Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo.
Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo.
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DOI：
10.1038/s41467-021-26518-y
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发表时间：
2021-11-01
影响因子：
16.6
通讯作者：
Sontheimer EJ
中科院分区：
文献类型：
--
作者：
Ibraheim R;Tai PWL;Mir A;Javeed N;Wang J;Rodríguez TC;Namkung S;Nelson S;Khokhar ES;Mintzer E;Maitland S;Chen Z;Cao Y;Tsagkaraki E;Wolfe SA;Wang D;Pai AA;Xue W;Gao G;Sontheimer EJ
腺相关病毒（AAV）载体是治疗性基因组编辑的重要递送平台，但受到货物限制的严重限制。同时递送多种媒介可限制剂量和效力，并增加安全风险。在这里，我们描述了单向量，~4.8 kb的AAV平台，表达Nme2Cas9和两个sgRNA进行片段缺失，或单个sgRNA与同源定向修复（HDR）模板。我们还使用抗crispr蛋白来实现通过Nme2Cas9切割自我失活的载体的生产。我们进一步介绍了一个基于纳米孔的测序平台，该平台旨在分析rAAV基因组，并作为载体均匀性的质量控制措施。我们证明这些平台可以通过基于hdr的疾病等位基因校正有效治疗小鼠的两种疾病模型[I型遗传性酪氨酸血症（HT-I）和粘多糖病I型（MPS-I）]。这些结果将使单载体aav的工程设计能够实现多种治疗性基因组编辑结果。在体内进行精确基因组编辑后，Cas9的长期表达可能会导致不良后果。在这里，我们展示了包含Cas9核酸酶、向导和DNA供体的单载体、自我失活的AAV系统可以在体内使用同源性定向修复来纠正疾病突变。
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. Long-term expression of Cas9 following precision genome editing in vivo may lead to undesirable consequences. Here we show that a single-vector, self-inactivating AAV system containing Cas9 nuclease, guide, and DNA donor can use homology-directed repair to correct disease mutations in vivo.
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影响因子：
16.6
作者：
Bolukbasi MF;Liu P;Luk K;Kwok SF;Gupta A;Amrani N;Sontheimer EJ;Zhu LJ;Wolfe SA
通讯作者：
Wolfe SA
影响因子：
9.2
作者：
de Koning W;Miladi M;Hiltemann S;Heikema A;Hays JP;Flemming S;van den Beek M;Mustafa DA;Backofen R;Grüning B;Stubbs AP
通讯作者：
Stubbs AP
影响因子：
8.8
作者：
Gao, Jian;Bergmann, Thorsten;Ehrhardt, Anja
通讯作者：
Ehrhardt, Anja
影响因子：
3.2
作者：
Deveau, Helene;Barrangou, Rodolphe;Moineau, Sylvain
通讯作者：
Moineau, Sylvain
影响因子：
46.9
作者：
Cho, Seung Woo;Kim, Sojung;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo