A new site-specific recombinase-mediated system for targeted multiple genomic deletions employing chimeric loxP and mrpS sites

A newly designed site-specific recombination system is presented which allows multiple targeted markerless deletions. The most frequently used tool for removing selection markers or to introduce genes by recombination-mediated cassette exchange is the Cre/loxP system. Many mutant loxP sites have been created for this purpose. However, this study presents a chimeric mutant loxP site denoted mroxP-site. The mroxP site consists of one Cre (loxP/2) and one MrpA (mrpS/2) binding site separated by a palindromic 6-bp spacer sequence. Two mroxP-sites can be recombined by Cre recombinase in head-to-tail as well as in head-to-head orientation. In the head-to-head orientation and the loxP half-sites inside, Cre removes the loxP half-sites during site-specific recombination, creating a new site, mrmrP. The new site is essentially a mrpS site with a palindromic spacer and cannot be used by Cre for recombination anymore. It does, however, present a substrate for the recombinase MrpA. This new system has been successfully applied introducing multiple targeted gene deletions into the Escherichia coli genome. Similar to Cre/loxP and FLP/FRT, this system may be adapted for genetic engineering of other pro- and eukaryotes.     

A randomized library approach to identifying functional lox site domains for the Cre recombinase

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool in a number of genetic engineering processes. The Cre recombinase has been shown to act on DNA sequences that vary considerably from that of its bacteriophage recognition sequence, loxP. However, little is known about the sequence requirements for functional lox-like sequences. In this study, we have implemented a randomized library approach to identify the sequence characteristics of functional lox site domains. We created a randomized spacer library and a randomized arm library, and then tested them for recombination in vivo and in vitro. Results from the spacer library show that, while there is great plasticity, identity between spacer pairs is the most important factor influencing function, especially in in vitro reactions. The presence of one completely randomized arm in a functional loxP recombination reaction revealed that only three wild-type loxP arms are necessary for successful recombination in Cre-expressing bacteria, and that there are nucleotide preferences at the first three and last three positions of the randomized arm for the most efficiently recombined sequences. Finally, we found that in vitro Cre recombination reactions are much more stringent for evaluating which sequences can support efficient recombination compared to the 294-CRE system.     

A genetic screen identifies novel non-compatible loxP sites

The ability of the Cre/lox system to make precise genomic modifications is a tremendous accomplishment. However, recombination between cis-linked heterospecific lox sites limits the use of Cre- mediated exchange of DNA to systems where genetic selection can be applied. To circumvent this problem we carried out a genetic screen designed to identify novel mutant spacer-containing lox sites displaying enhanced incompatibility with the canonical loxP site. One of the mutant sites recovered appears to be completely stable in HEK293 cells constitutively expressing Cre recombinase and supports recombinase-mediated cassette exchange (RMCE) in bacteria and mammalian cell culture. By preventing undesirable recombination, these novel lox sites could improve the efficiency of in vivo gene transfer.     

Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM

Cre recombinase selectively recognizes DNA and prevents non-specific DNA cleavage through an orchestrated series of assembly intermediates. Cre recombines two loxP DNA sequences featuring a pair of palindromic recombinase binding elements and an asymmetric spacer region, by assembly of a tetrameric synaptic complex, cleavage of an opposing pair of strands, and formation of a Holliday junction intermediate. We used Cre and loxP variants to isolate the monomeric Cre-loxP (54 kDa), dimeric Cre₂-loxP (110 kDa), and tetrameric Cre₄-loxP₂ assembly intermediates, and determined their structures using cryo-EM to resolutions of 3.9, 4.5 and 3.2 Å, respectively. Progressive and asymmetric bending of the spacer region along the assembly pathway enables formation of increasingly intimate interfaces between Cre protomers and illuminates the structural bases of biased loxP strand cleavage order and half-the-sites activity. Application of 3D variability analysis to the tetramer data reveals constrained conformational sampling along the pathway between protomer activation and Holliday junction isomerization. These findings underscore the importance of protein and DNA flexibility in Cre-mediated site selection, controlled activation of alternating protomers, the basis for biased strand cleavage order, and recombination efficiency. Such considerations may advance development of site-specific recombinases for use in gene editing applications.     

