Annals of Movement Disorders

: 2020  |  Volume : 3  |  Issue : 1  |  Page : 23--32

CRISPR/Cas9 technology in neurological disorders: An update for clinicians

Vishnu Swarup1, Vikas Kumar1, Mohammed Faruq2, Himanshu N Singh3, Inder Singh1, Achal K Srivastava1,  
1 Department of Neurology, All India Institute of Medical Sciences (AIIMS), New Delhi, India
2 Department of Genomics and Molecular Medicine, CSIR-Institute of Genomics and Integrative Biology (CSIR-IGIB), New Delhi, India
3 TAGC-Theories and Approaches of Genomic Complexity, Aix-Marseille University, INSERM, Marseille, France

Correspondence Address:
Prof. Achal K Srivastava
Clinical Neurophysiology Facility, Room No. 60 GF, CN Center, Department of Neurology, All India Institute of Medical Sciences (AIIMS), New Delhi 110029.


Gene therapy has proven its potential in treatment of several human diseases. Most recent method in a long line of genome-editing techniques is Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. This CRISPR–Cas9 technology uses a ribonucleic acid (RNA)-guided deoxyribonucleic acid (DNA) endonuclease, Cas9, which induces double-strand breaks (DSBs) in target site. These DSBs are repaired by various cellular DNA repair mechanisms leading to changes in target sites. This revolutionary technique has unraveled several mysteries not only in pathogenesis of several human diseases but also proved its high potential in developing disease models ranging from cell lines to large animals. The number of neurodegenerative disorders linked with mutations has been increasing every day. Several such monogenic disorders provide opportunities for gene therapy using CRISPR–Cas9 method. Translational gap toward developing highly precise and personalized medicine for several neurodegenerative disorders has been reduced by CRISPR–Cas9 technology. Recent advancements in this technique have reduced the adverse effects on targets also. In this review, we have summarized recent achievements of CRISPR–Cas9 technology in common neurological disorders aiming clinicians to understand the technology.

How to cite this article:
Swarup V, Kumar V, Faruq M, Singh HN, Singh I, Srivastava AK. CRISPR/Cas9 technology in neurological disorders: An update for clinicians.Ann Mov Disord 2020;3:23-32

How to cite this URL:
Swarup V, Kumar V, Faruq M, Singh HN, Singh I, Srivastava AK. CRISPR/Cas9 technology in neurological disorders: An update for clinicians. Ann Mov Disord [serial online] 2020 [cited 2021 Oct 18 ];3:23-32
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The CRISPR–Cas9-based genome editing is a most advanced, highly precise, programmable and affordable endonuclease-based powerful tool to interrogate and manipulate gene expressions. The CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. The Cas9 (CRISPR associated protein-9) is an endonuclease enzyme, which cuts the target deoxyribonucleic acid (DNA) site. This technology is derived from adaptive immune system present in archaea and eubacteria against foreign nucleic acids such as plasmids and bacteriophages.[1],[2] This immunity is heritable and leads to new resistant colonies against previously faced viruses.

This CRISPR–Cas9 complex is made of highly customizable single-guide ribonucleic acid (sgRNA or gRNA), which binds to Cas9 nuclease. The sgRNA has two components: a ~20 nucleotides long customizable sequence called “spacer” and scaffold sequence, which binds and holds Cas9 enzyme. The Cas9 recognizes protospacer adjacent motif (PAM), which is 5′-NGG or 5′-NAG sequence (N is any nucleotide) located in the target site.[3],[4] The Cas9 enzyme has two nuclease domains, RuvC and HNH, which cuts different strands of DNA generating double-stranded breaks (DSBs) that are repaired by cellular DNA repair machinery leading to certain (desired) changes.

