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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 )