1. Current Situation of AD patients

Alzheimer’s disease is a growing public health crisis in China. As the population ages rapidly, the number of people diagnosed with AD is increasing at an alarming rate. According to the Alzheimer’s Disease Chinese (ADC) research group, more than 10 million people in China are currently living with Alzheimer’s or related dementias — the highest number in the world ( Jia et al., 2020 ).

However, early detection remains a major challenge. Most diagnoses in China occur at moderate or late stages, when significant and irreversible brain damage has already occurred. Current diagnostic methods like cerebrospinal fluid (CSF) testing and PET imaging are either invasive, expensive, or unavailable in most primary care settings (Xu et al., 2021). This results in delayed intervention and missed opportunities for managing disease progression.

In a 2022 survey conducted by Alzheimer's Disease International, nearly 64% of Chinese respondents reported delays in receiving an accurate diagnosis, citing lack of awareness and limited access to advanced diagnostic tools (ADI, 2022). These findings highlight the urgent need for affordable, early, and accessible diagnostic solutions in China.

Pathogenesis and Diagnostic Methods of Alzheimer’s Disease:

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, primarily characterized by β-amyloid plaque accumulation, neurofibrillary tangles caused by abnormally phosphorylated tau protein, and neuronal cell death. These pathological features lead to memory loss, cognitive decline, and ultimately, a loss of independent living abilities (MUCKEL, 2009; JR et al., 2018).

In other words, the commonly accepted mechanism of AD suggests that β-amyloid deposition triggers a neuronal apoptosis cascade, resulting in the rupture and degradation of microtubules that connect neurons. As a consequence, tau proteins, which constitute microtubules, are released into the cerebrospinal fluid (CSF) and can cross the blood-brain barrier into the bloodstream (Arendt et al., 2003; Sunderland et al., 2020; Hesse et al., 2001). Total tau (T-Tau) levels in CSF have been proven to be a reliable biomarker for the neurodegenerative changes specific to AD (Mattsson et al., 2009).

Currently, there is no highly effective cure for AD (Alzheimer’s Dementia et al., 2025). Diagnosis remains a major clinical challenge. Early symptoms are often mistaken for normal aging, which leads to delays in seeking medical care and in obtaining an accurate diagnosis. Even within specialized medical environments, diagnostic sensitivity and specificity are limited. There is still a lack of efficient, user-friendly, and widely accessible early diagnostic tools. Current diagnostic methods typically rely on multidisciplinary clinical assessments supplemented by auxiliary examinations (Mistur et al., 2025).

For example, measuring tau levels in CSF requires an invasive lumbar puncture procedure, which significantly limits its utility in routine screening and monitoring. Furthermore, the correlation between T-Tau levels in blood and CSF is weak, making it difficult to predict AD status through direct measurement of blood T-Tau (Olsson et al., 2016).

Tau proteins include two types: BD-Tau (brain-derived tau) and P-Tau (phosphorylated tau). BD-Tau is released only when neurons undergo degenerative injury, while P-Tau can be triggered by other non-AD-related pathologies. Therefore, measuring BD-Tau levels in blood offers a feasible approach for detecting AD (Gonzalez-Ortiz et al., 2023). BD-Tau has been confirmed as a promising blood-based biomarker for AD-type neurodegeneration (Gonzalez-Ortiz et al., 2023).

Currently, literature reports that BD-Tau can be detected in blood using antibody-based methods. However, these are relatively expensive (Kyalu et al., 2023; Gonzalez-Ortiz et al., 2024). In contrast, DNA aptamers offer significant advantages such as high specificity, ease of modification, strong stability, and low cost, making them well-suited for large-scale applications (Teng et al., 2018).

This project aims to screen high-affinity DNA aptamers that can bind and inhibit BD-Tau protein, and distinguish it from T-Tau. These aptamers could be developed into low-cost, highly sensitive diagnostic tools for early screening of patients, thereby increasing the success rate of therapeutic interventions. Although previous studies have developed aptamers targeting P-Tau (Teng et al., 2018), no aptamers have yet been specifically designed for BD-Tau epitopes. Targeting BD-Tau’s unique epitopes to develop DNA aptamers with high sensitivity and specificity—while avoiding interference from peripheral tau isoforms—holds substantial promise.

Our team was driven by growing concern about the increasing burden of Alzheimer’s disease in China, where early diagnosis remains rare and often inaccessible. As students passionate about synthetic biology and its real-world applications, we were struck by the disconnect between the early molecular onset of AD and the lack of affordable, scalable diagnostic tools.

2. Application of CRISPR-Cas in AD Detection

The development and application of the CRISPR-Cas system has greatly advanced the field of biotechnology. One of its most well-known uses involves gene editing via Cas proteins guided by gRNAs to specifically cleave nucleic acid sequences, particularly the Cas9 protein, often referred to as "genetic scissors" (Zhou et al., 2023).

Beyond genetic manipulation, Cas proteins with trans-cleavage activity have been harnessed by researchers such as Feng Zhang and Jennifer Doudna to build cost-effective and portable nucleic acid diagnostic tools—such as SHERLOCK and DETECTR (Zhang et al., 2025). These CRISPR-Cas biosensing systems have been applied in pathogen detection, virus screening, genotyping, cancer mutation analysis, and single-nucleotide polymorphism identification (Pacesa et al., 2024; MondalR et al., 2023; ChenK et al., 2019).

