Site‐Specific DNA Recombinases as Instruments for Genomic Surgery

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Site‐Specific DNA Recombinases as Instruments for Genomic Surgery Aram Akopian and W. Marshall Stark Institute of Biomedical & Life Sciences University of Glasgow, Glasgow G11 6NU United Kingdom

I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

XII.

Introduction Inadequacy of Current Methods Site‐Specific Recombination Mechanisms of Site‐Specific Recombination Transposition Applied Site‐Specific Recombination and Transposition Systems Changing Recombination Site Specificity Mutagenesis‐Selection Strategies Structure‐Based Strategies: Z‐Resolvases Targeting Transposition to Specific Sequences General Considerations in Applications of Site‐Specific Recombinases and Transposases A. Recombinase and transgene “delivery” B. Chromatin C. Reversibility D. Nonspecific reactions Prospects and Conclusions References

Advances in Genetics, Vol. 55 Copyright 2005, Elsevier Inc. All rights reserved.

0065-2660/05 $35.00 DOI: 10.1016/S0065-2660(05)55001-6

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ABSTRACT Site‐specific DNA recombinases can “cut and paste” DNA. For example, they can promote excision of specific DNA segments or insertion of new DNA segments in specific places. However, natural recombinases act only at their cognate recombination sites, so current applications are limited to genetically modified organisms in which these sites have been introduced into the genome. Transposases also catalyze DNA rearrangements; they promote insertion of specific DNA sequences but at nonspecific locations. Applicability of site‐ specific recombinases and transposases in experimental genetics, biotechnology, and gene therapy would be much wider if they could be reengineered so as to act specifically at chosen sequences within an organism’s natural genome. This review will discuss progress towards the creation of such “designer” recombinases. ß 2005, Elsevier Inc.

I. INTRODUCTION As numerous genomes are sequenced and our understanding of genome function grows, the possibilities for changing genomic DNA sequence to our benefit become more apparent. We could add new useful bits of DNA in specific places and delete or change defective or undesirable bits. This “genomic surgery” could be applied to the treatment of disease as well as in biotechnology and experimental genetics. The development of realistic methods for genomic surgery will bring many opportunities for improving our health and well being, along with dangers and ethical problems that will be faced by science and society. In this review, we will discuss advances in the still‐primitive methods that are under consideration, focusing on the attempts to adapt natural enzymes that “cut and paste” DNA—recombinases and transposases—so that their activity can be directed to chosen sequences.

II. INADEQUACY OF CURRENT METHODS For many years, it has been possible to make artificial alterations to an organism’s genetic material. The alteration may be temporary (e.g., when foreign nonreplicating DNA is introduced into a cell or when gene expression is altered by RNAi) or permanent (e.g., when transgenic DNA integrates into a chromosome). Integration of a foreign DNA segment can be targeted to a specific genomic locus by methods based on homologous recombination (HR; see in later section). Why then do we need new methods to target DNA manipulation to particular genomic sites?

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The currently available methods for directing DNA modification to specific target sequences lack the versatility, efficiency, and specificity, which will be needed if genomic surgery is to take off (Yanez and Porter, 1998). For example, consider a scenario in which a new functional gene is to be integrated into a mammalian genome. Without targeting, a transfected DNA fragment will generally integrate inefficiently and at essentially random positions (Lacy et al., 1983; Smith, 2001). Transgene expression might therefore be subject to locus‐ specific downregulation by epigenetic silencing processes (Pannell and Ellis, 2001) or the integrated DNA might affect expression of nearby genes, thus potentially causing cell malfunction or oncogenesis (Palmiter and Brinster, 1986). Ideally, the transgene should be integrated at a locus where it will be expressed properly, and it will not interfere with other functions of the genome. Use of HR is the only current means of targeting integration to any chosen genomic locus. By embedding the gene to be transfected in the middle of a few kilobase‐pairs (kbp) of target site sequence, the cell’s HR machinery can be co‐ opted to promote integration at that site. This approach has been successfully applied as an experimental tool in some organisms for integration of new genes or to “knock out” resident genes (e.g., Colosimo et al., 2000; Vasquez et al., 2001). Unfortunately, integration by HR is inefficient, and specificity for the chosen target locus is low. Normally, the great majority of cells following transfection have not integrated the transfected DNA, and the transgene lands at the desired target site in only a small fraction of the remainder (Smith, 2001; Vasquez et al., 2001). Homologous recombination is therefore of practical use only if one can select the correctly modified cells and, if necessary, induce them to participate in embryogenesis. The options for improvement of HR‐based methods are limited because HR is crucial for the maintenance of the genome; tampering with its natural components will possibly have serious biological consequences and would probably only be feasible in experimental systems. A further technical limitation of HR is that the transgene must be flanked by long sequences homologous to the target. This can restrict the size of genes to be introduced with viral vectors, which can only carry a limited length of foreign sequence (Monahan and Samulski, 2000; Yanez and Porter, 1998). Methods for enhancing the efficiency of HR at chosen sites have been developed, based on the introduction of factors with sequence recognition specificity (DNA‐binding proteins or oligonucleotides) (Uil et al., 2003). However, it seems that, for more advanced genomic surgery, alternatives to HR will be required. In later sections, we consider whether site‐specific recombinases and transposases may be useful “surgical instruments.” Other strategies for alteration of specific genomic sequences are also being investigated (Belfort et al., 2002; Collins et al., 2003; Epinat et al., 2003; Guo et al., 2000; Lambowitz and Zimmerly, 2004; Monahan and Samulski, 2000; Portlock and Calos, 2003; Uil et al., 2003).

