Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells.
Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells.
复制标题
DOI：
10.1038/srep05105
复制
发表时间：
2014-05-29
影响因子：
4.6
通讯作者：
Gonçalves MA
中科院分区：
文献类型：
--
作者：
Maggio I;Holkers M;Liu J;Janssen JM;Chen X;Gonçalves MA
CRISPR/Cas9衍生的RNA引导的核酸酶（RGN）是DNA靶向系统，其正迅速被用于模式生物和细胞系中的基因调控和基因编辑目的。作为真正的基因递送载体，病毒载体可能特别适合于将RGN的适用性扩大到其他细胞类型，包括分裂和静止的原代细胞。在这里，腺病毒载体（AdVs）的适用性提供RGN组件到各种类型的细胞进行了研究。我们证明，AdV，即编码Cas9或将Cas9核酸酶寻址到AAVS 1“安全港”基因座或重组模型等位基因的单向导RNA（gRNA）分子的第二代纤维修饰的AdV，可以产生高滴度（高达20 × 1010个转导单位/ml）。重要的是，AdV介导的将gRNA：Cas9核糖核蛋白复合物转导到转化的和非转化的细胞中产生的靶向诱变速率类似于或接近于由编码靶向相同AAVSl染色体区域的TALEN的同基因AdV实现的那些。RGN诱导的基因破坏频率在各种细胞类型中的范围为18%至65%。我们的结论是，腺病毒构成了一个有价值的平台，将RGNS引入人体细胞，无论其转化状态。这种方法应该有助于研究RGN在许多实验环境中的潜力和局限性。
CRISPR/Cas9-derived RNA-guided nucleases (RGNs) are DNA targeting systems, which are rapidly being harnessed for gene regulation and gene editing purposes in model organisms and cell lines. As bona fide gene delivery vehicles, viral vectors may be particularly fit to broaden the applicability of RGNs to other cell types including dividing and quiescent primary cells. Here, the suitability of adenoviral vectors (AdVs) for delivering RGN components into various cell types is investigated. We demonstrate that AdVs, namely second-generation fiber-modified AdVs encoding Cas9 or single guide RNA (gRNA) molecules addressing the Cas9 nuclease to the AAVS1 “safe harbor” locus or to a recombinant model allele can be produced to high-titers (up to 20 × 1010 transducing units/ml). Importantly, AdV-mediated transduction of gRNA:Cas9 ribonucleoprotein complexes into transformed and non-transformed cells yields rates of targeted mutagenesis similar to or approaching those achieved by isogenic AdVs encoding TALENs targeting the same AAVS1 chromosomal region. RGN-induced gene disruption frequencies in the various cell types ranged from 18% to 65%. We conclude that AdVs constitute a valuable platform for introducing RGNs into human somatic cells regardless of their transformation status. This approach should aid investigating the potential and limitations of RGNs in numerous experimental settings.
登录
查看更多内容
影响因子：
17.3
作者：
Gaj, Thomas;Gersbach, Charles A.;Barbas, Carlos F., III
通讯作者：
Barbas, Carlos F., III
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
12.4
作者：
Goncalves, Manuel A. F. V.;Janssen, Josephine M.;de Vries, Antoine A. F.
通讯作者：
de Vries, Antoine A. F.
影响因子：
5.4
作者：
Ferrari, FK;Samulski, T;Samulski, RJ
通讯作者：
Samulski, RJ
影响因子：
14.9
作者：
Cradick TJ;Fine EJ;Antico CJ;Bao G
通讯作者：
Bao G