Exploring the Potential of CRISPR-Cas9 in Treating Neurodegenerative Diseases: Advances, Challenges, and Future Directions

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Adarsh Iyengar
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Abstract

Neurological disorders, such as Parkinson’s disease (PD), Huntington’s disease (HD), Alzheimer’s disease (AD), and Amyotrophic Lateral Sclerosis (ALS), are increasingly prevalent, posing significant challenges due to their progressive nature and lack of curative treatments. Current therapeutic options primarily focus on symptomatic management rather than addressing the underlying causes of the disease. This review explores the emerging role of gene editing, particularly CRISPR-Cas9, as a potential therapeutic strategy for these disorders. CRISPR-Cas9 offers a precise method to target genetic mutations associated with neurodegeneration, potentially halting or reversing disease progression. The review delves into the applications of gene editing in PD, HD, AD, and ALS, highlighting the successes, challenges, and ethical considerations of this technology. While CRISPR-Cas9 presents promising therapeutic potential, significant obstacles remain, including delivery to the central nervous system, off-target effects, and ethical concerns surrounding germline editing. This review synthesizes current literature on CRISPR-Cas9 applications in neurodegenerative diseases, examines the mechanisms underlying gene editing, discusses disease-specific genetic targets, and evaluates the technical and ethical challenges of CRISPR-Cas9.

Introduction

Neurodegenerative diseases represent a growing global health crisis, affecting millions of individuals worldwide and placing substantial burdens on healthcare systems. Parkinson’s disease (PD), Huntington’s disease (HD), Alzheimer’s disease (AD), and Amyotrophic Lateral Sclerosis (ALS) are characterized by progressive neuronal loss, leading to debilitating motor and cognitive impairments1. Despite decades of research, current treatments remain largely palliative, offering symptomatic relief without addressing the underlying pathophysiology2. The genetic basis of many neurodegenerative disorders has been increasingly elucidated, revealing specific mutations that contribute to disease onset and progression3. This genetic understanding has paved the way for novel therapeutic approaches, including gene editing technologies.

CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9) has emerged as a revolutionary tool in molecular biology, offering unprecedented precision in genome modification4. Originally discovered as a bacterial immune system, CRISPR-Cas9 has been adapted for mammalian genome editing, enabling researchers to correct disease-causing mutations, disrupt pathogenic gene expression, or insert therapeutic sequences5. The technology’s simplicity, efficiency, and versatility have accelerated its application across various fields, including the treatment of genetic disorders6.

Mechanism of CRISPR-Cas9 Gene Editing

The mechanism of CRISPR-Cas9 gene editing relies on two key components: the Cas9 endonuclease and a guide RNA (gRNA). The gRNA is a synthetic RNA molecule approximately 20 nucleotides in length that is designed to be complementary to a specific DNA sequence in the genome7. This gRNA directs the Cas9 protein to the precise genomic location for editing. Once the gRNA-Cas9 complex binds to the target sequence, Cas9 acts as a pair of scissors, creating a double-strand break (DSB) at the specified site8.

Following the DSB, the cell’s endogenous DNA repair mechanisms are activated. The two primary repair pathways are non-homologous end joining (NHEJ) and homology-directed repair (HDR)9,10. NHEJ is an error-prone process that often results in insertions or deletions, which can disrupt gene function, which is helpful for gene knockout applications. In contrast, HDR is a more precise mechanism that uses a provided DNA template to repair the break, allowing the correction of mutations or the insertion of new genetic sequences11. The choice between these repair pathways depends on the cell cycle stage and the presence of a donor template, making the optimization of CRISPR-Cas9 delivery crucial for therapeutic applications.

Delivery Systems of CRISPR-Cas9

Delivering CRISPR-Cas9 components to target cells, particularly in the central nervous system (CNS), presents significant technical challenges. Several delivery systems have been developed, each with distinct advantages and limitations. Viral vectors, particularly adeno-associated viruses (AAVs), are among the most widely used delivery vehicles for CRISPR-Cas9 in vivo12. AAVs offer several benefits, including low immunogenicity, broad tissue tropism, and the ability to transduce both dividing and non-dividing cells—a critical feature for targeting post-mitotic neurons13. However, AAVs have limited packaging capacity (approximately 4.7 kilobases), which can be restrictive when delivering the full-length Cas9 gene along with guide RNAs and regulatory elements14. To address this limitation, researchers have developed smaller Cas9 variants and split-AAV systems that deliver components across multiple viral particles15.