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III. SITE‐SPECIFIC RECOMBINATION Site‐specific recombinases rearrange DNA sequences by catalyzing cleavage and rejoining of DNA strands at specific short DNA sequences (sites) to which they bind. The outcome of the reaction can be excision of a DNA sequence segment bounded by two sites, or the reverse of this process, integration, or inversion of the orientation of a segment of DNA bounded by two sites (Fig. 1.1). An attractive feature of these systems as potential tools for genomic surgery is that they can be highly specific, efficient, and fast (Nash, 1996). Microbial site‐specific recombination systems have been widely exploited in mammals and other higher eukaryotes for experimental research or projected gene therapy/biotechnology applications (see Section VI). However, their use is currently limited to situations where one or more of the sites recognized by the recombinase have already been introduced into the genome by other methods. It would be a major advance if we could engineer recombinases to act with high efficiency and specificity at sequences that are already there. The best‐characterized site‐specific recombination systems come from bacteria and yeasts. Most of the systems can be assigned to one of two large families, according to the type of recombinase that is used. These two families, the tyrosine recombinases and the serine recombinases, are so‐called because the conserved nucleophilic amino acid residue that attacks and becomes linked to the DNA during strand exchange is either a tyrosine or a serine. The well‐ known enzymes Cre, FLP, and  integrase are tyrosine recombinases; examples from the serine recombinase family are  resolvase and C31 integrase (Nash, 1996). The two families are unrelated to each other, having different protein structures and reaction mechanisms.

Figure 1.1. Substrates and products of site‐specific recombination reactions. The diagrams show how the relationship of two recombining sites (small black and white arrows) determines the type of recombination product formed. On the left, excision of DNA between directly repeated sites and the reverse reaction, integration; on the right, inversion of the DNA segment between sites in inverted repeat.

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Natural site‐specific recombination systems have evolved to carry out specific “programmed” genetic rearrangements in their host organisms. Therefore, their activity is usually tightly regulated, and the type of rearrangement they bring about (excision, inversion, or integration) is usually specified. These constraints can cause problems for those who wish to subvert the recombinases to become tools for genetic manipulation.

IV. MECHANISMS OF SITE‐SPECIFIC RECOMBINATION The two DNA sites that are to recombine (typically a few tens of base‐pains(bp) long; Fig. 1.2B) must first be recognized by the recombinase protein. The protein‐bound sites must then be brought together (synapsed) prior to strand exchange, which involves cutting and rejoining of the DNA strands. During strand exchange, the recombinase transiently becomes covalently linked to a phosphate of the DNA backbone via the hydroxyl group of the nucleophilic tyrosine or serine residue (Hallet and Sherratt, 1997; Nash, 1996). The strands are broken at fixed points in the site sequence. Tyrosine recombinases exchange strands one pair at a time, and thus the reaction proceeds via an intermediate which is analogous to the Holliday junction of HR (Holliday, 1964). In contrast, serine recombinases make intermediates in which all four DNA strands of the two recombination sites are broken. These mechanisms are summarized in Fig. 1.2A. The recombination site generally has an asymmetric sequence and thus a “left” and a “right” end; each left end is joined to the right end of the partner site, so as to reconstitute similar sites in the recombination products. This polarity is often assured by an asymmetric “overlap” sequence between the staggered points of top and bottom strand exchange, so that recombination of misaligned sites would produce mismatched base pairs in the recombinants (Fig. 1.2B). The connectivity of the two recombining sites in the substrate DNA therefore defines the type of product formed: inversion, if the sites are oriented in opposite directions to each other; excision, if they are directly repeated (“head to tail”); and integration, if they are on separate molecules (Fig. 1.1). Some recombinases will act on pairs of sites in any relationship (e.g., Cre and Flp), whereas others are specific for one type of relationship (e.g.,  resolvase, which only catalyzes excision). Our understanding of the mechanisms of site‐specific recombination has been greatly advanced by a number of crystal structures showing recombinases bound to their cognate sites, and intermediates in the strand exchange process itself (Chen et al., 2000; Grindley, 2002; Guo et al., 1999; Van Duyne, 2002; Yang and Steitz, 1995). Structures of Cre (Fig. 1.3) and Flp show a recombinase tetramer synapsing two recombination sites. Each subunit of these tyrosine recombinases wraps around the DNA, making many contacts with it.