The CRISPR-based technology is more efficient and precise than other already known gene-editing systems such as TALENs (transcription activator-like effector nucleases) and ZFNs (zinc-finger nucleases).[5] These methods are costly and involve much higher off-target effects and cellular toxicity.[6],[7],[8] The successful delivery of these nucleases inside cells also poses a major challenge toward their efficacy. Moreover, targeting more than one gene for editing (i.e., multiplexing) is also difficult as compared with CRISPR-based methods, which altogether saves time, cost, and efforts.[9] The gene-editing performed by CRISPR is heritable as shown by rat offsprings when four gRNAs were simultaneously transfected.[10]

The high-throughput DNA and RNA sequencing has added new dimensions to the discovery of new mutations in most human diseases. List of new neurodegenerative diseases associated with genetic changes has been growing rapidly. However, most of genetic neurodegenerative disorders are non-treatable till date and only symptomatic treatment is given to patients. The complex mechanism of many of these disorders is still unclear, which makes developing new therapeutics a big challenge. Highly effective methods are required to correct genetic mutations in monogenic neurodegenerative diseases. The CRISPR-based gene-editing technology offers in both ways as it helps in understanding disease mechanisms by generating disease models as well as work as a “molecular tool” to modify the mutated genes acting as therapeutic tool. This advanced gene-editing technology has been used in developing animal models, cellular models, iPSCs (induced pluripotent stem cells), and targeting causative mutations in various neurodegenerative disorders. This review highlights the functionality of the CRISPR–Cas9 system, its applications, and summarizes the use of this technology in neurodegenerative diseases, especially in common neurological disorders.

 How Does this technology Work?

The process starts with designing sgRNAs against target DNA sequences. The target may be a coding or noncoding part of genome. The sgRNAs must fulfill two conditions: (1) the target sequence must be unique as compared to rest of genome and (2) it must be adjacent to PAM sites. Actually, the PAM sites are binding signal to enzyme Cas9.[11],[12] After designing and synthesizing sgRNAs, these are transfected into the target cells along with Cas9 enzyme as a complex ribonucleoprotein (RNP) or as plasmids containing their sequences. Various strategies are available to deliver these plasmids or RNP complexes into host cells by viral vectors (adeno-associated virus [AAV] or lentiviruses), lipofectamines (liposomes and nanoparticles), or via electroporation (creates temporary pores in cell membrane by applying high voltage).

The inserted plasmids translate multiple copies of sgRNA and Cas9, which interact to form sgRNA–Cas9 complex inside cell. The cleavage takes place at small RNA–DNA duplex, which is formed between spacer of sgRNA and its complementary DNA sequence near to PAM site. This cleaved DNA is repaired using either HDR (homology direct repair) or NHEJ (nonhomologous end joining) pathways. Here also, the user can decide which pathway to be followed inside cell. If some insertions/indels/frameshift are required at cleaved site, then NHEJ mode of repair is followed. However, these changes may disrupt the open reading frame (ORF) of gene resulting to activate or repress it[13] [Figure 1].{Figure 1}

The HDR pathway repairs the target sequence ranging from single to large nucleotide size. It uses a “repair template” containing the desired change in DNA, which has to be transfected along with sgRNA–Cas9 transfection into the cell. This template also carries homologous sequence up- and downstream of the modified sequence that complements the cut ends and helps the repairing enzymes.[13] Absence of PAM sequence in the repair template protects it from Cas9 editing. The efficiency of HDR-mediated repair is low and the DSBs are repaired by NHEJ method at several places of cleavage. Therefore, the resultant cells are mixture of unedited (wild type), NHEJ-mediated edited, and/or HDR-mediated edited cells [Figure 1].

