Epidermolysis Bullosa (EB), a rare skin disease caused by mutations in the COL7A1 structural protein gene, severely affects the survival and quality of life of newborns. Currently, there are no effective clinical treatments, making early screening and genetic intervention crucial for controlling birth defects. The main symptoms include blisters, blood blisters, or ulcers on the skin and mucous membranes after minor friction or collision. EB is caused by mutations in skin structural protein genes and can be classified into several types with varying degrees of severity. The core issue of EB is that gene mutations lead to abnormal structural proteins (such as keratin and collagen) between the epidermis and dermis. These proteins are responsible for maintaining the adhesion between the layers of the skin. After mutation, the skin becomes extremely fragile, and even minor external forces can cause epidermal separation, resulting in blisters or wounds. Based on the type and location of the defective protein, EB can be divided into subtypes such as simplex, junctional, and dystrophic, among which junctional and dystrophic types may involve internal organs and threaten life [1; 2; 3].
Dystrophic Epidermolysis Bullosa (DEB) is one of the major subtypes of Epidermolysis Bullosa (EB), characterized by skin fragility, blistering, and scarring [4]. Globally, DEB accounts for approximately 20-30% of all EB cases, making it the second most common subtype after Epidermolysis Bullosa Simplex (EBS). DEB is caused by mutations in the COL7A1 gene, which encodes type VII collagen, a key structural protein responsible for anchoring the epidermis to the dermis [5]. When this protein is defective or absent, the skin becomes highly fragile and prone to blistering. DEB can present in two main forms: Dominant DEB (DDEB) and Recessive DEB (RDEB), with recessive forms typically being more severe and involving widespread scarring and deformities [6].
The COL7A1 gene is responsible for encoding type VII collagen, a key protein in the integrity of the skin. Mutations in COL7A1 are the primary cause of dystrophic epidermolysis bullosa (DEB), with c.520G>A and c.6745C>T (46% of RDEB alleles in Spanish patients) being two high-frequency variants [7; 8]. The c.520G>A mutation and c.6745C>T mutation result in a glycine-to-glutamic acid substitution, disrupting collagen structure, affecting skin integrity, as well as impacting type VII collagen, contributing to skin fragility. These mutations are found in most DEB cases, respectively, and are particularly common in European and East Asian populations. Detecting these mutations is crucial for accurate DEB diagnosis, enabling early intervention and better management of the condition.
Globally, Dystrophic Epidermolysis Bullosa (DEB) accounts for approximately 20-30% of all Epidermolysis Bullosa (EB) cases, making it the second most common subtype after Epidermolysis Bullosa Simplex (EBS). DEB remains a significant global health concern due to its severe clinical impact. According to ForePharma [9], DEB is responsible for a considerable proportion of EB cases worldwide, with 10-20 cases per million people. This type of EB is particularly challenging because it often leads to severe complications, including chronic, painful blisters and skin scarring, which can cause disability and significantly affect the quality of life. Studies, including those by Laimer [10]; Thien et al. [11] in Italy, show that the incidence of DEB in many countries ranges between 1 in 50,000 and 1 in 100,000 people, underscoring its widespread impact. Figure 1 shows the incidence of epidermolysis bullosa in the Netherlands in recent years [12]. Hou et al. [13] emphasized that DEB’s clinical severity places a substantial burden on patients, as the disease is associated with a high risk of secondary complications like squamous cell carcinoma in older individuals, significantly impacting their life expectancy and overall well-being. The global occurrence of DEB, combined with its serious and debilitating consequences, underscores the urgent need for improved treatment options and comprehensive care for affected individuals [14].
Figure 1. Epidemiological outcomes of each major type of Epidermolysis Bullosa in the Netherlands for the time period 1988–2018,n = 490 [12].
In China, Dystrophic Epidermolysis Bullosa (DEB) follows a similar prevalence pattern to global data. It accounts for approximately 20-30% of all EB cases, positioning it as one of the major subtypes in the Chinese population. However, the prevalence of EB in China is slightly lower than in other regions, with estimates ranging from 8-12 per million people [15]. The genetic basis of DEB in China is primarily linked to mutations in the COL7A1 gene, along with the global situation. Recessive DEB (RDEB) tends to be more severe and involves extensive scarring, often beginning in early childhood, leading to significant functional and cosmetic challenges for affected individuals. Dominant DEB (DDEB), while also a concern, typically presents with less severe skin damage and fewer complications [16]. In terms of clinical manifestations, DEB in China mirrors global descriptions, with affected individuals showing a tendency for extensive wound formation, especially in response to minor trauma. Like in other regions, individuals with RDEB are at a high risk of developing skin cancer (especially squamous cell carcinoma) in adulthood due to chronic, non-healing wounds [17]. Several studies have focused on the clinical and genetic profiles of DEB in China, helping to enhance our understanding of the condition and improve early diagnosis and management strategies. However, challenges remain in providing comprehensive care for all individuals, particularly in rural or underserved areas [13].
