REFERENCES 1

ABSTRACT:
With the development of gene discovery methods and gene sequencing
technologies over the past few decades, many genetic variations that increase
the risk of neurodegenerative diseases or cause them have been identified.
However, the discovery of these pathogenic mutations and subsequent
mechanism-based studies have not always led to effective treatments, thus
necessitating alternative approaches for pinpointing rationally oriented
therapeutic targets. Nevertheless, with the evolution of gene targeting methods,
it is becoming increasingly obvious that even when how its biological function
is not fully understood, the disease-causing gene itself has the potential to be the
most promising therapeutic target. This is where the potential for CRISPR/Cas
gene-editing technology comes into play, which offers the promise of
permanent elimination or correction of the genetic mutations that cause
diseases, potentially overcoming key limitations of RNA-targeting approaches.
In the future, CRISPR/Cas-based strategies might allow the treatment of
diseases such as neurodegenerative diseases, which are challenging to treat with
other treatments. The purpose of this paper is to provide perspectives on
therapeutic gene editing for diseases of the nervous system by reviewing recent
reports of preclinical applications of CRISPR-Cas in Huntington, Alzheimer’s
and Parkinson type of neurodegenerative disorders.

KEYWORDS: CRISPR/cas9, neurodegenerative disease, Huntington Disease,

Alzheimer’s Disease, Parkinson Disease.
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INTRODUCTION:
In the field of neurodegenerative diseases, disease conditions characterized by
the loss of neurons in the nervous system are collectively referred to as
neurodegenerative diseases. Movement, balance, breathing, cognition, and
behaviour are affected by progressive neurodegenerative processes. As a result,
neurodegenerative diseases gradually lead to an individual's dependence,
resulting in a devastating experience for those afflicted and their families. The
lack of effective treatments for most neurodegenerative disorders, in spite of
multiple implicated underlying mechanisms and even known causal mutations,
can be explained by the difficulty in identifying rational drug targets by
mechanism-focused approaches. Although novel gene-editing techniques such
as CRISPR (clustered regularly interspaced short palindromic repeats)/Cas
(CRISPR-associated systems) are developing, they may not yet offer the same
benefits as traditional approaches
There are several clinical and preclinical uses of CRISPR/Cas, including gene
function analysis, disease modelling, and preclinical studies.6–13 Preclinical
investigations or clinical applications of CRISPR/Cas for disease treatment may
offer a few advantages over RNA-lowering approaches. The CRISPR/Cas
method overcomes several of the limitations of traditional RNAi and antisense
oligonucleotides (ASO) technologies, such as high off-target activity and a need
for repeated treatments, both of which may put patients suffering from chronic
progressive neurodegenerative diseases at risk for complications. Contrary to
this, most CRISPR/Cas approaches cause irreversible changes in the DNA (e.g.,
inactivation or correction), and therefore if the cell is properly targeted, it needs
only one treatment. These results suggest that CRISPR/Cas approaches may
have an application to the treatment of neurodegenerative diseases.
CRISPR/Cas techniques have been shown to edit genome content in postmitotic
neurons and mammalian brains. CRISPR/Cas is also highly flexible and
therefore suitable for treating neurodegenerative diseases caused by loss-of-
function mutations, providing a significant advantage over RNA-lowering
methods, which are primarily focused on the treatment of gain-of-function
mutations. A key difference between CRISPR/Cas and other gene-editing
techniques is that CRISPR/Cas is site-specific due to its interaction with the
guide RNA (gRNA). CRISPR/Cas does not require protein engineering, so this
method is highly feasible and affordable.
Preclinical studies have demonstrated therapeutic potential using various
CRISPR/ Cas strategies applied to models of neurodegenerative diseases.
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Neurodegenerative diseases can be treated by CRISPR/Cas in one of three

ways: (1) correction of disease-causing mutations; (2) inactivation of gain-of-
function mutations; and (3) modulation of transcription. We discuss recent
applications of CRISPR/Cas9 to neurodegenerative diseases in this review
article, with an emphasis on gene editing strategies based on CRISPR/Cas9.(32-
48)
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CRISPR:
Clustered regularly interspaced short palindromic repeats (CRISPR) and
CRISPR-associated proteins (Cas) systems are the RNA-mediated immune
device in prokaryotes that protects them in opposition to bacteriophage and
plasmids.

