would typically not be heritable [ 195 ]. Moreover, the ethical discourse extends to the utilization of human embryos in research, with varying viewpoints within the scientific community regarding their ethical status and appropriate use [ 196 ]. Despite these challenges, CRISPR/Cas9 is still a valuable tool for precise genome editing, offering considerable promise for treating genetic diseases and creating disease models for research purposes.

Solutions to improve CRISPR/Cas9 system

Overcoming challenges in CRISPR technology involves tackling issues such as off-target effects and developing effective delivery systems. Researchers are improving CRISPR specificity through novel Cas proteins and gRNA designs, aided by bioinformatics tools to predict and minimize off-target sites. In delivery, advancements include next-generation viral vectors with increased cargo ability and reduced immunogenicity, as well as innovations in non-viral systems using peptides and nanoparticles for enhanced efficiency and safety. These efforts promise to enhance CRISPR's medical applications and gene therapy.

Use of bioinformatics in CRISPR technology

Bioinformatics plays a key role in the analysis, prediction and determination of the CRISPR/Cas system. It enabled scientists to uncover the connection between various genes and their functions [ 197 ]. Off-target effects are unintentional alterations in DNA sequences that occur outside of the intended target site. The unintended impacts of these off-target effects can result in unforeseeable outcomes, such as triggering oncogenes or interfering with regular gene operations. Scientists are striving to reduce these effects by enhancing the precision of CRISPR systems, using newly developed Cas proteins and optimized gRNA designs. For instance, they demonstrated a clear link between the length of gRNA and the number of off-targets, determining the optimal gRNA size to minimize unnecessary targeting [ 198 ]. The three nucleotides adjacent to the PAM significantly contribute to minimizing off-target effects [ 199 ]. In addition, the chemical reactions between gRNA and tracRNA nucleotides and their spatial structure are also important and should be investigated. Finally, a lower percentage of GC nucleotides in gRNA is also important, because it influences the binding specificity of the CRISPR/Cas9 complex to DNA [ [40] , [200] , [201] ].

CRISPR/Cas9 screening

CRISPR/Cas9 screening is an effective method for discovering and confirming possible off-target locations [ 202 ]. This technique involves introducing a library of gRNAs into cells and screening for off-target effects using various assays. Researchers can systematically screen for unintended effects, enabling the identification and characterization of possible off-target locations. This approach helps refine gRNA design and formulate strategies to reduce unintended impacts. CRISPR/Cas9 screening offers important information on the accuracy and effectiveness of CRISPR technology, allowing scientists to enhance its preciseness and security [ 203 ]. It is advisable to sequence cells in both research and clinical settings before starting system designs. This approach can preempt potential issues caused by the presence of SNPs and mutations, thereby enhancing the reliability of the results.

Advancements in delivery systems

Non-viral carriers that deliver Cas9 RNPs have displayed potential in scientific and therapeutic uses due to their high effectiveness, safety and flexibility [ 172 ]. Carrier-mediated intracellular delivery needs forming a stable complex alongside the cargo molecule. The RNP complex holds nucleic acid components that have many negative charges and hydrogen bonding sites to interact with carriers. RNP delivery allows for the quickest genome editing by removing the requirement for intracellular transcription and translation [ [204] , [205] ]. Simultaneously, transient genome editing achieves significant editing efficiency while reducing off-target effects, insertion mutations and immune responses. It also eases enhancements in genome editing efficiency across diverse areas, including iPSCs, embryonic stem cells and tissue stem cells. Given the rapid growth of the RNP-based genome editing system and the demand for gene therapy in clinical settings, future research may concentrate on designing delivery vectors that enhance the effectiveness and responsiveness of RNP delivery systems while prioritizing safety.

Another field of study focuses on creating delivery systems capable of transporting CRISPR components in vivo to specific tissues or organs. This approach would cut the need for ex vivo manipulation of cells, enhancing efficiency and making it more practical for clinical applications. Various strategies, such as the use of tissue-specific promoters and viral vectors with tissue tropism, are being explored to achieve targeted in vivo delivery of CRISPR [ 206 ].

Choice of target cell types in CRISPR/Cas9 technique

Target cell choice, along with CRISPR/Cas delivery, stays the biggest bottleneck for genome editing. Different cells have been used for research and clinical purposes, which are summarized in Table 3 . It is important to note that transfecting cells and lines can be challenging in the pursuit of advancing genome editing for therapeutic purposes. While cancer lines can confirm methods, testing on patient stem cells expressing the edited gene is crucial. The CRISPR/Cas9 system is commonly employed to genetically alter pluripotent or multipotent stem cells, which are subsequently induced to develop into specific cell types for detailed study or possible clinical use [ [207] , [208] ]. Moreover, numerous attempts are being undertaken to create effective techniques for delivering CRISPR/Cas RNP without causing substantial harm to the viability of the target cell.