Mutually exclusive recombination of wild-type and mutant loxP sites in vivo facilitates transposon-mediated deletions from both ends of genomic DNA in PACs

Recombination of wild-type and mutant loxP sites mediated by wild-type Cre protein was analyzed in vivo using a sensitive phage P1 transduction assay. Contrary to some earlier reports, recombination between loxP sites was found to be highly specific: a loxP site recombined in vivo only with another of identical sequence, with no crossover recombination either between a wild-type and mutant site; or between two different mutant sites tested. Mutant loxP sites of identical sequence recombined as efficiently as wild-type. The highly specific and efficient recombination of mutant loxP sites in vivo helped in developing a procedure to progressively truncate DNA from either end of large genomic inserts in P1-derived artificial chromosomes (PACs) using transposons that carry either a wild-type or mutant loxP sequence. PAC libraries of human DNA were constructed with inserts flanked by a wild-type and one of the two mutant loxP sites, and deletions from both ends generated in clones using newly constructed wild-type and mutant loxP transposons. Analysis of the results provides new insight into the very large co-integrates formed during P1 transduction of plasmids with loxP sites: a model with tri- and possibly multimeric co-integrates comprising the PAC plasmid, phage DNA, and transposon plasmid(s) as intermediates in the cell appears best to fit the data. The ability to truncate a large piece of DNA from both ends is likely to facilitate functionally mapping gene boundaries more efficiently, and make available precisely trimmed genes in their chromosomal contexts for therapeutic applications.     

Enhanced error-prone RCA mutagenesis by concatemer resolution

Error-prone rolling circle amplification (RCA) is a promising alternative to error-prone PCR for random mutagenesis. The main disadvantage of error-prone RCA is the low transformation efficiency of the DNA concatemer produced in the amplification reaction. We improved the method by introducing loxP recombination site of bacteriophage P1 Cre recombinase into the target plasmid and reducing the concatemer by Cre recombinase to plasmid-sized units, increasing the number of transformants 50-fold in non-error-prone and 13-fold in error-prone conditions. The efficiency improvement was verified by obtaining 115 ± 57 ceftazidime resistant colonies per recombined RCA reaction from randomly mutated TEM-1 β-lactamase gene library whereas only 9 ± 11 colonies were gained without recombination. Supplementation of the error-prone RCA with Cre/loxP recombination is a simple and useful tool to increase the transformable library size.     

Cre-loxP Recombination System for Large Genome Rearrangements in Lactococcus lactis

We have used a new genetic strategy based on the Cre-loxP recombination system to generate large chromosomal rearrangements in Lactococcus lactis. Two loxP sites were sequentially integrated in inverse order into the chromosome either at random locations by transposition or at fixed points by homologous recombination. The recombination between the two chromosomal loxP sites was highly efficient (approximately 1 × 10⁻¹/cell) when the Cre recombinase was provided in trans, and parental- or inverted-type chromosomal structures were isolated after removal of the Cre recombinase. The usefulness of this approach was demonstrated by creating three large inversions of 500, 1,115, and 1,160 kb in size that modified the lactococcal genome organization to different extents. The Cre-loxP recombination system described can potentially be used for other gram-positive bacteria without further modification.     

Unidirectional Cre-mediated genetic inversion in mice using the mutant loxP pair lox66/lox71

The Cre/loxP recombination system is a commonly used tool to alter the mouse genome in a conditional manner by deletion or inversion of loxP-flanked DNA segments. While Cre-mediated deletion is essentially unidirectional, inversion is reversible and therefore does not allow the stable alteration of gene function in cells that constitutively express Cre. Site-directed mutagenesis yielded a pair of asymmetric loxP sites (lox66 and lox71) that display a favorable forward reaction equilibrium. Here, we demonstrate that lox66/lox71 mediates efficient and predominantly unidirectional inversion of a switch substrate targeted to the mouse genome in combination with either inducible or cell type-specific cre-transgenes in vivo.     

Replacing the wild type <Emphasis Type="Italic">loxP</Emphasis> site in BACs from the public domain with <Emphasis Type="Italic">lox66</Emphasis> using a <Emphasis Type="Italic">lox66</Emphasis> transposon

Background

Chromatin adjoining the site of integration of a transgene affects expression and renders comparisons of closely related transgenes, such as those derived from a BAC deletion series retrofitted with enhancer-traps, unreliable. Gene targeting to a pre-determined site on the chromosome is likely to alleviate the problem.

Findings

A general procedure to replace the loxP site located at one end of genomic DNA inserts in BACs with lox66 is described. Truncating insert DNA from the loxP end with a Tn10 transposon carrying a lox66 site simultaneously substitutes the loxP with a lox66 sequence. The replacement occurs with high stringency, and the procedure should be applicable to all BACs in the public domain. Cre recombination of loxP with lox66 or lox71 was found to be as efficient as another loxP site during phage P1 transduction of small plasmids containing those sites. However the end-deletion of insert DNA in BACs using a lox66 transposon occurred at no more than 20% the efficiency observed with a loxP transposon. Differences in the ability of Cre protein available at different stages of the P1 life cycle to recombine identical versus non-identical lox-sites is likely responsible for this discrepancy. A possible mechanism to explain these findings is discussed.

Conclusions

The loxP/lox66 replacement procedure should allow targeting BACs to a pre-positioned lox71 site in zebrafish chromosomes; a system where homologous recombination-mediated "knock-in" technology is unavailable.