Lentiviral vectors represent another viral delivery option, offering greater packaging capacity and stable genomic integration, but they carry higher immunogenicity risks and the potential for insertional mutagenesis16. Non-viral delivery methods, including lipid nanoparticles (LNPs), cell-penetrating peptides, and electroporation, have also been explored as alternatives to viral vectors17. LNPs, which have been successfully used for mRNA vaccine delivery, can encapsulate CRISPR-Cas9 ribonucleoprotein (RNP) complexes and protect them from degradation during systemic circulation18.

A major obstacle to CNS delivery is the blood-brain barrier (BBB), a selective permeability barrier that restricts the passage of molecules from the bloodstream into the brain parenchyma19. Recent advances in BBB-penetrating delivery systems, including focused ultrasound-mediated BBB disruption and receptor-mediated transcytosis, show promise for improving CRISPR-Cas9 delivery to the brain20.

ParamterAAV VectorLentiviral VectorLipid Nanoparticle (LNP)Cell Penetrating PeptidesElectroporation
Payload Capacity~4.7kb~9kbHighLimitedHigh
ImmunogenecityLowModerate-HighLow to ModerateLowLow
Targeting EfficiencyHighHighModerateVariableVariable
Integration RiskVery LowPotentialNoneNoneNone
Clinical StageAdvancedModeratePreclinicalPreclinicalPreclinical
Suitable for CNS?YesYesYesYesYes
Table 1 | Comparison of CRISPR-Cas9 Delivery Systems for CNS Applications

This review examines the current state of CRISPR-Cas9 research in treating neurodegenerative diseases, with a focus on PD, HD, AD, and ALS. We explore the disease-specific genetic targets, preclinical and clinical advances, and the technical and ethical challenges that must be overcome to realize the therapeutic potential of this technology.

Results

Parkinson’s Disease (PD)

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, characterized by the progressive loss of dopaminergic neurons in the substantia nigra, leading to motor symptoms such as tremor, rigidity, and bradykinesia21. While most PD cases are sporadic, approximately 10–15% have a genetic component, with mutations in several genes identified as causative or risk factors22. The most commonly implicated genes in familial PD include SNCA (α-synuclein), LRRK2 (leucine-rich repeat kinase 2), PARK2 (Parkin), PINK1 (PTEN-induced kinase 1), and DJ-123.

The SNCA gene encodes α-synuclein, a protein that misfolds and aggregates to form Lewy bodies, a pathological hallmark of PD24. Mutations in SNCA, including point mutations (A53T, A30P, E46K) and gene multiplications (duplications and triplications), lead to increased α-synuclein expression and enhanced aggregation propensity25. LRRK2 mutations, particularly the G2019S variant, are the most common genetic cause of PD, accounting for approximately 4% of familial cases and 1–2% of sporadic cases26. The G2019S mutation results in increased kinase activity, leading to neuronal dysfunction and death through mechanisms involving mitochondrial impairment and protein aggregation27. Loss-of-function mutations in PARK2, PINK1, and DJ-1 are associated with early-onset autosomal recessive PD and impair mitochondrial quality control pathways28.

CRISPR-Cas9 has been applied to model PD in vitro and in vivo, as well as to develop potential therapeutic strategies. Researchers have used CRISPR to introduce PD-associated mutations into cellular and animal models, enabling the study of disease mechanisms29. In therapeutic applications, CRISPR-Cas9 has been employed to silence or reduce SNCA expression in models with gene duplications or triplications30. Studies have demonstrated that CRISPR-mediated reduction of α-synuclein levels can decrease protein aggregation and neuronal toxicity in both cell culture and animal models31. Similarly, CRISPR strategies targeting LRRK2 have focused on either correcting pathogenic mutations or reducing overall LRRK2 expression to mitigate the toxic gain-of-function effects associated with variants like G2019S32.

