A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing

Alireza Edraki, Aamir Mir, Raed Ibraheim, Ildar Gainetdinov, Yeonsoo Yoon, Chun Qing Song, Yueying Cao, Judith Gallant, Wen Xue, Jaime A. Rivera-Pérez, Erik J. Sontheimer

Research output: Contribution to journalArticlepeer-review


CRISPR-Cas9 genome editing has transformed biotechnology and therapeutics. However, in vivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors), off-target editing, or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here, we exploited natural variation in the PAM-interacting domains (PIDs) of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis—Nme2Cas9—that recognizes a simple dinucleotide PAM (N 4 CC) that provides for high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA targeting Pcsk9 in adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Nme2Cas9 combines all-in-one AAV compatibility, exceptional editing accuracy within cells, and high target site density for in vivo genome editing applications.

Original languageEnglish (US)
Pages (from-to)714-726.e4
JournalMolecular Cell
Issue number4
StatePublished - Feb 21 2019


  • adeno-associated virus
  • anti-CRISPR
  • Neisseria
  • Nme2Cas9
  • off-target
  • PAM-interacting domain
  • protospacer adjacent motif
  • sgRNA

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology


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