A Cas9 with PAM recognition for adenine dinucleotides

CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif (PAM) requirements. We demonstrate a critical expansion of the targetable sequence space for a type II-A CRISPR-associated enzyme through identification of the natural 5-NAAN-3 ' PAM preference of Streptococcus macacae Cas9 (SmacCas9). To achieve efficient editing activity, we graft the PAM-interacting domain of SmacCas9 to its well-established ortholog from Streptococcus pyogenes (SpyCas9), and further engineer an increased efficiency variant (iSpyMac) for robust genome editing activity. We establish that our hybrids can target all adenine dinucleotide PAM sequences and possess robust and accurate editing capabilities in human cells. p id=Par Protospacer adjacent motif (PAM) requirements limit the target range of CRISPR endonucleases. Here, the authors graft the 5 ' -NAAN-3 ' PAM-interacting domain of SmacCas9 onto SpyCas9 to create adenine dinucleotide targeting chimeras.