CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors.
CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors.
复制标题
CRISPR介导的大基因盒使用AAV供体矢量整合。
DOI：
10.1016/j.celrep.2017.06.064
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发表时间：
2017-07-18
期刊：
影响因子：
8.8
通讯作者：
Porteus MH
中科院分区：
文献类型：
--
作者：
Bak RO;Porteus MH
CRISPR/Cas9系统最近已被证明在同源重组（HR）期间使用腺相关病毒（AAV）载体作为供体模板DNA促进高水平的精确基因组编辑。然而，约4.5千碱基的最大腺相关病毒包装容量限制了供体的大小。在这里，我们通过表明两个共转导的腺相关病毒载体可以在连续的HR事件中作为供体来整合大型转基因来克服这一限制。重要的是，该方法涉及适用于与治疗性基因组编辑相关的原代细胞的单步程序。我们在原代人T细胞和CD 34+造血干细胞和祖细胞中使用该方法来位点特异性整合表达盒，该表达盒作为单个供体载体将总计6.5个酶。这种方法现在提供了一种有效的方式，使用HR介导的基因组编辑与AAV载体将大的转基因盒整合到原代人细胞的基因组中。目前，使用CRISPR/Cas9和腺相关病毒供体载体将转基因整合到原代细胞基因组的特定位点受到腺相关病毒包装能力有限的阻碍。巴克和Porteus现在报道了一种有效整合超过单个AAV能力的大转基因的方法。
The CRISPR/Cas9 system has recently been shown to facilitate high levels of precise genome editing using adeno associated viral (AAV) vectors to serve as donor template DNA during homologous recombination (HR). However, the maximum AAV packaging capacity of ~4.5 kilobases limits the donor size. Here we overcome this constraint by showing that two co-transduced AAV vectors can serve as donors during consecutive HR events for integration of large transgenes. Importantly, the method involves a single-step procedure applicable to primary cells with relevance to therapeutic genome editing. We use the methodology in primary human T cells and CD34+ hematopoietic stem and progenitor cells to site-specifically integrate an expression cassette that as a single donor vector would otherwise amount to a total of 6.5 kilobases. This approach now provides an efficient way to integrate large transgene cassettes into the genomes of primary human cells using HR-mediated genome editing with AAV vectors. Integration of transgenes into specific sites of the genome of primary cells using CRISPR/Cas9 and AAV donor vectors is currently hampered by the limited packaging capacity of AAV. Bak and Porteus now report a method for efficient integration of large transgenes that exceed the capacity of a single AAV.
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影响因子：
64.8
作者：
Barzel, A.;Paulk, N. K.;Shi, Y.;Huang, Y.;Chu, K.;Zhang, F.;Valdmanis, P. N.;Spector, L. P.;Porteus, M. H.;Gaensler, K. M.;Kay, M. A.
通讯作者：
Kay, M. A.
影响因子：
64.8
作者：
Genovese, Pietro;Schiroli, Giulia;Escobar, Giulia;Di Tomaso, Tiziano;Firrito, Claudia;Calabria, Andrea;Moi, Davide;Mazzieri, Roberta;Bonini, Chiara;Holmes, Michael C.;Gregory, Philip D.;van der Burg, Mirjam;Gentner, Bernhard;Montini, Eugenio;Lombardo, Angelo;Naldini, Luigi
通讯作者：
Naldini, Luigi
影响因子：
46.9
作者：
Hendel A;Bak RO;Clark JT;Kennedy AB;Ryan DE;Roy S;Steinfeld I;Lunstad BD;Kaiser RJ;Wilkens AB;Bacchetta R;Tsalenko A;Dellinger D;Bruhn L;Porteus MH
通讯作者：
Porteus MH
影响因子：
46.9
作者：
Wang J;Exline CM;DeClercq JJ;Llewellyn GN;Hayward SB;Li PW;Shivak DA;Surosky RT;Gregory PD;Holmes MC;Cannon PM
通讯作者：
Cannon PM
影响因子：
82.9
作者：
Brown, Brian D.;Venneri, Mary Anna;Naldini, Luigi
通讯作者：
Naldini, Luigi