Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool

Abstract

Zinc Finger Targeter (ZiFiT) is a simple and intuitive web-based tool that facilitates the design of zinc finger proteins (ZFPs) that can bind to specific DNA sequences. The current version of ZiFiT is based on a widely employed method of ZFP design, the 'modular assembly' approach, in which pre-existing individual zinc fingers are linked together to recognize desired target DNA sequences. Several research groups have described experimentally characterized zinc finger modules that bind many of the 64 possible DNA triplets. ZiFiT leverages the combined capabilities of three of the largest and best characterized module archives by enabling users to select fingers from any of these sets. ZiFiT searches a query DNA sequence for target sites for which a ZFP can be designed using modules available in one or more of the three archives. In addition, ZiFiT output facilitates identification of specific zinc finger modules that are publicly available from the Zinc Finger Consortium. ZiFiT is freely available at http://bindr.gdcb.iastate.edu/ZiFiT/.

Figure 1.

Zinc Finger Modules and Binding Sites. (a) A canonical zinc finger module consists of two anti-parallel beta strands and an alpha helix, with a zinc ion coordinated by the conserved cysteine (C) and histidine (H) residues pairs. The numbered amino acid residues at positions −1, +2, +3 and +6, relative to the amino-terminal end of the alpha helix, can form important base-specific contacts in the major groove of the double-stranded target DNA (not shown). (b) A ZFP consisting of three linked ZF modules binds its target DNA site with amino acids of the recognition alpha helices (in the N- to C-terminal direction) contacting consecutive nucleotides in DNA running in the 3′ and 5′ direction. This can lead to confusion because the DNA target site is typically referred to in the 5′ and 3′ direction. Note that an ‘unnatural’ extended array is shown to better illustrate the critical amino acid-nucleotide contacts. Structure diagrams were generated using PyMol (http://www.pymol.org).

Figure 2.

Comparison of Single ZF Array and Dimeric ZF Nuclease Sites. (a) Zinc Finger Protein Bound to a Single ZF Array Target Site: A Single ZF Array Target Site consists of three to eight adjacent DNA triplets (9–24 nt) on the same strand of DNA. Each triplet of DNA is recognized by one “finger” or “ZF module” (light blue ovals). The cartoon illustrates an array with three fingers (ZF1, ZF2, ZF3) bound to a single ZF array target site, in this case, a 9 nt DNA sequence. (b) Zinc Finger Nuclease Bound to a Dimeric ZF Nuclease Target Site: A ZF nuclease target site consists of two single ZF array sites on complementary DNA strands, separated by a spacer of 5 or 6 nt. In this configuration, FokI endonuclease monomers (dark blue spheres), covalently linked to the C-terminal end of each ZF array domain, can form an active dimeric nuclease and induce a double-stranded break in the spacer DNA between two ZF array binding sites.

Figure 3.

ZiFiT Input Window. In this example, the user has chosen to search for potential dimeric zinc finger nuclease (ZFN) sites. The DNA sequence of interest can be supplied in either FASTA format or as a raw DNA sequence, in standard 5′ and 3′ direction. Above the sequence input box, check boxes allow users to specify which sets of characterized ZF modules should be considered in designing a ZFN to recognize the desired target site. Below the sequence input box, drop down menus allow users to specify how many DNA triplets should be included in each array site: a single array site may contain from 3 to 8 triplets; a dimeric nuclease site may contain 3 or 4 triplets in each array site (Left and Right), separated by a Spacer of either 5 or 6 nucleotides. Information that appears below the ‘Submit’ button in this screenshot corresponds to ‘Advanced Option’ parameters (see text). These have been revealed by selecting the ‘Advanced’ link (to the right of the Submit button) which toggles between ‘Basic’ and ‘Advanced’.

Figure 4.

Example of ZiFiT Output for a Dimeric ZF Nuclease Target Site. For each ‘hit’ identified by ZiFiT, the output consists of the DNA target site sequence and two tables that contain corresponding ZF protein modules that could be assembled to recognize the displayed target site. Each target site is displayed as a double-stranded DNA sequence, with triplets color-coded to facilitate identification of the corresponding ZF binding modules in the accompanying table. Each row in the ZF module table displays information for a single zinc finger module. Entries in the columns indicate the Finger position (Finger) of this module, the amino acids at positions −1 through +6 corresponding to its recognition helix (HELIX), the color-coded DNA triplet it recognizes (TRIPLET), a reference number to facilitate ordering the ZF molecule from the Zinc Finger Consortium (REFERENCE NUMBER), and the module set from which the indicated ZF module was derived (MODULE SOURCE). If more than one ZF module is known to recognize a given triplet sequence, all avaliable modules for that position are displayed. The screen shot shown here displays the complete output for one of several potential dimeric ZF nuclease sites identified within the input query DNA sequence.