 Application of CRISPR/Cas9 technology

Inhibition/activation of target gene: As aforementioned, Cas9 has two nuclease domains, RuvC and HNH, which cleave the opposite strands. The nuclease ability of Cas9 can be altered by inducing point mutations in these two regions. This dead Cas9 (dCas9) is capable of binding to the target DNA but unable to cut it.[14] If the target site is regulatory region of the gene of interest, then the dCas9–DNA complex blocks the transcription initiation leading to CRISPR-mediated inhibition of transcription (CRISPRi).[15] Alternatively, the dCas9 can also be fused with some enhancers that lead to high transcription of the target gene. This is known as CRISPRa.[16],[17]

Epigenetic modification: Epigenetic changes (tags) are not regulated by genetic codes; they affect the gene expression by reversible modification of the nucleotides of DNA and histone proteins. The epigenetic tags include adding or removing of methyl, acetyl, ubiquitin, and phosphate groups to nucleotides/histones. The dCas9 has been successfully fused with several epigenetic modifiers such as p300 (histone acetyltransferase), LSD1 (lysine-specific demethylase 1), and SMYD3 (SET and MYND Domain Containing 3) proteins to locate various epigenetic tags.[18],[19] The distinctive features of these tools are that do not create DSB but they modify the nucleotides of DNA (e.g., cytosine to methyl cytosine) or histone proteins (histone H3) switching on/off the target gene.

Multiplexing: This can be achieved by transfecting more than one sgRNA to the target cells. These sgRNAs either can be cloned into single plasmid or required number of sgRNAs (as RNPs) can be directly delivered into the cells.[9] The plasmid will multiply sgRNAs inside cells, whereas RNPs will directly act on the target sites. This in turn increases the editing of the target. The target sites of these multiple sgRNAs can be single or many or even large genome deleting/editing can also be aimed by removing the sequence between two target sites. As present, the multiplexing has been reported anywhere between 2 and 7 gene loci by cloning sgRNAs into single plasmid.[20]

Genome wide scans using CRISPR: This is performed by generating large population of cells containing desired mutation or activated/repressed state of some gene(s) of interest and subsequently these cells are used to identify the genetic perturbations that result in desired phenotypes. The forward genetic screens are particularly useful for studying diverse phenotypes, of which the underlying cause is not known. CRISPR technology is capable of making highly specific and permanent genetic modifications that ablate the functions of target genes.[21],[22] This technology has already been used extensively in screening of novel genes that regulate known phenotypes involved in drug resistance, cell viability, and metastatic tumor sites.

Visualize genomic loci using fluorophores: Here the dCas9 is tagged with fluorescent marker such as GFP (green fluorescent protein) and VFP (violent fluorescent protein).[23],[24] These tagged gRNAs bind to target genomic site and will emit fluorescence when irradiated with the light of specific wavelength. Live cells can also be visualized using CRISPR imaging. Multiple genetic loci can be targeted with user-friendly technology of sgRNA designing. FISH (fluorescent in situ hybridization) can also be integrated with CRISPR to divulge chromatin dynamics in live cells.[25] More than one type of fluorophore can be used making it multicolor. If the whole chromosome is targeted by multiple fluorophore-tagged sgRNAs, then it is called “chromosome painting.”[26],[27]

Purify genomic regions: The expression of any gene is regulated by several transcription factors (TFs) binding to the regulatory regions. Identifying these sequences (DNA) and TFs and modulating their features can help in controlling expression of those genes.[28] Like CRISPR imaging, tagging dCas9 by biotin, epitopes (3×FLAG) or anti-Cas9 antibodies can be used to bind any target gene sequences (genic or intergenic).[29],[30] The dCas9 bound to tags can be pulled by their attached tags. After purification, the bound fraction can be identified by next-generation sequencing (DNA) or mass spectrometry (TFs). This CRISPR-based purification is more useful in identifying DNA of interest and its bound proteins (TFs) in vitro.

 CRISPR/Cas9 toward Therapeutics

Numerous neurodegenerative diseases have been targeted by CRISPR technology either toward developing disease models or therapeutics. First report of use of this technology in neurodegenerative diseases was in prion disease. The PRP gene (PrP) was deleted to ablate its expression in N2a neuroblastoma cells, C2C12 myoblasts, and NMuMG epithelial cells.[31] In this review, we have highlighted the current status of CRISPR–Cas9 technology in most common neurological disorders.

