2) What is the provenance of the ZFN DNA binding domain ?
The binding domain comes form transcription factors, such as Zif268A and SP1.
3) What regions can they recognize in the genome? Can any spot in any gene be targeted?
The zinc fingers each recognize a 3 nucleotide substrate (three consecutive base pairs in the target DNA) and the nuclease domain requires a 6 nt stretch of DNA for steric fit. Staggered cuts leave 4-nt 5' sticky ends (just like FokI).
Because of the dimerization requirement of the nuclease, two sets of zinc finger triplets are used to bind to DNA, which brings up the recognition sequence to 18 nucleotides – enough to be unique in a mammalian genome (4exp18 = 6.9 x 10exp10). The sequence motif currently is of the form: < NNY NNY NNY > 6 nt < RNN RNN RNN >. This is because only GNN and ANN triplets have been rigorously tested for specific binding affinity by the Barbas and Sangamo groups. (See below attached list of zinc finger publications for more detail). New sequence specificities can be created by altering a few specific residues in the DNA binding domain of the ZFN protein. Randomization of these residues allows the selection of new fingers that have high affinity for arbitrarily selected DNA sequences.
4) How can we design a set of ZFNs for our favourite gene?
The PCR synthesis protocol of the zinc finger backbone can be found in a paper by D. Segal (see this link). The nuclease domain can be obtained by request from our lab.
5) How are the ZFNs tested for activity?
A brief in-vitro assay is performed on the purified protein (ZFNs): the plasmid containing the cDNA of the gene of choice is digested with different amounts of protein – by either of the zinc finger triplets (+ 1 nuclease domain) alone and in combination with each other (the full ZFN).
6) What is the next step?
The in-vitro assay is followed by in-vivo experiments, where typically the plasmids containing the ZFNs are injected into oocytes along with donor DNA, the successful individual carriers are crossed and their ZFNs expressed under the control of a heat shock promoter. We are also experimenting with direct protein injections, but we have not had much success so far.
Current literature on the subject of ZFN so far can be accessed here. (the file is in Word format)
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