Figure 5: Synthetic propagation circuits induce bi-directional spreading.

(A) Using a CRISPR-guided synthetic initiator (synI^dCas9) to investigate m6A propagation symmetry. synI^dCas9 is recruited to the center of the Clustered Reporter GATC array to establish a nucleation site and to observe synRW-induced m6A propagation away from the site (see Figures S3G, S3H).(B) synI^dCas9 enables targeted m6A enrichment near the center of the GATC reporter array. Quantification of m6A enrichment at a central GATC probe site (red; 716-bp downstream from ZF array) and sites located downstream (dark grey; +286-bp) and upstream (light grey; −122-bp) of probe site following transfection of Clustered Reporter lines with synI^dCas9 and single gRNA (or empty) cassette. m6A enrichment is obtained as previously described, normalizing to basal methylation induced by synI^dCas9 alone.(n=3; error bars, SD). (C) m6A profiles measured across the GATC array for Clustered Reporter cell lines transfected with: synI^dCas9 and target gRNA (grey); synI^dCas9, target gRNA, and synRW (purple); synI^dCas9, empty gRNA, and synRW (black). (n=3; error bars, SD). See also Figure S6.