The Chromatin Structure Differentially Impacts High-Specificity CRISPR-Cas9 Nuclease Strategies

Genome editing technologies based on RNA-guided nucleases (RGNs) derived from prokaryotic type II CRISPR-Cas9 adaptive immune systems, such as that from Streptococcus pyogenes1,2 and, more recently, Staphylococcus aureus,3,4 are becoming increasingly pervasive in both basic and applied research.5,6 RGNs are ribonucleoprotein complexes whose sequence-specific guide RNA (gRNA) moieties address a Cas9 nuclease to a DNA target site (Figure S1A). Base pairing between the 5′-terminal nucleotides of the gRNA (spacer) and DNA sequences connected to a protospacer-adjacent motif (PAM), triggers Cas9-mediated double-stranded DNA break (DSB) formation.