The primary objective of effectively managing infectious diseases and epidemics is the prompt and accurate detection of pathogens, with COVID-19 serving as a prime example. The significance and demand for point-of-care (POC) testing have been particularly emphasized during the ongoing pandemic.

According to the World Health Organization, POC testing should adhere to the ASSURED criteria, encompassing affordability, sensitivity, specificity, user-friendliness, robustness, equipment-free operation, and deliverability.

Developing pathogen-specific POC tests that fulfill all of these criteria poses a considerable challenge. Consequently, in vitro diagnostic companies have been focusing their efforts on combining existing technologies in recent years.

Among the most revolutionary and promising approaches is the integration of isothermal amplification with CRISPR technology, which not only meets the ASSURED criteria but also represents an ideal method for the development of a new generation of POC diagnostics.

This article will primarily provide a comprehensive overview of the CRISPR technology based on LAMP and explore the following key aspects:

1. LAMP and CRISPR technology
2. Rationale behind selecting the combination of LAMP and CRISPR technology
3. Application of LAMP-CRISPR technology in POC platforms

01 - LAMP (Loop-mediated isothermal amplification)

LAMP, or loop-mediated isothermal amplification, is a groundbreaking gene amplification method invented by Dr. Masahiro Notomi of Eiken Chemical Co., Ltd. in Japan in 2000. This technique has gained significant attention due to its simplicity, speed, accuracy, and cost-effectiveness in detecting pathogens across human, animal, and agricultural samples. During the COVID-19 pandemic, LAMP technology has experienced remarkable advancements with various improved methods used for detecting the SARS-CoV-2 virus, marking a golden age for LAMP.

The distinguishing feature of LAMP lies in its utilization of 4-6 primers that selectively target specific regions of DNA. The amplification process is initiated by the Bst enzyme, possessing strand-displacement activity, and two specially designed primers. This leads to the formation of loop structures, resulting in the exponential production of DNA after successive amplification cycles. Remarkably, LAMP can amplify 1-10 DNA copies into an abundant 10^9 - 10^10 copies within a timeframe of 15-60 minutes, showcasing its exceptional sensitivity and specificity.

One of the key advantages of LAMP is its simplicity, as it does not necessitate temperature cycling like qPCR. The amplification reaction is carried out at a constant temperature of 60-65°C, and the results can be observed using various methods such as turbidity, fluorescence, or probe-based techniques. This user-friendly approach enhances the practicality and versatility of LAMP in diverse diagnostic applications.

02 - CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

CRISPR, also known as Clustered Regularly Interspaced Short Palindromic Repeats, is an RNA-based adaptive immune defense system. It utilizes spacer sequences that exhibit similarity to viral or plasmid sequences, allowing it to employ target-specific RNA to guide Cas proteins in the precise recognition and correction of genetic errors in a wide range of organisms and cells.

The CRISPR/Cas system consists of a guide RNA (gRNA) sequence that serves as a navigational tool for Cas proteins (CRISPR-associated nucleases) to target specific regions within foreign genomic material:

The gRNA, comprising tracrRNA and crRNA regions, can be specifically designed to target desired regions, affording the CRISPR/Cas system its programmability.

Cas proteins, acting in accordance with the gRNA, act as nucleases capable of recognizing and cleaving target sequences in DNA or RNA.

The CRISPR/Cas system represents a remarkable and highly promising technology, applicable to gene editing and molecular diagnostics. Through its programmable gRNA sequence, it allows for precise targeting of any gene region. In the field of infectious disease detection, its primary role lies in pathogen identification and nucleic acid-based diagnostics facilitated by Cas proteins.

Cas proteins are classified into two main classes: Cas9, possessing target cleavage activity, and Cas12, Cas13, and Cas14, exhibiting collateral cleavage activity upon target recognition. Cas12 and Cas13 are commonly employed, with Cas12 cleaving non-target single-stranded DNA (ssDNA), and Cas13 cleaving single-stranded RNA (ssRNA).

CRISPR/Cas technology has revolutionized nucleic acid detection by combining it with pre-amplification or post-amplification steps. This synergy with PCR or isothermal amplification methods like LAMP, RPA, NASBA, and RCA has ushered in a new era of molecular diagnostics, enabling highly sensitive and specific detection of DNA and RNA.