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Figure 1.2. (A) Mechanisms of DNA strand exchange by serine recombinases and tyrosine recombinases. The pairs of thick or thin lines represent double‐stranded DNA. The small arrowheads mark the ends of a “site” of a few tens of basepairs (Fig. 1.2B), which is recognized specifically by the recombinase enzyme (not shown). The staggered lines show where the strands are broken at the centres of the sites. Note that the sites are aligned in parallel in the upper row (serine recombinases), but in antiparallel in the lower row (tyrosine recombinases), reflecting the proposed structures of reaction intermediates. See text for further details. (B) Recombination sites. The sequences of two simple recombination sites are shown: loxP, the site for Cre recombinase, and attB, one of the two nonidentical sites for C31 integrase. The loxP site has twofold (palindrome) symmetry, indicated by the long arrows, except in the central 8 bp; in contrast, C31 attB is quite asymmetric. The short black arrows indicate the positions of breakage and rejoining of the DNA strands. The asymmetric ‘overlap’ sequence between the two arrows is important in specifying that left and right half‐sites are joined in the recombinant products. The overlap is typically 2 bp for serine recombinase sites, as in C31 attB, and 5–8 bp for tyrosine recombinase sites (6 bp in loxP).

The basis of sequence‐specific binding by these proteins is still not clear. A structure of the serine recombinase  resolvase (Fig. 1.3) shows a recombinase dimer bound to a single site. A small C‐terminal domain of each resolvase subunit, thought to be responsible for much of the sequence specificity, binds in the major groove of the DNA. The larger N‐terminal domains, which contain the active site and make intersubunit interactions, contact the DNA in the minor groove on the opposite side of the double helix from the C‐terminal domains.

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Figure 1.3. Crystal structures of site‐specific recombinases bound to DNA. (A) A dimer of the serine recombinase  resolvase, bound to ‘site I’, the part of its recombination site where strand exchange occurs (Yang and Steitz, 1995). (B) A tetramer of the tyrosine recombinase Cre in a synapse with two loxP sites (Guo et al., 1999). The DNA (cream and blue) is in spacefill representation, and the protein (green and yellow) is in a ribbon representation. The two loxP sites in B are in approximately antiparallel alignment (see Fig. 1.1). The pictures were created with the program PYMOL.

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V. TRANSPOSITION Transposition has much in common with site‐specific recombination. A semiautonomous DNA element called a transposon moves from one locus to another in a reaction catalyzed by a transposase enzyme. The ends of the transposon must be recognized and acted upon by the transposase site‐specifically, but the transposon DNA is then inserted at new positions, which are generally not specific (Fig. 1.4). The mechanisms of transposition are diverse (Craig, 2002a) and can even involve RNA intermediates (the process being then called retrotransposition). DNA transposases (those that catalyze direct transfer of DNA from one locus to another) belong to several different structural groups. Some are related to the serine and tyrosine site‐specific recombinases introduced in Section IV. The best characterized are the so‐called “DDE” transposases, which are very widespread; transposons that encode them are found in bacteria, archaea, and eukarya. Like site‐specific recombinases, transposases have already attracted considerable interest as potential tools for gene therapy and biotechnology (Boeke, 2002). They share with site‐specific recombinases the ability to promote highly efficient, specific, and fast reactions. By their nature, transposases are best adapted to promote integration of a DNA segment, flanked by short “ends” from their cognate transposon, into target DNA. The transposase breaks either one or both strands at each end and then joins the broken ends to the target DNA. The complete process normally involves subsequent “tidying‐up” operations, which require host enzymes (Craig, 2002a). Usually, the transposase has little or no selectivity for specific target sequences, so transposon insertions can occur

Figure 1.4. Transposition. In the simplest case, as shown here, a DNA transposon (stippled rectangle) is excised from the DNA by its transposase enzyme acting at specific end sequences (arrowheads), and then inserted into a random site in target DNA (thicker lines).