Although currently evolved programmable modifying tools, inclusive of zinc

finger nucleases and transcription activator-like effector nucleases, have
extensively stepped forward the capability for particular genome modification,
those strategies have limitations. CRISPR (clustered often interspaced brief
palindromic repeats)/Cas9 era represents a enormous development over those
different next-technology genome modifying tools, accomplishing a brand new
degree of focused on, efficiency, and simplicity of use. The CRISPR/Cas9
gadget permits for site-precise genomic focused on in virtually any organism.
(Europe, 2018)
CRISPR-Cas9 Type II systems are widely used for targeted genome
enhancement in a variety of organisms including bacteria, yeast, and mammals
(Singh, 2020)
This type of CRISPR/Cas system uses noncoding RNAs to guide the Cas9
nuclease to cleave specific DNA segments at specific locations in the genome.
Damaged DNA is then repaired by other pathways. Both non-homologous quits
joining DNA repair (NHEJ) and homology-directed repair (HDR) are
mechanisms used to restore DNA in cells. (Europe, 2018)
It has been demonstrated that the CRISPR/Cas9 system can be harnessed to
mediate gene editing in mammals using RNA-programmable RNAs A gene
knockout can be produced (via insertions/deletions) or a knockin can be
generated (via HDR). The disruption of a gene involves a single DNA insertion
in the relevant gene (Figure 1). Cas9 nuclease is directed to a specific genomic
location by guide RNA (sgRNA). Double-strand breaks induced by Cas9 are
repaired by sgRNA by means of the NHEJ DNA repair pathway. Errors will
occur during the repair process causing insertions and deletions (INDELs) and
thereby disrupting genes expression. (Europe, 2018)

The principle of CRISPR/Cas9-mediated gene disruption. It contains a sequence

derived from a crRNA that is specific to DNA targets, and it's called a single
 REFERENCES 5

guide RNA (sgRNA) Cas9 protein is recombinantly expressed in inclusion

bodies that bind to a DNA endonuclease-activating version of the tracrRNA.
The A resultant complex is responsible for cleaving double-stranded DNA
specific to each target. The nonhomologous end-joining DNA will be used to
repair the cleavage site INDEL insertions or deletions may disrupt gene
function in the repair pathway, an error-prone process. (Europe, 2018)

The newest genome editing technology, CRISPR/Cas9, allows for genomic

targeting, efficiency, and precision previously unattainable simplicity. Guide-it
products further improve the usability of the CRISPR/Cas9 system by providing
a streamlined method for:

How gene therapy is different from CRISPR.

Genetic editing is the modification of a living organism's genetic make-up by
making highly specific changes to its DNA sequence. Enzymes, in particular
nucleases that are specifically designed to target specific DNA sequences, can
be used for gene editing, which involves making cuts in DNA strands to remove
existing DNA and then replacing it with replacement DNA. There are several
gene-editing technologies, and perhaps the one most important is CRISPR-
Cas9, a powerful tool discovered in 2012 by American scientists Jennifer
Doudna, Emmanuelle Charpentier, and colleagues. This tool has since been
improved by American scientists Feng Zhang and colleagues. Researchers were
able to precisely remove and insert DNA by using CRISPR-Cas9. (gene editing,
January 07, 2022)

Several approaches have previously been used to induce site-specific double-

strand breaks in DNA, including zinc-finger nucleases (ZFNs) and transcription
activator-like effector nucleases (TALENs). (gene editing, January 07, 2022)

CRISPR-Cas9 differs from ZFNs and TALENs in that RNA-DNA binding,

rather than protein-DNA binding, is employed to guide nuclease activity,
simplifying the design and allowing the broad application. Bacterial adaptive
immunity contributed to the development of CRISPR-Cas9. CRISPR refers to
clustered regularly interspaced short palindromic repeats found in the genomes
of the vast majority of bacteria. A sequence derived clearly from the genomes of
bacterial pathogens is sandwiched between the short palindromic repeats. An
older spacer is found at the distal end of a cluster, whereas a "newer" spacer,
which represents a pathogen that has been encountered more recently, is found
at the proximal end of the cluster. (gene editing, January 07, 2022)
 REFERENCES 6

