March 2, 2024

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The application prospect of CRISPR/Cas9 in Alzheimer’s disease

The application prospect of CRISPR/Cas9 in Alzheimer’s disease



 

The application prospect of CRISPR/Cas9 in Alzheimer’s disease.

Alzheimer’s disease ( AD ) is a progressive, irreversible neurodegenerative disease characterized clinically by cognitive impairment, behavioral abnormalities, and social deficits.

It is predicted that by 2050, the number of dementia patients aged 65 and over in the United States may reach 13.8 million. 

 

At present, the pathogenesis of AD is still unclear. β-Amyloid deposition and hyperphosphorylation of tau protein are widely considered to be the neurobiological mechanisms of AD pathogenesis.

In addition, other age-related, protective, and disease-promoting factors may interact with the core mechanisms of AD and may participate in the pathogenesis of AD.

 

In recent years, the gene editing technology of CRISPR/Cas9 has developed rapidly, showing great potential in the fields of basic research and disease treatment.

Recently, gene editing technology has been evaluated as a promising approach for AD research and treatment.

CRISPR/Cas9 has great application potential in AD model construction, disease-causing gene screening, and targeted therapy.

 

CRISPR/Cas9 system

The CRISPR/Cas9 system is essentially a bacterial defense mechanism against foreign DNA. CRISPR , originally discovered in the late 1980s, is an unusual genetic construct consisting of alternating repeating and non-repeating DNA sequences.

Genomic analysis revealed that CRISPR and Cas proteins function as an adaptive immune system and protect prokaryotic DNA from phage and plasmid DNA through an RNA-guided DNA cleavage system.

 

When foreign DNA invades bacteria, segments of its DNA are incorporated into CRISPR sites as spacers.

This site is then transcribed into a CRISPR pre-RNA ( crRNA ), which attaches to a constitutively formed trans-activating RNA ( tracrRNA ), modified by a CRISPR-associated protein into a gRNA.

When the gRNA binds to the REC I domain of the inactive Cas9 complex, the complex is activated, resulting in heteroduplex formation of the gRNA and its complementary single-stranded DNA.

The HNH and RuvC nuclease domains of Cas9 then cleave complementary and noncomplementary DNA strands, respectively.

 

Cas9 belongs to type II of the class 1 CRISPR-Cas system and is derived from Streptococcus pyogenes.

Coupling of Cas9 to the target sequence requires a protospacer adjacent motif ( PAM ), a small 3–8 bp DNA sequence in the invasive genome, and PAM is critical in distinguishing bacterial self from non-self DNA and successfully binding Cas9.

The application prospect of CRISPR/Cas9 in Alzheimer's disease

 

 

Unlike ZFNs and TALENs, which rely on the refined protein product of each specific gene sequence, the CRISPR/Cas9 system relies on guide RNAs, making it a more flexible platform. The Cas9 protein remains unchanged, while the gRNA can be easily customized for each gene.

Another advantage of the CRISPR/Cas9 system is that it enables simultaneous gene editing at multiple loci, providing a more efficient and scalable platform than previous systems.

 

 

 

CRISPR/Cas9 applied to the construction of AD model

Because cell models do not involve ethical issues, the experimental period is relatively short, and the cost is low, they have been widely used in the research of various neurological diseases including AD.

Over the past few decades, many in vitro cell models of AD have been established.

Cell lines commonly used in AD research to date include human neuroblastoma cells SH-SY5Y and SK-N-SH, mouse hippocampal neuronal cell line HT22 and glial cell line BV2, and mouse glioblastoma cell N2a .

The emergence of CRISPR/Cas9 gene editing technology can facilitate more efficient development of AD cell models.

 

For example, downregulation of thioredoxin-interacting protein ( Txnip ) levels in HT22 cells by CRISPR/Cas9 system can effectively attenuate β-amyloid-induced oxidative modification of protein cysteine.

The findings suggest that Txnip may be a therapeutic target for the treatment of AD.

 

In addition to constructing AD cell models, CRISPR/Cas9 technology can also be used to construct AD animal models.

In 2020, Serneels et al . created a new model called Apphu/hu by using the CRISPR/Cas9 strategy to generate humanized aβ sequences ( G676R, F681Y, and R684H ) in the APP gene of mice and rats.

By inserting three amino acids into the rodent Aβ sequence, the levels of Aβ were more than three-fold increased compared to the original wild-type strain.

 

 

 

Application of CRISPR/Cas9 in the screening of AD pathogenic genes

Alzheimer’s disease is divided into sporadic ( SAD ) and familial ( FAD ). APP, PSEN1 and PSEN2 are the main pathogenic genes that cause FAD, and the mutation of each gene may lead to the occurrence of FAD.

However, more than 95% of AD cases are sporadic, and the screening of SAD causative genes is crucial for understanding the molecular pathogenesis, early diagnosis, risk prediction and treatment of AD.

 

The development of high-throughput sequencing and CRISPR/Cas9 has provided great help for the screening of SAD pathogenic genes.

