PT  - JOURNAL ARTICLE
AU  - J.E. van Dongen
AU  - J.T.W. Berendsen
AU  - J.C.T. Eijkel
AU  - L.I. Segerink
TI  - A CRISPR/Cas12a-assisted platform for identification and quantification of single CpG methylation sites
AID  - 10.1101/2021.04.06.438612
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.04.06.438612
4099  - http://biorxiv.org/content/early/2021/04/06/2021.04.06.438612.short
4100  - http://biorxiv.org/content/early/2021/04/06/2021.04.06.438612.full
AB  - Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated nuclease (Cas) systems have repeatedly shown to have excellent performance in nucleotide sensing applications1–5. 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. 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 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.Competing Interest StatementWe would like to thank The Weijerhorst team from the University of Twente and the AmsterdamUMC for the fruitful collaboration. The Weijerhorst Foundation is acknowledged for their financial support.