One notable study used CRISPR-Cas9 to correct the LRRK2 G2019S mutation in patient-derived induced pluripotent stem cells (iPSCs), successfully reversing the pathological phenotype in differentiated dopaminergic neurons33. This proof-of-concept work demonstrates the potential for personalized gene editing approaches in PD. However, translating these findings to clinical applications faces significant hurdles, including the efficient delivery of CRISPR components to affected neurons in the adult brain and ensuring the safety of edited cells.

Huntington’s Disease (HD)

Huntington’s disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an expansion of CAG trinucleotide repeats in the huntingtin (HTT) gene34. Individuals with 40 or more CAG repeats invariably develop HD, while those with 36–39 repeats have incomplete penetrance35. The expanded polyglutamine tract in the mutant huntingtin protein (mHTT) causes it to misfold and aggregate, leading to neuronal dysfunction and death, particularly in the striatum and cortex36. HD presents with a triad of motor, cognitive, and psychiatric symptoms, typically manifesting in mid-life and progressing over 15–20 years37.

The genetic simplicity of HD—a single gene disorder with a known mutation—makes it an attractive target for gene editing therapies. CRISPR-Cas9 strategies for HD have focused on selectively inactivating or reducing the expression of the mutant HTT allele while preserving the wild-type allele38. Several approaches have been developed, including allele-specific targeting based on single-nucleotide polymorphisms (SNPs) linked to the expanded CAG repeat, as well as non-selective HTT reduction39.

In preclinical studies, CRISPR-Cas9-mediated disruption of the HTT gene has shown promising results. Researchers have successfully reduced mHTT expression in HD mouse models and patient-derived cells, leading to decreased protein aggregation and improved cellular phenotypes40. One study demonstrated that AAV-mediated delivery of CRISPR Cas9 targeting HTT in the striatum of HD mice resulted in reduced mHTT levels and improved motor function41. Another approach involves targeting the CAG repeat expansion itself, using CRISPR-Cas9 to excise the expanded repeat region or disrupt the repeat tract through NHEJ-mediated indels42. This strategy has shown efficacy in reducing toxic mHTT fragments and preventing neurodegeneration in animal models43.

Despite these advances, challenges remain in achieving sufficient editing efficiency in the brain, minimizing off-target effects, and ensuring long-term safety. The dominantly inherited nature of HD also raises the question of whether partial reduction of mHTT is sufficient for therapeutic benefit or whether complete knockout is necessary.

Alzheimer’s Disease (AD)

Alzheimer’s disease (AD) is the most common cause of dementia, affecting over 55 million people worldwide44. AD is characterized by progressive cognitive decline, memory loss, and behavioral changes, associated with the accumulation of amyloid-beta (Aβ) plaques and neurofibrillary tangles composed of hyperphosphorylated tau protein45. While most AD cases are late-onset and sporadic with complex genetic and environmental contributions, approximately 1–5% are early-onset familial AD (FAD) caused by autosomal dominant mutations46.

The three genes primarily responsible for FAD are APP (amyloid precursor protein), PSEN1 (presenilin 1), and PSEN2 (presenilin 2)47. Mutations in APP lead to increased production or altered processing of Aβ peptides, promoting their aggregation into neurotoxic oligomers and plaques48. PSEN1 and PSEN2 encode components of the γ-secretase complex, which cleaves APP to generate Aβ peptides. Mutations in these genes cause aberrant γ-secretase activity, resulting in increased production of the highly aggregation-prone Aβ42 isoform49. The most common PSEN1 mutations include E280A, M146L, and L286V, while PSEN2 mutations are less frequent but equally pathogenic50.

CRISPR-Cas9 has been used to model FAD by introducing pathogenic mutations into cellular and animal models, facilitating the study of disease mechanisms and the testing of potential therapies51. Therapeutic applications of CRISPR-Cas9 in AD focus on correcting FAD-causing mutations in APP, PSEN1, or PSEN2 genes in patient-derived iPSCs52. Studies have demonstrated that correction of APP or PSEN1 mutations can normalize Aβ production and reduce pathological features in neuronal cultures derived from gene-edited iPSCs53. Additionally, CRISPR has been employed to modulate genes involved in Aβ clearance, such as APOE (apolipoprotein E), with the APOE4 allele being the strongest genetic risk factor for late-onset AD54.

