Editing TALEN Real Assembly (section)

== TALEN Design  ==

Identification of potential TALEN target sites using web-based ZiFiT&nbsp;Targeter software<br>1, Users are encouraged to first identify any repeat sequence within the query sequence using RepeatMasker Web Server (http://www.repeatmasker.org/). Any sequences with repeats should be avoided. 

2, Visit the ZiFiT Targeter website at&nbsp;http://zifit.partners.org. http://zifit.partners.org/ZiFiT/ChoiceMenu.aspx<br> 

3, Click on the ZiFiT option on the top menu followed by the&nbsp;“Design TALE Nucleases” option under the “TALE Assembly” menu.&nbsp; 

4, Paste the query sequence into the text box&nbsp;labeled&nbsp;‘Sequence’. The sequences may be entered as raw data or in FASTA&nbsp;format. If multiple queries are entered they need to be in FASTA format. All numbers and characters that are not G, A, T, or C will be&nbsp;ignored. The user must indicate the nucleotide position at which they&nbsp;wish the break to occur by framing it with brackets (e.g.--[A] or&nbsp;[G]). ZiFiT Targeter will attempt to identify sites such that this nucleotide falls within the spacer sequence between the TALEN binding half-&nbsp;sites.&nbsp; 

5,&nbsp; Check the option “Ignore redundant sites” to ensure that the&nbsp;sites identified differ from each other by at least 3 nucleotides (6 total) 

6,&nbsp; By default, ZiFiT Targeter will only return the five highest-&nbsp;ranked sites it identifies using criteria described in Supplementary&nbsp;Discussion of Sander et al. http://www.nature.com/nbt/journal/v29/n8/full/nbt.1934.html). Users can display all the potential target&nbsp;sites identified by the program by checking the box for “Display&nbsp;all”. With this option checked, ZiFiT Targeter will return all&nbsp;potential TALEN target sites that consist of half-sites of lengths&nbsp;12-22 base pairs and spacer&nbsp;sequences of lengths 12-23 base pairs.&nbsp;This option is not available while ZiFiT Targeter is operating in batch mode. 

7,&nbsp; Click the Submit button. The output reports the top five TALEN&nbsp;target sites below the sequence entry box. If multiple queries are submitted the potential target sites for each query sequence is reported in a different tab. The position where the break is intended to occur is now demarcated with an asterisk. 

8, To obtain a customized graphical guide for assembling a particular&nbsp;TALEN, click on the target site.&nbsp;ZiFiT generates graphical guides for assembling TALENs using either REAL or REAL-Fast by default. Users can access either guide by clicking on the appropriate buttons at the top of the page.