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anywhere in the genome; for exceptions that do have strongly preferred target sites or insert in specific regions of the genome, see Bushman, 2003; Craig, 2002b. The long terminal repeat (LTR) retrotransposons and LTR retroviruses reverse‐transcribe their RNA genome into DNA before integrating it into the host genome. Their integrase enzymes belong to the DDE transposase family, and the mechanism of integration is similar to that of typical DNA transposons with DDE transposases (Craig, 2002a). Integration by the non‐LTR retrotransposons involves different types of enzymes and direct interaction of RNA molecules with the target DNA. Non‐LTR retrotransposition systems may also have potential for targeted gene integration (Eickbush, 2002). In Section X, we will briefly review attempts to target insertion mediated by DDE transposases/retroviral integrases.

VI. APPLIED SITE‐SPECIFIC RECOMBINATION AND TRANSPOSITION SYSTEMS Recombinases have become very popular tools for manipulating DNA in vitro and in vivo (Boeke, 2002; Branda and Dymecki, 2004; Gorman and Bullock, 2000; Groth and Calos, 2003; Kilby et al., 1993; Kolb, 2002; Nagy, 2000). The most frequently used enzymes have been Cre recombinase from bacteriophage P1 and Flp recombinase from Saccharomyces cerevisiae. The 38‐kDa Cre protein promotes recombination between two 34‐bp loxP sites, and the 43‐kDa Flp recombinase acts on 34‐bp FRT sites. Advantages of these systems are their short DNA recombination sites, enzyme stability in vivo, and the robustness of their activity even when acting upon chromatin‐associated DNA (Jayaram et al., 2002; Sauer, 2002). Numerous other recombinases have been used in biotechnology or investigated as possible biotechnology tools (Boeke, 2002). Similarly, several DNA transposition systems have become popular for sequencing, mutagenesis, and transgene integration purposes (Boeke, 2002); and retroviral integration is being widely investigated for possible uses in gene therapy (see Sections X and XI).

VII. CHANGING RECOMBINATION SITE SPECIFICITY Hundreds of different natural site‐specific recombination systems have been identified, so there is a large natural “library” of sites with different sequences, acted upon by known recombinases. However, it is still very unlikely that any of these sequences will be found by chance in a useful place in a genome of interest, as most natural recombination sites are at least about 25 bp long. Furthermore,

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characterization of a new system to the stage where it could be used as a research tool requires a considerable amount of work. A more practical approach is to mutate or redesign a well‐characterized recombinase so that it recognizes a new sequence. Changing the sequence recognition properties of site‐specific recombinases has been a subject of investigation for many years; initially the aims were to provide insight on structure and mechanism and to create useful experimental tools. Recognition by serine recombinases related to  resolvase has been altered by point mutation (Grindley, 1993) or by replacing part or all of the entire DNA‐binding domain with the equivalent sequence from a related protein (Ackroyd et al., 1990; Avila et al., 1990; Schneider et al., 2000). Similar mutation and domain‐swap approaches were used to change the specificities of tyrosine recombinases (Dorgai et al., 1995; Shaikh and Sadowski, 2000; Yagil et al., 1995). Recently, researchers have begun to develop more systematic strategies for altering recombinase site recognition, with the long‐term goal of creating more useful and adaptable systems for genetic manipulation in vivo. Two types of strategy are discussed in Sections IV and V. As hinted previously, it would be a great advance if integration could be targeted efficiently and specifically to even one good natural site in a genome of interest (e.g., human), where gene expression is optimal and there would be no adverse effects. One approach is therefore to choose a natural sequence with some similarity to the site for a useful recombinase, often one that has been shown to act as a “pseudosite” (where the recombinase occasionally catalyzes recombination). One can then attempt to produce variants of the recombinase that act there efficiently and specifically by multiple rounds of mutagenesis and selection. A more radical approach is the attempt to design a recombinase whose sequence specificity can be altered at will by changing a “DNA recognition module.”

VIII. MUTAGENESIS‐SELECTION STRATEGIES C31 Int, a serine recombinase, mediates integration by recombination of phage attP (39 bp) and bacterial genomic attB (34 bp) sites (Groth and Calos, 2003). Integration is irreversible in the reconstituted system lacking factors encoded by the phage C31 or its Streptomyces host. The relatively short sites and irreversible reaction make the C31 Int system a very attractive candidate for applications. There is no high‐resolution structural information as yet on C31 Int or any of its close relatives, but current biochemical evidence suggests that DNA recognition involves multiple domains within these large proteins. Systematic redesign of sequence specificity by “protein engineering” is therefore not an option at present. Although there are high‐resolution structures of the tyrosine recombinases Cre and Flp bound to their cognate sites, a protein‐engineering