Development of CRISPR-CAS9 system for genome editing/ Application:

1. Microbial genome editing using CRISPR-Cas9 system

It is currently being developed and used for targeting genome editing of

pathogenic microorganisms for the control of infections and diseases using the
CRISPR-Cas9 system. Additionally, it has been used to improve the production
of biofuels, metabolites, chemicals, and antibiotics by modifying beneficial
microorganisms. CRISPR-Cas9 has been employed in the treatment of
infectious diseases. (Singh, 2020) (Sierra, 2015) (Bikard, 2014).

CRISPR-Cas9 has been used to knock out drug resistance genes and virulence
factors, among many others. Using this system, targeted genome editing has
been achieved in various microbes. (Wenyan Jiang, 2013).

Researchers developed a dual-RNA: Cas9 system whose specificity could be

changed by changing nucleotides present in crRNA. To produce multiplex
mutagenesis, two crRNAs were used simultaneously and tested in Streptococcus
pneumoniae and E. coli. This approach resulted in 100% efficiency with 60
desired mutations. (Wenyan Jiang, 2013)

Additionally, (Bikard, 2014) developed a CRISPR-Cas9 system capable of

removing the targeted pathogen from a mixed population based on a sequence-
specific method. By inserting Cas9 and tracrRNAs into the staphylococcal
vector, they were able to generate crRNA.

Staphylococcus aureus was targeted and killed by the vector via the aph-3 gene
(kanamycin resistance) while the non-pathogenic S. aureus population was not
affected. For the purpose of testing the functionality of CRISPR-Cas9
multiplexing, phagemids have been engineered with an array of CRISPR and
have been used to either target a portion of the mecA gene or the enterotoxin's
superantigen sek gene. The strain became lethal as a result of this. (Bikard,
2014)

There is a growing problem of antibiotic resistance worldwide. The most

effective way to kill the resistant strains is to target them precisely.
A new technique called CRISPR has been created to kill only pathogenic
bacteria without harming beneficial bacteria. Using the CRISPR-Cas9 platform,
we targeted and killed extended spectrum beta-lactamases (ESBLs)-producing
E. coli. Multi-drug resistance (MDR) is commonly associated with ESBLs, and
it is a form of horizontal transmission of antibiotic resistance mediated by
plasmids. Antibiotic resistance genes were specifically targeted to re-sensitize
the pathogen to its appropriate antibiotic. (Bikard, Using CRISPR-Cas systems
as antimicrobials, 2017) (Beisel, 2014) (Gomaa, 2014)
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2. Viral genome editing using CRISPR-Cas9 system Viruses

All organisms have the potential to become infected with viruses, which are
small particles. Most viruses are responsible for causing diseases in animals and
plants. Antiretroviral therapy (ART) has been used to control viral infections in
the past, but it is not currently able to cure the infection. There are instances in
which the viral genome may merge with the animal genome and become
latently present, causing a number of serious diseases. As an anti-viral therapy,
CRISPR/Cas9 has revolutionized.

In animals and plants, it has been used to eradicate viral infections as well as the
CeC chemokine receptor type 5 (CCR5) present on the surface of white blood
cells. It has been observed that individuals having a mutation in this gene
(CCR5D32) are resistant to HIV infection. CCR5 is essential for HIV entry and
facilitates the entry of HIV particles. The CRISPR/Cas9 system knocked down
the expression of the CCR5 gene, rendering the cells resistant to HIV infection.
(Singh, Recent Advances in CRISPR-Cas9 Genome Editing Technology for
Biological and Biomedical Investigations, 2018)

(Ye, 2014) used CRISPR-Cas9 to eradicate the 32nd bp (CCR5D32) of the CCR5
gene using CRISPR. The modified iPSCs were observed to be resistant to HIV
infection. In addition to editing the HIV genome with CRISPR-Cas9, there is a
method of eliminating HIV by targeting the long terminal repeat (LTR) (Ebina,
2013). Additionally, (Hu, 2014)targeted the LTR U3 region of the HIV-1
genome by using single and multiplex short guide RNAs in order to prevent
infection with HIV.

