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A Cas9 with PAM recognition for adenine dinucleotides

  • Chatterjee, Pranam
  • Lee, Jooyoung
  • Nip, Lisa
  • Koseki, Sabrina R. T.
  • Tysinger, Emma
  • Sontheimer, Erik J.
  • Jacobson, Joseph M.
  • Jakimo, Noah
Publication Date
May 18, 2020
[email protected]
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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[Formula: see text]-NAAN-3[Formula: see text] 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.

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