Endonuclease-Assisted PAM-free Recombinase Polymerase Amplification Coupling with CRISPR/Cas12a (E-PfRPA/Cas) for Sensitive Detection of DNA Methylation

Abstract

DNA methylation is considered as a potential cancer biomarker. The evaluation of DNA methylation level will contribute to the prognosis and diagnosis of cancer. Herein, we propose a novel assay based on endonuclease-assisted protospacer adjacent motif (PAM)-free recombinase polymerase amplification coupling with CRISPR/Cas12a (E-PfRPA/Cas) for sensitive detection of DNA methylation. The methylation-sensitive restriction enzyme (MSRE) is first used to selectively digest unmethylated DNA, while the methylated target remains structurally intact. Therefore, the methylated target can initiate the RPA reaction to generate a large amount of double-stranded DNA (dsDNA). To avoid the dependence of PAM site of CRISPR/Cas12a, one of the RPA primers is designed with 5′phosphate terminuses. After treating with Lambda, the sequence with 5′-phosphate modification will be degraded, leaving the singlestranded DNA (ssDNA). The CRISPR/Cas12a can accurately locate ssDNA without PAM, then initiating its trans-cleavage activity for further signal amplification. Meanwhile, non-specific amplification can be also avoided under Lambda, effectively filtering the detection background. Benefiting from the specificity of MSRE, the high amplification efficiency of Lambda-assisted RPA, and the self-amplification effect of CRISPR/Cas, the E-PfRPA/Cas assay shows outstanding sensitivity and selectivity, and as low as 0.05% of methylated DNA can be distinguished. Moreover, the lateral flow assay is also introduced to exploit the point-of-care diagnostic platform. Most importantly, the proposed method shows high sensitivity for determination of genomic DNA methylation from cancer cells, indicating its great potential for tumor-specific gene analysis.

FINDeM: A CRISPR-based, molecular method for rapid, inexpensive, and field-deployable organism detection.

“…The minimal requirements of FINDeM -a reliably amplified gene with a 20bp to 24bp-long sequence that does not contain a 5'-AAA-3' and is adjacent to a 5'-TTTV-3' PAM site (Zetsche et al 2015; Chen et al 2018) -are likely common within the genomes of most, if not all, taxa; a statement empirically supported by the presence of two such sites within the ˜146bp target gene in this study. However, even if such sites did not exist or were not specific to a single taxon, researchers have begun to identify variants of LbCas12a which recognize additional PAM sites (i.e., 'impLbCas12a'; Tóth et al 2020) and have developed approaches which can even operate in a PAM-independent manner (Zhou et al 2022), potentially allowing for an even greater number of target sequences and, as a result, a greater number of target taxa. Therefore, it appears the applications for which field-friendly, CRISPR-based detection of species is limited only by the imaginations of its users.…”

ALP-assisted chemical redox cycling signal amplification for ultrasensitive fluorescence detection of DNA methylation

“…The sensitivity is comparable or superior to the reported fluorescence methods using other signal amplification strategies (shown in Table S3†). 9,39–41 More important is that the proposed fluorescence method does not require any fluorescent tags. Additionally, it is worth noting that the LOD obtained by this work is much lower than that of other affinity based assays for DNA methylation detection.…”

“…Unfortunately, the bisulfite treatment requires strict conditions such as an acidic environment and high temperature, which may lead to the degradation of the target DNA and thus inaccurate results. 9 In contrast, the methylation-sensitive restriction enzyme digestion method can be performed under mild conditions to prevent DNA degradation. However, the restriction enzyme can only recognize the methylation sites in a specific sequence.…”

Construction of Genetically Encoded Light-Up RNA Aptamers for Label-free and Ultrasensitive Detection of CircRNAs in Cancer Cells and Tissues

“…RPA is an efficient DNA amplification strategy with high sensitivity (10 copies of genomic DNA), simplicity (few and easy hands-on manipulations), isothermality (37–42 °C), and a short reaction time (10–20 min). The integration of RPA with various approaches including the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system and lateral flow device , greatly improves the detection accuracy, sensitivity, and selectivity. However, the involvement of fluorescent modifications and antibody labels may increase the experimental cost.…”