Fig. 5: Enrichment of specific genomic regions using a CRISPR-based method.

a The enrichment protocol is divided into three major steps: (1) Flanking the region of interest with two Cas12a complexes, followed by digestion of all remaining genomic DNA with exonucleases. (2) Second, the generation of 3’ ssDNA overhangs at each end of the fragment used to assemble the hairpins are created using the dead Cas9, followed by lambda exonuclease digestion. (3) Finally, this fragment is used to assemble the hairpin structure required for our platform. A more detailed protocol is available in Supplementary Fig. 8. b qPCR of enrichment of four E. coli targets. A control excluding the exonucleases was included (to account for purification loss) and a positive control for digestion was performed by quantifying off-target DNA. Protection was measured for each target after the Cas12a (dark blue) and dCas9 (light blue) steps. Bars represent the average protected material from three biological replicates +s.e.m, n = 3. c Repartition of target molecules analyzed on the MT platform from three biological replicates (shades of blue) and their average (red bars). d Detection of m⁵C and m⁶A on the same enriched dam molecules (each column represents a single molecule, and in each panel, the same column corresponds to the same bead). Gray points indicate detected binding events and the expected modification positions are indicated on the right axis. Blue crosses indicate detected blockages corresponding to the modification and red crosses indicate expected positions where methylation was not detected. e Analysis of m⁵C methylation of all the isolated E. coli fragments for all three biological replicates. The CCwGG site positions within the hairpins are indicated, as well as their rate of methylation.