One innovative approach involves using CRISPR-Cas9 to convert the APOE4 allele to the less risky APOE3 variant in patient cells, which has shown promise in reducing AD-related pathology55. However, the sporadic and polygenic nature of most AD cases complicates the application of gene editing, as therapeutic strategies may need to target multiple genes or pathways simultaneously. The late-onset nature of AD also poses challenges for the timing of therapeutic intervention.

Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of motor neurons in the brain and spinal cord, leading to muscle weakness, paralysis, and eventual respiratory failure56. Most ALS cases are sporadic (approximately 90%), while 10% are familial, with mutations in over 30 genes identified as causative or contributory57. The most commonly mutated genes in familial ALS include SOD1 (superoxide dismutase 1), C9ORF72 (chromosome 9 open reading frame 72), FUS (fused in sarcoma), and TARDBP (TAR DNA-binding protein 43)58.

SOD1 mutations account for approximately 20% of familial ALS cases and 1–2% of sporadic cases59. Over 180 different SOD1 mutations have been identified, most of which cause disease through a toxic gain-of-function mechanism involving protein misfolding and aggregation60. The C9ORF72 hexanucleotide repeat expansion (GGGGCC) is the most common genetic cause of both ALS and frontotemporal dementia (FTD), accounting for approximately 40% of familial ALS and 7% of sporadic ALS cases61. The expanded repeat forms toxic RNA foci and produces dipeptide repeat proteins through non-canonical translation, both contributing to neurodegeneration62. Mutations in FUS and TARDBP disrupt RNA metabolism and lead to the formation of cytoplasmic protein aggregates, impairing cellular function63.

CRISPR-Cas9 has been extensively used to develop ALS models and explore therapeutic strategies. Researchers have employed CRISPR to introduce disease-causing mutations into cell and animal models, enabling mechanistic studies and drug testing64. Therapeutic applications focus on silencing or correcting mutant SOD1 alleles, excising or disrupting the C9ORF72 repeat expansion, and modulating genes involved in RNA metabolism and protein homeostasis65. Studies have shown that CRISPR-mediated reduction of mutant SOD1 expression can slow disease progression and extend survival in ALS mouse models66. For C9ORF72-related ALS, CRISPR strategies have targeted the expanded repeat itself, using Cas9 to induce DSBs that result in repeat contraction or excision through NHEJ repair67. This approach has demonstrated efficacy in reducing toxic RNA foci and dipeptide repeat proteins in patient-derived cells68.

The heterogeneity of genetic causes in ALS presents a challenge for developing universal gene editing therapies, as treatments may need to be tailored to specific mutations. Additionally, the progressive and often rapid course of ALS necessitates early intervention, which requires reliable biomarkers for disease detection and monitoring.

DiseaseKey Gene TargetsMutation Type
Parkinson’s Disease (PD)SNCA, LRRK2, PARK2Point Mutation and Gene Duplications
Huntington’s Disease (HD)HTTCAG Repeat
Alzheimer’s Disease (AD)APP, PSEN1, PSEN2Gene Duplications and Missense Mutations
Amyotrophic Lateral Sclerosis (ALS)SOD1, C9orf72, TARDBP, FUSRepeat Expansion and missense mutations
Table 2 | Key Genetic Targets in Neurodegenerative Diseases

Challenges and Limitations

Off-Target Effects

One of the most significant concerns with CRISPR-Cas9 gene editing is the potential for off-target effects, where the Cas9 endonuclease cleaves DNA at unintended genomic sites that share sequence similarity with the target site69. Off-target editing can lead to unintended mutations, chromosomal rearrangements, or disruption of essential genes, potentially causing cellular dysfunction or oncogenic transformation70. The specificity of CRISPR-Cas9 is primarily determined by the sequence of the gRNA, which typically requires a 20-nucleotide target sequence adjacent to a protospacer adjacent motif (PAM)71. However, Cas9 can tolerate mismatches between the gRNA and the target DNA, particularly in the PAM-distal region, leading to off-target cleavage72.