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approach is still problematic for them because it is difficult to distinguish residues involved in DNA recognition from those involved in catalysis (Section IV). The mutagenesis‐selection approach for altering site recognition, which does not depend on a deep understanding of the recombinase structure, has therefore been adopted for Cre, Flp, and C31 Int. A typical procedure employs an in vivo test substrate, which is designed so that recombination at the sites under investigation alters the expression of an observable marker gene, such as a fluorescent protein or the lacZ‐encoded ‐fragment of ‐Gal (e.g., by excision of the gene, separating it from its promoter; Fig. 1.5). The substrate is established in the organism of choice, then challenged by introduction of DNA encoding a “library” of recombinase mutants (which may be generated by any appropriate method, such as error‐ prone PCR). Cells containing active mutants are then selected (e.g., by a change in color [Santoro and Schultz, 2002; Sclimenti et al., 2001]). A variation in the procedure, which facilitates the isolation of active mutants, is to combine the recombinase gene and the test construct in one plasmid (Buchholz and Stewart, 2001). Recombinase mutants obtained in the first cycle of mutagenesis‐ selection may then be used as the starting point for further cycles, either selecting for increased recombination efficiency on the same site or activity on a site that contains further changes from the wild‐type sequence. Mutants obtained by mutagenesis‐selection procedures, which can recombine at sites at which there are some differences compared with the wild‐ type site, generally have activity on the latter (normal) site (i.e., they have

Figure 1.5. An example of an assay for recombinase activity. Excision separates the reading frame of a marker gene (in this example green fluorescent protein, GFP) from its promoter, abolishing expression of the gene. Cells in which recombination has occurred can thus be detected (in this case, by loss of fluorescence). See text for further details.

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relaxed specificity). Therefore, some groups have developed methods to select, either simultaneously or sequentially, for activity on a variant site and loss of activity on the wild‐type site (Buchholz and Stewart, 2001; Voziyanov et al., 2002). A frequent elaboration of the mutagenesis‐selection procedure is to use “DNA shuffling,” a method that allows the generation of random combinations of a number of existing mutants (Stemmer, 1994), along with further mutagenesis; this potentially speeds up the selection process (Buchholz and Stewart, 2001; Santoro and Schultz, 2002; Sclimenti et al., 2001; Voziyanov et al., 2002, 2003). Impressive changes to the sequence specificities of Cre, Flp, and C31 Int have been achieved by these methods (see references previously cited), and specific activity at certain natural sequences in mammalian genomic DNA has been demonstrated (Buchholz and Stewart, 2001; Sclimenti et al., 2001; Thyagarajan et al., 2001). However, the strategy is not yet applicable to the targeting of sequences other than those that have been shown to be pseudosites or whose sequence is quite similar to the recombinase’s proper site. One might predict that, as more “specificity mutants” are identified, this approach would highlight the recombinase residues that are most important for sequence recognition. Further mutagenesis could then focus on these residues. Currently, it seems that many residues in Cre and Flp, along the whole length of the primary amino acid sequence, contribute to specificity. Some residues do appear as “hot spots” for mutations in the altered‐specificity variants, but as yet there is no clear pattern in the published data that would allow a more rational approach to the creation of new variants (Hartung and Kisters‐Woike, 1998; Voziyanov et al., 2003).

IX. STRUCTURE‐BASED STRATEGIES: Z‐RESOLVASES Crystallography of the serine recombinase  resolvase reveals a modular structure. The 140‐residue N‐terminal domain, which contains all residues known to be involved in catalysis, is structurally and spatially distinct from the 40‐residue C‐terminal “helix‐turn‐helix” domain that is the primary determinant of sequence‐specific DNA binding (Fig. 1.3; Abdel‐Meguid et al., 1984; Grindley, 2002; Yang and Steitz, 1995). The two domains are connected by a short linker sequence that associates with the DNA minor groove.  Resolvase and its very close relative Tn3 resolvase have been extensively studied in vitro (Grindley, 2002). The length of the tripartite recombination site (res; 114 bp), together with strong selectivities for supercoiling and pairs of sites that are in direct (“head‐to‐tail”) repeat on the same DNA molecule, renders these enzymes unsuitable for most of the envisaged applications of site‐specific recombinases. However, recent studies have led to “hyperactive” resolvase variants that

