University of Massachusetts Medical School Faculty Publications
Title
All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo
UMMS Affiliation
RNA Therapeutics Institute; Program in Molecular Medicine
Publication Date
2018-05-10
Document Type
Article Preprint
Disciplines
Amino Acids, Peptides, and Proteins | Enzymes and Coenzymes | Genetic Phenomena | Genetics and Genomics | Molecular Biology | Nucleic Acids, Nucleotides, and Nucleosides
Abstract
Clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted forin vivo genome engineering applications: SpyCas9, SauCas9 and CjeCas9. However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors, especially those in which Cas9 and sgRNA are combined into a single vector genome. Here, we present an additional in vivo editing platform using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct PAM, making it an excellent candidate for safe gene therapy applications. We find that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogrammed the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we delivered NmeCas9 with its single-guide RNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded >35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo. Our findings indicate that NmeCas9 can facilitate future efforts to correct disease-causing mutations by expanding the targeting scope of RNA-guided nucleases.
Keywords
viruses, Neisseria meningitidis Cas9, genome editing, gene therapy, mice, RNA, cholesterol, mutations, Molecular Biology
Rights and Permissions
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
DOI of Published Version
10.1101/295055
Source
bioRxiv 295055; doi: https://doi.org/10.1101/295055. Link to preprint on bioRxiv service.
Journal/Book/Conference Title
bioRxiv
Repository Citation
Ibraheim, Raed; Song, Chun-Qing; Mir, Aamir; Amrani, Nadia; Xue, Wen; and Sontheimer, Erik J., "All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo" (2018). University of Massachusetts Medical School Faculty Publications. 1500.
https://escholarship.umassmed.edu/faculty_pubs/1500
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.
Included in
Amino Acids, Peptides, and Proteins Commons, Enzymes and Coenzymes Commons, Genetic Phenomena Commons, Genetics and Genomics Commons, Molecular Biology Commons, Nucleic Acids, Nucleotides, and Nucleosides Commons