WO2000060091A2 - Methode de selection de variantes de recombinase a specificite modifiee - Google Patents

Methode de selection de variantes de recombinase a specificite modifiee Download PDF

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WO2000060091A2
WO2000060091A2 PCT/US2000/009154 US0009154W WO0060091A2 WO 2000060091 A2 WO2000060091 A2 WO 2000060091A2 US 0009154 W US0009154 W US 0009154W WO 0060091 A2 WO0060091 A2 WO 0060091A2
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recombination
sites
recombination sites
variant
dna
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WO2000060091A3 (fr
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Brian Lee Sauer
Andreas Walter Rufer
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Oklahoma Medical Research Foundation
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Priority to EP00921779A priority patent/EP1169458A2/fr
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Publication of WO2000060091A2 publication Critical patent/WO2000060091A2/fr
Publication of WO2000060091A3 publication Critical patent/WO2000060091A3/fr

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • recombinases mediate the site-specific recombination of DNA. These recombinases were first identified in phage that integrate into host chromosomes. Such integration allows the phage to remain latent in the cell as a prophage.
  • Cre sites-specific recombinases catalyze conservative DNA rearrangements at specific target sequences.
  • Cre acts on a 34 bp sequence located on both ends of the linear PI genome, that is called lox? (locus of crossover of PI; Sternberg and Hamilton, J. Mol. Biol, 150:467-486 (1981)), lox? consists of two 13 bp inverted repeats flanking a non-palindromic 8 bp core that defines the assigned direction of the sequence (as shown on the upper part of Figure 1). Depending on this direction, recombination catalyzed by Cre leads to excision of insertion of DNA flanked by lox? sites orientated in the same direction (indicated by lox? ), but leads to inversion when oriented in the opposite direction ( Figure 2).
  • Cre-recombination involves the following four events: (i) DNA binding, (ii) synapsis (as defined below), (iii) cleavage, and (iv) strand exchange.
  • mutants defective for each step have been isolated using several screening procedures (Wierzbicki et al., J. Mol. Biol. , 195 :785-794 ( 1987)).
  • crystal structure of Cre complexed with an artificial suicide substrate has been recently resolved, providing additional insights into site-specific recombination (Guo et al., Nature, 389:40-46 (1997)).
  • this first strand exchange is asymmetric, since the bottom strand ( Figure 1) is always exchanged first (Hoess et al., Proc. Natl. Acad. Sci. USA, 84:6840-6844 (1987)).
  • the second strand is exchanged and Cre released from its substrate.
  • Cre assisted site-specific recombination has become an important tool for efficient, specific, and conditional manipulations of eukaryotic genomes (Lakso et al., Proc. Natl. Acad. Sci.
  • Cre-related technologies that include the following: (/) lox sites need to be introduced by homologous recombination at the desired region into the genome before Cre can be used, (ii) the frequency of correct site-specific recombination due to Cre expression is not 100%, and consequently, (iii) selectable markers are necessary in most strategies involving Cre for genome manipulation in higher eukaryotes. These markers, e.g. neo or TK, may introduce problems in subsequent studies, particular in those related to animal development. The number of available selectable markers that can be used in limited also. Additional site-specific recombinases that also function efficiently in eukaryotic systems, but recognize different sites from lox would be helpful. Similar inconveniences limit the usefulness of other recombinases.
  • the method involves producing mutant recombinases and testing the mutant recombinases with specially designed constructs.
  • the constructs contain variant recombination sites that are not recognized by non-mutant recombinase but will undergo recombination in the presence of a mutant recombinase with altered specificity.
  • Recombination at the variant recombination sites can be monitored or detected by any suitable means. It is preferred that recombination is detected by screening or selection based on the expression or lack of expression of a reporter gene.
  • constructs containing a reporter gene associated with the variant recombination sites such that the reporter gene is rearranged or deleted, or a spacer sequence interrupting the reporter gene is rearranged or deleted, as a result of recombination at the recombination sites. Recombination of such constructs will result in a loss of expression of the reporter gene, where the construct contained a functional reporter gene, or in a gain in expression of the reporter gene, where the construct contained a non-functional reporter gene.
  • the disclosed method also involves determining whether a variant recombinase retains the ability to mediate recombination at recombination sites recognized by non-variant recombinase. This can be accomplished by using constructs containing recombination sites recognized by non-variant recombinase. Recombination at these recombination sites can be monitored or detected by any suitable means. It is preferred that recombination is detected by screening or selection based on the expression or lack of expression of a reporter gene.
  • constructs containing a reporter gene associated with the recombination sites recognized by non-variant recombinase such that the reporter gene is rearranged or deleted, or a spacer sequence interrupting the reporter gene is rearranged or deleted, as a result of recombination at the recombination sites. Recombination of such constructs will result in a loss of expression of the reporter gene, where the construct contained a functional reporter gene, or in a gain in expression of the reporter gene, where the construct contained a non-functional reporter gene.
