A CRISPR/Cas12a-assisted in vitro diagnostic tool for identification and quantification of single CpG methylation sites.

The excellent specificity and selectivity of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated nuclease (Cas) is determined by CRISPR RNA's (crRNA's) interchangeable spacer sequence, as well as the position and number of mismatches between target sequence and the crRNA sequence. Some diseases are characterized by epigenetic alterations rather than nucleotide changes, and are therefore unsuitable for CRISPR-assisted sensing methods. Here we demonstrate an in vitro diagnostic tool to discriminate single CpG site methylation in DNA 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 are determined. The modularity of both Cas12a and MSREs provides 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.