All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo [preprint]
Document Type
PreprintPublication Date
2018-05-10Keywords
virusesNeisseria meningitidis Cas9
genome editing
gene therapy
mice
RNA
cholesterol
mutations
Molecular Biology
Amino Acids, Peptides, and Proteins
Enzymes and Coenzymes
Genetic Phenomena
Genetics and Genomics
Molecular Biology
Nucleic Acids, Nucleotides, and Nucleosides
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Show full item recordAbstract
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.Source
bioRxiv 295055; doi: https://doi.org/10.1101/295055. Link to preprint on bioRxiv service.
DOI
10.1101/295055Permanent Link to this Item
http://hdl.handle.net/20.500.14038/29271Rights
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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.Distribution License
http://creativecommons.org/licenses/by/4.0/ae974a485f413a2113503eed53cd6c53
10.1101/295055
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Except where otherwise noted, this item's license is described as 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.