Figure 4.

Effect of T4 DNA modifications on type II-A CRISPR–Cas sgRNA mediated DNA targeting. (A) Schematic of DNA targeting by Cas9. Modified cytosine residues are indicated in red. Cleavage sites are indicated by black arrows. (B) Cleavage assay of Cas9 on 98 bp modified targets (indicated by black arrow). Cas9 is loaded with either targeting sgRNA (T sgRNA) or non-targeting sgRNA (NT sgRNA). Restriction products of Cas9 are 61 and 37 bp. (C) EMSA of dCas9 on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows.