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>.

Inter-domain linker

Inter-domain linker connects DNA-binding domain with DNA-cleavage domain. Length of the linker is responsible for the shaping of spacer. Spacer is short sequence between both recognition sites for DNA-binding domains, which enables precise definition of cleavage site. Wilson and colleagues have demonstrated that sites with 5 bp spacer lengths showed the most efficient ZFN mediated gene targeting when the linker is 2 or 4 amino acids and that sites with 3 or 4 bp spacers could not be targeted efficiently. Group also demonstrated that ZFN variants with 5 amino acids linker can efficiently target sites with a 7 bp spacer. However, the ideal linker with corresponding spacer can only be determined by taking into consideration both ZFN activity on the chromosomal target site and ZFN associated toxicity <ref name="ref7">Wilson, K.A.; McEwen, A.E.; Pruett-Miller, S.M.; Zhang, J.; Kildebeck, E.J.;, Porteus, M.H. Expanding the Repertoire of Target Sites for Zinc Finger Nuclease-mediated Genome Modification. Mol Ther Nucleic Acids., 2013, 2, e88.</ref>.

DNA-cleavage domain

DNA-cleavage domain of ZFN originates from FokI catalytic domain. FokI is type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and non-specific DNA-cleavage C-terminal domain. In order to cleave DNA two FokI catalytic domains must form dimer. Each subunit contains one catalytic centre that is responsible for cleaving one strand of DNA duplex. This characteristic of FokI catalytic domain is crucial for successful genome editing with ZFN. Dimerization requires two independent binding events, which must occur in correct orientation and spacing between monomers. DNA-cleavage domain is usually fused to the C-terminus of DNA-binding domain, thus two individual ZFNs must bind to opposite strands of DNA. This enables specific targeting of long and potentially unique recognition sites</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>.

ZFN-mediated genome editing

ZFNs create double-stranded breaks (DSBs) in DNA at sequence specific site. DSBs can be repaired by cell’s endogenous mechanisms: homology-directed repair (HDR) or non-homologous end joining (NHEJ). There are few different possible outcomes of DSB repair. If the break is repaired by NHEJ, it can lead to gene disruption, tag ligation or large deletion. Gene disruption occurs when two broken ends are ligated back together that can lead to small insertions or deletions at the break site causing target gene disruption. Tag ligation occurs when adaptor with sticky ends is provided and is ligated into the chromosome producing tagged allele. When two different ZFNs cleave simultaneous same chromosome, entire segment between them is deleted. If the break is repaired by HDR, which occurs in the presence of donor DNA, it can result in gene correction, targeted gene addition or transgene stacking. If donor with small change, for example single bp, is provided, editing of endogenous allele results in gene correction. This enables easier functional genomics studies, modelling of disease caused by point mutation and gene therapy research. If provided donor carries transgene and has specific homology arms, transgene can be efficiently integrated into chromosome at break site, which results in targeted gene addition. Transgene stacking occurs when provided donor carries multiple linked transgenes between the homology arms. With this approach not only gene addition can be done, but also gene correction if DSB is created within or near gene in question and provided donor carries modified gene sequence</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><ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. Genetics., 2011, 188(4), 773-782.</ref><ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol., 2013, 31(7), 397-405.</ref>.

Applications:

Model organisms

ZFNs enable introduction of targeted modification in several model organisms used in common biological researches. Benefits of DSB repaired by cell’s endogenous mechanisms include: simple production of knock-out or knock-in cell lines, permanent and heritable mutation, compatibility with variety of species<ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol., 2013, 31(7), 397-405.</ref>.

Therapeutic application of ZFNs

ZFN-mediated gene modification by HDR has been used to correct mutations associated with X-linked severe combined immune deficiency (SCID), haemophilia B, sickle-cell disease and α1-antitrypsin deficiency. ZFN induced gene targeting by NHEJ repair has been shown as potential treatment for HIV/AIDS, either by targeting cellular co-receptors for HIV in primary T cells and hematopoietic stem/progenitor cells (clinical trials) or by targeting proviral HIV DNA<ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol., 2013, 31(7), 397-405.</ref>.

Potential problems

If the DNA-binding domain is not specific enough or target site is not unique in genome, ZFN may cleave at undesired location in the genome. Another potential problem is formation of ZFN homodimers that are by products of ZFN heterodimer that can cleave off-target. Besides cleaving off-target, ZFN homodimers mediate toxic effect. Undesired cleavage can lead to unwanted insertions or deletions at additional cleavage sites or vast number of DSBs can cause cell death. This can be avoided by extensive bioinformatics research beforehand, construction of ZFP that recognises longer and consequently rarer target sequences, usingf suitable linkers and cleavage domains that do not form unwanted homodimers<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>.