Fetal DNA screening of the patient's next generation in early pregnancy has an accuracy rate of up to 98% and is currently the most effective means of preventing and treating DEB [18]. Existing prenatal detection methods, such as amniocentesis combined with sequencing, though accurate, have significant drawbacks [19]. They are costly, technically complex, and highly invasive, carrying a risk of miscarriage. The limitations make them impractical for widespread use, particularly in economically underdeveloped regions or among low-risk populations. As a result, many families only learn of the condition after birth, missing the chance for early intervention and informed decision-making. Consequently, the need for non-invasive diagnostic methods—low-cost and with minimal equipment dependency—has become increasingly urgent. Non-invasive prenatal testing (NIPT) through blood samples from pregnant women can identify gene mutations related to Epidermolysis Bullosa (EB), offering a non-invasive and promising solution for patients [20]. However, reliable non-invasive EB detection methods have yet to be reported.
Non-invasive diagnostic methods are critical for early detection and intervention in diseases like dystrophic epidermolysis bullosa (DEB), reducing both family and societal burdens [21; 22]. The development of such technologies, particularly using the CRISPR-Cas12a system, can overcome the limitations of current invasive methods and improve accessibility to timely care, especially for high-risk populations [23]. CRISPR-Cas12a offers several advantages for clinical diagnostics: its high sensitivity, specificity, and ability to detect mutations with low input DNA make it ideal for early disease detection. For instance, Cas12a has been successfully used for diagnosing genetic disorders like sickle cell anemia and certain cancers in clinical settings [24]. However, there has been limited research applying Cas12a in non-invasive prenatal testing for EB, highlighting a significant gap in the field.
In summary, early DNA screening is the most effective approach for preventing and managing dystrophic epidermolysis bullosa (DEB). However, current prenatal methods, such as amniocentesis and high-throughput sequencing, are limited by high costs, invasiveness, and lengthy procedures, restricting their use in primary healthcare and high-risk populations. Research in prenatal genetic screening has progressively shifted from invasive sampling to non-invasive prenatal testing (NIPT). The CRISPR-Cas system, known for its high sensitivity and specificity, has shown promise in diagnosing various diseases, yet non-invasive prenatal detection for rare skin disorders like Epidermolysis Bullosa (EB) remains underexplored. This project focuses on developing the PrenatalEB-Detect platform based on CRISPR-Cas12a, offering a pioneering, non-invasive method for detecting DEB-causing mutations, with significant potential for both technological innovation and clinical application.
Cas12a (Cpf1) possesses trans-cleavage activity, making it suitable for highly sensitive DNA detection. Initially, we utilize synthetic biology techniques to construct the AsCpf1-related vector, which is then transformed into E. coli BL21 cells to achieve high-level expression of Cas12a. The purified Cas12a protein is subsequently used for downstream detection.
In line with the concept of non-invasive detection, we select the method of non-invasive extraction of cell-free fetal DNA (cfDNA) from maternal peripheral blood for sampling. The DNA samples are first amplified using Recombinase Polymerase Amplification (RPA) to provide the initial signal amplification, followed by a secondary signal amplification through Cas12a cleavage of the RPA products. The detection results are presented via fluorescence signals and lateral flow assay strips.
Thus, we have constructed a fluorescence detection platform and a lateral flow assay detection platform based on RPA and Cas12a, targeting two single-base mutation sites, c.520G>A and c.6745C>T, in the COL7A1 gene.
All elements are specifically designed, as outlined below:
The construction process is illustrated in Figure 2, which includes PCR amplification of the AsCpf1 gene, double enzyme digestion to linearize the pET-28a vector, and homologous recombination to ligate the linearized vector with the AsCpf1 gene fragment.
- PCR Amplification of the AsCpf1 Gene
We amplified the AsCpf1 gene through PCR technology. In the design of the primers, we ingeniously introduced sequences homologous to the pET-28a vector to facilitate subsequent vector construction. This step ensures that the Cas12a gene can be accurately inserted into the expression vector, laying the foundation for subsequent protein expression.
- Double digestion-mediated linearization of the pET-28a vector
Two restriction enzymes (EcoRI and BamHI) are used to double-digest the pET-28a vector, linearizing it. This step enables the vector to effectively connect with the Cas12a gene, forming a recombinant plasmid. The selection of the double digestion was based on the multiple cloning sites of the vector and the sequence characteristics of the Cas12a gene, ensuring that the ends of the digested vector and the Cas12a gene have complementary sticky ends.
- Homologous recombination-based ligation
By using homologous recombination technology, we ligated the Cas12a gene with the linearized pET-28a vector to form a recombinant plasmid. Homologous recombination is an efficient and precise method for gene connection, which ensures that the Cas12a gene is inserted into the vector in the correct direction and position, providing a guarantee for subsequent protein expression.
Figure 2. Schematic diagram of the construction process for the pET-28a-AsCpf1c recombinant plasmid.
