A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells

Zinc-finger recombinases (ZFRs) represent a potentially powerful class of tools for targeted genetic engineering. These chimeric enzymes are composed of an activated catalytic domain derived from the resolvase/invertase family of serine recombinases and a custom-designed zinc-finger DNA-binding domain. The use of ZFRs, however, has been restricted by sequence requirements imposed by the recombinase catalytic domain. Here, we combine substrate specificity analysis and directed evolution to develop a diverse collection of Gin recombinase catalytic domains capable of recognizing an estimated 3.77 × 10⁷ unique DNA sequences. We show that ZFRs assembled from these engineered catalytic domains recombine user-defined DNA targets with high specificity, and that designed ZFRs integrate DNA into targeted endogenous loci in human cells. This study demonstrates the feasibility of generating customized ZFRs and the potential of ZFR technology for a diverse range of applications, including genome engineering, synthetic biology and gene therapy.     

Expanding the zinc-finger recombinase repertoire: directed evolution and mutational analysis of serine recombinase specificity determinants

The serine recombinases are a diverse family of modular enzymes that promote high-fidelity DNA rearrangements between specific target sites. Replacement of their native DNA-binding domains with custom-designed Cys₂–His₂ zinc-finger proteins results in the creation of engineered zinc-finger recombinases (ZFRs) capable of achieving targeted genetic modifications. The flexibility afforded by zinc-finger domains enables the design of hybrid recombinases that recognize a wide variety of potential target sites; however, this technology remains constrained by the strict recognition specificities imposed by the ZFR catalytic domains. In particular, the ability to fully reprogram serine recombinase catalytic specificity has been impeded by conserved base requirements within each recombinase target site and an incomplete understanding of the factors governing DNA recognition. Here we describe an approach to complement the targeting capacity of ZFRs. Using directed evolution, we isolated mutants of the β and Sin recombinases that specifically recognize target sites previously outside the scope of ZFRs. Additionally, we developed a genetic screen to determine the specific base requirements for site-specific recombination and showed that specificity profiling enables the discovery of unique genomic ZFR substrates. Finally, we conducted an extensive and family-wide mutational analysis of the serine recombinase DNA-binding arm region and uncovered a diverse network of residues that confer target specificity. These results demonstrate that the ZFR repertoire is extensible and highlights the potential of ZFRs as a class of flexible tools for targeted genome engineering.     

Effects of DNA binding of the zinc finger and linkers for domain fusion on the catalytic activity of sequence-specific chimeric recombinases determined by a facile fluorescent system

Artificial zinc finger proteins (ZFPs) consist of Cys(2)-His(2)-type modules composed of ～30 amino acids with a ββα structure that coordinates a zinc ion. ZFPs that recognize specific DNA target sequences can substitute for the binding domains of enzymes that act on DNA to create designer enzymes with programmable sequence specificity. The most studied of these engineered enzymes are zinc finger nucleases (ZFNs). ZFNs have been widely used to model organisms and are currently in human clinical trials with an aim of therapeutic gene editing. Difficulties with ZFNs arise from unpredictable mutations caused by nonhomologous end joining and off-target DNA cleavage and mutagenesis. A more recent strategy that aims to address the shortcomings of ZFNs involves zinc finger recombinases (ZFRs). A thorough understanding of ZFRs and methods for their modification promises powerful new tools for gene manipulation in model organisms as well as in gene therapy. In an effort to design efficient and specific ZFRs, the effects of the DNA binding affinity of the zinc finger domains and the linker sequence between ZFPs and recombinase catalytic domains have been assessed. A plasmid system containing ZFR target sites was constructed for evaluation of catalytic activities of ZFRs with variable linker lengths and numbers of zinc finger modules. Recombination efficiencies were evaluated by restriction enzyme analysis of isolated plasmids after reaction in Escherichia coli and changes in EGFP fluorescence in mammalian cells. The results provide information relevant to the design of ZFRs that will be useful for sequence-specific genome modification.     

Role of arginine-43 and arginine-69 of the Hin recombinase catalytic domain in the binding of Hin to the hix DNA recombination sites

The Hin recombinase mediates the site-specific inversion of a segment of the Salmonella chromosome between two flanking 26 bp hix DNA recombination sites. Mutations in two amino acid residues, R43 and R69 of the catalytic domain of the Hin recombinase, were identified that can compensate for loss of binding resulting from elimination of certain major and minor groove contacts within the hix recombination sites. With one exception, the R43 and R69 mutants were also able to bind a hix sequence with an additional 4 bp added to the centre of the site, unlike wild-type Hin. Purified Hin mutants R43H and R69C had both partial cleavage and inversion activities in vitro while mutants R43L, R43C, R69S, and R69P had no detectable cleavage and inversion activities. These data support a model in which the catalytic domain plays a role in DNA-binding specificity, and suggest that the arginine residues at positions 43 and 69 function to position the Hin recombinase on the DNA for a step in the recombination reaction which occurs either at and/or prior to DNA cleavage.     