Alzheimer’s disease

Alzheimer’s disease (AD) is a progressive and most common neurodegenerative disorder and known as leading cause of death among elderly people. Although majority of AD cases are sporadic, several genetic mutations have been linked to onset of AD. Mutations in presenilins (PSEN1 and PSEN2), catalytic subunits of γ-secretase, and amyloid precursor protein (APP) are linked with early onset of AD, whereas mutations in apolipoprotein-E (ApoE) have been associated with late onset of the disease.[6],[32] Abnormal cleavage of APP protein by β-secretase-1 enzyme (coded by BACE1 gene) is crucial for amyloid β (Aβ) peptide production amyloid plaques, which are characteristic features of AD.[33]

Significant reduction in amyloid plaques accumulation and decreased amyloid β secretion from postmitotic neurons of hippocampus were observed when BACE1 gene was targeted via nanoparticles coated Cas9–sgRNA complex in AD knock-in (KI) mouse models. Multiple deliveries of nanoparticles into hippocampus region were found to be more effective in suppressing BACE1 gene.[34] Earlier in 2018, two Swedish mutations were knocked out (KO) in human fibroblasts using CRISPR technology reducing levels of mutant APP protein without changing its wild-type form[35] [Figure 2]. Subsequently, this was tried on mice models carrying same mutation where animal viruses (vectors) were used to deliver CRISPR–Cas9 complex directly into hippocampus of adult mice and results were same as those of in vitro study. This was the first study of viral-based delivery of CRISPR in vivo in AD disease models. Successful rescue of the PSEN1 mutations by CRISPR, APP-KO in human stem-cell-derived cortical neurons has been recently achieved.[36] Mutations in APP and PSEN1 cause major defects in lysosome function and autophagy in iPSC-derived human neurons. Deletion of APP in PSEN1 Y115C neurons reduced LAMP1 protein and increased the axonal transport of lysosomes when compared with isogenic PSEN1 Y115C neurons. In an another study, it was shown that CRISPR/Cas9-mediated correction of the PSEN2 point mutation in forebrain cholinergic neurons abolished the electrophysiological deficit and thereby restoring the maximal number of spikes and spike height as compared to the levels observed in the controls. Moreover, increased Aβ42/40 was also normalized post-CRISPR/Cas9-mediated correction of the PSEN2N141I mutation.[37]{Figure 2}

The polymorphism of KIBRA (KIdney and BRAin expressed protein) is well associated with cognitive performance in AD. Song et al.[38] recently developed a CRISPR-based KIBRA-KO mouse to investigate its role in AD. They found high neuronal loss in KIBRA-KO mice in hippocampus by apoptosis, whereas the overexpression of KIBRA in neuronal cell lines significantly promoted its proliferation and inhibited Aβ-induced apoptosis. This showed that KIBRA functions as a neural protective gene that promotes cell survival and inhibits Aβ-induced apoptosis making it a potentially therapeutic target for AD treatment.

Parkinson’s disease

Parkinson’s disease (PD) is progressive neurodegenerative disorder, which is caused by death of dopaminergic neurons in substantia nigra. Sporadic as well as genetic causes such as mutations in genes PINK1 (PTEN-induced kinase 1), SNCA (α-synuclein), and LRRK2 (leucine-rich repeat kinase 2) have been established in PD. The hallmark features of PD are presence of Lewy bodies filled with mutant α-synuclein protein in dying or dead neurons. Multiple copies of SNCA gene fasten the synucleopathy causing severe disease. CRISPRi was used to reduce the expression of SNCA gene in neuronal and other cell lines and found high suppression up to 60% of the same.[39] Later, Kantor et al.[40] tried to modulate methylation (epigenetic) tags located in intron1 of SNCA gene that controls its expression. They used “all-in-one” viral vector, that is, fused dCas9 with catalytic domain of DNA methyltransferase-3A enzyme to transfect iPSC-derived dopaminergic neurons containing triplicate copies of SNCA gene. Reduced levels SNCA mRNA and protein were noticed and other parameters such as mitochondrial ROS and cellular viability were improved. Activated mitochondrial uniporter (MCU; calcium channels in neurons) and calcium-mediated mitochondrial dysfunction also stimulate the death of dopaminergic neurons in PD.[41] In this study, Lee et al.[41] used PD model of zebrafish, which had PINK gene deleted (PINK–/–) and showed abnormal mitochondrial functioning. When the MCU gene was deactivated by inducing a frameshift mutation in its exon 3 of this PD model using CRISPR technology, the mitochondrial functioning was restored. This showed protective effects of MCU gene inactivation in PD models and projects this gene to develop new therapeutics[42] [Figure 2].

