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

Mol Cell. 2019 Feb 21;73(4):714-726.e4. doi: 10.1016/j.molcel.2018.12.003. Epub 2018 Dec 20.

Abstract

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 (N4CC) 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.

Keywords: CRISPR; Neisseria; Nme2Cas9; PAM-interacting domain; adeno-associated virus; anti-CRISPR; off-target; protospacer adjacent motif; sgRNA.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA / genetics*
  • DNA / metabolism
  • Dependovirus / genetics
  • Embryo Transfer
  • Female
  • Gene Editing / methods*
  • Genetic Vectors
  • HEK293 Cells
  • Humans
  • K562 Cells
  • Liver / enzymology*
  • Mice, Inbred C57BL
  • Neisseria meningitidis / enzymology*
  • Nucleotide Motifs
  • Proprotein Convertase 9 / genetics*
  • Proprotein Convertase 9 / metabolism
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • Substrate Specificity
  • Zygote / metabolism

Substances

  • RNA, Guide
  • DNA
  • CRISPR-Associated Protein 9
  • Pcsk9 protein, mouse
  • Proprotein Convertase 9