A CRISPR/Cas12a-assisted platform for identification and quantification of single CpG methylation sites

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated nuclease (Cas) systems have repeatedly shown to have excellent performance in nucleotide sensing applications. High specificity and selectivity of Cas effector proteins is determined by the CRISPR RNAs (crRNAs) interchangeable spacer sequence, as well as position and number of mismatches between target sequence and the crRNA sequence. Some diseases are characterized by epigenetic alterations rather than nucleotide change, and are therefore unsuitable for CRISPR assisted sensing methods. Here we demonstrate a method to discriminate single CpG site methylation in DNA, which is an epigenetic alteration, by the use of methylation sensitive restriction enzymes (MSREs) followed by Cas12a assisted sensing. Non-methylated sequences are digested by MSREs, resulting in fragmentation of the target sequence that influences the R loop formation between crRNA and target DNA. We show that fragment size, fragmentation position and number of fragments influence the subsequent collateral trans-cleavage activity towards single-stranded DNA (ssDNA), enabling deducting the methylation position from the cleavage activity. Utilizing MSREs in combination with Cas12a, single CpG site methylation levels of a cancer gene were for the first time determined. The modularity of both Cas12a and MSREs provide a high level of versatility to the Cas12a MSRE combined sensing method, which opens the possibility to easily and rapidly study single CpG methylation sites for disease detection.

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

“…MSRE is a class of enzymes, including HpaII, AciI and MspI, which can recognize and cut DNA containing unmethylated cytosine sites, but it does not digest methylated DNA. [ 105–107 ] Instead, GlaI specifically cleaves a specific methylation site(5′‐G5 m CG5 m C‐3′) but does not cut unmodified DNA and N4‐methylcytosines DNA, [ 108 ] effectively avoiding the interference of incomplete digestion of methylation sequences. Traditionally, MSRE has been integrated with quantitative polymerase chain reaction (qPCR) amplification for quantification.…”

“…Recently, an in vitro diagnostic tool has been discovered to distinguish methylation at individual CpG sites in DNA by using MSRE and Cas12a auxiliary sensing. [ 107 ] In the experiments, the author synthesized a 31 bp dsDNA sequence containing PAM with three MSRE recognition sites, which can be selectively cleaved by MSRE. Without MSRE treatment, the target dsDNA is able to activate the trans‐cleavage of Cas12a.…”