Recently, another IPSC line of PD-containing tyrosine hydroxylase (TH) gene tagged with GFP has been developed using CRISPR/Cas9 technique. The TH enzyme performs rate-limiting step during biosynthesis of dopamine and has been established as a reliable marker for dopaminergic neurons.[43] This GFP-tagged gene construct was very helpful in monitoring transfection efficiency.[44] In another report, the IPSC-derived astrocytes were developed containing mutation (G2019S mutation) in LRRK2 gene, which has been reported in autosomal-dominant PD cases. These PD astrocytes showed impaired autophagic machinery accumulation of endogenous α-synuclein in PD astrocytes.[45]

Higher organisms share much similar cellular physiology with humans and animals such as pigs and chimpanzees. Benefits of using such animal disease models are that they process the diseased proteins in almost same manner and produce same symptoms and prognosis approximately as that of humans. A PD model of Guangxi Bama Minipigs has been generated in which three mutations (E46K, H50Q, and G51D) in SCNA were introduced using CRISPR technology. These heterozygous mutations were reconfirmed at genomic level (DNA sequencing) and transcriptomic level (mRNA analysis). Although the genetically modified animals did not show disease symptoms at 3 months of age, aging might introduce disease symptoms in these animals.[46]

Huntington’s disease

Huntington’s disease (HD) is another well-known genetic neurodegenerative disorder caused by the overexpansion of cytosine-adenine-guanine (CAG) repeats in huntingtin (HTT) gene. It is an autosomal-dominant phenotype, which means that heterozygous expansion will cause phenotype and making these repeats as therapeutic targets using CRISPR–Cas9 technology.

CRISPR-based deletion of 44-kb region including promoter, transcription start site, and the CAG expansion mutation of the mutant HTT gene was carried out to completely inactivate the mutant allele. This resulted in complete inhibition of mHTT mRNA and protein without impacting the normal allele[47] [Figure 2]. In another study, the 5′-UTR (untranslated region) and exon-1-intron boundary was targeted by CRISPR–Cas9 method to interfere with their transcription. The plasmid-based delivery of CRISPR–Cas9 was carried out in mesenchymal stem cells (MSCs) extracted from the bone marrow of YAC128 mice, an HD model. The translation of mutant HTT protein was reduced in MSC cells stepping toward HD therapeutics.[48] A most recent protocol based on use of Cas9 nickase (which cuts only one strand of target DNA and does not produce DSB) to cut and replace the expanded CAG repeats with normal CAG repeats has shown reduction in protein levels by 70%.[49] In another report, delivery of Cas9 via AAV was done directly into striatum to disrupt mutant HTT in HD mice model, R6/2. The disruption of mutant protein reduced the formation of neurotoxic inclusions by twofold and improved motor deficits leading to longer life span of the animal as compared to non-transfected mice.[50]

Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal-recessive phenotype caused by homozygous expansion of guanine-adenine-adenine (GAA) trinucleotides in the first intron of FXN gene, which codes a crucial mitochondrial protein frataxin. Levels of frataxin protein are inversely related to disease severity. It is an early-onset disorder characterized by gait ataxia, difficulty in speech and swallowing, and hypertrophic cardiomyopathy leading patient to wheelchair bound and death in early 20s.

