CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors

被引:99
作者
Bak, Rasmus O. [1 ]
Porteus, Matthew H. [1 ]
机构
[1] Stanford Univ, Dept Pediat, Stanford, CA 94305 USA
关键词
HEMATOPOIETIC STEM-CELLS; MESSENGER-RNA; IN-VIVO; GENOME; DNA; RECOMBINATION; SIZE; EXPRESSION; NUCLEASES; FRAGMENT;
D O I
10.1016/j.celrep.2017.06.064
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
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 similar to 4.5 kb limits the donor size. Here, we overcome this constraint by showing that two cotransduced AAV vectors can serve as donors during consecutive HR events for the 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 kb. 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.
引用
收藏
页码:750 / 756
页数:7
相关论文
共 32 条
[1]   Universal Real-Time PCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2-Derived Inverted Terminal Repeat Sequences [J].
Aurnhammer, Christine ;
Haase, Maren ;
Muether, Nadine ;
Hausl, Martin ;
Rauschhuber, Christina ;
Huber, Ingrid ;
Nitschko, Hans ;
Busch, Ulrich ;
Sing, Andreas ;
Ehrhardt, Anja ;
Baiker, Armin .
HUMAN GENE THERAPY METHODS, 2012, 23 (01) :18-28
[2]   Promoter less gene targeting without nucleases ameliorates haemophilia B in mice [J].
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. .
NATURE, 2015, 517 (7534) :360-U476
[3]  
Basiri M, 2017, CELL J, V18, P532
[4]   Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer [J].
Brown, Brian D. ;
Venneri, Mary Anna ;
Zingale, Anna ;
Sergi, Lucia Sergi ;
Naldini, Luigi .
NATURE MEDICINE, 2006, 12 (05) :585-591
[5]   Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids [J].
Chamberlain, Kyle ;
Riyad, Jalish Mahmud ;
Weber, Thomas .
HUMAN GENE THERAPY METHODS, 2016, 27 (01) :1-12
[6]   An Efficient Low Cost Method for Gene Transfer to T Lymphocytes [J].
Chicaybam, Leonardo ;
Sodre, Andressa Laino ;
Curzio, Bianca Azevedo ;
Bonamino, Martin Hernan .
PLOS ONE, 2013, 8 (03)
[7]   CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease [J].
De Ravin, Suk See ;
Li, Linhong ;
Wu, Xiaolin ;
Choi, Uimook ;
Allen, Cornell ;
Koontz, Sherry ;
Lee, Janet ;
Theobald-Whiting, Narda ;
Chu, Jessica ;
Garofalo, Mary ;
Sweeney, Colin ;
Kardava, Lela ;
Moir, Susan ;
Viley, Angelia ;
Natarajan, Pachai ;
Su, Ling ;
Kuhns, Douglas ;
Zarember, Kol A. ;
Peshwa, Madhusudan V. ;
Malech, Harry L. .
SCIENCE TRANSLATIONAL MEDICINE, 2017, 9 (372)
[8]   CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells [J].
Dever, Daniel P. ;
Bak, Rasmus O. ;
Reinisch, Andreas ;
Camarena, Joab ;
Washington, Gabriel ;
Nicolas, Carmencita E. ;
Pavel-Dinu, Mara ;
Saxena, Nivi ;
Wilkens, Alec B. ;
Mantri, Sruthi ;
Uchida, Nobuko ;
Hendel, Ayal ;
Narla, Anupama ;
Majeti, Ravindra ;
Weinberg, Kenneth I. ;
Porteus, Matthew H. .
NATURE, 2016, 539 (7629) :384-389
[9]   Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells [J].
DeWitt, Mark A. ;
Magis, Wendy ;
Bray, Nicolas L. ;
Wang, Tianjiao ;
Berman, Jennifer R. ;
Urbinati, Fabrizia ;
Heo, Seok-Jin ;
Mitros, Therese ;
Munoz, Denise P. ;
Boffelli, Dario ;
Kohn, Donald B. ;
Walters, Mark C. ;
Carroll, Dana ;
Martin, David I. K. ;
Corn, Jacob E. .
SCIENCE TRANSLATIONAL MEDICINE, 2016, 8 (360)
[10]   An mRNA surveillance mechanism that eliminates transcripts lacking termination codons [J].
Frischmeyer, PA ;
van Hoof, A ;
O'Donnell, K ;
Guerrerio, AL ;
Parker, R ;
Dietz, HC .
SCIENCE, 2002, 295 (5563) :2258-2261