Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo

Raed Ibraheim, Phillip W. L. Tai, Aamir Mir, Nida Javeed, Jiaming Wang, Tomás C. Rodríguez, Suk Namkung, Samantha Nelson, Eraj Shafiq Khokhar, Esther Mintzer, Stacy Maitland, Zexiang Chen, Yueying Cao, Emmanouela Tsagkaraki, Scot A. Wolfe, Dan Wang, Athma A. Pai, Wen Xue, Guangping Gao and Erik J. Sontheimer ()
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Raed Ibraheim: University of Massachusetts Medical School
Phillip W. L. Tai: University of Massachusetts Medical School
Aamir Mir: University of Massachusetts Medical School
Nida Javeed: University of Massachusetts Medical School
Jiaming Wang: University of Massachusetts Medical School
Tomás C. Rodríguez: University of Massachusetts Medical School
Suk Namkung: University of Massachusetts Medical School
Samantha Nelson: University of Massachusetts Medical School
Eraj Shafiq Khokhar: University of Massachusetts Medical School
Esther Mintzer: University of Massachusetts Medical School
Stacy Maitland: University of Massachusetts Medical School
Zexiang Chen: University of Massachusetts Medical School
Yueying Cao: University of Massachusetts Medical School
Emmanouela Tsagkaraki: University of Massachusetts Medical School
Scot A. Wolfe: University of Massachusetts Medical School
Dan Wang: University of Massachusetts Medical School
Athma A. Pai: University of Massachusetts Medical School
Wen Xue: University of Massachusetts Medical School
Guangping Gao: University of Massachusetts Medical School
Erik J. Sontheimer: University of Massachusetts Medical School

Nature Communications, 2021, vol. 12, issue 1, 1-17

Abstract: Abstract Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. 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 by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes.

Date: 2021
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DOI: 10.1038/s41467-021-26518-y

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