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

Abstract

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...

Effects of DNA Methylation on Gene Expression and Phenotypic Traits in Cattle: A Review

“…The optical biosensor detects the light generated when the target analyte is captured by the biometric layer, while the electrochemical biosensor detects the changes in electrical parameters before and after biometric recognition of the target analyte [90,91]. Among these methods, CRISPR-Casassisted sensor systems have shown great promise and attracted widespread interest in site-specific DNA methylation detection based on their high specificity of binding and signal amplification capabilities [92]. However, although these biosensors have the advantages of simplicity, low cost, high sensitivity and high detection efficiency, they have limitations in the application of clinical samples [93].…”

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

“…In recent years, the discovery of the clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has opened up new avenues for DNA methylation detection. , Because of its strict base-complementation-dependent cleavage principle (single-base resolution) and programmability, it is a helpful tool for the highly specific detection of nucleic acids. − Particularly, in addition to the crRNA-guided endonuclease activity, the trans -cleavage activity of Cas12a allows signal amplification by cleaving the abundant nonspecific single-stranded DNA (ssDNA) after being triggered by an activator. − It is worth noting that CRISPR/Cas12a is often combined with various tools to further improve its accuracy and sensitivity. In view of this, a few studies have combined the MSRE, CRISPR/Cas12a, and signal amplification strategies for methylation detection.…”