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

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

Author information

1
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA.
2
Department of Pediatrics, Division of Genes and Development, University of Massachusetts Medical School, Worcester, MA 01605, USA.
3
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
4
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address: erik.sontheimer@umassmed.edu.

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

Comment in

PMID:
30581144
PMCID:
PMC6386616
[Available on 2020-02-21]
DOI:
10.1016/j.molcel.2018.12.003
[Indexed for MEDLINE]

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