Notable examples include:

• CRISPR/Cas9 combined with NASBA technology, successfully applied for Zika virus detection and lineage identification.
• CRISPR-Cas9 typing PCR (ctPCR), a pre-amplification process for detecting HPV subtypes.
• The SHERLOCK system, gaining popularity, which merges Cas13 with RPA in CRISPR/Cas technology, facilitating molecular diagnostics of viruses like dengue fever and Zika virus.
• Cas14 enzyme combined with RCA, enabling detection of microRNA (miRNA) and single nucleotide polymorphisms (SNPs).
• HOLMES, a fusion of CRISPR/Cas with PCR.
• Cas12 combined with LAMP and RPA methods for pathogen-specific detection. Initially known as the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) method, this combined technique has gained traction in point-of-care (POC) diagnostics. The application and development of DETECTR have led to an increasing adoption of LAMP-based CRISPR/Cas technologies in pathogen research.

These combined approaches exemplify the versatility and immense potential of CRISPR/Cas technology in advancing molecular diagnostics for accurate pathogen detection.

03 - LAMP-CRISPR (Loop-mediated isothermal amplification combined with CRISPR)

LAMP-CRISPR represents a cutting-edge molecular diagnostic technology that has emerged in recent years for pathogen detection. This innovative approach combines the strengths of both techniques, effectively overcoming their individual limitations.

LAMP, known for its rapid amplification capabilities, can be susceptible to false-positive results. Factors such as incorrect primer design, DNA contamination, and suboptimal reaction conditions (such as buffer pH, Mg concentration, or interfering dyes) can compromise the assay's specificity and sensitivity.

CRISPR/Cas technology, on the other hand, leverages a reporter system to detect specific targets, offering a powerful solution to mitigate the limitations of LAMP and minimize the occurrence of false-positive results. This integration results in a highly sensitive and specific diagnostic platform.

By combining LAMP with CRISPR/Cas technology, visualizing the results becomes feasible through fluorescence chromatographic strips or direct visual inspection. This eliminates the need for quantitative measurements typically required in other nucleic acid detection methods. Consequently, this streamlined approach enables efficient, sensitive, specific, and user-friendly point-of-care (POC) testing without the requirement for complex equipment.

In the combination of CRISPR/Cas with LAMP technology, Cas12 endonucleases are particularly favored due to their collateral cleavage activity.

Cas12 enzymes encompass several subtypes, including Cas12a, Cas12c, Cas12d, Cas12e, Cas12b, Cas12h, Cas12i, and Cas12g.

Among these subtypes, Cas12a and Cas12b are the most commonly employed in LAMP-CRISPR detection, with Cas12a being the preferred choice. This preference stems from the fact that Cas12b requires a lengthy single-guide RNA (sgRNA) of 111 nucleotides, which encompasses the crRNA and tracrRNA regions. The extended sgRNA may potentially overlap with the LAMP primers designed to target the specific region, leading to false-positive results. In contrast, Cas12a utilizes a shorter gRNA of 41 nucleotides, which was originally employed in the development of the DETECTR method.

Cas13 has also found utility in LAMP-CRISPR applications. As Cas13 cleaves single-stranded RNA (ssRNA), an additional T7 transcription step is necessary when utilizing Cas13. This step allows for the conversion of the pre-amplified DNA products into RNA molecules.

LAMP-CRISPR/Cas technology has been extensively utilized for detecting a wide range of infectious viruses and bacteria. Remarkably, this approach has exhibited exceptional specificity and sensitivity, reaching up to 100% in pathogen detection assays. Moreover, LAMP-CRISPR outperforms the gold standard qPCR by significantly reducing turnaround times, rendering it highly suitable for rapid testing applications.

However, it is important to acknowledge that LAMP-CRISPR/Cas also has certain limitations, primarily due to the requirement of two separate steps: the amplification process and the CRISPR/Cas process. These steps involve different chemical components and reaction conditions. LAMP amplification utilizes the Bst enzyme at temperatures around 60-65 degrees Celsius, while the CRISPR reaction with Cas enzymes typically occurs at approximately 37 degrees Celsius. This necessitates the use of two different temperatures, which can potentially increase the risk of contamination.

