Dual Methylation-Sensitive Restriction Endonucleases Coupling with an RPA-Assisted CRISPR/Cas13a System (DESCS) for Highly Sensitive Analysis of DNA Methylation and Its Application for Point-of-Care Detection

Abstract

High-performance detection of DNA methylation possesses great significance for the diagnosis and therapy of cancer. Herein, for the first time, we present a digestion strategy based on dual methylation-sensitive restriction endonucleases coupling with a recombinase polymerase amplification (RPA)-assisted CRISPR/Cas13a system (DESCS) for accurate and sensitive determination of site-specific DNA methylation. This dual methylation-sensitive restriction endonuclease system selectively digests the unmethylated target but exhibits no response to methylated DNA. Therefore, the intact methylated DNA target triggers the RPA reaction for rapid signal amplification. In contrast, the digested unmethylated target initiates no RPA reaction. RPA products with a T7 promoter can execute the T7 transcription in the presence of T7 RNA polymerase to generate a large number of single-stranded RNA (ssRNA). This ssRNA can be recognized by CRISPR/Cas13a to induce the ssRNase activity of Cas13a, showing the indiscriminate cleavage of the collateral FQ reporter to release the fluorescence signal. With such a design, by combining the unique features of dual methylation-sensitive restriction endonucleases with RPA-assisted CRISPR/Cas13a, the DESCS system not only presents the rapid and powerful signal amplification for the determination of methylated DNA with ultrahigh sensitivity but also effectively eliminates the false positive influences from incomplete digestion of the unmethylated target. More importantly, 0.01% methylation level can be effectively distinguished with the existence of excess unmethylated DNA. In addition, the DESCS assay is integrated into the lateral flow biosensor (LFB) for the point-of-care determination of DNA methylation. In view of the superiorities in high sensitivity, outstanding selectivity, and ease of operation, the DESCS system will provide a reliable assay for site-specific analysis of methylation.

Multiplexed lateral flow assay integrated with orthogonal CRISPR-Cas system for SARS-CoV-2 detection

“…In the effort to avoid the reliance on fluorescence equipment and achieve better POCT feasibility, lateral flow assays are further combined with proposed CRISPR platforms for visual readout [23] , [24] , [25] . Albeit the significant progress in developing field-deployable lateral flow strips for SARS-CoV-2 detection, the readout capability is usually constrained to 1 gene per test, which severely impacts the accuracy and efficiency of the results [26] , [27] , [28] . During the diagnosis of COVID-19, multiplexed gene detection are recommended (for example, ORF1ab and N genes) to avoid the false negative or positive results caused by gene degradation or amplification errors [29] , [30] .…”

Detection of small-sized DNA fragments in a glassy nanopore by utilization of CRISPR-Cas12a as a converter system

“…The integration of the CRISPR-Cas12a and nucleic acid amplification techniques has been proved by several works. 22,60,63–65 These results demonstrated that our nanopore sensor using a DNA tetrahedron as a signal transducer based on the CRISPR-Cas12a conversion mechanism could detect targets specifically, which we believe could become a promising alternative to biomolecular detection.…”

Powerful CRISPR-Based Biosensing Techniques and Their Integration With Microfluidic Platforms

“…CRISPR-based biosensors also showed promising potential for the detection of non-infectious human diseases, such as cancer, based on the features with disease-related gene mutations, single-nucleotide polymorphism, DNA methylation, and so on ( Aman et al, 2020 ; Li B. et al, 2021 ; Wang et al, 2021c ; Chen Y. et al, 2021 ; Sheng et al, 2021 ). For example, a Cas12a-based biosensor showed sensitive detection of gene-PIK3CA H1047R mutation low at 0.001%, which has great potential to predict early-stage breast cancer ( Deng et al, 2021 ).…”

“…Currently, clustered regularly interspaced short palindromic repeats (CRISPR) based biosensors have been developed as powerful tools for nucleic acid sensing and widely applied to the rapid diagnosis of infectious pathogens ( Kellner et al, 2019 ; Qin et al, 2019 ; Broughton et al, 2020 ; Ding R. et al, 2021 ; Song F. et al, 2021 ; Chen et al, 2021c ; Escalona-Noguero et al, 2021 ; Ge et al, 2021 ) and the detection of DNA or miRNAs associated with diseases such as cancer ( Li B. et al, 2021 ; Wang et al, 2021c ; Chen Y. et al, 2021 ). These biosensors rely on the CRISPR systems containing CRISPR-associated proteins (Cas) with nonspecific endonuclease activity to efficiently cleave specific targets via guide RNAs (gRNAs) ( Cong et al, 2013 ; Mali et al, 2013 ; van der Oost, 2013 ).…”