Systemic delivery of viral vectors was carried out in YG8sR and YG8R mice models of FRDA. These mice models contain one and two tandem copies of human FXN transgene, respectively. In CRISPR–Cas9-transfected YG8sR mice, the levels of frataxin protein were increased, whereas in YG8R mice the protein levels were reduced [Figure 2]. This could be explained by presence of two copies of FXN gene in the later; deletion of one copy led to reduced protein expression. However, the successful delivery of Cas9 complex in mice models paved the promising path of future therapeutics of FRDA.[51]

Recently, a new inducible cellular model of FRDA, HEK-CFXN, has been developed by KO of endogenous FXN gene of HEK293 cells via CRISPR–Cas9 method and adding an “inducible” FXN gene. Different levels of frataxin protein could be produced by switching it “on.” Interestingly, this study also showed that overexpression of frataxin protein might led to high oxidative stress and labile iron pool, which were as toxic as in cells of patients with FRDA. However, this new cellular model provides useful cellular model to investigate role of frataxin further in cellular physiology.

Fragile-X syndrome

Fragile-X syndrome (FXS) is an X-linked dominant phenotype caused by cytosine-guanine-guanine (CGG) trinucleotide repeat expansion in 5′-UTR of FMR1 gene. Full-mutation alleles contain more than 200 CGG repeats and hypermethylation upstream to these repeats leads to a loss of FMR1-encoded protein (FMRP). Presence of abnormal repeat expansion upstream to coding region provides nice opportunity to edit these sequences to develop therapeutics. The hypermethylated state of upstream sequence was spontaneously corrected (i.e., demethylated) when CGG repeats were excised in both embryonic stem cells and iPSCs derived from patients with FXS [Figure 2]. The FMRP expression was restored and maintained in precursor and mature neuron cells.[52] The restoration of FMRP was also achieved in somatic hybrid cell line (Y75) and patient-derived IPSCs when these pathogenic CGG repeats were excised using CRISPR technology. The status of methylation upstream to these repeats was not investigated.[53] In another study, Cas9 was tagged with a transcriptional activator VP192 aiming to enhance FMR1 transcription, which is pathogenically reduced due to expansion of CGG repeats. This tagged CRISPR–Cas9 complex was able to increase FMRP levels in hESCs (human embryonic stem cells) model of FXS.[54] It was interesting to see that the hypermethylation of upstream sequence did not affect the activating effects of tagged Cas9.

The nanoparticle-based delivery of CRIPSR-Cas9 complex (RNP), named as CRISPR–gold, was used to transfect primary cultured hippocampal neurons of mouse models of FXS. The cytotoxicity was very low and transfection did not show adverse effects on neuronal membrane health or neuronal excitability. Subsequently, the same CRISPR–gold was used to deliver RNP complex targeting Grm5 gene inside brain of FMR1–/– (FMR1 gene deficient) mice, an FXS mouse model. The Grm5 gene codes glutamate metabotropic receptor 5 (mGluR5), which is exaggerated and has been well associated with FXS pathophysiology.[55] The transfected mice showed reduction in mGluR5 levels (40%–50%) with no significant adverse effects. The behavioral changes (excessive digging and jumping behavior) were also normalized in these mice.[56] Recently, a homozygous FMR1-KO hESCs line have been generated using CRISPR–Cas9 technology containing a homozygous 280 nucleotide deletion at exon 1, removing the start codon. This hESC line was able to differentiate and retained pluripotency and normal karyotype.[57]

Spinocerebellar ataxias

Spinocerebellar ataxia type 2 (SCA2) is an autosomal-dominant disorder caused by expansion of (CAG) trinucleotides in the first exon of ATXN2 gene. Patients with SCA2 develop gait ataxia, double vision, and problem in speech with time. Research group of Marthaler used CRISPR–Cas9 technology to generate “corrected” cell lines of SCA2. They replaced pathogenic (CAG) repeats (n = 36) to develop iPSC line of SCA2 and named H196.[58] These corrected cells showed normal karyotype and did not carry any frameshift or any other mutation in those cells [Figure 2]. They used cells of other patients and similarly developed two more iPSC cell lines, named H271 and H266, in which they replaced (CAG) 44 repeats with normal (CAG)22 repeats.[59],[60] These cell lines will provide new and true control iPSC cell lines for SCA2 investigation.