However, these applications are still largely confined to nucleic acid detection. The inability to detect non-nucleic acid analytes—such as small molecules or proteins—presents a major limitation. Therefore, it is of great significance to break through this bottleneck and expand the detection scope of the CRISPR system to develop simple, efficient, low-cost, and stable biosensing platforms. Such platforms would benefit various fields including scientific research, environmental monitoring, food safety, and clinical diagnostics.

To overcome this limitation and expand the CRISPR-Cas system’s detection capabilities beyond nucleic acids and small molecules, we propose to combine the ssDNA trans-cleavage activity of Cas12a with a recognition element that can bind a wide range of target analytes. Our research indicates that nucleic acid aptamers fulfill this requirement. Aptamers are short synthetic single-stranded DNA or RNA sequences capable of binding a wide array of targets—including metal ions, small molecules, proteins, viruses, cells, and pathogens—with dissociation constants (Kd) in the picomolar to nanomolar range.

Therefore, this project integrates the high affinity of aptamers with the non-specific ssDNA trans-cleavage activity of Cas12a to build a biosensing platform capable of detecting non-nucleic acid analytes, thereby expanding the detection range of CRISPR-based diagnostics.

1. How will your project address the problem through synthetic biology?

Our project proposes a two-cycle synthetic biology-based diagnostic platform that enables early, accurate, and minimally invasive detection of Alzheimer’s disease by targeting BD-Tau in blood samples.

• Cycle 1 focuses on the development of an aptamer-based molecular probe using SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This aptamer is designed to bind specifically and strongly to the BD-Tau protein — a blood-based biomarker shown to highly correlate with Alzheimer’s pathology.
• Cycle 2 integrates this aptamer into a CRISPR-Cas12a-powered biosensor system. When BD-Tau is present in a patient sample, the aptamer binds the protein and releases a DNA signal sequence. This activates the trans-cleavage function of Cas12a, which in turn cleaves a fluorophore-quencher-labeled DNA probe — generating a fluorescence signal proportional to the concentration of BD-Tau.

Figure 1. Schematic Diagram of Biosensor

Biotin-labeled ComDNA binds directly to streptavidin-coated magnetic beads via strong biotin-streptavidin interactions, anchoring it firmly to the magnetic beads. The aptamer–dsDNA complex is then indirectly immobilized on the magnetic beads through complementary base pairing with the biotin-labeled ComDNA. This forms the composite "streptavidin magnetic bead–biotinylated ComDNA–aptamer-linked dsDNA (containing the Cas12a cleavage site)" complex, which constitutes the biosensor(Figure 1).

Once the biosensor is introduced into a sample containing the target analyte, the analyte binds specifically to the aptamer, inducing the aptamer to fold into a secondary structure. Due to the higher binding affinity between the analyte and the aptamer compared to that between the aptamer and ComDNA, the aptamer dissociates from the ComDNA. This causes the aptamer–dsDNA complex that now contains the analyte to detach from the magnetic bead surface and be released into the solution.The supernatant containing the released aptamer–dsDNA is then introduced into the Cas12a signal reporting system for detection.

When the dsDNA containing the Cas12a cleavage site enters the reporting system, the crRNA forms a complex with the Cas12a protein, guiding it to target and cleave the dsDNA. Once cleavage occurs, Cas12a undergoes a conformational change, activating its trans-cleavage activity toward single-stranded DNA (ssDNA). This rapidly cleaves the fluorophore-quencher (FQ)-labeled reporter probe, separating the fluorophore from the quencher and generating a fluorescent signal. In the absence of dsDNA, the fluorophore remains quenched and no fluorescence is emitted. Therefore, when the aptamer–dsDNA complex in the supernatant is added to the Cas12a reporting system, the resulting fluorescence intensity correlates positively with the concentration of dsDNA, which in turn is positively correlated with the concentration of the target analyte.

Throughout this process, the fluorescence intensity directly reflects the amount of aptamer–dsDNA released into the supernatant, which depends on the amount of analyte present. Consequently, the fluorescence signal intensity is positively correlated with analyte concentration, enabling quantitative and qualitative analysis of the target molecule through fluorescence detection.

This approach leverages synthetic biology tools to build a highly specific, highly sensitive, and low-cost diagnostic system. It has the potential to be translated into a portable, real-time blood test — suitable for use in community clinics, hospitals, and even home testing in China.

1. What problem will you solve？

We aim to solve the problem of delayed and inaccessible diagnosis of Alzheimer’s disease in China by creating a tool that enables early-stage, non-invasive detection using a blood-based diagnostic platform.

2. What kind of product do you want to develop?

Our final product will be a CRISPR-Cas12a-based biosensor diagnostic kit that detects BD-Tau levels in human blood samples. This kit will be:

• Highly specific due to aptamer-target interaction
• Highly sensitive due to CRISPR-Cas12a signal amplification
• Non-invasive and easy to use
• Cost-effective and scalable for use in primary care and remote settings

By combining the power of molecular recognition with synthetic biology diagnostics, our project aspires to contribute a novel, accessible tool to Alzheimer’s care — with particular relevance to China’s healthcare needs.

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