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Prospects of CRISPR/Cas9 in Alzheimer’s Disease
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Prospects of CRISPR/Cas9 in Alzheimer’s Disease.
Alzheimer’s disease ( AD ) is a progressive, irreversible neurodegenerative disease clinically manifested by cognitive impairment, behavioral abnormalities and social deficits.
It is predicted that by 2050, the number of people 65 and older with dementia in the United States could reach 13.8 million.
At present, the pathogenesis of AD remains unclear. β-Amyloid deposition and tau hyperphosphorylation are widely recognized as 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 be involved in AD pathogenesis.
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, pathogenic gene screening and targeted therapy.
The CRISPR/Cas9 system is essentially a bacterial defense mechanism against foreign DNA.
First discovered in the late 1980s, CRISPR is an unusual genetic structure composed 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 attack by phage and plasmid DNA through an RNA-guided DNA cleavage system.
When foreign DNA invades bacteria, its DNA fragments are incorporated into the CRISPR site as spacers.
This site is then transcribed into CRISPR pre-RNA ( crRNA ), which attaches to a constitutively formed transactivating RNA ( tracrRNA ), which is modified into gRNA by CRISPR-associated proteins.
When the gRNA binds to the REC I domain of the inactive Cas9 complex, the complex is activated, resulting in the formation of heteroduplexes between the gRNA and its complementary single-stranded DNA.
The HNH and RuvC nuclease domains of Cas9 subsequently cleave complementary and non-complementary 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 invading genome, and PAM is critical in distinguishing bacterial self and non-self DNA and successfully binding Cas9.
Unlike ZFNs and TALENs, which rely on refined protein products for each specific gene sequence, the CRISPR/Cas9 system relies on gRNAs, making it a more flexible platform.
The Cas9 protein remains the same, while the gRNA can be easily tailored to 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
Cell models have been widely used in the research of various neurological diseases, including AD, because they do not involve ethical issues, the experimental period is relatively short, and the cost is low.
Over the past few decades, many in vitro cellular 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 lines HT22 and glial BV2, and mouse glioblastoma cell N2a .
The advent of CRISPR/Cas9 gene editing technology could facilitate more efficient development of AD cell models.
For example, down-regulation of thioredoxin-interacting protein ( Txnip ) levels in HT22 cells by the CRISPR/Cas9 system can effectively attenuate β-amyloid-induced protein cysteine oxidative modification.
The findings suggest that Txnip may be a therapeutic target for the treatment of AD.
In addition to building AD cell models, CRISPR/Cas9 technology can also be used to build AD animal models.
In 2020, Serneels et al . created a new model called Apphu/hu by using a CRISPR/Cas9 strategy to generate humanized aβ sequences ( G676R, F681Y, and R684H ) in the APP gene in mice and rats.
By inserting three amino acids into the rodent Aβ sequence, Aβ levels were more than tripled 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 mutations in each gene may lead to the occurrence of FAD.
However, more than 95% of AD cases are sporadic, and 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, we found that knockdown of TREM2 in IPSCs by the CRISPR system severely affected microglial survival, clearance of apolipoprotein E, and SDF-1α/CXCR4-mediated chemotaxis, ultimately resulting in increased response to beta amyloid. Injury response of plaque-like plaques.
In addition, Duan et al. designed an imaging-based array CRISPR to study genes associated with AD traits. It will also provide a platform to explore AD biology and an opportunity for drug discovery.
CRISPR/Cas9 for targeted therapy of AD
CRISPR/Cas9 targets APP gene mutations
Mutations in the APP gene lead to increased β-secretase cleavage of the Aβ precursor protein, resulting in dominantly inherited AD.
Gyorgy et al. reported that Aβ protein expression was reduced when the APP allele was knocked out using CRISPR/Cas9 technology.
Therefore, the CRISPR/Cas9 system may provide a gene therapy strategy for AD patients with APP mutations.
CRISPR/Cas9 targets key enzymes of Aβ protein
Aβ protein is formed by sequential modification of APP by BACE1 and γ-secretase.
Therefore, targeting BACE1 and γ-secretase is a potential therapeutic strategy for AD. Park et al. reported successful reduction of BACE1 expression by using CRISPR/Cas9 in two AD mouse models.
γ-secretase is regulated by γ-secretase activating protein ( GSAP ), and Wong et al. utilized CRISPR-Cas9 technology to knock down 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 genotypes
The APOE4 subtype is the strongest genetic risk factor for SAD. It is known that APOE is mainly expressed by astrocytes of the central nervous system.
Wadhwani et al. on the therapeutic target of APOE4 showed that when the E4 allele in IPSCs of two AD patients was corrected to the E3/E3 genotype by the CRISPR/Cas9 approach, E3 neurons were resistant to ionomycin-induced cells Toxicity was less sensitive and showed a decrease in tau phosphorylation.
In addition, Lin et al. identified the function of APOE4 using hiPSC and CRISPR/Cas9 technologies, and their results indicated 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 immune response is also a major pathway of AD etiology.
Growing evidence points to the importance of chronic neuroinflammation in AD. CD33 is an immunomodulatory receptor, expressed at high levels on neutrophils and at low levels on microglia, with distinct roles in regulating phagocytosis in AD pathology.
The latest study by Bhattacherjee et al. shows that disruption of the CD33 gene via CRISPR/Cas and replacement with a protective variant of hCD33 mitigates Aβ pathology and neurodegeneration.
Glial maturation factor ( GMF ), a newly discovered pro-inflammatory molecule, 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 the p38 MAPK signaling pathway and oxidative toxicity.
Raikwar et al. successfully reduced GMF expression in BV2 cells by a CRISPR/Cas9 approach, thereby inhibiting pp38 MAPK to modulate GMF-induced microglial pro-inflammatory responses.
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, increasing evidence has shown that CysLT1R is closely related to the occurrence and development of AD and can mediate inflammatory responses through the NF-κB pathway. Chen et al. demonstrated that deletion of CysLT1R via the CRISPR/Cas9 system reduced amyloidosis and reduced neuroinflammation in APP/PS1 mice.
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 related 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 mismatch and specific tissue targeting in this technology, the ultimate clinical application of CRISPR-Cas9 in 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, despite considering some shortcomings, the CRISPR-Cas9 system still has broad application prospects in the clinical treatment of AD.
1. Application of CRISPR/Cas9 in Alzheimer’s Disease. Front Neurosci. 2021; 15: 803894.
Prospects of CRISPR/Cas9 in Alzheimer’s Disease
(source:internet, reference only)