In spinocerebellar ataxia type 3 (SCA3), Ouyang et al.[61] reported deletion of pathogenic (CAG) 78 repeats in ATX3 gene from patient-derived iPSCs. The corrected cells retained pluripotency and were able to differentiate further into neuronal cells.

Duchene muscular dystrophy

Duchene muscular dystrophy (DMD) is a severe and most prevalent neuromuscular disease. Diagnosed between 3 and 5 years of age, it starts with weakening of truncal and lower limb muscles and progresses with abnormal gait and motor delay. In 10 years of onset, the patients become wheelchair bound.[62] It is an X-linked recessive disease that is caused by mutation in short arm of X-chromosome and therefore limited to boys. The DMD gene, dystrophin (DMD), is the longest gene known containing 2.3 × 106 base pairs. More than 4000 mutations have been reported till date in which two-thirds are large deletions spread over exons 2–20 and 44–53. The DMD gene has been targeted in vitro and in vivo by various technologies in numerous studies. Most of these reports focused on restoring mutated regions in exons [Figure 2].

The DMD protein function was restored up to 18% in mdx mice model of DMD (mdx mice possess mutation in exon 23).[63],[64] Both NHEJ and HDR variation of CRISPR were tried in these studies aiming to normalize cC3185T mutation of exon 23 in mdx mice model. Furthermore, restoration of target protein was obtained up to 80% of wild-type mice in another DMD mice model lacking exon 50 (∆exon 50)[65] [Figure 2]. Viral (AAV) delivery of Cas9–gRNA complex was used to attain this high restoration frequency. Use of gold-coated nanoparticles in CRISPR (CRISPR–gold) was injected intramuscular in mdx mice. Results showed that dystrophin levels were restored and muscle strength was also improved. Zhang et al.[66] used a new Cas9 nuclease, Cpf1, to correct mutated DMD gene in iPSCs of mice model (∆exon 50). The Cpf1 nuclease differs from Cas9 that it produces blunt ends and requires T-rich PAMs.

Myotonic dystrophy

Myotonic dystrophy (DM) is most common dystrophy among adults caused by nucleotide expansion mutation in causative genes. Abnormal expansion of cytosine-thymine-guanine (CTG) trinucleotide repeats in 3′-UTR of DMPK gene on 19th chromosome causes type-1 myotonic dystrophy (DM1), whereas expansion of (CCTG) tetranucleotide in intron-1 of CNBP gene leads to type-2 myotonic dystrophy (DM2). The disease starts with weakening and wasting of muscles, myotonia, and difficulties in respiration. Pathogenic nucleotide expansions have been targets of gene-editing tools in DMPK/CNBP genes. Interestingly, complete excision of DMPK gene did not affect survival of cells. The edited cells (myoblasts) showed no sign of pathogenic ribonuclear foci in their nuclei. Later, modified dCas9 was successful in removing mutated mRNA resulting in reduction of up to 90% in DM1 myoblasts[67] [Figure 2]. In another study, dCas9 was delivered by viral vectors (AAV) in temporal vein of DM1 mice model. This led to improvement of myotonia in most of mice.[68]

 Clinical Trials

CRISPR-based clinical trial has not yet been started in neurodegenerative diseases. Ongoing majority of ongoing clinical trials follow immunotherapy in cancers. CRISPR-based editing of antigen receptors of T cells (CAR-T technology) is being tried for treatment of melanoma, sarcoma, and multiple myeloma in the USA.[69] Another clinical CAR-T has been started on B-cell leukemia and lymphoma, metastatic non-small-cell lung carcinoma (NSCLC), and invasive bladder cancer in China.[70] In addition to cancers, locally injectable CRISPR–Cas9-based eye treatment for Leber congenital amaurosis (LCA) is undergoing clinical trial.[71] The most common mutation in causative CEP20 gene is under target by Cas9 aiming to improve function of photoreceptor cells before disease progresses to vision loss.

