Nature communications · 2021
Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo.
Оценочное исследованиеСтатьяПоддержка NIH: внешняя программаПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Ibraheim Raed, Tai Phillip W L, Mir Aamir, Javeed Nida, Wang Jiaming, Rodríguez Tomás C, Namkung Suk, Nelson Samantha, Khokhar Eraj Shafiq, Mintzer Esther, Maitland Stacy, Chen Zexiang, Cao Yueying, Tsagkaraki Emmanouela, Wolfe Scot A, Wang Dan, Pai Athma A, Xue Wen, Gao Guangping, Sontheimer Erik J
Аннотация
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.