A novel methyl-dependent DNA endonuclease GlaI coupling with double cascaded strand displacement amplification and CRISPR/Cas12a for ultra-sensitive detection of DNA methylation

Zhou, Shiying; Sun, Human; Huo, Danqun; Wang, Xianfeng; Qi, Na; Peng, Lan; Yang, Mei; Lü, Peng; Hou, Changjun

doi:10.1016/j.aca.2022.339914

Cited by 14 publications

(3 citation statements)

References 43 publications

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“…Recently, Zhou et al proposed a method which combined methyl-sensitive DNA endonuclease GlaI with double cascade strand displacement amplification and CRISPR-Cas12a (GlaI-DC-SDA-CRISPR-Cas12a) to detect DNA methylation at specific sites. [129] As shown in Figure 9, first, the author chose a specific site of 5′-G m CG m C-3′/3′m CG m CG-5′ (77373474 site) in Septin 9 gene. Under the action of GlaI, the methylated sequences were cleaved into several different DNA fragments with 3′-OH.…”

Section: Combination With Isothermal Amplificationmentioning

confidence: 99%

Locus‐Specific Detection of DNA Methylation: The Advance, Challenge, and Perspective of CRISPR‐Cas Assisted Biosensors

Cao

et al. 2023

Small Methods

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which is considered to be the main epigenetic modification in mammals. It regulates many essential biological events such as chromatin stability, control of tissue-specific gene expression, and stem cell differentiation. [4,5] Therefore, it is crucial to specifically identify DNA methylation using a rapid, sensitive method for screening diseases such as cancer.Conventional DNA methylation detection techniques include PCR-based methods, epityper DNA methylation analysis and genome sequencing, which have been widely used to study the biological function of DNA methylation. [6][7][8][9] But for genome-wide methylation analysis, they hold some disadvantages, such as crossreactivity, complex and time-consuming procedures, expensive instruments, need of skilled technicians and complex data analysis processes. Since the amount of DNA methylation in the actual sample is extremely low, a relatively perfect assay to detect DNA methylation should be fast, sensitive, site-specific and affordable. In recent years, clustered regularly interspaced short palindromic repeats-associated nuclease (CRISPR-Cas) systems have largely updated the way we detect nucleic acids. [10] The CRISPR-Cas system consists of a Cas protein acting as a DNA/RNA enzyme and gRNA/crRNA specifically recognizing target sequences. The assays based on CRISPR-Cas systems have some notable features, such as mild reaction temperature, good recognition specificity, and high signal amplification ability. [11][12][13] This versatility underscores the great promise of CRISPR-Cas systems for future biomedical applications, especially in site-specific DNA methylation detection.In this review, we first summarized the generation, clinical significance, and traditional detection methods of DNA methylation. Then, the basic features, applications, and advantages of the CRISPR-Cas systems for detecting site-specific DNA methylation were summarized in detail. Next, the emerging strategies of the CRISPR-Cas systems for site-specific DNA methylation determination combined with multiple detection techniques were carefully analyzed and summarized. These strategies were divided into five categories, including bisulfite conversion, methylation-sensitive restriction endonuclease (MSRE) digestion, methyltransferase modification, isothermal amplification and nanopore sequencing. Finally, the challenges of CRISPR-Cas systems for DNA methylation Deoxyribonucleic acid (DNA) methylation is one of the epigenetic characteristics that result in heritable and revisable phenotype changes but without sequence changes in DNA. Aberrant methylation occurring at a specific locus was reported to be associated with cancers, insulin resistance, obesity, Alzheimer's disease, Parkinson's disease, etc. Therefore, locus-specific DNA methylation can serve as a valuable biomarker for disease diagnosis and therapy. Recently, Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are applied to develop biosensors for DNA, ribonucleic acid, proteins, and small molecules detection. Bec...

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Section: Combination With Isothermal Amplificationmentioning

confidence: 99%

Locus‐Specific Detection of DNA Methylation: The Advance, Challenge, and Perspective of CRISPR‐Cas Assisted Biosensors

Cao

et al. 2023

Small Methods

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“…In view of this, a few studies have combined the MSRE, CRISPR/Cas12a, and signal amplification strategies for methylation detection. For example, methylation was identified by using GlaI, followed by strand displacement amplification (SDA) to generate a large number of activators and finally, a second signal amplification and output of the detection signal using the trans -cleavage of Cas12a . However, these studies still suffer from two shortcomings.…”

mentioning

confidence: 99%

“…For example, methylation was identified by using GlaI, followed by strand displacement amplification (SDA) to generate a large number of activators and finally, a second signal amplification and output of the detection signal using the trans-cleavage of Cas12a. 30 However, these studies still suffer from two shortcomings. First, as SDA and other nucleic acid amplification techniques are often difficult to accurately distinguish single-base mismatches, a large number of nonspecific products may be amplified and may activate Cas12a, generating erroneous signals.…”

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confidence: 99%

Ratiometric CRISPR/Cas12a-Triggered CHA System Coupling with the MSRE to Detect Site-Specific DNA Methylation

Ding,

Cao,

et al. 2024

ACS Sens.

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The precise determination of DNA methylation at specific sites is critical for the timely detection of cancer, as DNA methylation is closely associated with the initiation and progression of cancer. Herein, a novel ratiometric fluorescence method based on the methylation-sensitive restriction enzyme (MSRE), CRISPR/ Cas12a, and catalytic hairpin assembly (CHA) amplification were developed to detect site-specific methylation with high sensitivity and specificity. In detail, AciI, one of the commonly used MSREs, was employed to distinguish the methylated target from nonmethylated targets. The CRISPR/Cas12a system was utilized to recognize the site-specific target. In this process, the protospacer adjacent motif and crRNA-dependent identification, the single-base resolution of Cas12a, can effectively ensure detection specificity. The trans-cleavage ability of Cas12a can convert one target into abundant activators and can then trigger the CHA reaction, leading to the accomplishment of cascaded signal amplification. Moreover, with the structural change of the hairpin probe during CHA, two labeled dyes can be spatially separated, generating a change of the Forster resonance energy transfer signal. In general, the proposed strategy of tandem CHA after the CRISPR/Cas12a reaction not only avoids the generation of false-positive signals caused by the target-similar nucleic acid but can also improve the sensitivity. The use of ratiometric fluorescence can eradicate environmental effects by self-calibration. Consequently, the proposed approach had a detection limit of 2.02 fM. This approach could distinguish between colorectal cancer and precancerous tissue, as well as between colorectal patients and healthy people. Therefore, the developed method can serve as an excellent site-specific methylation detection tool, which is promising for biological and disease studies.

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CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly for ultrasensitive fluorescence detection of HIV-1 DNA

Zhao

Xia

Wang

et al. 2022

Analytica Chimica Acta

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A novel methyl-dependent DNA endonuclease GlaI coupling with double cascaded strand displacement amplification and CRISPR/Cas12a for ultra-sensitive detection of DNA methylation

Cited by 14 publications

References 43 publications

Locus‐Specific Detection of DNA Methylation: The Advance, Challenge, and Perspective of CRISPR‐Cas Assisted Biosensors

Locus‐Specific Detection of DNA Methylation: The Advance, Challenge, and Perspective of CRISPR‐Cas Assisted Biosensors

Ratiometric CRISPR/Cas12a-Triggered CHA System Coupling with the MSRE to Detect Site-Specific DNA Methylation

CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly for ultrasensitive fluorescence detection of HIV-1 DNA

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