In many countries around the world, the human papillomavirus (HPV) can
cause sexually transmitted infections and cervical cancer. Currently, there are
100 different types of HPV, of which 40 HPVs are transmitted through sexual
contact, affecting the genital, mouth, and throat areas. (Sharma, 2014)

In particular, HPV E6 and E7 are two major oncogenes that play a significant
role in the development of cancer. 

Using CRISPR-Cas9, (Kennedy, 2014) targeted the E6 and E7 genes in HPV16

and controlled the infection. Not very long ago, CRISPR-Cas9-based antiviral
agents were used for the treatment of a variety of viral infections, including
Kaposi's sarcoma herpesvirus (KSHV) (Avey, 2015) (Johnson, 2014) (Gomaa,
Programmable removal of bacterial strains by use of genome- targeting
CRISPR-cas systems, 2014), herpes simplex virus (HSV-1 and HSV-2). (18)
(19)
 REFERENCES 8

3. Mammalian cells genome editing using CRISPR-Cas9 system for

therapeutic applications

Mammalian cells were used for genome editing with the CRISPR-Cas9 system
in 2013. A CRISPR-Cas9 system was developed by using the Cas9 gene from
Streptococcus thermophilus, where they used 293FT cells along with the
CRISPR system (SpCas9, SPRNase III, tracrRNA, pre-crRNA) for targeting
mammalian genomes which worked efficiently. The CRISPR-Cas9 system was
used in to manipulate genes at the endogenous AAVS1 locus by creating a
chimeric crRNA-tracrRNA, commonly called sgRNA. Mutations were found in
293T cells (10e24%), K562 cells (13-8%), and iPSCs (2e4%) in the current
study. Mammalian genome editing has been made possible through both of
these studies. Using liposome-mediated transfection/electroporation, delivered
Cas9/sgRNA ribonucleoprotein (RNP) complexes into several mammalian cells.
Jurkat T cells and iPSCs were both found to contain 94% and 87% indels,
respectively. Indel rates were 93% and 65% when more than two loci were
targeted. CRISPR-Cas9 is being used in several studies to prevent and treat
diseases in animals. (Mali, 2013) (Cong, 2013) (Liang, 2015)

4. Cancer therapy

Our lives have been impacted positively and negatively by lifestyle changes,
physical activity, foods, and drugs. A high level of exposure to contaminants
may cause serious health problems. One of them is cancer, which is a leading
cause of death worldwide. A chimeric antigen receptor (CAR-T)
immunotherapy was approved by the US Food and Drug Administration in
2017. Using CRISPR-Cas9, a gene that is believed to be connected with T-cell
expression of CAR has been knocked out, which inactivates the programmed
death-1 (PD-1) receptor. PD-1 is involved in regulating the immune response
against cancer cells. By generating knock-outs, cancer cells were prevented
from producing receptor blocking ligands. There are a number of studies that
have demonstrated that targeting the PD-1 gene with CRISPR increased the
production of anti-tumor cytotoxic T-cells (in both solid and hematologic
cancers). The study suggests that CRISPR-Cas9 might be able to cure cancer
and help to better understand how cancer works. (Torre) (Okada, 2017) (Shao,
2017)

5. Duchenne muscular dystrophy therapy

The genetic disorder Duchenne muscular dystrophy (DMD) affects the muscles.
The disorder results in progressive muscle weakness and degeneration. DMD is
caused by dystrophin, which is a protein whose gene expression results in
DMD. The degradation of muscle strength results from a mutation in the
dystrophin gene, which is a model for DMD. Recent studies have used
 REFERENCES 9

CRISPR-Cas9 for repairing the dystrophin gene mutation, restoring its function.
In vitro and in vivo testing has confirmed the efficiency and safety of CRISPR-
Cas9 for treating DMD. Numerous studies have been conducted on mice. The
future of DMD treatment in humans will require more research. (Kim, 2017)

