ZiFiT V3.0 FAQ
Problem: Should I use modular assembly or OPEN to design zinc
finger proteins?
Explanation: ZiFiT currently supports two methods for zinc finger
design: Oligomerized Pool ENgineering
(OPEN) and modular assembly. Modular
assembly is simpler to implement but is often unreliable. OPEN takes more time and requires
considerable molecular biology expertise, but typically generates more active
zinc finger proteins.
Solution 1: Both methods currently use incomplete triplet libraries. Check to ensure which method has the
requisite triplet library to target your sequence.
Solution 2: For modular assembly, use scoring methods to determine the likelihood of success using modular
assembly. Low modular assembly scores
are indicative of high failure rates in modular design.
Problem: ZiFiT found no target sites in my sequence.
Explanation: The target sequence you provided does not have a
target site with the number of adjacent triplets you requested for the module
sets you have chosen.
Solution 1: If using modular assembly, choose a different module
set
Solution 2: If applicable choose a smaller target site.
Solution 3: Use less stringent “Triplet Composition”
requirements
Problem: The target sequence contains a site for the selected
module set but ZiFiT does not return that site.
Explanation: One of the modules contains an Asp in the +2
position of the recognition helix. An
Asp in the +2 position specifies an additional 3’ base. If this occurs in the module in the Finger 1
position, it specifies a longer target site (i.e., a single 3’ base extension
of the canonical target site). This
additional base must be present to be considered a valid target site. In any position other than the Finger 1
position, the additional base may conflict with the base specified by an
adjacent module. If a conflict is detected,
ZiFiT considers the site invalid.
Because a nuclease site consists of two neighboring arrays on opposite
strands, it is possible that one or both arrays could specify an additional 3’
base. An additional 3’ base on the
complementary strand corresponds to an additional 5’ base in the submitted
sequence.
Solution 1: If ZiFiT failed to return a target site due an in to
an unspecified 3’ base required by an Asp in the +2 of position, include that
base and resubmit the sequence.
Problem: There are too many potential target sites for the
submitted sequence.
Solution 1: Shorten the input sequence.
Solution 2: Use triplet composition to use primarily GNN triplets
as they have been more thoroughly validated.
Solution 3: Focus on high scoring sites. (Scoring is
currently available only for three module proteins - See Instructions)
Problem: ZiFiT returns multiple modules for a given
position. Which ones should I choose?
Solution: Currently, there is no consensus among zinc finger
experts as to which module set if any is “superior”, and no direct comparisons
of different ZF modules sets have been published. It is recommended that users try to use
modules from the same set before mixing and matching between sets.
Problem: Why can I only design nuclease arrays with three or
four modules?
Solution: Although it is certainly
possible to design ZFN monomers with more than 4 fingers, doing so is not
necessary for specifying a unique site in most eukaryotic genomes, and there is
little available data regarding the function of ZFN arrays with more than 4
fingers.
Last modified 7/24/2008