Future prospects of CRISPR in medicine

The future of CRISPR technology in medicine is exceptionally promising, with advancements expected to revolutionize the treatment of genetic diseases and beyond. One of the most expected applications is in the correction of monogenic disorders, such as cystic fibrosis, sickle cell anemia, and Huntington’s disease. By precisely targeting and repairing defective genes, CRISPR has the potential to offer curative therapies that were previously unattainable. Ongoing clinical trials for diseases like sickle cell anemia and beta-thalassemia using ex vivo gene editing approaches have already shown significant progress, paving the way for broader clinical applications [ [71] , [209] , [210] , [211] , [212] , [213] ]. Essentially, CRISPR/Cas9 represents a significant advancement in the treatment of SCD, particularly targeting the erythrocyte-specific enhancer region of the BCL11A gene [ 214 ]. The genetic modifications induced by Cas9 have led to elevated levels of HbF and a substantial reduction in vascular complications [ 215 ]. This innovative approach not only drops the need for frequent blood transfusions but also promises to significantly enhance the quality of life for individuals dealing with the myriad challenges posed by SCD.

LNPs have appeared as a safer delivery vehicle for CRISPR REGV131-LNP1265 systems compared to other viral vectors. Several earlier preclinical hemophilia studies in phases 1 and 2, utilizing LNP-CRISPR ( NCT06379789 ), have proved sufficient and sustained therapeutic effects. These findings suggest that LNP-CRISPR-mediated genome editing therapy could be a practical way for treating hemophilia. However, our research on this disease indicates that CRISPR-mediated editing may be less effective in hemophilia B than in hemophilia A, due to the genomic characteristics of the respective genes [ 94 ]. Beyond monogenic disorders, CRISPR holds great promise in oncology, where it can be used to engineer immune cells for more effective cancer immunotherapies. For example, CRISPR can enhance the precision of CAR-T cell therapies by editing T cells to improve their specificity and reduce off-target effects ( NCT04037566 , NCT03545815 , NCT03399448 , NCT04035434 ). Additionally, it can be employed to disable tumor-specific immune evasion pathways, potentially improving patient outcomes. The ability to edit multiple genes simultaneously through multiplexing makes CRISPR an ideal tool for addressing the complexities of cancer biology [ [213] , [216] ].

Another area of significant interest is the use of CRISPR in infectious diseases. The technology has shown potential in developing antiviral therapies, such as targeting latent reservoirs of HIV or inhibiting viral replication in diseases like hepatitis B and herpes simplex ( NCT03164135 ). CRISPR-based diagnostic tools, like SHERLOCK and DETECTR, are also expected to play a pivotal role in early disease detection and monitoring, offering rapid, sensitive, and cost-effective solutions [ 217 ]. Despite its potential, several challenges must be overcome to realize CRISPR’s full potential in medicine. Off-target effects, delivery efficiency, and immune responses to CRISPR components are still key concerns. Innovative delivery systems, such as lipid nanoparticles (LNP1265) and viral vectors, are being actively developed to address these challenges and ensure precise and efficient gene editing. Advances in base and prime editing technologies are also expected to enhance CRISPR’s accuracy and expand its therapeutic applications [ 218 ]. As the regulatory and ethical landscape continues to evolve, the integration of CRISPR into mainstream medicine will require robust oversight and public engagement. The technology’s transformative potential must be balanced with careful consideration of its societal and ethical implications, particularly in germline editing. Nevertheless, with ongoing innovation and collaboration, CRISPR is poised to become a cornerstone of personalized medicine, offering hope for previously incurable diseases and fundamentally transforming healthcare [ 219 ].

Although beta-thalassemia and SCD are well-known blood disorders treated by CRISPR/Cas9 system, this revolutionary technology offers possibilities beyond these conditions. This groundbreaking gene-editing tool holds the potential for treating other blood disorders, inspiring hope among both patients and scientists. Despite existing obstacles, the future applications of CRISPR in medicine appear highly promising. The potential uses of CRISPR technology are extensive and diverse, ranging from treating genetic disorders, infectious diseases, and targeting cancer cells. The precision offered by CRISPR in genome editing opens new pathways for personalized medicine and targeted therapies. As research progresses, advancements in CRISPR technology are expected to overcome current challenges and limitations. Scientists are actively striving to enhance its specificity and efficiency, develop new delivery systems, and refine the ethical frameworks surrounding gene editing. These concerted efforts pave the way for the safe and effective application of CRISPR-based therapies.

Conflicts of interest

The authors declare no conflict of interest.

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