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Nucleic Acids Res. 2014 Apr;42(6):e42. doi: 10.1093/nar/gkt1326. Epub 2013 Dec 30.

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

Author information

1
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.

Abstract

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.

PMID:
24381193
PMCID:
PMC3973315
DOI:
10.1093/nar/gkt1326
[Indexed for MEDLINE]
Free PMC Article

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