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131015 Journal Seminar T.Hirota Nucleic Acids Research Advance Access published September 3, 2013 Nucleic Acids Research, 2013, 1–7 doi:10.1093/nar/gkt789 E-TALEN: a web tool to design TALENs for genome engineering Florian Heigwer, Grainne Kerr, Nike Walther, Kathrin Glaeser, Oliver Pelz, Marco Breinig and Michael Boutros* German Cancer Research Center (DKFZ), Division of Signaling and Functional Genomics and Heidelberg University, Department of Cell and Molecular Biology, Medical Faculty Mannheim, D-69120 Heidelberg, Germany Received June 28, 2013; Revised August 2, 2013; Accepted August 11, 2013 ABSTRACT Use of transcription activator-like effector nucleases (TALENs) is a promising new technique in the field of targeted genome engineering, editing and reverse genetics. Its applications span from introducing knockout mutations to endogenous tagging of proteins and targeted excision repair. Owing to this wide range of possible applications, there is a need for fast and user-friendly TALEN design tools. We developed E-TALEN (http://www.e-talen.org), a webbased tool to design TALENs for experiments of varying scale. E-TALEN enables the design of TALENs against a single target or a large number of target genes. We significantly extended previously published design concepts to consider genomic context and different applications. ETALEN guides the user through an end-to-end design process of de novo TALEN pairs, which are specific to a certain sequence or genomic locus. Furthermore, E-TALEN offers a functionality to predict targeting and specificity for existing TALENs. Owing to the computational complexity of many of the steps in the design of TALENs, particular emphasis has been put on the implementation of fast yet accurate algorithms. We implemented a user-friendly interface, from the input parameters to the presentation of results. An additional feature of E-TALEN is the in-built sequence and annotation database available for many organisms, including human, mouse, zebrafish, Drosophila and Arabidopsis, which can be extended in the future. E-TALEN workflow. E-TALEN workflow. The E-TALEN workflow can be divided into three parts: the web service/interface, the implemented design algorithm and the output in various file formats and an html report. The web service can be sub-divided into the two different aims of de novo design of TALENs against a specific target and the re-evaluation of existing TALENs to find/re-check their target or genomic context. Depicted on the right are the pre-calculated databases that supply the design algorithm with genomic or sequence information, enabling fast and efficient information management during the design procedure. The main part of E-TALEN consists of design algorithms that find and validate putative TALEN targets, followed by providing comprehensive information on resulting TALENs and their target sites. These two parts of the workflow are hidden from the user. The last part of the E-TALEN workflow is the generation of an output that comprises various computer and human-readable file formats, which are known from high-throughput sequencing, and a visual report shown in the user’s browser. Heigwer F et al. Nucl. Acids Res. 2013;nar.gkt789 © The Author(s) 2013. Published by Oxford University Press. TALEN design output with exon 2 of Pten in different organisms as target. TALEN design output with exon 2 of Pten in different organisms as target. TALENs were designed against the tumour suppressor gene Pten with the aim to target the second exon. Shown are independent design runs for the three organisms human, zebrafish and fruit fly. Different transcripts of the gene are coloured orange, different coding sequences are coloured light green and TALENs are shown in yellow. Note that a TALEN is considered valid if any transcript’s second exon is targeted. Targeting the second exon is likely to introduce knockout mutations in the Pten gene. Heigwer F et al. Nucl. Acids Res. 2013;nar.gkt789 © The Author(s) 2013. Published by Oxford University Press. Different transcripts of the gene are coloured orange, different coding sequences are coloured light green and TALENs are shown in yellow. The green box shows that a 5‘ CpG island does not allow TALEN designs in this region in human. In addition, one gene can comprise several very distinct transcripts which all have to be considered. The red box highlights that in vertebrates the first coding exon is very often spanned by a CpG island and, thus, difficult to target (22). The blue box shows three independent TALEN designs, that all target the first coding exon of all transcripts of a fly’s Pten gene. Research Seminar Ribosomal RNA Depletion & RNA-seq Experimental flow 1st & 2nd Xenotransplantation ↓ Total RNA extraction ↓ Kusabira-Orange check for PCR ↓ Total RNA analysis with Agilent 2100 Bioanalyzer ↓ Ribosomal RNA depletion ↓ RNA-seq 7 Result of total RNA analysis Sample ID Setting density (ng/µl) Dilution Meadured density (ng/µl) Real density (µg/µl) RIN Ir-PC3_1 1.80 1/1071 4.59 4.92 9.5 Ir-PC3_2 1.80 1/193 1.16 0.22 8.1 Ir-PC3_3 1.80 1/586 4.10 2.40 9.4 Control_1 1.80 1/529 4.18 2.21 8.8 Control_2 2.50 1/575 3.86 2.22 8.1 Control_3 1.80 1/196 4.01 0.79 8.5 8 Result of analysis after ribosomal RNA depletion Sample ID Measured density (ng/µl) dilution Real density (ng/µl) Volume (µl) Total (ng) Ir-PC3_1 2.87 1/10 28.7 34 975.8 Ir-PC3_2 0.76 1/10 7.6 34 258.4 Ir-PC3_3 0.74 1/10 7.4 34 251.6 Control_1 2.25 1/10 22.5 34 765 Control_2 1.35 1/10 13.5 34 459 Control_3 0.88 1/10 8.8 34 299.2 Future plan • RNA-seq (4th-Oct) • Write a master’s thesis (Materials and Metods )