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recombine two copies of a short dimer‐binding site (the 28 bp site I of res) and no longer require supercoiling or directly repeated sites (Arnold et al., 1999; Burke et al., 2004). These variants might be put to the same kinds of uses as Cre and Flp. Mutant versions of relatives of Tn3/  resolvase have also been shown to act at simple dimer‐binding recombination sites without the need of additional factors (Johnson, 2002). Some minor alterations in sequence selectivity of this group of serine recombinases have been made by mutation, or by substituting the DNA‐binding domain with that of another member of the group (see Section VII). However, it became apparent that the sequence specificity of hyperactive resolvase variants might be more radically altered by replacing the DNA‐binding domain with one from an unrelated protein. The zinc‐finger DNA‐binding domain of the mouse transcription factor Zif268 was an especially attractive choice for this purpose. The Zif268 DNA‐binding domain is small (~90 amino acids), is monomeric in its functional form, and recognizes a short (9‐bp) sequence with high specificity. Its structure in a complex with DNA has been solved (Elrod‐Erickson et al., 1996; Pavletich and Pabo, 1991). Significantly, it is the focus of a campaign, involving a number of research groups, to create engineered zinc‐finger proteins, which can recognize any defined short DNA sequence (Beerli and Barbas, 2002; Pabo et al., 2001). Variants of the Zif268 domain that recognize some specific sequences in mammalian and viral genomes have already been created (Reynolds et al., 2003). The domain is composed of three similar zinc‐finger modules, each recognizing 3–4 bp of its 9‐bp target sequence. In each finger, only a few residues make base‐specific contacts with the DNA. It is therefore possible to simplify the task of selection for recognition of a new sequence by focusing on one finger at a time, and on the most important residues for recognition (Beerli and Barbas, 2002). The so‐called “phage display” technique has been adapted for the efficient selection of Zif268 domain variants that bind a chosen DNA sequence with very high affinity (Pabo et al., 2001), and other methods are being developed to select for high specificity (Hurt et al., 2003). These Zif268 domain variants with novel sequence specificity can act as artificial transcription factors, repressing or activating transcription from promoters close to their binding sites (Reynolds et al., 2003). They can also be used to tether an enzyme to a specific site on DNA. Novel chimeric nucleases have been created in which a nonspecific DNA cleavage domain is linked to a Zif268‐derived site‐specific DNA‐binding domain (Chandrasegaran and Smith, 1999). Cleavage mediated by the DNA‐bound chimeric enzyme occurs selectively at the chosen recognition site, and this can increase the in vivo efficiency of HR at that site (Bibikova et al., 2002, 2003; Porteus and Baltimore, 2003). Chimeric recombinases (“Z‐resolvases”) in which the catalytic domain of a hyperactive Tn3 resolvase variant is linked to the DNA‐binding domain of

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Figure 1.6. Cartoon of a Z‐site þ Z‐resolvase complex. Two 9 bp sequences (boxes with three arrows) are each recognized by a 3‐zinc‐finger DNA‐binding domain (dark grey shape) derived from that of Zif268. The 22 bp intervening sequence (hatched) interacts with the dimerized catalytic domains of a mutant version of Tn3 resolvase (light grey ovals) (see text for details). The Zif268 domain and the resolvase catalytic domain are linked by a short peptide (curved lines). The positions of strand breakage and rejoining are indicated by the staggered thin line.

Zif268 have been shown to recombine in vivo and in vitro specifically at “Z‐sites” (Akopian et al., 2003). Z‐sites consist of appropriately spaced pairs of 9‐bp sequence motifs recognized by the Zif268 domain, flanking a central sequence which is acted upon by the catalytic domains (Fig. 1.6). Each Z‐site binds a dimer of Z‐resolvase. The Z‐resolvase/Z‐site specificity was very high, and no activity on the natural target sequence of the catalytic domain (res site I) was detected. The rate of recombination was shown to depend on structural features of the Z‐resolvase and on attributes of the Z‐site sequence. The distance between the two 9‐bp motifs recognized by the Zif268 domains was critical, 22 bp being optimal. The reasons for this distance requirement are unknown as yet. The length and sequence of the peptide linking the two domains of Z‐resolvase also affected activity. In principle, any sequence of about 40 bp might be regarded as a potential Z‐site. Its ends could be recognized by variant Zif268 domains, and its central sequence could be cut and rejoined by resolvase catalytic domains. However, useable sequences might be relatively scarce. Thirteen basepairs of the original 28‐bp Tn3 res site I sequence were retained at the centre of all the Z‐sites tested so far (Fig. 1.6). Although current evidence suggests that resolvase contacts only a few basepairs of this sequence (Hatfull et al., 1988; Yang and Steitz, 1995), very different central sequences might be unsuitable. Sequences resembling that of the centre of res site I are likely to be the best targets, especially those with a central TATA motif, because these 4 bp contain the bonds that are broken and rejoined by resolvase. The 2‐bp “overlap” sequence in the TATA motif (AT) is palindromic, so a TATA‐centered Z‐site does not