6. Beta-thalassemia therapy

The disease B-thalassemia is the result of uncontrolled production of

haemoglobin. There is a deficiency in the expression of B-globin due to a
mutation in the globin gene. A protein containing iron, haemoglobin, is present
in red blood cells (RBCs) and transports oxygen throughout the body.
Nevertheless, in cases of b-thalassemia, low haemoglobin expression results in
reduced oxygen concentration in many parts of the body. Using CRISPR-Cas9,
a mutation in the globin gene was repaired, encouraging an adequate supply of
oxygen into the cells through globin. In summary, it could be a potential
treatment for patients with b-thalassemia, but further experiments and in vivo
studies are necessary for its use in the clinic. (Galanello, 2010) (reardon, 2015)
(Antony et al., 2018)

7. Blindness therapy

Blindness is a disease caused by retinal degeneration worldwide. According to

estimates, 196 million people worldwide may contract this disease by 2020. It
has already been widely tried to use drugs, gene therapy, and transplantation to
treat retinal degeneration. Recently, CRISPR-Cas9 technology was used to treat
retinitis pigmentosa, a degenerative disorder that results in the loss of cone
photoreceptors and ultimately blindness. It represents one of the most promising
therapeutic opportunities for the treatment of blindness. CRISPR-Cas9 based on
AAV has been developed to deliver Cas9 DNA to post-mitotic photoreceptor
cells targeting the Nrl gene, which is a rod fate determinant during
photoreceptor development. Blindness may be treated by disrupting Nrl, a
solution that has been proven effective in the past. (27) (28)

8. Cardiovascular disease therapy

CVD is the leading cause of death worldwide due to its heart-related nature. An
iPSC-derived patient with familial hypercholesterolemia heterozygous (HoFH)
has recently been treated with CRISPR-Cas9 for correction of a homozygous
deletion of three bases in exon 4 of low-density lipoprotein cholesterol receptors
(LDLRs). The cellular metabolism of cholesterol could be normalized by this
therapy. Cardiovascular disease is associated with the G-protein-coupled
oestrogen receptor (Gper1). The deletion of Gper1 using CRISPR-Cas9 altered
the microbiota of salt-sensitive hypertensive rats, along with changes in the
level of short chain fatty acids (SCFAs), which reversed the cardioprotective
 REFERENCES 10

effect exerted by deletion of Gper1. According to the study, Gper1 plays a role
in accelerating improved vascular relaxation. From hypertension GPER1*/*
rats, we transplanted the microbiota. The alterations in microbiota that cause
cardiovascular disease are studied. CRISPR-Cas9 has been used for the
treatment of many serious animal diseases; however, there are several
challenges and issues that must be resolved before it can be successfully
implemented in the clinic. (29) (30).
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Recent developments in CRISPR interference platform

In addition to gene repression and activation, CRISPRi has revolutionized high

throughput screening, imaging, epigenetic modification, and many other
biomedical processes. To do this, Cas9 has been mutated in the active region
(RuvC (D10A) and HNH (H840A)) in order to weaken it but retain its ability to
bind. Dead Cas9, also known as dCas9 (CRISPRi) is a modified version of the
Cas9 protein that has been extensively used with many types of cells and
organisms.

1. CRISPRi

CRISPRi has been repurposed for gene regulation in many organisms. In

addition to targeting the promoter region and coding gene that interfere with the
expression of the gene, sgRNA is also designed to activate the endogenous gene
in order to increase its function. The initial tests were conducted on bacteria and
mammalian cells before being expanded to other organisms. In (Bikard,
Development of sequence-specific antimicrobials based on programmable
CRISPR-Cas nucleases, 2014)E. coli, sgRNA was used as an antisense
molecule and caused a 1000-fold reduction of transcription with no off-target
effects. A study found that researchers could suppress sfGFP (superfolder green
fluorescent protein) and mRFP (monomeric red fluorescent protein). In
HEK293 cells (Human Emblematic Kidney 293 cells), eGFP (enhanced green
fluorescent protein) was targeted, a gene under the control of the SV40
promoter. Results showed up to 46% repression, which has been improved by
testing other sgRNAs at different gene locations. (Chen, 2014)