DNA recognition via mutual-induced fit by the core-binding domain of bacteriophage lambda integrase

Bacteriophage lambda integrase (lambda-Int), a phage-encoded DNA recombinase, cleaves its substrate DNA to facilitate the formation and later resolution of a Holliday junction intermediate during recombination. The core-binding and catalytic domains of lambda-Int constitute a bipartite enzyme that mediates site-specific DNA cleavage through their interactions with opposite sides of the recognition sequence. Despite minimal direct contact between the domains, the core-binding domain has been shown to facilitate site-specific DNA cleavage when provided in trans, indicating that it plays a role beyond enhancing binding affinity. Biophysical characterization of the core-binding domain and its interactions with DNA reveal that the domain is poorly structured in its free form and folds upon binding to DNA. Folding of the protein is accompanied by induced-fit structural changes in the DNA ligand. These data support a model by which the core-binding domain plays a catalytic role by reshaping the substrate DNA for effective cleavage by the catalytic domain.     

Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases

Artificial endonucleases consisting of a FokI cleavage domain tethered to engineered zinc-finger DNA-binding proteins have proven useful for stimulating homologous recombination in a variety of cell types. Because the catalytic domain of zinc-finger nucleases (ZFNs) must dimerize to become active, two subunits are typically assembled as heterodimers at the cleavage site. The use of ZFNs is often associated with significant cytotoxicity, presumably due to cleavage at off-target sites. Here we describe a structure-based approach to reducing off-target cleavage. Using in silico protein modeling and energy calculations, we increased the specificity of target site cleavage by preventing homodimerization and lowering the dimerization energy. Cell-based recombination assays confirmed that the modified ZFNs were as active as the original ZFNs but elicit significantly less genotoxicity. The improved safety profile may facilitate therapeutic application of the ZFN technology.     

102 Genome engineering nucleases derived from GIY-YIG homing endonucleases

Efficient targeted manipulation of complex genomes requires highly specific endonucleases to generate double-strand breaks at defined locations (Bibikova et al., 2003; Bogdanove and Voytas, 2011). The predominantly engineered nucleases, zinc-finger nucleases (ZFNs), and TAL effector nucleases (TALENs) use the catalytic domain of FokI as the nuclease portion. This domain, however, functions as a dimer to nonspecifically cleave DNA meaning that ZFNs and TALENs must be designed in head-to-head pairs to target a desired sequence. To overcome this limitation and expand the toolbox of genome editing reagents, we used the N-terminal catalytic domain and interdomain linker of the monomeric GIY-YIG homing endonuclease I-TevI to create I-TevI-zinc-fingers (Tev-ZFEs), and I-TevI-TAL effectors (Tev-TALs) (Kleinstiver et al. 2012). We also made I-TevI fusions to LAGLIDADGs homing endonucleases (I-Tev-LHEs). All the three fusions showed activity on model substrates on par with ZFNs and TALENs in yeast-based recombination assays. These proof-of-concept experiments demonstrate that the catalytic domain of GIY-YIG homing endonucleases can be targeted to relevant loci by fusing the domain to characterize DNA-binding platforms. Recent efforts have focused on improving the Tev-TAL platform by (1) understanding the spacing requirements between the nuclease cleavage site and the DNA binding site, (2) probing the DNA binding requirements of the I-TevI linker domain, and (3) demonstrating activity in mammalian systems.     

Site-specific photo-cross-linking between lambda integrase and its DNA recombination target

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and is composed of three domains as follows: an amino-terminal domain that binds with high affinity to "arm-type" sequences within the recombination target DNA (att sites), a carboxyl-terminal domain that contains all of the catalytic functions, and a central domain that contributes significantly to DNA binding at the "core-type" sequences where DNA cleavage and ligation are executed. We constructed a family of core-type DNA oligonucleotides, each of which contained the photoreactive analog 4-thiodeoxythymidine (4-thioT) at a different position. When tested for their respective abilities to promote covalent cross-links with Int after irradiation with UV light at 366 nm, one oligonucleotide stood out dramatically. The 4-thioT substitution on the DNA strand opposite the site of Int cleavage led to photo-induced cross-linking efficiencies of approximately 20%. The efficiency and specificity of Int binding and cleavage at this 4-thioT-substituted core site was shown to be largely uncompromised, and its ability to participate in a full site-specific recombination reaction was reduced only slightly. Identification of the photo-cross-linked residue as Lys-141 in the central domain provides, along with other results, several insights about the nature of core-type DNA recognition by the bivalent recombinases of the lambda Int family.     