 Challenges and Limitations of CRISPR–Cas9 Technology in Neurological Disorders

This is a comparatively new and more advanced technology of gene editing but there are several setbacks: first being the host genome itself. Targets to modify may have enough sequence resemblance (similarity) with other part of genome that leads to unintended cuts in host genome. Thus, undesired mutations are generated, which may affect overall health and even survival of host organism adversely. In vivo delivery is another limiting factor. Crossing blood–brain barrier is an utmost challenge for components of this gene-editing technology.[72] Although several mechanisms (aforementioned) have already been developed, safety of host is a major concern. Viral-mediated delivery in vivo is still questionable as long-term effects of using such animal viruses and the ramifications of stripping down the CRISPR components on their effectiveness in the brain are still under investigation. Precise and safe delivery to target tissues, sparing healthy ones, is also a major concern for liposome/nanoparticle-based transfection of CRISPR–Cas9 components. Further, the HDR-mediated gene editing requires efficient DNA repair machinery, which may be less active in postmitotic cells such as neurons.[73] Allele-specific gene correction is another big concern to overcome. Although several attempts to correct pathogenic allele have been made in HD, other disorders such as SCA types -1, -2, and -3 pose a big challenge toward the implementation of this technology in clinical settings.[47]

Immune response to CRISPR–Cas9 and the presence of preexisting antibodies against Cas9 (a protein) could be a significant hurdle especially for in vivo gene editing. It is highly desirable to exercise more rigorous assessment of possible immunological responses to the microbial origin of the system.[74] However, delivery of RNP complexes and codon optimization may hold the key to overcome such hurdles.[75]

 Future Prospects in Neurological Disorders

Several single-nucleotide polymorphisms (SNPs) have been found in different neurodegenerative diseases. The role of such SNPs can be investigated by combining dCas9 with nucleotide-modifying enzymes such as cytidine deaminase or adenosine deaminase, which converts the single nucleotide to its derivative without causing DSBs.[76] Remodeling of promoters, enhancers, and repressors that decide the levels of the genes needs more investigation using different applications of CRISPR technology such as CRISPRi, CRISPRa, or dCas9. Role of noncoding moieties such as microRNAs, Piwi-interacting RNA (piRNAs), and long non-coding RNAs (ncRNAs) has to be interrogated further, which has been involved in several neurodegenerative diseases. Furthermore, new animal models expressing the diseases more accurately have to be derived from CRISPR–Cas9-generated iPSCs of neurodegenerative diseases, which has been carried out to create iPSC-derived kidney[77] and intestinal organoids.[78] In above of all, the off-target effects which Cas9 induces while binding and cleaving the nontarget sequences need to be addressed by enhancing the efficiency of Cas9. However, newer gene-editing enzymes such as Cpf1, Cas12a, and CasX possessing better editing efficiency and lesser off-target effects have also been investigated in various diseases.[79],[80]


The advantages of CRISPR–Cas9 technology such as high precision, easy customization, better multiplexing, and cost-effectiveness over other conventional gene-editing method have been proven in numerous studies. However, the delivery of sgRNAs and Cas9 moieties inside target cells and off-target effects thereof are still challenges toward success of this method. Although this technology has unraveled several new pathogenic mechanisms in neurological disorders, several disease models have been prepared for these to target that defective gene in monogenic disorders is still not completely achieved in many such cases. Targeting host sequence in a particular cell type in tissue/organ and reducing off-target effects is difficult but will be achievable in near future using more advanced version editing enzymes. However, editing sequence of gene is irreversible and therefore utmost precautions must be taken to minimize harmful genetic changes by scheduling timing of CRISPR–Cas9 delivery and its dosage. These trials will pave the way of CRISPR–Cas9 technology to establish itself in routine clinic providing an opportunity to save lives.

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Conflicts of interest

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