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| Image created by Dr. Michael J. Miller |
Researchers at the University of Florida (UF) have developed a testing method that utilizes CRISPR gene-editing technology to detect HPV in blood or saliva samples that could promote earlier cervical cancer detection.
The researchers discussed their method in the American Chemical Society's journal ACS Applied Materials & Interfaces. The nucleic acid assay the team developed uses Cas12a to analyze viral DNA and can be paired with inexpensive devices using smartphones for testing. It therefore has the potential to increase access to easier diagnosis of the virus that causes most cervical cancers, especially in resource-limited areas.
While CRISPR technology has been used effectively in diagnostics, it often requires preamplification to increase the target concentration before the virus can be detected, which adds complexity to the process and increases the risk of contamination during handling. Thus, the UF team aimed to design a preamplification-free CRISPR-Cas assay with high sensitivity, no need for preamplification, and a simple workflow.
For the assay, dubbed Solid-Phase Extraction and Enhanced Detection Assay integrated by CRISPR-Cas12a (SPEEDi-CRISPR), magnetic beads coated with Cas12a/crRNA complexes called ribonucleoproteins capture and extract the target DNA. The ribonucleoproteins then generate a signal that can be read through lateral flow assay or paired with a portable device such as the team's smartphone-based fluorescence detector.
SPEEDi-CRISPR was able to detect two HPV subtypes, HPV-16 and HPV-18, which are responsible for about 70% of all cervical cancers, with high sensitivity and specificity, as well as Parvovirus B19.
Because it is primarily caused by a virus for which a vaccine exists, cervical cancer is one of the most preventable forms of cancer. While effective vaccines against HPV infection are available, vaccination rates remain low. Moreover, early diagnosis through screening and HPV detection greatly improves chances of successful treatment of cervical cancer. A simple, accurate test for HPV that can be delivered at the point of care could help significantly with cervical cancer prevention.
In a statement, the UF researchers said that the assay could be adapted for other diseases caused by viruses in future work.
Reference
Solid-Phase Extraction and Enhanced Amplification-Free Detection of Pathogens Integrated by Multifunctional CRISPR-Cas12a. Zimu Tian, He Yan, and Yong Zeng, ACS Applied Materials & Interfaces 2024 16 (12), 14445-14456, DOI: 10.1021/acsami.3c17039
Abstract
Public healthcare demands effective and pragmatic diagnostic tools to address the escalating challenges in infection management in resource-limited areas. Recent advances in clustered regularly interspaced short palindromic repeat (CRISPR)-based biosensing promise the development of next-generation tools for disease diagnostics, including point-of-care (POC) testing for infectious diseases. The currently prevailing strategy of developing CRISPR/Cas-based diagnostics exploits only the target identification and trans-cleavage activity of a CRISPR-Cas12a/Cas13a system to provide diagnostic results, and they need to be combined with an additional preamplification reaction to enhance sensitivity. In contrast to this dual-function strategy, here, we present a new approach that collaboratively integrates the triple functions of CRISPR-Cas12a: target identification, sequence-specific enrichment, and signal generation. With this approach, we develop a nucleic acid assay termed Solid-Phase Extraction and Enhanced Detection Assay integrated by CRISPR-Cas12a (SPEEDi-CRISPR) that negates the need for preamplification but significantly improves the detection of limit (LOD) from the pM to fM level. Specifically, using Cas12a-coated magnetic beads, this assay combines efficient solid-phase extraction and enrichment of DNA targets enabled by the sequence-specific affinity of CRISPR-Cas12a with fluorogenic detection by activated Cas12a on beads. SPEEDi-CRISPR, for the first time, leverages the possibility of employing CRISPR/Cas12a in nucleic acid extraction and integrates the ability of both enrichment and detection of CRISPR/Cas into a single platform. Our proof-of-concept studies revealed that the SPEEDi-CRISPR assay has great specificity to distinguish HPV-18 from HPV-16, and Parvovirus B19, in addition to being able to detect HPV-18 at a concentration as low as 2.3 fM in 100 min and 4.7 fM in 60 min. Furthermore, we proved that this assay can be coupled with two point-of-care testing strategies: the smartphone-based fluorescence detector and the lateral flow assay. Overall, these results suggested that our assay could pave a new way for developing CRISPR diagnostics.