Multiple strategies have been developed to minimize off-target effects and enhance the precision of CRISPR-Cas9 editing. High-fidelity Cas9 variants, such as SpCas9-HF1, eSpCas9, and HypaCas9, have been engineered to reduce non-specific DNA binding and improve on-target specificity without compromising editing efficiency73. These variants contain mutations in the Cas9 protein that weaken interactions with the DNA backbone, making the enzyme more stringent in its target recognition74. Another approach involves using truncated gRNAs (17–18 nucleotides instead of the standard 20), which have been shown to reduce off-target activity while maintaining on-target editing75. Base editors and prime editors represent alternative gene editing technologies that do not rely on DSB formation, thereby reducing the risk of off-target indels and large deletions76. Comprehensive off-target detection methods, including whole-genome sequencing, GUIDE-seq, and CIRCLE-seq, are essential for evaluating the safety profile of CRISPR-Cas9 therapeutics before clinical application77.

Delivery Systems for CRISPR-Cas9

Delivering CRISPR-Cas9 components to the CNS remains one of the most formidable technical barriers to clinical translation. The blood-brain barrier (BBB) serves as a protective shield that prevents approximately 98% of small molecules and nearly all large molecules, including viral vectors and nanoparticles, from entering the brain parenchyma78. This selective permeability is maintained by tight junctions between endothelial cells, efflux transporters, and limited transcytosis79. Consequently, systemic delivery of CRISPR-Cas9 therapeutics results in poor brain penetration and requires extremely high doses, which increase the risk of off-target effects and immune responses in peripheral tissues80.

Several strategies are being developed to overcome BBB limitations. Direct intracerebral or intrathecal injection bypasses the BBB but is highly invasive, carries risks of infection and inflammation, and achieves limited distribution within the brain81. Focused ultrasound (FUS) combined with microbubble administration can transiently disrupt the BBB in a spatially targeted manner, allowing systemic delivery of CRISPR-Cas9 vectors to specific brain regions82. This approach has shown promise in preclinical studies but requires careful optimization to avoid tissue damage and inflammation83.

Engineering viral vectors with enhanced BBB-crossing capabilities represents another avenue. AAV variants with improved CNS tropism, such as AAV9 and AAV-PHP.eB, have demonstrated increased brain transduction following systemic administration in rodent models84. However, these variants show reduced efficiency in larger animals and humans, highlighting species-specific differences in BBB permeability85. Receptor-mediated transcytosis, utilizing engineered vectors that bind to receptors expressed on brain endothelial cells (such as transferrin receptor or insulin receptor), offers a non-invasive approach for BBB crossing86. Lipid nanoparticles functionalized with targeting ligands and cell-penetrating peptides are also under investigation for CNS delivery of CRISPR-Cas9 RNP complexes87.

Immune Responses

The immunogenicity of CRISPR-Cas9 components poses another significant challenge for therapeutic applications. The bacterial-derived Cas9 protein can elicit both innate and adaptive immune responses in mammalian hosts88. Studies have revealed that a substantial proportion of the human population has pre-existing adaptive immunity to Cas9 orthologs, particularly Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9), likely due to prior exposure to these common bacterial species89,90. Pre-existing antibodies against Cas9 could neutralize the therapeutic agent, reducing efficacy, or trigger adverse immune reactions. Additionally, the innate immune system can recognize foreign DNA and RNA introduced during CRISPR delivery, activating inflammatory pathways that may lead to the clearance of edited cells or systemic inflammation91.

Strategies to mitigate immune responses include immunosuppression protocols during treatment, engineering of Cas9 variants with reduced immunogenicity, transient delivery of CRISPR components as ribonucleoprotein (RNP) complexes rather than DNA-encoded vectors, and screening patients for pre-existing immunity before treatment92. The development of Cas9 orthologs from bacterial species with lower seroprevalence in humans also offers promise for reducing adaptive immune responses93.

Ethical and Regulatory Considerations

The application of CRISPR-Cas9 in treating neurodegenerative diseases raises several ethical and regulatory issues that must be carefully addressed. A primary concern is the distinction between somatic and germline gene editing94. Somatic gene editing, which targets non-reproductive cells and therefore does not affect future generations, is generally considered more ethically acceptable and is the focus of current therapeutic development95. In contrast, germline editing, which modifies genes in reproductive cells or embryos, results in heritable changes that are passed to offspring, raising profound ethical questions about consent, unintended consequences, and the potential for eugenics96. The international scientific community has called for a moratorium on clinical applications of heritable human genome editing until appropriate ethical and regulatory frameworks are established97.