2. CRISPRa

In CRISPRi, activator domains fuse with dCas9 to activate gene function. The
C- and N-terminal regions of dCas9 were fused to u-subunit RNA polymerase
for degradation of LacZ in E. coli. The activation of dCas9-u at the C-terminal
region was found to be 2.8-fold as a result of its presence. It has been shown
that 7.2e23 fold activation in gene expression has been found when the GFP-
mut2 has been targeted at different locations. An activator known as VP16 is
well known for its ability to increase mammalian gene activation. The dCas9
gene was fused with four copies of VP16 (VP64) and one copy of p65AD in a
study. In order to target Gal4 UAS (upstream activation sequence), it was
transfected into a cell line (HEK293). Activation by dCas9-VP64 and dCas9-
 REFERENCES 12

p65D yielded 25-fold and 12-fold increases, respectively. Numerous studies

suggest that CRISPRi can be used in eukaryotes and prokaryotes for a variety of
purposes. Currently, CRISPRi offers the opportunity to easily alter gene
function in a cost-effective, rapid, and versatile manner. (Bikard, Development
of sequence-specific antimicrobials based on programmable CRISPR-Cas
nucleases, 2014) (Gomaa, Programmable removal of bacterial strains by use of
genome- targeting CRISPR-cas systems, 2014)

3. Loci imaging

Mapping and studying genes on the chromosome require tracking and

visualization. Currently, CRISPRi is used for mapping genes on chromosomes
as well as visualizing them in cells. For live imaging, we have developed a
CRISPRi (dCas9-eGFP fusion) that targets the repetitive elements present in
telomeres. They could During telomere disruption and elongation, they could
identify and study the dynamics Additionally, they were able to visualize the
localization of MUC4 loci on sister chromatiBy fusing eGPF with dCas9,
CRISPRi was constructed to target telomeric repeats pericentrically and
centricThe engineered multi-colour CRISPRi targets multiple loci on
chromosomes using three orthogonal dCas9s.the many loci on chromoThe
distance between the two loci could be easily determined by measuring and
physicaFurther development of CRISPRi could lead to a better understanding of
gene dynamics and regulation by mapping and visualizing gene positions on
chromosomes namics and regulation.
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NEUROGENERATION:

There is a huge economic burden associated with neurodegenerative diseases,

which is devastating for patients and their families. A cure for
neurodegenerative diseases does not exist as of today, let alone a treatment that
slows the progression of these diseases. Our insufficient understanding of the
cellular and molecular mechanisms driving neurodegeneration is largely
responsible for this lack of therapeutic options.

It is interesting to note that certain types of neurons are selectively susceptible

to neurodegeneration, whereas other types are not. Neurodegenerative diseases
exhibit different patterns of selective vulnerability. Knowing the cellular factors
that determine selective vulnerability would contribute to a better understanding
of neurodegeneration's cellular mechanisms, as well as point to potential
therapeutic targets. Pharmacologically targeting the factors controlling selective
vulnerability could lead to the transformation of susceptible neurons into
resilient neurons, therefore slowing or perhaps preventing the progression of
neurodegenerative diseases.
In order to understand the determinants of selective vulnerability, transcriptional
analysis has primarily been employed to characterize vulnerable and resilient
cells. Although both transcriptomics and proteomics are capable of cataloguing
the differences in gene expression between neurons that are vulnerable and
those that are resilient, the list of differentially expressed genes is lengthy and
only correlates with the underlying genes. Moreover, it does not establish causal
relations between the genes. To establish the causal role of genes in selective
vulnerability and to have the ability to use them as therapeutic targets, a
functional approach is required that is able to control the neuronal expression of
genes with a high degree of precision. (49-53).
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Correction of disease-causing DNA repeats and single-nucleotide variants

There are several forms of neurodegenerative diseases caused by genetic

mutations1, 5, 27 and CRISPR/Cas strategies have been employed in different
models to correct these mutations.

