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.

Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators

“…target TALE binding and TALEN activity (Hockemeyer et al 2011;Osborn et al 2013;Fine et al 2014;Guilinger et al 2014). Notably, both technologies showed similar exceptional levels of specificity of gene activation by RNA-seq ( Fig.…”

“…Previous studies of the specificity of TALEand CRISPR/Cas9-based genome engineering technologies have largely relied on predictive methods, using bioinformatic algorithms (Hockemeyer et al 2011;Fu et al 2013;Hsu et al 2013;Cho et al 2014;Fine et al 2014), assays of purified protein activity in vitro (Pattanayak et al 2013;Guilinger et al 2014), and/or reporter assays (Mali et al 2013a) to inform the selection of off-target sites for direct interrogation. Recently developed methods for unbiased genome-wide specificity analysis are dependent on nuclease activity and therefore cannot directly assess the specificity of gene regulation tools (Frock et al 2015;Kim et al 2015;Tsai et al 2015;Wang et al 2015).…”

“…Studies of TALE nuclease (TALEN) specificity in human cells have readily detected activity at off-target sites with sequence homology to the intended target site using both bioinformatic predictions (Hockemeyer et al 2011;Fine et al 2014) and approaches for genetically labeling sites of nuclease activity within cells (Osborn et al 2013); however, clonal populations without modification of the exome can be easily obtained (Ousterout et al 2013). Other assays of DNA-recognition properties of purified TALEs or TALENs showed that although the proteins are highly specific for the intended target site, there are significant levels of activity at sites containing sequence mismatches (Meckler et al 2013;Guilinger et al 2014).…”

Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection

“…To test for potential nonspecific mutations induced by the introduction of the TALENs, we studied 22 potential off-target loci of TALEN pairs (Table S2), predicted using a bioinformatics-based approach, the ranking algorithm PROGNOS (22). All of the potential off-target sites were located in intron and intergenic regions; 21 out of the 22 consisted of inverted repeats of the right TALEN target sites, making these sites vulnerable to cleavage by the wild-type Fok1 homodimer (23).…”

Rescue of the activity of HNH nuclease mutants - towards controlled enzymes for gene therapy

“…Trials to establish controlled artificial nucleases through lightactivation [62,63], DNA binding [64], cold-shock technique [65], protein-protein interaction [66], DNA modification [67] or metal ion dependent DNA binding [68] were also executed. Bioinformatics was utilized to predict ZFN and TALEN off-target cleavage [69].…”