TALEN Targeter Tutorial
TALEN Targeter designs pairs of custom TAL effectors for use as TAL effector nucleases (TALENs) by identifying good candidate binding sites of the proper length and with the proper spacing and orientation between the two sites.
Each TALEN monomer consists of the TAL effector DNA binding domain with a Fok1 catalytic domain fused to its C terminus. The monomers are designed to bind to candidate target sites (red on the image above) oriented from 5' to 3' on opposite strands of DNA. The spacer region between the sites must be sufficiently large enough for the two Fok1 domains to dimerize and cut the DNA, but not so long that they do not come into contact.
TALEN Targeter assists users in identifying pairs of TALEN monomer binding sites in the proper spacing and orientation for the TALEN pair to function.
- Upload the DNA sequence you want to target.
To follow along with this tutorial, use the button at the top of the page to load the sample data set:
To load your own DNA sequence, cut and paste FASTA formatted sequences into the text box or upload a file containing the sequences. All sequences should include a FASTA identifier line (">long_sequence" in the image above) and sequences are limited to the characters ACGTN. For more details on correct formatting, see the Help page.
- Select an architecture or customize spacer and array lengths.
Different TAL effector/TALEN construction methods use different portions of the TAL effector protein flanking the repeat region. These different "architectures" have different optimal ranges for spacer lengths and number of repeats. Spacer length and array length options allow you to customize your query for different TALEN architectures or construction methods.
Four architectures, with preset ranges for optimal spacer length and number of repeats, are available.
If none of the preset architectures meet your needs, click on the tab "Provide Custom Spacer/RVD Lengths". Enter your desired ranges for spacer size and number of repeats in the text boxes.
To follow along with the tutorial, click on the tab "Use a Preset Architecture". Choose the architecture "Cermak et al., (2011)". These settings work with the Golden Gate TALEN and TAL Effector Kit available from Addgene.
- Choose filter options.
Please note that the guidelines proposed in "Cermak, et al.(2011) are no longer available, because a recent paper by Reyon et al. (2011) provided evidence that following the guidelines had little effect on TALEN efficiency.
If you still wish to design sites according to these guidelines, the old version of TALEN Targeter is available here.
Users may choose one of three options to filter their results:
- Show TALEN pairs (hide redundant TALENs)
- returns up to one TALEN pair targeting each base in the sequence. A base is "targeted" if it is at the center of the spacer. Most bases will be located in the center of the spacer for multiple TALEN pairs. This option returns one TALEN pair per base by choosing the pair with the smallest average number of RVDs and the shortest spacer (within the selected spacer range).
- Show all TALEN pairs (include redundant TALENs)
- returns the complete list of TALEN pairs. Multiple TALEN pairs that target the same site may be returned; redundant pairs are not filtered out of the output. This option will give a very long results list.
- Show TALEN pairs targeting a specific site
- allows users to specify a single position that they are interested in targeting. This option will return all TALEN pairs that have this specific position at the center of the spacer. Choosing this option will bring up text box that allows you to specify (by number) the position you want to target.
For this tutorial, select "Show TALEN pairs (hide redundant TALENs)" to return a filtered list of TALEN pairs targeting nucleotides throughout the input sequence.
- Select an upstream base.
Users can choose which nucleotide(s) will be required to be upstream of returned sites. In nature, the majority of TAL effector targets are preceded by a T. However, at least one example of a TAL effector targeting a site preceded by C has been found in nature (Yu et al., 2011), and custom TAL effectors targeting sites preceded by C have been reported in the literature (Miller et al., 2010).
In our hands, sites preceded by a C were significantly less active than those preceded by a T. Therefore, we recommend that users select the default setting for sites preceded by a T only.
For the purposes of this tutorial, set the upstream base to T only (the default option). This T will be shown in the output.
- Submit your query.
Optionally, you may enter your email address to receive an email with a link to your results when your job is complete.
You must also choose the length of time your results will be available for download from our server.
When you are finished, hit the Submit button.
- Retrieve your results.
After hitting Submit, you will be taken to a page detailing the progress of your job.
Do not navigate away from this page; or bookmark the page so you can return and download your results later!
When the job finishes, your results will appear in a table on the webpage.
If you entered an email address, you will receive an email notification with a link to this page. There is also a link to download your results as a tab-delimited text file.
- Interpret your results.
From your output, select TALEN pairs for construction. All TALEN pairs returned by the program are designed to work with equal efficiency. Note that all target sites are directly preceded by a T (or C, if that option was selected) at the 5' end. This T (or C) is shown in the output in the target sequence column.
To create a double stranded break (DSB) in a specific region, look for pairs with your target region centered in the spacer. The column "Cut Site" indicates the base at the center of the spacer.
You may also wish to choose TALEN pairs with unique restriction enzyme sites in the spacer (shown in the last column of the output). The identified enzymes cut exactly once in the spacer and nowhere else in the 250 bases upstream and 250 bases downstream of the spacer. These unique sites can be useful for screening for TALEN activity.
If no sites targeting your region of interest are identified, you can increase the number of sites by using the "Provide Custom Spacer/RVD Lengths" tab to broaden your ranges for spacer length and number of RVDs.
If you chose to "Find TALEN Configurations for a Specific Cut Site", and no sites were returned, try moving your specified position a few base to the right or left.
To see the complete list of sites, repeat your search using the option to "Find All TALEN Configurations for All Cut Sites".