  • the first reporter gene can be associated with the variant recombination sites and the second reporter gene can be associated with recombination sites recognized by non-variant recombinase.
  • Recombination between two recombination sites requires (1) that the recombinase recognize the sites as recombination sites, and (2) that the sequences of the two sites is sufficiently similar. It has been discovered that recombination between two recombination sites (both of which are recognized by a recombinase) can be substantially reduced or prevented by using different compatibility sequences for the recombination sites (the recognition sequences can also differ if the recombinase can recognize different sequences). Thus, it is also preferred that the variant recombination sites be made incompatible with the recombination sites recognized by non-variant recombinase by using different compatibility sequences for the two sets of recombination sites.
  • Compatibility sequences in a recombination site are those sequences in the recombination site, other than the sequences required for recognition of the site by the recombinase, that must be similar in a pair of recombination sites for recombination to occur between them.
  • Many recombination sites contain repeats of a characteristic sequence separated by spacer sequences. In such recombination sites, the spacer sequences are generally compatibility sequences and the repeats (or parts of the repeats) are recognition sequences.
  • Recombinases require specific recognition sequences but allow wide variation in compatibility sequences. Thus, recombination sites that are recognized by a given recombinase but are incompatible with each other can be readily designed using the disclosed principles.
  • variant recombinases made or identified by the disclosed method that have broadened specificity for the site of recombination.
  • the disclosed variants mediate recombination between sequences other than recombination sites on which the wild type recombinase is active.
  • the disclosed recombinase variants can mediate efficient recombination between recombination sites that wild type recombinase can act on (referred to as wild type recombination sites), between variant recombination sites not efficiently utilized by wild type recombinase (referred to as variant recombination sites), and between a wild type recombination site and a variant recombination site.
  • the disclosed variant recombinases can be used in any method or technique where wild type recombinases can be used.
  • the disclosed variant recombinases allow different alternative recombinations to be performed since the variant recombinases can allow much more efficient recombination between wild type recombination sites and variant recombination sites. Control of such alternative recombination can be used to accomplish more sophisticated sequential recombinations to achieve results not possible with wild type recombinases.
  • the disclosed variant recombinases also allow recombination at specific genomic sites without the need to first introduce a recombination site.
  • variants of Cre recombinase that have broadened specificity for the site of recombination.
  • the disclosed variants mediate recombination between sequences other than the loxP sequence and other lox site sequences on which wild type Cre recombinase is active.
  • the disclosed Cre variants mediate efficient recombination between lox sites that wild type Cre can act on (referred to as wild type lox sites), between variant lox sites not efficiently utilized by wild type Cre (referred to as variant lox sites), and between a wild type lox site and a variant lox site.
  • methods of recombining nucleic acids using the disclosed Cre variants are also disclosed.
  • the disclosed Cre variants can be used in any method or technique where Cre recombinase (or other, similar recombinases such as FLP) can be used.
  • the disclosed Cre variants allow different alternative recombinations to be performed since the Cre variants allow much more efficient recombination between wild type lox sites and variant lox sites. Control of such alternative recombination can be used to accomplish more sophisticated sequential recombinations to achieve results not possible with wild type Cre recombinase.
  • Figure 1 is a comparison of three different lox sites.
  • loxP is the original recombination site for Cre recombinase.
  • loxKl and loxK2 are variant lox sites.
  • Figure 2 is a diagram of two different forms of construct and the resulting recombination products.
  • Figure 3 is a diagram of an example of a random mutagenesis using DNA shuffling.
  • Figure 4 is a diagram of the selection plasmid for loxK2 recombination, pBS584. Recombination of two loxK2 sites by a potent Cre mutant will result in the excision o EGFP and the transcriptional terminator rrnBT ⁇ T ⁇ . Subsequently, neo transcription can take place, rendering E. coli resistant to kanamycin. Note that the promoter (pRSV) even though of eukaryotic origin was shown to be functional in E. coli (Antonucci et al., J. Biol. Chem., 264:17656-17659 (1989)).
  • Figure 5 depicts gels of nucleic acid fragments and PCR products generated during the DNA shuffling process.
  • Figure 6 is a diagram of plasmid pBAD33 used for expression of mutant cre pools.
  • Figure 7 is a diagram of the construction of selection plasmids pBS568 and pBS569.
  • Figure 8 is a diagram of the construction of selection plasmids pBS583 and pBS584.
  • Figure 9 is a diagram of control plasmid pBS613.
  • Figure 10 is a diagram of the construction of screening plasmids pBS601 and pBS602.
  • Figure 11 is a diagram of examples of basic types of constructs useful in the disclosed method. These types of constructs are: (1) interrupted constructs where the gene is interrupted by a nucleic acid segment (which is flanked by recombination sites) that is deleted during recombination, (2) flanked constructs where the gene as a unit is flanked by recombination sites and the gene is deleted by recombination, and (3) inverted constructs where a portion of the gene is on an inverted nucleic acid segment and recombination causes the segment to invert and reconstitute the intact gene. The type of recombination is indicated in parentheses.