Protein cofactor-dependent acquisition of novel catalytic activity by the RNase P ribonucleoprotein of E. coli

Escherichia coli RNase P derivatives were evolved in vitro for DNA cleavage activity. Ribonucleoproteins sampled after ten generations of selection show a >400-fold increase in the first-order rate constant (k(cat)) on a DNA substrate, reflecting a significant improvement in the chemical cleavage step. This increase is offset by a reduction in substrate binding, as measured by K(M). We trace the catalytic enhancement to two ubiquitous A-->U sequence changes at positions 136 and 333 in the M1 RNA component, positions that are phylogenetically conserved in the Eubacteria. Furthermore, although the mutations are located in different folding domains of the catalytic RNA, the first in the substrate binding domain, the second near the catalytic core, their effect on catalytic activity is significantly influenced by the presence of the C5 protein. The activity of the evolved ribonucleoproteins on both pre-4.5 S RNA and on an RNA oligo substrate remain at wild-type levels. In contrast, improved DNA cleavage activity is accompanied by a 500-fold decrease in pre-tRNA cleavage efficiency (k(cat)/K(M)). The presence of the C5 component does not buffer this tradeoff in catalytic activities, despite the in vivo role played by the C5 protein in enhancing the substrate versatility of RNase P. The change at position 136, located in the J11/12 single-stranded region, likely alters the geometry of the pre-tRNA-binding cleft and may provide a functional explanation for the observed tradeoff. These results thus shed light both on structure/function relations in E. coli RNase P and on the crucial role of proteins in enhancing the catalytic repertoire of RNA.     

Zinc finger recombinases with adaptable DNA sequence specificity

Site-specific recombinases have become essential tools in genetics and molecular biology for the precise excision or integration of DNA sequences. However, their utility is currently limited to circumstances where the sites recognized by the recombinase enzyme have been introduced into the DNA being manipulated, or natural 'pseudosites' are already present. Many new applications would become feasible if recombinase activity could be targeted to chosen sequences in natural genomic DNA. Here we demonstrate efficient site-specific recombination at several sequences taken from a 1.9 kilobasepair locus of biotechnological interest (in the bovine β-casein gene), mediated by zinc finger recombinases (ZFRs), chimaeric enzymes with linked zinc finger (DNA recognition) and recombinase (catalytic) domains. In the "Z-sites" tested here, 22 bp casein gene sequences are flanked by 9 bp motifs recognized by zinc finger domains. Asymmetric Z-sites were recombined by the concomitant action of two ZFRs with different zinc finger DNA-binding specificities, and could be recombined with a heterologous site in the presence of a third recombinase. Our results show that engineered ZFRs may be designed to promote site-specific recombination at many natural DNA sequences.     

Identification of two topologically independent domains in RAG1 and their role in macromolecular interactions relevant to V(D)J recombination

V(D)J recombination is instigated by the recombination-activating proteins RAG1 and RAG2, which catalyze site-specific DNA cleavage at the border of the recombination signal sequence (RSS). Although both proteins are required for activity, core RAG1 (the catalytically active region containing residues 384-1008 of 1040) alone displays binding specificity for the conserved heptamer and nonamer sequences of the RSS. The nonamer-binding region lies near the N terminus of core RAG1, whereas the heptamer-binding region has not been identified. Here, potential domains within core RAG1 were identified using limited proteolysis studies. An iterative procedure of DNA cloning, protein expression, and characterization revealed the presence of two topologically independent domains within core RAG1, referred to as the central domain (residues 528-760) and the C-terminal domain (residues 761-980). The domains do not include the nonamer-binding region but rather largely span the remaining relatively uncharacterized region of core RAG1. Characterization of macromolecular interactions revealed that the central domain bound to the RSS with specificity for the heptamer and contained the predominant binding site for RAG2. The C-terminal domain bound DNA cooperatively but did not show specificity for either conserved RSS element. This domain was also found to self-associate, implicating it as a dimerization domain within RAG1.     

