We are using our novel selection tools to engineer zinc finger proteins with novel DNA-binding specificity for use in Zebrafish to knock-out specific target genes in collaboration with Nathan Lawson (UMMS – PGFE). Zinc Finger nucleases (ZFNs) are a fusion between Cys2His2 Zinc finger DNA-binding domains and the nuclease domain of FokI (where the DNA-binding domain of this enzyme has been removed). The FokI nuclaese domain is only active in the context of a dimer, consequently two ZFNs must come together on the DNA with an appropriate orientation and spacing (5 or 6 bps) to generate a double-stranded break.
Figure legend: A) Two nuclease domains (FokI) are brought together over a desired target sequence in the genome by two zinc finger proteins (grey and green fingers). We utilize heterodimeric FokI (+/-) domains that were created by Miller and colleagues at Sangamo Biosciences. B) If the ZFNs have the appropriate spacing each nuclease monomer will cleave one strand of the DNA. C) This results in a double-stranded break, which if repaired by Non-homologous end joining (NHEJ) can lead to insertions or deletions due to the error prone nature of this process. If this error mediated repair occurs in a coding sequence it can functionally inactivate the gene.
Using a combination of design and selection via the omega-based B1H system we create zinc finger proteins with DNA-binding specificity appropriate for the desired target sequences. The specificity of these proteins is verified using the B1H binding site selection system and then they are introduced into zebrafish embryos. We have just completed our first successful gene knockout using this system (recently described in Nature Biotechnology).
The reagents (list) necessary to use the omega-based B1H system for the selection of Zinc Finger Proteins with novel specificity are now available at Addgene. We have a basic outline for testing the B1H reagents in your hands to confirm that they work before you get started. Below is the link for the on-line tool that can be used to identify target sites in your gene of interest. If you have any questions about these tools and reagents please contact us.