High-throughput detection of single nucleotide polymorphisms (SNPs) via CRISPR-based diagnostic platform

This CRISPR-based technology enables precise detection of single nucleotide polymorphisms (SNPs) in nucleic acids by using programmed mismatches between CRISPR RNA and target nucleic acids, facilitating high-throughput, quantitative, and point-of-care diagnostics.

Background

Single nucleotide polymorphisms (SNPs) play a crucial role in disease susceptibility, pathogenesis, and drug efficacy, as evidenced by their impact on the transmission of the SARS-CoV-2 omicron variant. Detecting these genetic variations is essential for personalized medicine, yet current methods like PCR-based techniques and whole genome sequencing are often limited by the need for specialized laboratory equipment and are time-consuming.

CRISPR-based diagnostics offer a promising alternative due to their ability to rapidly discriminate nucleic acids. These systems, which rely on the sequence-specific endonuclease activity of CRISPR-associated (Cas) enzymes guided by CRISPR RNA (crRNA), have been adapted for various applications, including genome editing and nucleic acid detection.

However, existing CRISPR-Cas systems face challenges in distinguishing between closely related nucleic acid sequences, such as SNPs, which are critical for accurate diagnostics and therapeutic interventions. Therefore, there is a need for more efficient methods and systems that can precisely differentiate between target nucleic acids and their variants in specific regions.

Technology description

This technology utilizes a CRISPR-based method for detecting single nucleotide polymorphisms (SNPs) in nucleic acids by employing programmed mismatches between CRISPR RNA (crRNA) and target nucleic acids. The system leverages a CRISPR/Cas complex, specifically using Cas13 or its variant Cas13d, which cleaves a nucleic acid probe when the crRNA is fully complementary to the target nucleic acid. The presence of mismatches, especially in the proximal region of the crRNA, reduces cleavage activity, enabling differentiation between the target and its variants.

This method supports high-throughput, quantitative detection and can be adapted for multiplex formats, making it suitable for point-of-care diagnostics. The platform includes a nucleic acid probe that emits distinct signals upon cleavage, facilitating SNP identification in diverse samples from biological or environmental sources.

This technology stands out due the use of Cas13d, a smaller and more flexible enzyme that does not require a protospacer flanking sequence, enhances the system’s adaptability and ease of production. By allowing for rapid, point-of-care diagnostics, this technology offers significant advantages in personalized medicine, enabling timely and precise detection of pathogen variants or disease-associated genetic mutations. Its multiplex capability further reduces analysis time and costs, making it a valuable tool in clinical and environmental settings.