For example, it was found that by knocking out TREM2 in IPSCs by the CRISPR system, the survival of microglial cells, the clearance of apoE, and the chemotaxis mediated by SDF-1α/CXCR4 were all severely affected, which eventually led to the β-amyloid Plaque-like damage response.

In addition, Duan et al. designed imaging-based array CRISPR to study genes associated with AD traits. This will also provide a platform for exploring AD biology and opportunities for drug discovery.

 

Application of CRISPR/Cas9 in Targeted Therapy of AD


CRISPR/Cas9 targeting APP gene mutation

Mutations in the APP gene lead to increased β-secretase cleavage of the Aβ precursor protein, leading to dominantly inherited AD. Gyorgy et al. reported that when the APP allele was knocked out using CRISPR/Cas9 technology, the expression of Aβ protein was reduced. Therefore, the CRISPR/Cas9 system may provide a gene therapy strategy for AD patients with APP mutations.

 

CRISPR/Cas9 targets the key enzyme of Aβ protein

Aβ protein is formed by the sequential modification of APP by BACE1 and γ-secretase. Therefore, targeting BACE1 and γ-secretase is a potential therapeutic strategy for the treatment of AD. Park et al. reported successful reduction of BACE1 expression in two mouse models of AD by using CRISPR/Cas9. γ-secretase is regulated by γ-secretase activating protein ( GSAP ). Wong et al. used CRISPR-Cas9 technology to knock out GSAP in HEK293 cells stably expressing APP, resulting in a significant decrease in Aβ secretion and γ-secretase activity.

 

CRISPR/Cas9 targeted editing of apolipoprotein E genotype

APOE4 subtype is the strongest genetic risk factor for SAD. It is well known that APOE is mainly expressed by astrocytes in the central nervous system.

A study by Wadhwani et al. on the therapeutic target of APOE4 showed that when the E4 allele was corrected to the E3/E3 genotype in the IPSCs of two AD patients by the CRISPR/Cas9 approach, E3 neurons were significantly more resistant to ionomycin-induced cell Less sensitive to toxicity and showed a reduction in tau phosphorylation.

In addition, Lin et al. identified the function of APOE4 using hiPSC and CRISPR/Cas9 technology, and their results showed that APOE4 affects Aβ metabolism in different ways. These findings suggest that APOE4 may be a promising target for the treatment of AD.

 

CRISPR/Cas9 targets pro-inflammatory molecules

Human gene association studies have shown that the immune response is also a major pathway in the etiology of AD. Accumulating evidence points to the importance of chronic neuroinflammation in AD. CD33, an immunomodulatory receptor expressed at high levels on neutrophils and at low levels on microglia, has differential roles in regulating phagocytosis in AD pathology.

A recent study by Bhattacherjee et al. showed that disruption of the CD33 gene by CRISPR/Cas and replacement with a protective variant of hCD33 can attenuate Aβ pathology and neurodegeneration.

 

Glial maturation factor ( GMF ) is a newly identified pro-inflammatory molecule that is mainly expressed in reactive glial cells surrounding amyloid plaques and is highly expressed in various AD brain regions.

Overexpression of GMF usually leads to neuronal cell death through activation of p38 MAPK signaling pathway and oxidative toxicity.

Raikwar et al. successfully reduced GMF expression in BV2 cells by CRISPR/Cas9 approach, thereby inhibiting pp38 MAPK to regulate GMF-induced microglial pro-inflammatory response.

 

Cysteine ​​leukotrienes ( Cys-LTs ) are a group of inflammatory lipid molecules that initiate inflammatory signaling cascades through two major G protein-coupled receptors ( CysLT1R and CysLT2R ). In recent years, more and more evidences have shown that CysLT1R is closely related to the occurrence and development of AD, and can mediate inflammatory response through NF-κB pathway. Chen et al. demonstrated that deletion of CysLT1R by the CRISPR/Cas9 system reduced amyloidosis and attenuated neuroinflammation in APP/PS1 mice.

 

 


Summary


Gene editing has entered a period of vigorous development in recent years due to its broad and effective application prospects in scientific research and disease treatment.

Recently, there have been many studies on CRISPR/Cas9-mediated AD, mainly involving the use of this technology to construct AD models, screen disease-causing genes, and treat AD through specific target genes (such as APP, BACE1, APOE4, CD33, GMF, and CysLT1R) .

However, considering the potential non-targeted mismatches and specific tissue targeting in this technology, the ultimate application of CRISPR-Cas9 in the clinical treatment of AD still faces many challenges.

 

In conclusion, CRISPR/Cas9, as a breakthrough in gene editing technology in this century, has opened up new avenues for clinical gene therapy.

Compared with other gene editing technologies, the CRISPR-Cas9 system has the advantages of short cycle, low cytotoxicity, low price, and simple delivery. Therefore, the CRISPR-Cas9 system still has broad application prospects in the clinical treatment of AD, although considering that there are still some shortcomings.

 

 

 

 

references:

1. Application of CRISPR/Cas9 in Alzheimer’sDisease. Front Neurosci. 2021; 15: 803894.

The application prospect of CRISPR/Cas9 in Alzheimer’s disease

(source:internet, reference only)


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