     

Reactivation of an integrated disabled viral vector using a Cre-loxP recombination system in Arabidopsis thaliana

Summary We developed an inactivated DNA replicon of Turnip Mosaic Virus (TuMV), which was reactivated by a recombination event based on the Cre-loxP system. Viral replication was prevented by the insertion of a translation terminator sequence flanked by two loxP sites at the junction of the P1–HCPro-coding genes. In vitro recombination was tested with purified Cre, which excised the floxed sequence from the TuMV DNA, leaving a single loxP site in the reactivated viral genome, and restored the open reading frame of the replicon. Arabidopsis thaliana plants were made transgenic for the inactivated TuMV replicon. Removal of the translation terminator sequence was achieved by the controlled expression of Cre. Delivery of the Cre recombinase to the transgenic plants was obtained by three methods: agroinfiltration, PVX-based production, or transgenic chemical-inducible expression. In each case, reactivation of TuMV replication was observed.     

Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination

The Cre recombinase mediates precise site-specific recombination between a pair of loxP sequences through an intermediate containing Holiday junction. The recombination junction in the loxP sequence is located within the asymmetric 8-nucleotide spacer region. To examine the role of each nucleotide sequence of the spacer region in the recombination process, we synthesized a complete set of 24 loxP spacer mutants with single-base substitutions and 30 loxP spacer mutants with double-base substitutions. Each synthesized loxP mutant was ligated at both ends of a linear DNA or to one end of a DNA-containing wild-type loxP at the other end and their recombination efficiencies were analyzed with an in vitro system. The sequence identity of the right two nucleotides and left four nucleotides in the central six bases of the spacer region was found to be essential for formation and resolution, respectively, of the intermediate product. Furthermore, even when homology was maintained, the recombination efficiencies were lower than that of wild-type loxP and varied among mutants. Based on this knowledge, we identified two loxP mutants with double-base substitutions, mutants 5171 and 2272, which recombine efficiently with an identical mutant but not with the other mutant or wild-type loxP.     

DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages

Sequencing of the 7 kb immC region from four P1-related phages identified a novel DNA recombinase that exhibits many Cre-like characteristics, including recombination in mammalian cells, but which has a distinctly different DNA specificity. DNA sequence comparison to the P1 immC region showed that all phages had related DNA terminase, C1 repressor and DNA recombinase genes. Although these genes from phages P7, ϕw39 and p15B were highly similar to those from P1, those of phage D6 showed significant divergence. Moreover, the D6 sequence showed evidence of DNA deletion and substitution in this region relative to the other phages. Characterization of the D6 site-specific DNA recombinase (Dre) showed that it was a tyrosine recombinase closely related to the P1 Cre recombinase, but that it had a distinct DNA specificity for a 32 bp DNA site (rox). Cre and Dre are heterospecific: Cre did not catalyze recombination at rox sites and Dre did not catalyze recombination at lox sites. Like Cre, Dre catalyzed both integrative and excisive recombination and required no other phage-encoded proteins for recombination. Dre-mediated recombination in mammalian cells showed that, like Cre, no host bacterial proteins are required for efficient Dre-mediated site-specific DNA recombination.     

Extent of sequence homology required for bacteriophage lambda site-specific recombination

Bacteriophage lambda integration and exicision occur by reciprocal recombination within a 15-base homologous core region present in the recombining attachment (att) sites. Strand exchange within the core occurs at precise nucleotide positions, which define an overlap region in which the products of recombination contain DNA strands derived from different parents. In order to define the role of sequence homology during recombination we have constructed point mutations within the core and assayed their effects in vivo and in vitro on site-specific recombination. Two of the mutations are located at position ?3 of the core, which is one base-pair outside of the overlap region where strand exchange occurs. These mutations do not affect integrative or excisive recombination, thereby suggesting that homology outside the overlap region is not required for recombination. Two other mutations are located at position ?2 of the core, which is one base-pair within the overlap region. These mutations show severely depressed integrative and excisive recombination activities in vitro and in vivo when recombined against wild-type att sites. However, the ?2 mutations show normal recombination activity when recombined against att sites containing the homologous mutation, thereby suggesting that homology-dependent DNA interactions are required within the overlap region for effective recombination. In vitro recombination between homoduplex attP sites and heteroduplex attB sites demonstrated that the DNA interactions require only one strand of the attB overlap region to be homologous to attP in order to promote recombination.     