Fortunately, there are strategies available to overcome this limitation. For example, in HOLMESv2 detection, a solution is found by utilizing a thermostable Cas12b enzyme derived from Acidophilic Bacillus, enabling a single-tube reaction and eliminating the need for different reaction temperatures.

Another approach to minimize technical demands and contamination risks for the operator involves sealing the LAMP reagents at the bottom of the reaction tube with oil and covering the CRISPR reagents with a reaction tube cap, facilitating a single-tube reaction. After LAMP amplification, the tube is inverted to mix the contents and initiate the CRISPR/Cas12a process.

CRISPR/Cas technology can also be combined with PCR and other isothermal methods, such as NASBA, RCA, and RPA. However, each of these approaches has its limitations.

PCR, for instance, is not ideal for point-of-care (POC) diagnostic platforms due to the need for complex thermal cycling equipment.

Similarly to LAMP, other isothermal amplification methods do not require complex equipment. However, they typically involve the use of two primers and two or more enzymes. NASBA, for example, relies on three enzymes for amplifying RNA or single-stranded DNA. Furthermore, the combination of CRISPR and NASBA generally exhibits lower sensitivity compared to other methods. In contrast, LAMP technology offers advantages in terms of lower costs and operational complexity by utilizing a single Bst enzyme. Moreover, LAMP provides higher specificity and efficiency by utilizing multiple primers.

RPA is widely used as well but requires an additional step to convert the amplification products into RNA molecules. As a result, it is often combined with the Cas13 enzyme. One of RPA's main advantages is its reaction temperature, which closely matches that of the CRISPR reaction. However, it has a major limitation in the lower concentration of amplification products compared to CRISPR, leading to decreased sensitivity when the target concentration is low. When combined with CRISPR, LAMP can generate high concentrations of amplification products. Therefore, LAMP is preferred over other isothermal methods for integration with CRISPR technology in POC diagnostic platforms.

04 - LAMP-CRISPR in POC Applications

Inspired by LAMP technology's success in point-of-care (POC) platforms, LAMP-CRISPR POC devices streamline the process into three stages:

• DNA/RNA extraction,
• LAMP amplification, and
• CRISPR/Cas reaction.

By integrating these steps into a single tube, user convenience is maximized. Various POC platforms have successfully implemented this approach. The key concept involves integrating LAMP and CRISPR/Cas reactions on a microfluidic chip and using fluorescence detection or lateral flow chromatography for result analysis.

For instance, a portable POC instrument is designed as a compact, rechargeable, and semi-automated device measuring only 3.5cm x 3cm x 13cm. It finds applications in mobile laboratories, airports, and quarantine areas. The instrument includes a temperature-adjusting button to facilitate RT-LAMP amplification at 65°C and CRISPR/Cas12a reaction at 37°C. Within a rapid 35-minute timeframe, it can detect up to 10 samples, with results visually interpreted using chromatographic paper.

05 - Solution of SBS Genetech

SBS Genetech is proud to present our innovative range of solutions designed to revolutionize nucleic acid amplification and molecular diagnosis. Leveraging our world-class platform, we offer state-of-the-art products that harness the potential of LAMP (Loop-mediated Isothermal Amplification) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technologies. These groundbreaking solutions pave the way for accurate, rapid, and sensitive pathogen detection as well as precise gene editing applications.

Bst DNA/RNA Polymerase: Powering High-Efficiency Isothermal Amplification

At the core of our platform lies the Bst DNA/RNA Polymerase, a game-changing enzyme that facilitates isothermal amplification reactions for both DNA and RNA templates. This versatile polymerase exhibits exceptional efficiency and specificity, enabling the detection of low-sensitivity nucleic acid templates with remarkable accuracy. Through a meticulous preparation process, our Bst DNA/RNA Polymerase boasts a rapid amplification rate and high tolerance to impurities, ensuring reliable results even in challenging conditions. Notably, its extreme thermostability and sensitive reverse transcriptase activity contribute to higher sensitivity, particularly at high Ct values, as demonstrated in a recent international assessment study [1].

