Title

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

UMMS Affiliation

RNA Therapeutics Institute; Department of Pediatrics, Division of Genes and Development; Program in Molecular Medicine

Publication Date

2019-02-21

Document Type

Article

Disciplines

Amino Acids, Peptides, and Proteins | Bioinformatics | Biotechnology | Genetics and Genomics | Investigative Techniques | Molecular Biology | Nucleic Acids, Nucleotides, and Nucleosides | Therapeutics

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

DOI of Published Version

10.1016/j.molcel.2018.12.003

Source

Mol Cell. 2019 Feb 21;73(4):714-726.e4. doi: 10.1016/j.molcel.2018.12.003. Epub 2018 Dec 20. Link to article on publisher's site

Journal/Book/Conference Title

Molecular cell

Related Resources

Link to Article in PubMed

PubMed ID

30581144

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