Huntington’s disease

There are many neurodegenerative diseases, such as Huntington’s

Disease (HD), spinocerebellar ataxias] mainly result from the expansion of
unstable nucleotide repeats in some genes.28 It has been hypothesized that
reducing the size of these repeats via CRISPR/Cas could alleviate the
pathogenesis of associated diseases. An expanded CAG trinucleotide repeat in
the first exon of the Huntington gene (HTT) is related to HD, a dominantly
inherited neurodegenerative disease.
Human HTT CAG repeats are highly polymorphic in the normal population;
once their length reaches a certain level, a number of characteristic neurological
symptoms result. iPSCs derived from an individual suffering from HD (carrying
72 and 19 CAGs) were the first to be used in a clinical trial for reducing the size
of the disease-causing CAG repeat using a BAC containing the entire HTT
combined with a normal CAG repeat (21 CAGs).
According to expression profiling analysis in addition to apoptosis assays,
genetically corrected iPSC clonal lines normalized cellular pathological
signalling pathways (e.g., cadherin, TGF-*, and BDNF) and disease phenotypes
(e.g., susceptibility to cell death), suggesting its therapeutic benefits. The
CRISPR/Cas9 technology has since been used to enhance homologous
recombination efficiency in HD cell models. Recent studies have shown that
larger HTT CAG repeats in HD patient-derived iPSC lines can be corrected by
CRISPR/Cas9 in combination with a new piggyBac-based selection system
through HDR33; impaired neural rosette formation and enhanced cell death
after growth factor removal were reversed in corrected HD iPSC lines.
CRISPR/Cas9 D10A nickase was also reported to be capable of expanding
CAG/CTG repeats without using a repair template. The repeat sequence was
directly targeted by CRISPR/Cas9, causing it to expand and contract. Due to the
activation of distinct DNA repair mechanisms, the repeat in the reporter vector
contracted when single-strand breaks were introduced via Cas9 D10A nickase.
For CRISPR/Cas methods directly targeting disease-causing repeats, the target
 REFERENCES 15

gene specificity should be assessed thoroughly to avoid altering other repeat-

containing genes permanently. (54)(55-62)
 REFERENCES 16

Parkinson’s disease and Alzheimer’s disease

In addition, a mutant form of Cas9 was used, which was paired with various
fusions, to modulate the expression levels of genes related to neurodegenerative
diseases. Combined with a gRNA that targeted transcription start sites, the use
of dCas9 led to a significant reduction in the expression of several disease-
associated genes, such as SNCA, MAPT, and APP. By using the dCas9-KRAB
and dCas9-VPR effector domains fusion features of the CRISPR/Cas9 systems,
it was possible to up-and downregulate the levels of mRNA and protein
expression of SNCA in human iPSC-derived neurons. (54)
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Challenges and opportunities

Various CRISPR/Cas strategies tested in cellular models of neurodegenerative

diseases have been shown to successfully engage targets and ameliorate disease-
associated phenotypes, supporting their therapeutic potential in clinical trials.
Although CRISPR/Cas strategies have been reported in the scientific literature,
most of them are not immediately useful for treating patients with
neurodegenerative diseases. Before CRISPR/Cas can be used therapeutically on
humans, a number of technical hurdles and biological questions must be
resolved.

Off-targeting:

Since the majority of CRISPR/Cas methods aim to generate permanent changes

in DNA, off-target effects are among the major concerns of CRISPR/Cas
applications in medicine. The Cas9 enzyme requires extensive homology
between target DNA and gRNA sequences in order to initiate cleavage.
Nevertheless, Cas9 can bind to off-target sequences that have mismatches, and
such transient binding may cause different off-target activities. In other words,
the level of off-targeting will depend on the CRISPR/Cas strategy, and the
possibility of developing one without any off-target activity may be very
difficult. There is, however, the possibility of significantly reducing levels of
off-target activities by (1) exploiting optimized gRNA design methods (2) using
truncated gRNA (3) using Cas9 with improved fidelity, (4) using a Cas9 nickase
mutant (5) using low levels of Cas923 and (6) using controllable Cas9. In
combination with other approaches, CRISPR/Cas may further reduce its off-
target activity to the point that therapeutic benefits outweigh its off-targeting-
mediated side effects.(63-71)

Delivery:

