An online bioinformatics tool predicts zinc finger and TALE nuclease off-target cleavage

被引：95

作者：

Fine, Eli J.

Cradick, Thomas J.

Zhao, Charles L.

Lin, Yanni

Bao, Gang ^{[1
]}

机构：

[1] Georgia Inst Technol, Dept Biomed Engn, Atlanta, GA 30332 USA

来源：

NUCLEIC ACIDS RESEARCH | 2014年 / 42卷 / 06期

基金：

美国国家科学基金会; 美国国家卫生研究院;

关键词：

EFFECTOR NUCLEASES; HUMAN-CELLS; GENOME; SPECIFICITIES; DISRUPTION; PROTEINS; MUTATION; DESIGN; SITES;

D O I：

10.1093/nar/gkt1326

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Although engineered nucleases can efficiently cleave intracellular DNA at desired target sites, major concerns remain on potential 'off-target' cleavage that may occur throughout the genome. We developed an online tool: predicted report of genome-wide nuclease off-target sites (PROGNOS) that effectively identifies off-target sites. The initial bioinformatics algorithms in PROGNOS were validated by predicting 44 of 65 previously confirmed off-target sites, and by uncovering a new off-target site for the extensively studied zinc finger nucleases (ZFNs) targeting C-C chemokine receptor type 5. Using PROGNOS, we rapidly interrogated 128 potential off-target sites for newly designed transcription activator-like effector nucleases containing either Asn-Asn (NN) or Asn-Lys (NK) repeat variable di-residues (RVDs) and 3- and 4-finger ZFNs, and validated 13 bona fide off-target sites for these nucleases by DNA sequencing. The PROGNOS algorithms were further refined by incorporating additional features of nuclease-DNA interactions and the newly confirmed off-target sites into the training set, which increased the percentage of bona fide off-target sites found within the top PROGNOS rankings. By identifying potential off-target sites in silico, PROGNOS allows the selection of more specific target sites and aids the identification of bona fide off-target sites, significantly facilitating the design of engineered nucleases for genome editing applications.

引用

页数：13

共 40 条

[31] Chimeric nucleases stimulate gene targeting in human cells [J].

Porteus, MH ;

Baltimore, D .

SCIENCE, 2003, 300 (5620) :763-763

[32]

Rozen S, 2000, Methods Mol Biol, V132, P365

[33] In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites [J].

Sander, Jeffry D. ;

Ramirez, Cherie L. ;

Linder, Samantha J. ;

Pattanayak, Vikram ;

Shoresh, Noam ;

Ku, Manching ;

Foden, Jennifer A. ;

Reyon, Deepak ;

Bernstein, Bradley E. ;

Liu, David R. ;

Joung, J. Keith .

NUCLEIC ACIDS RESEARCH, 2013, 41 (19) :e181

[34] In Situ Genetic Correction of the Sickle Cell Anemia Mutation in Human Induced Pluripotent Stem Cells Using Engineered Zinc Finger Nucleases [J].

Sebastiano, Vittorio ;

Maeder, Morgan L. ;

Angstman, James F. ;

Haddad, Bahareh ;

Khayter, Cyd ;

Yeo, Dana T. ;

Goodwin, Mathew J. ;

Hawkins, John S. ;

Ramirez, Cherie L. ;

Batista, Luis F. Z. ;

Artandi, Steven E. ;

Wernig, Marius ;

Joung, J. Keith .

STEM CELLS, 2011, 29 (11) :1717-1726

[35] TAL effector RVD specificities and efficiencies [J].

Streubel, Jana ;

Bluecher, Christina ;

Landgraf, Angelika ;

Boch, Jens .

NATURE BIOTECHNOLOGY, 2012, 30 (07) :593-595

[36] Knockout rats generated by embryo microinjection of TALENs [J].

Tesson, Laurent ;

Usal, Claire ;

Menoret, Severine ;

Leung, Elo ;

Niles, Brett J. ;

Remy, Severine ;

Santiago, Yolanda ;

Vincent, Anna I. ;

Meng, Xiangdong ;

Zhang, Lei ;

Gregory, Philip D. ;

Anegon, Ignacio ;

Cost, Gregory J. .

NATURE BIOTECHNOLOGY, 2011, 29 (08) :695-696

[37] A flexible and efficient template format for circular consensus sequencing and SNP detection [J].

Travers, Kevin J. ;

Chin, Chen-Shan ;

Rank, David R. ;

Eid, John S. ;

Turner, Stephen W. .

NUCLEIC ACIDS RESEARCH, 2010, 38 (15) :e159-e159

[38] The genome of black cottonwood, Populus trichocarpa (Torr. & Gray) [J].

Tuskan, G. A. ;

DiFazio, S. ;

Jansson, S. ;

Bohlmann, J. ;

Grigoriev, I. ;

Hellsten, U. ;

Putnam, N. ;

Ralph, S. ;

Rombauts, S. ;

Salamov, A. ;

Schein, J. ;

Sterck, L. ;

Aerts, A. ;

Bhalerao, R. R. ;

Bhalerao, R. P. ;

Blaudez, D. ;

Boerjan, W. ;

Brun, A. ;

Brunner, A. ;

Busov, V. ;

Campbell, M. ;

Carlson, J. ;

Chalot, M. ;

Chapman, J. ;

Chen, G. -L. ;

Cooper, D. ;

Coutinho, P. M. ;

Couturier, J. ;

Covert, S. ;

Cronk, Q. ;

Cunningham, R. ;

Davis, J. ;

Degroeve, S. ;

Dejardin, A. ;

dePamphilis, C. ;

Detter, J. ;

Dirks, B. ;

Dubchak, I. ;

Duplessis, S. ;

Ehlting, J. ;

Ellis, B. ;

Gendler, K. ;

Goodstein, D. ;

Gribskov, M. ;

Grimwood, J. ;

Groover, A. ;

Gunter, L. ;

Hamberger, B. ;

Heinze, B. ;

Helariutta, Y. .

SCIENCE, 2006, 313 (5793) :1596-1604

[39] Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases [J].

Watanabe, Takahito ;

Ochiai, Hiroshi ;

Sakuma, Tetsushi ;

Horch, Hadley W. ;

Hamaguchi, Naoya ;

Nakamura, Taro ;

Bando, Tetsuya ;

Ohuchi, Hideyo ;

Yamamoto, Takashi ;

Noji, Sumihare ;

Mito, Taro .

NATURE COMMUNICATIONS, 2012, 3

[40] Zinc-finger nuclease mediated disruption of Rag1 in the LEW/Ztm rat [J].

Zschemisch, Nils-Holger ;

Glage, Silke ;

Wedekind, Dirk ;

Weinstein, Edward J. ;

Cui, Xiaoxia ;

Dorsch, Martina ;

Hedrich, Hans-Juergen .

BMC IMMUNOLOGY, 2012, 13

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