- Transformation into competent cells
The recombinant plasmid was introduced into the competent Escherichia coli BL21 cells through heat shock transformation. Heat shock transformation is a commonly used method for introducing exogenous DNA into bacterial cells. Through a brief heating treatment, the cell membrane permeability is increased, allowing the recombinant plasmid to enter the cell interior.
- Protein Expression
In the successfully transformed BL21 cells, we used IPTG as an inducer to express the Cas12a protein. The Cas12a protein was designed to carry a 6-His tag, which makes the subsequent protein purification easier and more efficient. The 6-His tag can specifically bind to nickel ions, thus allowing the Cas12a protein to be purified from the bacterial cells through nickel column purification.
We released the Cas12a protein by lysing the cells and then separated it from the complex cell lysate using nickel column purification. Nickel column purification is based on the principle of affinity chromatography, which achieves efficient purification of the Cas12a protein through the specific binding of the 6-His tag to nickel ions. The purified Cas12a protein serves as the Cas12a enzyme for our detection, with high activity and specificity.
Through the above steps, we successfully constructed the vector for expressing the Cas12a protein and achieved efficient expression and purification of the Cas12a protein. This design work provides a key enzymatic basis for our test strip detection platform and fluorescence detection platform, enabling us to utilize the trans-cleavage activity of Cas12a for efficient and sensitive EB prenatal detection.
Compared to conventional nucleic acid amplification technologies, RPA amplification can be performed under isothermal conditions, which further reduces reliance on complex equipment and enhances the portability of the detection products.
The c.520G>A and c.6745C>T mutations in the COL7A1 gene are single-base mutations. After primer design based on the mutation sequences, there is a single-base difference at the mutation sites between normal samples and samples with the mutation sequence. Although crRNA requires stringent recognition of the first five bases of the PAM-proximal sequence for target DNA, it does not guarantee that a single-base mismatch in the normal sequence will completely block crRNA recognition and lead to a total loss of Cas12a cleavage activity. The increase in the number of mismatch sites results in a decrease in Cas12a cleavage activity. This change is not gradual or linear but abrupt and sudden. To create a significant signal difference between the normal and mutation sequences, we gradually increased the number of mismatched bases in the RPA primers. We expect that at a certain number of mismatched sites, the normal sequence (which will always have one more mismatch than the mutation sequence because the crRNA design is based on the mutation sequence) will hardly produce any Cas12a cleavage signal, while the mutation sequence can still be recognized by crRNA, preserving most of the cleavage signal. Based on this, different RPA primers designed for the normal and mutation sequences were used for RPA amplification, and the preferred RPA primers were selected through the fluorescence signals generated by Cas12a cleavage.
A schematic diagram of RPA primer design for different mutation sites is shown in Figure 3.
Figure 3 Primer design diagram
The Cas12a cleavage reaction system includes RPA amplification products, Cas12a, crRNA, and ssDNA reporters. The crRNA is designed based on the mutation sequence, searching for the PAM sequence (TTN) near the mutation site, with the requirement that the mutation site is within five bases after the PAM sequence. The first half of the crRNA sequence is specific to Cas12a, while the latter half is complementary to the 19–24 bases after the selected PAM site on the Target sequence. The relative position of the crRNA to the mutation site is illustrated in Figure 3. Additionally, ssDNA with group labels on both ends is used as the signal reporter.
In the fluorescence detection system, the ssDNA is modified with FAM (fluorescent group) and BHQ1 (quencher group) and named FAM-ssDNA-BHQ1. Upon detecting the mutation sequence, fluorescence can be observed in the system. In the lateral flow test strip detection system, the ssDNA is modified with FAM (as a regular antigen) and biotin (as another antigen), named FAM-ssDNA-Biotin. When the mutation sequence is detected, the corresponding test line (T line) appears on the detection strip. The antibodies on the test strip correspond to the FAM and Biotin antigens on the FAM-ssDNA-Biotin.
Schematic diagrams of the detection process for the fluorescence detection system and the lateral flow test strip detection system are shown in Figures 4 and 5, respectively.
Figure 4. Schematic diagram of the fluorescence detection principle.
Figure 5. Schematic diagram of the test strip detection principle.
The objective of this project is to develop a highly sensitive, non-invasive single-base mutation detection platform utilizing the trans-cleavage activity of Cas12a (Cpf1). By employing synthetic biology techniques, we aim to construct and efficiently express the AsCpf1 protein in E. coli BL21 cells, followed by purification for subsequent detection applications. The project will leverage Recombinase Polymerase Amplification (RPA) for the initial amplification of cell-free fetal DNA (cfDNA) extracted from maternal peripheral blood, followed by secondary signal amplification through Cas12a-mediated cleavage. This will enhance the sensitivity of detection. Focusing on two common single-base mutations, c.520G>A and c.6745C>T in the COL7A1 gene, the platform will incorporate both fluorescence signal detection and lateral flow assay-based detection methods, providing a rapid, accurate, and non-invasive approach to genetic mutation diagnostics. This approach offers an innovative solution for precision medicine and non-invasive prenatal diagnostics.
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