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have a polarity. A substrate with two of these sites could recombine to give both excision and inversion products (see Section IV and Fig. 1.2B). Polarity can be restored by an asymmetric overlap sequence (e.g., AC), which prevents ligation of two left half‐sites or two right half‐sites (Stark et al., 1991). A pair of recombining sites must have the same overlap sequence; if not, recombination is blocked because the products would contain mismatched basepairs. Because resolvase assembles on DNA as a dimer, two Zif268 domain variants might have to be created, one to bind to each “half‐site” of a chosen genomic sequence (Fig. 1.6). In vivo targeting of a heterodimeric enzyme, with each subunit being fused to a different Zif268 domain variant, has a precedence in the studies on chimeric nucleases (see earlier section; Bibikova et al., 2003; Porteus and Baltimore, 2003). More generally, up to four Z‐resolvases, each with the same catalytic domain but a different Zif268 domain variant, could theoretically be used to recombine between two different natural Z‐sites (e.g., to excise a chosen segment of genomic DNA). However, strong recognition of just one half‐site by one subunit of a Z‐resolvase homodimer can suffice to target recombination (Akopian, A., unpublished results). Therefore, it is likely to be advantageous to use rare sequences as targets for each of the Zif268‐derived domains to minimize reactions at sequences other than the chosen Z‐site. The as‐yet hypothetical task of selecting a new site specificity for a Z‐resolvase can be split into parts, because of the enzyme’s modular nature. Zinc‐ finger domain variants that recognize the outer sequences of the Z‐site, and catalytic domains with optimal activity on the central sequence, would be created separately. Candidate recombinases targeted to the full chosen sequence would then be assembled by linking the selected domains. Enhancement of recombination activity on the chosen site could be achieved by mutagenesis‐ selection, as described in the Section VIII.

X. TARGETING TRANSPOSITION TO SPECIFIC SEQUENCES The problem of targeting transposition to specific genomic sites is somewhat different from the analogous problem for site‐specific recombination. A recombinase must be redirected from its natural site to a new target sequence, at which it would normally be inactive. In contrast, transposases act specifically at their cognate transposon ends but typically can insert them into many target sequences; the problem is to direct insertion to a chosen target site and ideally prevent insertion at any others. Whereas a “designer” site‐specific recombinase is generally intended to have activity at a single new site, transposase activity can potentially be targeted to larger regions of genomic DNA. One option is to “tether” the transposase to a specific binding site (e.g., by using an attached DNA‐binding domain), in order

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to favor transposition nearby. Alternatively, the transposase may be redesigned so as to interact with a specific DNA‐binding protein, so transposition occurs in regions of genomic DNA where the target protein is present. This strategy echoes the behavior of some natural retroviral integrases, which interact similarly with endogenous DNA‐binding proteins (Bushman, 2003; Sandmeyer, 2003). The published studies are all based on retroviral integrases, but similar approaches could be adopted for classical transposases. Fusions of HIV integrase with the DNA‐binding protein LexA (Goulaouic and Chow, 1996; Katz et al., 1996), Zif268 (Bushman and Miller, 1997), or E2C (Tan et al., 2004), and of Moloney murine leukemia virus integrase with Sp1 (Peng et al., 2002), have all been studied—the idea being that tethering the transposition intermediate to an appropriate binding site in the DNA will enhance insertion nearby. However, the results have been rather discouraging; the extent of integration near the target site was only a few times higher than that expected if target choice is random. Recent work by Voytas’ group (Zhu et al., 2003) showed how the yeast retrotransposon Ty5 is naturally targeted to heterochromatin by interaction with the protein Sir4p but will also insert close to artificially introduced ectopic Sir4p binding sites. Furthermore, the integrase protein can be redesigned so as to interact with other DNA‐binding protein partners. Target selection by some natural site‐specific DNA transposons also involves protein–protein interactions (e.g., Tn7; Craig, 2002b), but so far there have been no attempts to retarget them to new insertion sites. Despite these advances, it seems likely that competing transposon insertion at random sites will continue to be a problem until strategies that restrict transposase activity to the chosen locus are devised.

XI. GENERAL CONSIDERATIONS IN APPLICATIONS OF SITE‐SPECIFIC RECOMBINASES AND TRANSPOSASES A. Recombinase and transgene “delivery” In current applications of site‐specific recombination, DNA encoding the recombinases(s) and cognate recombination site(s) is either transfected or is preintegrated in the genome of the organism under study. Targeting of natural genomic sequences would normally still require transfection of DNA encoding the modified recombinase(s) along with any foreign sequences to be integrated. Recombinase‐based gene therapies would therefore have to surmount the same DNA delivery problems as all other gene therapies (Pfeifer and Verma, 2001). Of course, the difficulties are considerably less if transfection can be carried out

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in vitro or “ex vivo,” as would be the case for most nontherapeutic applications. Site‐specific recombination may be promoted by direct introduction of recombinase protein into cells (Baubonis and Sauer, 1993). It might even be practicable to introduce protein–DNA complexes in which the recombinase or transposase proteins required for targeted integration are already bound to the DNA (Goryshin et al., 2000). These ambitious approaches would avoid any undesirable side effects of prolonged production of recombinase from a transgenic expression construct (see Section D).

