Single TALE Targeter designs custom TAL effectors for use as transcription factors or in other applications.

1. 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.

2. 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, unless they have a specific reason to do otherwise.

   For the purposes of this tutorial, set the upstream base to T only (the default option). This T will be shown in the output.

3. Customize the array length range.
   The array length fields allow you to specify a range for the number of RVDs that will be considered when designing custom TAL effectors. This option allows you to customize your query for different TALEN architectures or construction methods. To follow along with the tutorial, use the default settings.

   

   The default settings (shown above) work with the Golden Gate TALEN and TAL Effector Kit available from Addgene.

4. Choose other search options.
   The remaining options correspond to design guidelines discussed in Cermak, et al.(2011). Relaxing these rules may allow you to find more TALEN pairs for your sequence.

   By default, TAL Effector Targeter designs TAL effectors that obey the positional and composition biases of TAL effectors observed in nature. Although following these rules may not be necessary, it certainly will not reduce the TAL effectors' functionality! If you do not want to require your TAL effectors to obey these rules, or if the default settings do not find enough potential TAL effector binding sites in your DNA sequences, you can check one or more of the boxes to relax the design rules.

   A recent paper by Reyon et al. (2011) provided evidence that following the guidelines had little effect on TALEN efficiency. However, the effect of the guidelines on TAL effector transcriptional activator activity. Therefore, we have left the guidelines available for TAL Effector Targeter. If you do not with to follow one or more guidelines, uncheck the appropriate box to turn the guideline off.

   

   To follow along with the tutorial, leave all options checked (default settings).

   Additionally, you can choose whether or not to search the reverse complement of the submitted sequence for custom TAL effector sites.

   For the tutorial, leave this option unchecked (default settings).

5. 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.

6. 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. You also be provided with a link to a downloadable tab-delimited text file containing your results. If you entered an email address, you will receive an email notification with a link to this page so you can retrieve your results.

7. Interpret your results.
   From your output, select custom TAL effectors for construction. All custom TAL effectors 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 this option is chosen) at the 5' end. This T is shown in the output, separated from the target sequence by a space.

   

   Note that the strand column indicates if the target is on the sequence as entered (Plus) or the reverse complement (Minus, only if the option to search the reverse complement was selected). If you checked the option to search the reverse complement of the entered sequence, the table will have an additional column containing the Plus Strand Sequence (the target sequence for plus strand sites and the reverse complement of the target for minus strand sites). The TAL_start column indicates the position on the entered sequence to which the first RVD will be bind.