ZiFiT Targeter Version 4.2




ZiFiT V4.2 FAQ

For CRISPR/Cas:

Problem: ZiFiT finds several target sites but crashes when I click "Find Orthogonality".

Explanation: There are too many potential off-target sites.

The most likely explanation for this problem is:

i. The site identified are part of a repeat element
Solution : Use Repeat Masker to ensure that this is not the case

Solution: Move the query sequence a few 100 bps up or downstream


Problem: ZiFiT is unable to find any CRISPR/Cas target sites within my query sequence.

Explanation: The target site is required to have the following structure: GG-(N)18-NGG. The guide RNA is synthesized using a T7 expression system which requires the 5' GG dinucleotide. As described in Hwang et al., Nat Biotechnol. 2013 (doi:10.1038/nbt.2501), the target site also needs to be followed by a 'NGG' protospacer adjacent motif (PAM) (resulting in the requirement for a 3' GG dinucleotide).

There are several reason for this problem:

i. The sequence is too short
Solution : Increase the length of the query sequence

ii. Sequence composition of the region is not ideal
Solution: Move the query sequence a few 100 bps up or downstream





For TALENS:

Problem: ZiFiT is unable to find any TALEN target sites within my query sequence.

Explanation: There are several potential reasons:

i. The sequence is too short (i.e. less than 100 bp)
Solution: Increase the length of the query sequence

ii. Composition of the region flanking the locus where the DSB should occur (the NT in brackets) is not ideal (extrememly G rich)
Solution: Move the locus where the DSB should occur




Problem: For a given query sequence, the target sites identified by ZiFiT in batch mode (FLASH) are different from the target sites identified by ZiFiT while designing in REAL/REAL-Fast mode

Explanation:

In batch mode, ZiFiT returns 1 target site per query. This site has the the following configuration:
i. Length of binding site ranges from 16 to 18 bases
ii. The length of the spacer region between the TALEN pairs ranges between 16 and 18 bases.
These parameters are used because they were extensively tested in Reyon et al. (doi:10.1038/nbt.2170).

If you wish to see all the potential target sites please use the REAL/REAL-Fast option which allows you to search upto 12 sequences at a time.





Problem: ZiFiT is finds < 5 TALEN target sites within my query sequence?

Explanation:There are several potential reasons:

i. Target sites found were very similar to each other.
ZiFiT only reports sites that are different from each other by at least 6 base pairs (3 on each binding site)
Solution: Uncheck the "Mask redundant sites" options

ii. Contraints too stringent
By default ZiFiT tries find only sites of length 16:18 and spacer length of 12:23.But if you would like to see all potential target sites you could check the "Relax constraints" option.
Solution: Check the "Relax constraints" option.




For Engineered Zinc Fingers:


Problem: Which method should I use to engineer zinc finger proteins?

Explanation:ZiFiT currently supports two methods for zinc finger design: Context Dependent Assembly (CoDA) and Oligomerized Pool ENgineering (OPEN). CoDA is both simple to use and has high success rates. OPEN takes more time and requires considerable molecular biology expertise, but typically generates more active zinc finger proteins. The following are some factors to consider when choosing which method to use for your target gene of interest.

Solution 1: We recommend starting with CoDA method if possible.

Solution 2: If your gene can not be targeted using CoDA, we recommend that you consider using OPEN to target your gene. OPEN yields high quality ZFNs and has a higher success rate. For OPEN, use the ZiFOpt scoring method provided in ZiFiT to determine the likelihood of success for any given target site.




Problem: ZiFiT found no target sites in my sequence.

Explanation: The target sequence you provided does not have a potential ZFN site that can be targeted by the method you selected.

Solution 1: The three methods have different targeting capabilities. The sequence may be targetable by another method.

Solution 2: Use less stringent "Triplet Composition" requirements.




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 (if using OPEN - See Instructions)