DNA Substrates Influence the Recombination Efficiency Mediated by FLP Recombinase Expressed in Mammalian Cells

The FLP recombinase derived from Saccharomyces cerevisiae mediates precise site‐specific recombination between a pair of FLP recognition targets (FRTs). Like the Cre/loxP system derived from bacteriophage P1, the FLP/FRT system has recently been applied to gene regulation systems using an FLP‐expressing recombinant adenovirus (rAd) (Nakano et al, Nucleic Acids Res. 29: e40, 2001). In an attempt to improve the FLP/FRT system by altering its DNA substrates, we compared the recombination efficiency among different substrates by a quantitative in vitro assay using FLP expressed in mammalian cells. Unexpectedly, we found that one linearized DNA substrate showed 4‐ to > 20‐fold lower recombination efficiency than other substrates, which phenomenon has not been observed in the Cre/loxP system. The quantitative in vitro assay using truncated DNA substrates suggested that the recombination efficiency seemed to be influenced not only by the linearized position of the substrate, but also by the length between a pair of FRTs. Such substrate preference of FLP expressed in mammalian cells should probably be noted when designing versatile applications of the FLP/FRT system as a gene regulation system in mammalian systems. Fortunately, however, we demonstrated that no substrate preference was observed when using a particular substrate (pCAFNF5) and the preference was reduced when using a certain pair of mutant FRTs (f72), which will also be a promising tool for simultaneous gene regulation in combination with wild‐type FRT.     

A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent

Conditional gene inactivation using the Cre/loxP system is widely used, but the difficulty in properly regulating Cre expression remains one of the bottlenecks. One approach to regulate Cre activity utilizes a mutant estrogen hormone-binding domain (ER^T) to keep Cre inactive unless the non-steroidal estrogen analog 4-hydroxytamoxifen (OHT) is present. Here we describe a mouse strain expressing Cre-ER^T from the ubiquitously expressed ROSA26 (R26) locus. We demonstrate efficient temporal and spatial regulation of Cre recombination in vivo and in primary cells derived from these mice. We show the existence of marked differences in recombination frequencies between different substrates within the same cell. This has important consequences when concurrent switching of multiple alleles within the same cell is needed, and highlights one of the difficulties that may be encountered when using reporter mice as indicator strains.     

Cre-loxP-mediated gene replacement: a mouse strain producing humanized antibodies

Background: The bacteriophage-derived Cre–loxP recombination system operates efficiently in mammalian cells. This system is particularly useful in gene-targeting experiments in the mouse, and has already been used to generate ‘clean’ deletions of target genes in the germ line, as well as to inactivate target genes in a conditional manner (based on regulated expression of the Cre recombinase). In principle, Cre–loxP-mediated recombination should also allow gene replacement, and thus the introduction of virtually any kind of mutation into the genome.Results We used the Cre–loxP system, in mouse embryonic stem cells, to replace the mouse gene Cγ 1, which encodes the constant region of the heavy chain of IgG1 antibodies, with its human counterpart. The mutation was transmitted through the mouse germ line, and the resulting mutant mice were crossed with mice expressing κ light chains with a human, instead of a mouse, constant region. Mice homozygous for both mutations produce humanized, κ-chain-bearing IgG1 antibodies at the same level and efficiency as wild-type mice produce murine IgG1 antibodies. These animals should enable the ex vivo production of humanized, chimeric monoclonal antibodies specific for any antigen to which the mouse can respond.Conclusion Cre–loxP-mediated gene replacement is a simple and efficient general method of targeted mutagenesis in the mouse.     

Modulation of the active complex assembly and turnover rate by protein-DNA interactions in Cre-LoxP recombination

Cre promotes recombination at the 34 bp LoxP sequence. Substitution of a critical C-G base pair in LoxP with an A-T base pair, to give LoxAT, reduced Cre binding in vitro and abolished recombination in vivo [Hartung, M., and Kisters-Woike, B. (1998) J. Biol. Chem. 273, 22884-22891].We demonstrated that LoxAT can be recombined in vitro. However, Cre discriminates against this substrate both before and after DNA binding. The preference for LoxP over LoxAT is the result of reduced binding and a slower turnover rate, amplified by changes in cooperativity of complex assembly. With LoxAT, similar levels of substrate turnover required 2-2.5-fold higher protein-DNA concentrations compared to LoxP, but the sigmoidal behavior of the concentration dependence was more pronounced. Further, the Cre-LoxAT complexes reacted 4-5-fold more slowly. In the 2.3 A resolution Cre-LoxAT complex structure, the major groove Arg259-guanine interaction was disrupted, explaining the reduced binding. Overall structural shifts and mobility changes indicate more favorable interactions between subunits, providing a hypothesis for the reduced turnover rate. Concomitant with the displacement of Arg259 from the DNA, adjacent charged residues Glu262 and Glu266 shifted to form salt bridges with the Arg259 guanidinium moiety. Substitution of Glu262 and Glu266 with glutamine increased Cre complex assembly efficiency and reaction rates with both LoxAT and LoxP, but diminished Cre's ability to distinguish them. The increased rate of this variant suggests that DNA substrate binding and turnover are coupled. The improved efficiency, made at some expense of sequence discrimination, may be useful for enhancing recombination in vivo.