DNA arms do the legwork to ensure the directionality of lambda site-specific recombination

The integrase protein of bacteriophage lambda (Int) catalyzes site-specific recombination between lambda phage and Escherichia coli genomes. Int is a tyrosine recombinase that binds to DNA core sites via a C-terminal catalytic domain and to a collection of arm DNA sites, distant from the site of recombination, via its N-terminal domain. The arm sites, in conjunction with accessory DNA-bending proteins, provide a means of regulating the efficiency and directionality of Int-catalyzed recombination. Recent crystal structures of lambda Int tetramers bound to synaptic and Holliday junction intermediates, together with new biochemical data, suggest a mechanism for the allosteric control of the recombination reaction through arm DNA binding interactions.     

DNA ligases in the repair and replication of DNA

DNA ligases are critical enzymes of DNA metabolism. The reaction they catalyse (the joining of nicked DNA) is required in DNA replication and in DNA repair pathways that require the re-synthesis of DNA.Most organisms express DNA ligases powered by ATP, but eubacteria appear to be unique in having ligases driven by NAD(+). Interestingly, despite protein sequence and biochemical differences between the two classes of ligase, the structure of the adenylation domain is remarkably similar. Higher organisms express a variety of different ligases, which appear to be targetted to specific functions. DNA ligase I is required for Okazaki fragment joining and some repair pathways; DNA ligase II appears to be a degradation product of ligase III; DNA ligase III has several isoforms, which are involved in repair and recombination and DNA ligase IV is necessary for V(D)J recombination and non-homologous end-joining. Sequence and structural analysis of DNA ligases has shown that these enzymes are built around a common catalytic core, which is likely to be similar in three-dimensional structure to that of T7-bacteriophage ligase. The differences between the various ligases are likely to be mediated by regions outside of this common core, the structures of which are not known. Therefore, the determination of these structures, along with the structures of ligases bound to substrate DNAs and partner proteins ought to be seen as a priority.     

Identification of basic residues in RAG2 critical for DNA binding by the RAG1-RAG2 complex.

In V(D)J recombination, the RAG1 and RAG2 proteins are the essential components of the complex that catalyzes DNA cleavage. RAG1 has been shown to play a central role in DNA binding and catalysis. In contrast, the molecular roles of RAG2 in V(D)J recombination are unknown. To address this, we individually mutated 36 evolutionarily conserved basic and hydroxy group containing residues within RAG2. Biochemical analysis of the recombinant RAG2 proteins led to the identification of a number of basic residue mutants defective in catalysis in vitro and V(D)J recombination in vivo. Five of these were deficient in binding of the RAG1-RAG2 complex to its cognate DNA target sequence while interacting normally with RAG1. Our findings provide support for the direct involvement of RAG2 in DNA binding during all steps of the cleavage reaction.     

Mutations at residues 282, 286, and 293 of phage lambda integrase exert pathway-specific effects on synapsis and catalysis in recombination

Bacteriophage lambda integrase (Int) catalyzes site-specific recombination between pairs of attachment (att) sites. The att sites contain weak Int-binding sites called core-type sites that are separated by a 7-bp overlap region, where cleavage and strand exchange occur. We have characterized a number of mutant Int proteins with substitutions at positions S282 (S282A, S282F, and S282T), S286 (S286A, S286L, and S286T), and R293 (R293E, R293K, and R293Q). We investigated the core- and arm-binding properties and cooperativity of the mutant proteins, their ability to catalyze cleavage, and their ability to form and resolve Holliday junctions. Our kinetic analyses have identified synapsis as the rate-limiting step in excisive recombination. The IntS282 and IntS286 mutants show defects in synapsis in the bent-L and excisive pathways, respectively, while the IntR293 mutants exhibit synapsis defects in both the excision and bent-L pathways. The results of our study support earlier findings that the catalytic domain also serves a role in binding to core-type sites, that the core contacts made by this domain are important for both synapsis and catalysis, and that Int contacts core-type sites differently among the four recombination pathways. We speculate that these residues are important for the proper positioning of the catalytic residues involved in the recombination reaction and that their positions differ in the distinct nucleoprotein architectures formed during each pathway. Finally, we found that not all catalytic events in excision follow synapsis: the attL site probably undergoes several rounds of cleavage and ligation before it synapses and exchanges DNA with attR.     