Bst P DNA/RNA Polymerase: Advancing Amplification Performance

Building upon the success of our Bst DNA/RNA Polymerase, we proudly present the Bst P DNA/RNA Polymerase—an upgraded version achieved through cutting-edge in silico design and in vitro evolution screening. This enhanced polymerase is specifically tailored for LAMP or RT-LAMP amplification of DNA or RNA. Key performance improvements include:

• Hot start Aptamer: With an efficiency of over 95% in blocking enzyme activity below 30°C and complete activity release within 1 minute above 60°C, the hot start Aptamer ensures precise control over the reaction system. By enabling the establishment of the system at room temperature, it significantly reduces non-specific amplification at lower temperatures.
• Elevated reaction temperature: By raising the reaction temperature to 70°C, the formation of primer dimers is substantially reduced, enhancing amplification specificity. Additionally, this temperature increase promotes more efficient nucleic acid release from crude samples, further improving overall performance.
• Inclusion of Helicase: The incorporation of Helicase enables Premium LAMP amplification (pLAMP) without the need for F3/B3 primers. This feature not only simplifies the reaction setup but also assists in strand unwinding, reducing the concentration of FIP/BIP primers and minimizing non-specific amplification. The result is amplified homogeneity and superior performance.

Cas12a (Cpf1) and Cas13: Empowering Precise Gene Editing and RNA Cleavage

In addition to our LAMP solutions, we offer Cas12a (Cpf1) and Cas13, two cutting-edge CRISPR effector proteins that revolutionize gene editing and targeted RNA cleavage. These class II, type VI CRISPR systems hold immense potential in various fields, including microbiology, agriculture, and animal genetics.

• Cas12a (Cpf1): This endonuclease binds to specific sites of target DNA guided by single-stranded guide RNA, facilitating precise gene editing in microorganisms, plants, and animals. Our lyophilized version of Cas12a can be transported at room temperature, eliminating the need for costly dry ice transportation.
• Cas13: As a novel CRISPR protease, Cas13 offers targeted RNA cleavage capabilities. Guided by RNA guide sequences, it recognizes and cleaves target RNA, while activating collateral cleavage activity to efficiently cleave non-specific single-stranded RNA (ssRNA). Similar to Cas12a, our lyophilized version of Cas13a can be conveniently transported at room temperature, eliminating the requirement for expensive dry ice transportation.

When you choose SBS Genetech's solutions, you benefit from a range of advantages that set us apart:

• Cutting-edge Technology: Our products incorporate the latest advancements in LAMP and CRISPR technologies, ensuring superior performance and accuracy in nucleic acid amplification and molecular diagnosis.
• Enhanced Sensitivity: Our Bst DNA/RNA Polymerase and Bst P DNA/RNA Polymerase offer high sensitivity, even for low-concentration nucleic acid templates, enabling reliable detection and analysis.
• Improved Specificity: With optimized reaction conditions and innovative features like hot start Aptamer and elevated reaction temperature, our solutions minimize non-specific amplification and primer dimer formation, guaranteeing amplified homogeneity and exceptional specificity.
• Ease of Transportation: Our lyophilized versions of Cas12a and Cas13a can be conveniently transported at room temperature, reducing costs associated with dry ice shipping and enabling hassle-free delivery.
• Application Versatility: Whether you require pathogen detection, gene editing, or RNA cleavage, our solutions offer broad applications across various scientific fields, including medical research, diagnostics, agriculture, and more.
• Scientific Excellence: Backed by extensive research and development, our products undergo rigorous testing to ensure optimal performance and reliability in real-world scenarios.

At SBS Genetech, we are committed to providing cutting-edge solutions that drive advancements in nucleic acid amplification and molecular diagnosis. With our powerful Bst DNA/RNA Polymerase and Bst P DNA/RNA Polymerase, you gain access to exceptional sensitivity, specificity, and performance in LAMP-based amplification. Additionally, our Cas12a and Cas13 effector proteins empower precise gene editing and targeted RNA cleavage, opening new avenues for scientific discovery and innovation.

Reference

[1] Lu S, Duplat D, Benitez-Bolivar P, León C, Villota SD, Veloz-Villavicencio E, et al. (2022) Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application. PLoS ONE 17(5): e0268340. https://doi.org/10.1371/journal.pone.0268340