B. Chromatin Sequences that are inaccessible due to chromatin structure are likely to be poor targets for any recombinase‐based system. There is no obvious solution to this problem, although some enzymes may prove to be better than others in reaching protected sequences. Chromatin structure might also have effects on the sequence specificity of site‐specific recombinases or transposases (Portlock and Calos, 2003).

C. Reversibility Many site‐specific recombinases (e.g., Cre and Flp) promote recombination between two identical sites, so there are also two identical sites in the products. The reaction is therefore reversible (Kilby et al., 1993; Fig. 1.2B). The back reaction can be minimized if the recombinase is present only transiently (Baubonis and Sauer, 1993; Kilby et al., 1993). Some site sequences favor the forward reaction (Bouhassira et al., 1997; Hoess et al., 1986; Thomson et al., 2003), so it may be possible to choose pairs of sites for which this is the case. Certain recombinases, such as the phage integrases, recombine nonidentical sites, and their reactions can be essentially unidirectional (Groth and Calos, 2003). Similar problems might arise with DDE transposase‐mediated integration. The same transposase might promote excision of the integrated DNA. Such problems might be alleviated by strategies similar to those mentioned in Section A (e.g., transient availability of the transposase).

D. Nonspecific reactions All recombinases have the potential to cause “collateral damage” if they act at sequences other than the intended targets, and there is evidence that this happens in vivo. Cre can recombine at a number of pseudo‐loxP sites in the human and mouse genomes, which are quite divergent from the standard (“wild‐ type”) loxP sequence (Thyagarajan et al., 2000). Similar illegitimate recombination events, due to overexpression of Cre in transgenic mouse cells, can lead to

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growth inhibition, male sterility, cell‐cycle arrest, and DNA damage (Adams and van der Weyden, 2001; Loonstra et al., 2001; Schmidt et al., 2000). Such nonspecific activity would be very undesirable and potentially dangerous in gene therapy applications. The dangers of untargeted integration were tragically highlighted by two cases of leukemia in children participating in a trial for gene therapy of X‐linked severe combined immunodeficiency (X‐SCID). The therapeutic transgene was introduced ex vivo into cells that had been temporarily removed from the patients, by infection with a modified retrovirus whose integration was not targeted. Some of the retroviral integration events that ensued activated a nearby gene, resulting in oncogenesis (Kohn et al., 2003). Minimization of risk of analogous genetic damage would entail optimization of target site specificity, cell type‐specific recombinase gene delivery, cell type‐ specific promoters, and temporary recombinase expression. The tendency for retargeted recombinases to have relaxed specificity compared with their parent enzyme might restrict their applicability. Development of methods to select for restoration of high specificity is therefore most important. Of course, therapeutic modification of gene expression might be achieved in many circumstances by targeting but not altering DNA sequences (e.g., with DNA‐binding proteins or triplex‐forming oligonucleotides) (Uil et al., 2003).

XII. PROSPECTS AND CONCLUSIONS We still have a long way to go, but what will we be able to do when we have created efficient and highly specific “designer recombinases?” Perhaps the most exciting (but distant) prospects are in the field of gene therapy, where we can hope to bring about safe and efficient integration of transgenes at chosen genetic locations that support optimal expression. We might also knock out genes, or alter their expression levels, by integration of DNA sequences at suitable positions. Complete removal of a gene or other segment of DNA by recombinase‐ mediated excision will be very demanding technically because sites at both ends of the segment would have to be targeted by different versions of the recombinase. However, the task may be simpler in some special cases where an undesirable DNA segment is flanked by similar or identical sequences—the most obvious of these being the integrated proviral DNA of LTR retroviruses such as HIV. In the field of biotechnology, we can hope to improve and simplify procedures for making transgenic organisms for production of valuable proteins. Integration could be targeted to loci where gene expression will be high and only in the desired tissues (e.g., to a mammalian casein gene‐expression site, for production of proteins in milk).

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The uses of designer recombinases will not be restricted to mammals; similar strategies could be applied in any organism. In view of the concerns about introduction of transgenic DNA into food plants and wild species, the possibility that genetic changes at specific sites might be brought about by injecting designer recombinase proteins into cells, without any foreign DNA at all, may be appealing. Finally, the uses of these systems in experimental genetics, as tools for the specific genetic modification of laboratory organisms, are limited only by the scientists’ imagination.

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