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

Genome editing technologies based on RNAguided nucleases (RGNs) derived from prokaryotic type II CRISPR-Cas9 adaptive immune systems, such as that from Streptococcus pyogenes and, more recently, Staphylococcus aureus, are becoming increasingly pervasive in both basic and applied research. 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 50-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. RGNs based on S. pyogenes Cas9 (SpCas9) and on S. aureus Cas9 (SaCas9) typically have spacers with a length of 20 and 21–24 nucleotides, respectively. The PAMof SpCas9 is NGG, while that of SaCas9 is NNGRRT. The repair of RGN-induced targeted DNA lesions by non-homologous end joining (NHEJ) or homologous recombination can result in either the deletion or addition of genetic information in cells from virtually any organism.