A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing
RNA Therapeutics Institute; Department of Pediatrics, Division of Genes and Development; Program in Molecular Medicine
Amino Acids, Peptides, and Proteins | Bioinformatics | Biotechnology | Genetics and Genomics | Investigative Techniques | Molecular Biology | Nucleic Acids, Nucleotides, and Nucleosides | Therapeutics
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.
CRISPR, Neisseria, Nme2Cas9, PAM-interacting domain, adeno-associated virus, anti-CRISPR, off-target, protospacer adjacent motif, sgRNA
DOI of Published Version
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
Edraki, Alireza; Mir, Aamir; Ibraheim, Raed; Ildar Gainetdinov; Yoon, Yeonsoo; Song, Chun-Qing; Cao, Yueying; Gallant, Judith; Xue, Wen; Rivera, Jaime A.; and Sontheimer, Erik J., "A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing" (2019). Pediatric Publications and Presentations. 261.