Adeno-associated viruses (AAVs) offer the best prospects for CRISPR/Cas

delivery for neurodegenerative diseases, as they are relatively safe and efficient
in delivering a gene of interest, without integrating into the genome. AAV
vectors may benefit from the use of smaller endonucleases such as Cpf176–78
and saCas975 due to their limited transgene capacity. Even so, dual systems of
AAV vectors expressing spCas9 and gRNA successfully induced DNA
modifications in mouse brains. It is important to note that prolonged expression
of high levels of Cas9 may result in greater levels of off-target activity. In the
 REFERENCES 18

context of protein therapy, the Cas9 protein can be delivered to target DNA
effectively while causing minimal off-target effects.(54)

Homology-directed repair versus non- homologous end joining

As a consequence, it is likely that CRISPR/Cas strategies for genomic

correction via HDR may be less efficient and can instead induce NHEJ, which
may result in the knockout of the target gene. A CRISPR/Cas strategy that
attempts to correct the mutant allele may, in the worst-case scenario, also
inactivate the normal allele. A screening approach was employed in preclinical
studies to select genetically corrected cell clones that would be molecularly
characterized in further studies. In addition to the fact that selection is not
desirable during therapeutic applications of CRISPR/Cas, it is also essential to
develop genome editing strategies that will result in high-fidelity HDR with
minimal NHEJ.23,81 Using CRISPR/Cas strategies that are designed for HDR
of the mutant allele will also contribute to minimizing NHEJ of the normal
allele.(54)

Requirement of personalization for allele- specific CRISPR/Cas

In order to target genetic variations associated with the mutations using

CRISPR/Cas strategies tailored exactly to each patient's mutation, specialized
designs are needed, since each patient may have different combinations of
mutant and normal allele haplotypes. Personalized CRISPR/Cas strategies
tailored to specific alleles may seem unrealistic with the cost associated with
clinical trials. Nevertheless, certain diseases subjects share the founder
mutation, and therefore alleles on the ancestral haplotype that have low allele
frequencies in the normal population may allow CRISPR/Cas gene editing with
a single mutant allele to be applicable to the largest number of patients who
share the founder mutation. Moreover, in cases where distributions of
haplotypes of the mutant allele and the normal allele are not quite identical (i.e.,
in the case of HD), a number of CRISPR/Cas strategies specific to the mutant
allele can be developed for the majority of patients. A better understanding of
the general applicability of allele-specific target sites would facilitate the testing
of human CRISPR/Cas strategies for efficacy.(54)

Limitations of transcriptional modulation

Due to the lack of DNA modification caused by dead Cas9, this use would only
generate temporary effects, and this remains to be investigated to see whether
CRISPR-mediated transcription modulation strategies utilizing dead Cas9
(CRISPRi) have advantages over conventional RNA-lowering approaches such
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as ASO. As well, the likelihood of applying allele-specific technology would

vary depending on the amount of genetic variation near transcription start sites
of target genes that are available. However, transcriptional activation using the
dCas9–VPR fusion demonstrates the versatility of CRISPR approaches and
confirms its applicability to treating human diseases such as haploinsufficiency.
(54)
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Conclusions
In terms of treating neurodegenerative disorders, CRISPR/Cas offers both
promise and limitations. Before CRISPR/Cas strategies can be applied to the
treatment of human diseases, there are several technical and safety issues that
need to be addressed. Although preliminary studies have examined conceptual
foundations and the key components necessary for CRISPR/Cas to be applied to
neurodegenerative diseases, and the potential for integrative approaches has also
been demonstrated through preclinical interventions. With the advent of
personal genomics, the discovery of disease-causing genes and the identification
of targets for CRISPR/Cas are both faster and more affordable than ever before.
Alternatively, the development of widely applicable and efficient CRISPR/Cas
genome engineering tools will enable the identification of disease-causing
mutations through personal genomics, facilitating genetic tests, and enabling the
application of therapeutic CRISPR/Cas genome engineering techniques.
CRISPR/Cas will eventually be used in precision medicine for
neurodegenerative diseases and more due to the synergistic interactions between
two disciplines which will greatly contribute to understanding diseases and, in
turn, improving overall human health as a result of this.
Conflict of interest statement
The authors declare that there is no conflict of interest.
 REFERENCES 21

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