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

Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA
Nucleic Acids Research (Impact Factor: 9.11). 12/2013; 42(6). DOI: 10.1093/nar/gkt1326
Source: PubMed


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.

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Available from: Thomas James Cradick, Apr 15, 2014
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    • "Activity and cytotoxicity of designer nucleases are mainly determined by the target site specificity of the DNA-binding domain [26], and nuclease-induced toxicity most likely results from off-target cleavage [25]. As compared to the ZFNs, the TALEN pair revealed considerably less cleavage activity at off-target sites predicted by bioinformatics [36]. The impact of both higher specificity and reduced nuclease-associated cytotoxicity was emphasized by the number of correctly targeted iPSC clones, which was consistently higher in TALEN than in ZFN-corrected samples. "
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    ABSTRACT: X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of the immune system. It is characterized by a defect in the production of reactive oxygen species (ROS) in phagocytic cells due to mutations in the NOX2 locus, which encodes gp91phox. Because the success of retroviral gene therapy for X-CGD has been hampered by insertional activation of proto-oncogenes, targeting the insertion of a gp91phox transgene into potential safe harbor sites, such as AAVS1, may represent a valid alternative. To conceptually evaluate this strategy, we generated X-CGD patient-derived induced pluripotent stem cells (iPSCs), which recapitulate the cellular disease phenotype upon granulocytic differentiation. We examined AAVS1-specific zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for their efficacy to target the insertion of a myelo-specific gp91phox cassette to AAVS1. Probably due to their lower cytotoxicity, TALENs were more efficient than ZFNs in generating correctly targeted iPSC colonies, but all corrected iPSC clones showed no signs of mutations at the top-ten predicted off-target sites of both nucleases. Upon differentiation of the corrected X-CGD iPSCs, gp91phox mRNA levels were highly up-regulated and the derived granulocytes exhibited restored ROS production that induced neutrophil extracellular trap (NET) formation. In conclusion, we demonstrate that TALEN-mediated integration of a myelo-specific gp91phox transgene into AAVS1 of patient-derived iPSCs represents a safe and efficient way to generate autologous, functionally corrected granulocytes. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Full-text · Article · Aug 2015 · Biomaterials
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    • "e a relatively small number of position - dependent mismatches ( Juillerat et al . , 2014 ) . A study in human cells demonstrated that off - target effects were extremely rare even if there was only one nucleotide mismatch ( Mussolino et al . , 2011 ) . We identified several potential off - target sites for T - OsBADH2b using the PROGNOS program ( Fine et al . , 2013 ) with less stringent criteria that allowed up to a 6 - bp mismatch and 10 - to 30 - bp spacers ( Table S1 ) . We then examined three of the most likely off - target sites with 9 - bp ( OffT - 1 ) , 11 - bp ( OffT - 5 ) and 10 - bp ( OffT - 17 ) mismatches in their recognition sequences in the six T0 plants and found none . This suggest"
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    ABSTRACT: Fragrant rice is favoured worldwide because of its agreeable scent. The presence of a defective badh2 allele encoding betaine aldehyde dehydrogenase (BADH2) results in the synthesis of 2-acetyl-1-pyrroline (2AP), which is a major fragrance compound. Here, transcription activator-like effector nucleases (TALENs) were engineered to target and disrupt the OsBADH2 gene. Six heterozygous mutants (30%) were recovered from 20 transgenic hygromycin-resistant lines. Sanger sequencing confirmed that these lines had various indel mutations at the TALEN target site. All six transmitted the BADH2 mutations to the T1 generation; and four T1 mutant lines tested also efficiently transmitted the mutations to the T2 generation. Mutant plants carrying only the desired DNA sequence change but not the TALEN transgene were obtained by segregation in the T1 and T2 generations. The 2AP content of rice grains of the T1 lines with homozygous mutations increased from 0 to 0.35-0.75 mg/kg, which was similar to the content of a positive control variety harbouring the badh2-E7 mutation. We also simultaneously introduced three different pairs of TALENs targeting three separate rice genes into rice cells by bombardment and obtained lines with mutations in one, two and all three genes. These results indicate that targeted mutagenesis using TALENs is a useful approach to creating important agronomic traits. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
    Full-text · Article · Jan 2015 · Plant Biotechnology Journal
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    • "We find that mutant embryos derived from all such crosses exhibit the same phenotype and that this phenotype segregates with the hoxb1b mutation (Additional file 3: Table S2 and Additional file 4: Figure S2), suggesting that it is due to disruption of the hoxb1b gene. Furthermore, the PROGNOS on-line tool [37] revealed five exonic sites in the top fifty potential off-target sites for Zb1b-3, but neither of these sites resides on the same chromosome as the hoxb1b gene, and they would therefore segregate independently of the hoxb1b mutation in our crosses. Further analysis of hoxb1bum197/um197 mutant embryos revealed expression of pax2, hoxb3a and hoxd4a in the expected domains (Figure 2G, O). "
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    ABSTRACT: Background The developing vertebrate hindbrain is transiently segmented into rhombomeres by a process requiring Hox activity. Hox genes control specification of rhombomere fates, as well as the stereotypic differentiation of rhombomere-specific neuronal populations. Accordingly, germ line disruption of the paralog group 1 (PG1) Hox genes Hoxa1 and Hoxb1 causes defects in hindbrain segmentation and neuron formation in mice. However, antisense-mediated interference with zebrafish hoxb1a and hoxb1b (analogous to murine Hoxb1 and Hoxa1, respectively) produces phenotypes that are qualitatively and quantitatively distinct from those observed in the mouse. This suggests that PG1 Hox genes may have species-specific functions, or that anti-sense mediated interference may not completely inactivate Hox function in zebrafish. Results Using zinc finger and TALEN technologies, we disrupted hoxb1a and hoxb1b in the zebrafish germ line to establish mutant lines for each gene. We find that zebrafish hoxb1a germ line mutants have a more severe phenotype than reported for Hoxb1a antisense treatment. This phenotype is similar to that observed in Hoxb1 knock out mice, suggesting that Hoxb1/hoxb1a have the same function in both species. Zebrafish hoxb1b germ line mutants also have a more severe phenotype than reported for hoxb1b antisense treatment (e.g. in the effect on Mauthner neuron differentiation), but this phenotype differs from that observed in Hoxa1 knock out mice (e.g. in the specification of rhombomere 5 (r5) and r6), suggesting that Hoxa1/hoxb1b have species-specific activities. We also demonstrate that Hoxb1b regulates nucleosome organization at the hoxb1a promoter and that retinoic acid acts independently of hoxb1b to activate hoxb1a expression. Conclusions We generated several novel germ line mutants for zebrafish hoxb1a and hoxb1b. Our analyses indicate that Hoxb1 and hoxb1a have comparable functions in zebrafish and mouse, suggesting a conserved function for these genes. In contrast, while Hoxa1 and hoxb1b share functions in the formation of r3 and r4, they differ with regards to r5 and r6, where Hoxa1 appears to control formation of r5, but not r6, in the mouse, whereas hoxb1b regulates formation of r6, but not r5, in zebrafish. Lastly, our data reveal independent regulation of hoxb1a expression by retinoic acid and Hoxb1b in zebrafish.
    Full-text · Article · Jun 2014 · BMC Developmental Biology
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