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 applications1– 8 5 9 . High specificity and selectivity of Cas effector proteins is determined by the CRISPR RNA’s (crRNA’s) interchangeable spacer sequence, as well as position and number of mismatches between target sequence and the crRNA sequence1 11 . Some diseases are characterized by epigenetic alterations rather than nucleotide changes, 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 fragmentsinfluence 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.