Additional ethical considerations include ensuring equitable access to CRISPR-Cas9 therapies, which are likely to be expensive and initially available only in advanced healthcare settings98. The potential for unequal access could exacerbate healthcare disparities and create a divide between those who can afford genetic enhancements and those who cannot99. Informed consent is another critical issue, particularly for neurodegenerative disease patients who may have cognitive impairments affecting their decision-making capacity100. Long-term monitoring of gene-edited patients is essential to detect any delayed adverse effects, but this raises questions about patient privacy and the duration of surveillance101. Regulatory agencies, including the FDA and EMA, are developing frameworks for evaluating the safety and efficacy of gene editing therapies, but guidelines continue to evolve as the technology advances102.

Future Directions

The field of CRISPR-Cas9 gene editing for neurodegenerative diseases is rapidly advancing, with several promising directions for future research and clinical development. Next generation gene editing technologies, including base editors, prime editors, and CRISPR interference (CRISPRi) and activation (CRISPRa) systems, offer enhanced precision and expanded capabilities beyond traditional Cas9-mediated DSB formation103. Base editors can directly convert one DNA base to another without creating DSBs, enabling the correction of point mutations with reduced risk of off-target indels104. Prime editors provide even greater versatility by allowing targeted insertions, deletions, and base substitutions without requiring DSBs or donor DNA templates105. These technologies may overcome some limitations of conventional CRISPR-Cas9 in treating neurodegenerative diseases caused by specific point mutations.

Combination therapies that integrate CRISPR-Cas9 with other therapeutic modalities, such as small molecule drugs, antibody-based treatments, or stem cell therapies, may provide synergistic benefits106. For example, CRISPR-edited iPSC-derived neurons could be transplanted into patients to replace lost neurons in PD or HD, combining gene correction with cell replacement therapy107. Advances in machine learning and computational biology are improving gRNA design algorithms, enabling more accurate prediction of on-target efficiency and off-target activity108. These tools will help researchers select optimal target sites and gRNA sequences, enhancing the safety and efficacy of CRISPR-Cas9 therapeutics.

Clinical trials of CRISPR-Cas9-based therapies for various genetic diseases are underway, with several showing encouraging preliminary results109,110. The first in vivo CRISPR therapy, targeting transthyretin amyloidosis, demonstrated successful gene editing in the liver and clinical benefit, paving the way for similar approaches in neurological disorders. As delivery technologies improve and safety concerns are addressed, CRISPR-Cas9 is poised to become a transformative tool in the treatment of neurodegenerative diseases.

Conclusion

CRISPR-Cas9 gene editing represents a paradigm shift in the approach to treating neurodegenerative diseases, offering the potential to address the genetic root causes of PD, HD, AD, and ALS rather than merely managing symptoms. The precision and versatility of CRISPR-Cas9 enable targeted correction of disease-causing mutations, modulation of gene expression, and exploration of disease mechanisms in unprecedented detail. Preclinical studies have demonstrated proof-of-concept for CRISPR-Cas9 therapies in cellular and animal models of these disorders, with promising results in reducing pathological protein aggregation, improving cellular function, and slowing disease progression.

However, significant challenges must be overcome before CRISPR-Cas9 can be safely and effectively applied in clinical settings. Off-target effects, delivery barriers—particularly the blood-brain barrier—immune responses, and ethical considerations all require careful attention and innovative solutions. The development of high-fidelity Cas9 variants, advanced delivery systems, immunomodulatory strategies, and robust regulatory frameworks will be essential for the successful translation of CRISPR-Cas9 therapies from bench to bedside.

As the field continues to evolve, interdisciplinary collaboration among molecular biologists, neurologists, bioengineers, ethicists, and policymakers will be crucial for navigating the complex landscape of gene editing therapeutics. With continued research and technological innovation, CRISPR-Cas9 holds the promise of transforming the treatment of neurodegenerative diseases and improving the lives of millions of patients worldwide.

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