title: An online bioinformatics tool predicts zinc finger and TALE nuclease off-target cleavage.
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.
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
authors.forename: Eli J
authors.initials: EJ
authors.lastname: Fine
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Cradick
authors.forename: Charles L
authors.initials: CL
authors.lastname: Zhao
authors.forename: Yanni
authors.initials: Y
authors.lastname: Lin
authors.forename: Gang
authors.initials: G
authors.lastname: Bao
mesh_terms: D000465:Algorithms; D019295:Computational Biology; D053837:DNA Cleavage; D059766:DNA End-Joining Repair; D003851:Deoxyribonucleases / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D054643:INDEL Mutation; D020407:Internet; D019713:Receptors, CCR5 / Q000235:genetics; D012984:Software; D016335:Zinc Fingers
chemical_list: D019713:Receptors, CCR5; D003851:Deoxyribonucleases