V(D)J recombination: site-specific cleavage and repair

V(D)J recombination is a site-specific gene rearrangement process that contributes to the diversity of antigen receptor repertoires. Two lymphoid-specific proteins, RAG1 and RAG2, initiate this process at two recombination signal sequences. Due to the recent development of an in vitro assay for V(D)J cleavage, the mechanism of cleavage has been elucidated clearly. The RAG complex recognizes a recombination signal sequence, makes a nick at the border between signal and coding sequence, and carries out a transesterification reaction, resulting in the production of a hairpin structure at the coding sequence and DNA double-strand breaks at the signal ends. RAG1 possesses the active site of the V(D)J recombinase although RAG2 is essential for signal binding and cleavage. After DNA cleavage by the RAG complex, the broken DNA ends are rejoined by the coordinated action of DNA double-strand break repair proteins as well as the RAG complex. The junctional variability resulting from imprecise joining of the coding sequences contributes additional diversity to the antigen receptors.     

Catalysis by site-specific recombinases

Site-specific recombination reactions bring about controlled rearrangements of DNA molecules by cutting the DNA at precise points and rejoining the ends to new partners. The recombinases that catalyse these reactions can be grouped into two families by amino acid sequence homology. We describe our current understanding of how these proteins catalyse recombination, and show how the catalytic mechanisms of the two families differ.     

Two DNA-binding and nick recognition modules in human DNA ligase III

Human DNA ligase III contains an N-terminal zinc finger domain that binds to nicks and gaps in DNA. This small domain has been described as a DNA nick sensor, but it is not required for DNA nick joining activity in vitro. In light of new structural information for mammalian ligases, we measured the DNA binding affinity and specificity of each domain of DNA ligase III. These studies identified two separate, independent DNA-binding modules in DNA ligase III that each bind specifically to nicked DNA over intact duplex DNA. One of these modules comprises the zinc finger domain and DNA-binding domain, which function together as a single DNA binding unit. The catalytic core of ligase III is the second DNA nick-binding module. Both binding modules are required for ligation of blunt ended DNA substrates. Although the zinc finger increases the catalytic efficiency of nick ligation, it appears to occupy the same binding site as the DNA ligase III catalytic core. We present a jackknife model for ligase III that posits conformational changes during nick sensing and ligation to extend the versatility of the enzyme.     

Sandwiched zinc-finger nucleases demonstrating higher homologous recombination rates than conventional zinc-finger nucleases in mammalian cells

We previously reported that our sandwiched zinc-finger nucleases (ZFNs), in which a DNA cleavage domain is inserted between two artificial zinc-finger proteins, cleave their target DNA much more efficiently than conventional ZFNs in vitro. In the present study, we compared DNA cleaving efficiencies of a sandwiched ZFN with those of its corresponding conventional ZFN in mammalian cells. Using a plasmid-based single-strand annealing reporter assay in HEK293 cells, we confirmed that the sandwiched ZFN induced homologous recombination more efficiently than the conventional ZFN; reporter activation by the sandwiched ZFN was more than eight times that of the conventional one. Western blot analysis showed that the sandwiched ZFN was expressed less frequently than the conventional ZFN, indicating that the greater DNA-cleaving activity of the sandwiched ZFN was not due to higher expression of the sandwiched ZFN. Furthermore, an MTT assay demonstrated that the sandwiched ZFN did not have any significant cytotoxicity under the DNA-cleavage conditions. Thus, because our sandwiched ZFN cleaved more efficiently than its corresponding conventional ZFN in HEK293 cells as well as in vitro, sandwiched ZFNs are expected to serve as an effective molecular tool for genome editing in living cells.     

The catalytic residues of Tn3 resolvase

To characterize the residues that participate in the catalysis of DNA cleavage and rejoining by the site-specific recombinase Tn3 resolvase, we mutated conserved polar or charged residues in the catalytic domain of an activated resolvase variant. We analysed the effects of mutations at 14 residues on proficiency in binding to the recombination site (‘site I’), formation of a synaptic complex between two site Is, DNA cleavage and recombination. Mutations of Y6, R8, S10, D36, R68 and R71 resulted in greatly reduced cleavage and recombination activity, suggesting crucial roles of these six residues in catalysis, whereas mutations of the other residues had less dramatic effects. No mutations strongly inhibited binding of resolvase to site I, but several caused conspicuous changes in the yield or stability of the synapse of two site Is observed by non-denaturing gel electrophoresis. The involvement of some residues in both synapsis and catalysis suggests that they contribute to a regulatory mechanism, in which engagement of catalytic residues with the substrate is coupled to correct assembly of the synapse.