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Precision Cas9 Genome Editing in vivo with All-in-one, Self-targeting AAV Vectors

By Raed Ibraheim, Phillip W. L. Tai, Aamir Mir, Nida Javeed, Jiaming Wang, Tomas C Rodriguez, Samantha Nelson, Eraj Khokhar, Esther Mintzer, Stacy Maitland, Yueying Cao, Emmanouela Tsagkaraki, Scot Wolfe, Dan Wang, Athma Pai, Wen Xue, Guangping Gao, Erik J. Sontheimer

Posted 09 Oct 2020
bioRxiv DOI: 10.1101/2020.10.09.333997

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits, especially for large effectors like Cas9s. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. The use of compact effectors has enabled single-AAV delivery of Cas9s with 1-3 guides for edits that use end-joining repair pathways, but many precise edits that correct disease-causing mutations in vivo require homology-directed repair (HDR) templates. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs to produce segmental deletions, or a single sgRNA with an HDR template. We also examine the utility of Nme2Cas9 target sites in the vector for self-inactivation. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice. These results will enable single-vector AAVs to achieve diverse therapeutic genome editing outcomes. ### Competing Interest Statement The authors have filed patent applications on technologies related to this work. G.G. is a scientific co-founder of Voyager Therapeutics, Adrenas Therapeutics, and Aspa Therapeutics and holds equity in these companies. G.G. is an inventor on patents with potential royalties licensed to Voyager Therapeutics, Aspa Therapeutics, and other biopharmaceutical companies. E.J.S. is a co-founder and scientific advisor of Intellia Therapeutics.

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