Probing low abundant DNA methylation by CRISPR-Cas12a-assisted cascade exponential amplification

Zhang, Liangliang; Zhao, Xianxian; Hu, Xiaolin; Zhang, Yi; Liu, Ruining; Peng, Hai; Chen, Youhao; Zhang, Hong; Luo, Yang

doi:10.1039/d2an00170e

Cited by 10 publications

(4 citation statements)

References 28 publications

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“…The trans -cleavage of CRISPR has been integrated with nucleic acid isothermal amplification techniques, including RCA [ 25 , 69 , 70 ], LAMP [ [71] , [72] , [73] ], RPA [ 74 , 75 ], and RAA [ [76] , [77] , [78] ] to realize various cascade amplifications.…”

Section: Isothermal Enzyme-activated Cascade Amplificationmentioning

confidence: 99%

Recent advances in cascade isothermal amplification techniques for ultra-sensitive nucleic acid detection

Jiang

et al. 2023

Talanta

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Section: Isothermal Enzyme-activated Cascade Amplificationmentioning

confidence: 99%

Recent advances in cascade isothermal amplification techniques for ultra-sensitive nucleic acid detection

Jiang

et al. 2023

Talanta

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“…Recently, Zhang et al studied a CRISPR-Cas12a assisted methylation (CAM) method: bisulfite treatment integrated rolling cycle amplification and CRISPR-Cas12a assisted cascade amplification to detect locus-specific DNA methylation. [103] First, the methylated and unmethylated SEPT9 gene fragments were treated with bisulfite. Then, the methylated fragments were bound to a padlock oligonucleotide probe, initiating the RCA reaction to produce long ssDNA in the presence of T4 ligase, Phi29 and dNTPs.…”

Section: Bisulfite Conversionmentioning

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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“…Utilizing the trans-cleavage activity of Cas proteins, various biosensors had been established to detect different nucleic acid targets, including pathogenic microorganisms, [13][14][15][16] cancer diagnose, 17,18 genetically modified food, 19,20 and others. 21 To increase detection sensitivity, nucleic acid amplification methods, such as polymerase chain reaction (PCR) and loopmediated isothermal amplification (LAMP), were combined with CRISPR Cas system. 22,23 Nevertheless, these systems still required temperature control equipment to realize the exponential increase of the target concentration.…”

Section: Introductionmentioning

confidence: 99%

CRISPR Cas12a-enabled biosensors coupled with commercial pregnancy test strips for the visible point-of-care testing of SARS-CoV-2

Shen

Huang

et al. 2023

Analyst

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Probing low abundant DNA methylation by CRISPR-Cas12a-assisted cascade exponential amplification

Abstract: Aberrant DNA methylation plays a pivotal role in tumor development and metastasis, and is regarded as a valuable non-invasive cancer biomarker. However, the sensitive and accurate quantification of DNA methylation...

Cited by 10 publications

References 28 publications

Recent advances in cascade isothermal amplification techniques for ultra-sensitive nucleic acid detection

Recent advances in cascade isothermal amplification techniques for ultra-sensitive nucleic acid detection

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

CRISPR Cas12a-enabled biosensors coupled with commercial pregnancy test strips for the visible point-of-care testing of SARS-CoV-2

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