An improved zinc-finger nuclease architecture for highly specific genome editing

Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J.An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol., 2007, 25(7), 778-785.

(Eva Knapič)

Genome editing

Genome editing is a type of genetic engineering in which DNA is inserted, removed or replaced from genome using artificially designed nucleases, enzymes that cleave the phosphodiester bonds between nucleotides. The principle of method is based on nuclease cleavage at a desired site of the genome in order to create specific double-stranded break, then put to use the cell’s endogenous mechanisms to repair the induced break by homology-directed repair or non-homologous end joining. There are a few different classes of engineered nucleases that are currently being used: zinc-finger nucleases, transcription activator-like effector nucleases and the CRISPR/Cas system. In this seminar we will focus on zinc finger nucleases. Firstly, we will take a look at basics of zinc-finger nucleases and their mechanism. Then we will discuss improved zinc-finger nuclease architecture for more specific efficiency described by Miller and colleagues in their paper published in Nature Biotechnology in 2007<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol., 2007, 25(7), 778-785.</ref>.

Zinc-finger nuclease

Zinc-finger nuclease (ZFN) is an artificially designed endonuclease that can be customised to cleave at sequence specific site on the DNA. The enzyme consists of two domains: DNA-binding domain composed of zinc finger structural motifs and DNA-cleavage domain of restriction enzyme FokI. Domains are connected with short inter-domain linker. This structure combines key qualities of DNA binding specificity, flexibility of zinc-finger motifs and cleavage activity of FokI catalytic domain that is robust but restricted. Furthermore all three elements can be optimised for retargeting and improving efficiency<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. Nat Rev Genet., 2010, 11(9), 636-646.</ref>.

DNA-binding domain

As mentioned above, zinc finger motifs represent DNA-binding domain of ZFN. Zinc fingers are small protein structural motifs distinguished by coordinated zinc ions that stabilise the protein fold. Each zinc-finger recognises 3 base pairs (bp) of DNA. Contacts between the zinc fingers and DNA are made by α-helix that binds in the DNA major groove<ref name="ref3">Zinc finger ; Wikipedia the free encylopedia [cited 2.1.2015]. http://en.wikipedia.org/wiki/Zinc_finger.</ref>. Proteins that contain any number of zinc finger motifs are called zinc-finger proteins (ZFPs). Engineered ZFPs consist of three to six zinc finger motifs thus enabling binding of 9 bp to 18 bp targets. Longer recognition sequence improves specificity and precision of ZFPs. ZFP based DNA-binding domains can be coupled to various effector domains, which then cuts the DNA sequence determined by the ZFP<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. Nat Rev Genet., 2010, 11(9), 636-646.</ref>.

There are multiple approaches to designing new ZFPs with binding specificities chosen by user. The simplest and widely used method to generate new zinc finger sets is modular assembly. Candidate ZFPs for target sequence are obtained by determining individual zinc fingers that bind each triplet and assembling them together thus recognising the target sequence<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. Nat Rev Genet., 2010, 11(9), 636-646.</ref>. Alternative methods for designing new ZFPs include two-finger modules, oligomerised pool engineering (OPEN) and Context-Dependent Assembly (CoDA). In two-finger modules instead of individual fingers, construct of two-fingers is used for recognising target DNA sequence. Advantage of this approach is better optimisation of finger junctions and speed of finding and assembling suitable zinc fingers, a limitation is extension of initial characterisation of two-finger units <ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. Genetics., 2011, 188(4), 773-782.</ref>. The OPEN system approach is carried out in two steps, firstly appropriate finger pools are recombined to create small combinatorial library, then members of library that efficiently bind to the target site are isolated using bacterial two-hybrid selection method in which binding of a zinc finger domain to its target activates expression of selected marker gene<ref name="ref5">Maeder M. L. et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell., 2008, 31(2), 294–301.</ref>. CoDA is publicly available platform of reagents and software. With this approach three-finger sets are assembled using N- and C- terminal fingers that have been previously identified in other arrays with common middle finger. CoDA does not treat fingers as independent modules but instead explicitly accounts for context-dependent effects between adjacent fingers, increasing the probability of efficiency <ref name="ref6">Sander J.D. et al. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nat Methods., 2011, 8(1), 67-69.</ref>. Regardless of which approach is used for designing new ZFPs in vitro, their application in living cells is not always successful. Reason is the complexity of genome that often contains naturally occurring multiple copies of sequence that are either identical or very similar to target sequence and these copies can act as additional targets for ZFNs. Another complication is chromatin structure at target sites that may obstruct cleavage<ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. Genetics., 2011, 188(4), 773-782.</ref>.