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 A, Mir A, Ibraheim R, I, Yoon Y, Song C, Cao Y, Gallant J, Xue W, Rivera JA, Sontheimer EJ. (2019). A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing. Pediatric Publications and Presentations. https://doi.org/10.1016/j.molcel.2018.12.003. Retrieved from https://escholarship.umassmed.edu/peds_pp/261