  • Figures 12 A, 12B, and 12C are diagrams of examples of constructs and their expected recombination when used in the disclosed method.
  • Figure 12A shows examples of deletion constructs (flanked and interrupted).
  • Figure 12B shows examples of inverted constructs.
  • Figure 12C shows examples of constructs that combine through recombination to reconstitute an intact gene.
  • Figure 13 is a diagram showing the identified amino-acid changes in the six selected Cre mutants are listed according to their position in the protein's secondary structure (silent mutations in parenthesis). Only one amino acid change, E262G, is common to all mutants with remarkably increased /oxK2 activity (R3M1, 2, 3, 5, and 6), suggesting that this mutation is essential for the observed phenotype.
  • Figure 14A is a table comparing recombination frequencies in vivo obtained with a variety of lox sites altered at positions 11 and 12.
  • Figure 14B is a table comparing recombination frequencies in vivo obtained with identical and mixed lox sites. Wild type Cre and five different mutant enzymes were tested for their performance on different o " substrates, as indicated. Given are the obtained percentages of recombination in vivo based on the described negative selection.
  • Figure 15 is a table comparing recombination frequencies in vitro obtained with a variety of lox sites altered at positions 11 and 12.
  • Figure 16 is a graph of percent of various Cre recombinases (wt, G, GA, GN, GS, R3M3) bound to various lox sites (loxP, loxK2, loxKl).
  • Figure 17 shows wildtype and target FRT sites.
  • Figure 18 shows the strategy for selection of altered specificity FLP mutants.
  • Figure 19 shows an alternate target mutant FRT site.
  • the design and rationale for design of the target mutant FRT site is as described in Figure 17, but the mutant FRT-M2 site differs from FRT-M by carrying a different mutational alteration in the repeat elements.
  • the method involves producing mutant recombinases and testing the mutant recombinases with specially designed constructs.
  • the constructs contain variant recombination sites that are not recognized by non-mutant recombinase but will undergo recombination in the presence of a mutant recombinase with altered specificity.
  • the disclosed method also involves determining whether a variant recombinase retains the ability to mediate recombination at recombination sites recognized by non- variant recombinase.
  • variant recombinases When variant recombinases are tested for activity on both variant recombination sites and recombination sites recognized by non-variant recombinase in the same system or at the same time, it is preferred that two different reporter genes which can be separately detected or monitored be used.
  • a first reporter gene can be associated with the variant recombination sites and a second reporter gene can be associated with recombination sites recognized by non-variant recombinase.
  • the variant recombination sites be made incompatible with the recombination sites recognized by non-variant recombinase by using different compatibility sequences for the two sets of recombination sites. This allows separate assessment of the ability of a variant recombinase to mediate recombination between variant recombination sites and recombination sites recognized by non- variant recombinase.
  • variant recombinases made or identified by the disclosed method that have broadened specificity for the site of recombination. Also disclosed are methods of recombining nucleic acids using the disclosed variant recombinases.
  • the disclosed variant recombinases can be used in any method or technique where wild type recombinases can be used.
  • the disclosed variant recombinases allow different alternative recombinations to be performed since the variant recombinases can allow much more efficient recombination between wild type recombination sites and variant recombination sites. Control of such alternative recombination can be used to accomplish more sophisticated sequential recombinations to achieve results not possible with wild type recombinases.
  • variants of Cre recombinase that have broadened specificity for the site of recombination.
  • the disclosed variants mediate recombination between sequences other than the loxP sequence and other lox site sequences on which wild type Cre recombinase is active.
  • Preferred forms of the disclosed Cre variants have the amino acid sequence SEQ ID NO: 1 (top sequence, Table 11) altered by one or more amino acid substitutions, deletions, or insertions, where the glutamic acid at amino acid 262 has been substituted with an amino acid other than glutamic acid, and where the Cre variant recognizes (that is, mediates recombination at) a variant lox recombination site.
  • Useful Cre variants include proteins that recognize a variant lox recombination site and have the amino acid sequence SEQ ID NO:l altered by substitution of the glutamic acid at amino acid 262 with an amino acid other than glutamic acid and one or more of the following amino acid substitutions: isoleucine at amino acid 16, alanine at amino acid 29, glutamine at amino acid 101, glycine at amino acid 138, asparagine at amino acid 189, serine at amino acid 198, glutamine at amino acid 220, glutamine at amino acid 223, isoleucine at amino acid 227, glycine at amino acid 254, arginine at amino acid 255, glutamine at amino acid 284, leucine at amino acid 307, and serine at amino acid 316.
  • Preferred amino acid substitutions at amino acid position 262 include alanine, tryptophan, or glycine.
  • Examples of preferred Cre variants include proteins having the amino acid sequence SEQ ID NOT altered by substitutions E262G and D189N; proteins having the amino acid sequence SEQ ID NO:l altered by substitutions E262G and T316S; proteins having the amino acid sequence SEQ ID NOT altered by substitutions E262G and D29A; proteins having the amino acid sequence SEQ ID NOT altered by substitutions E262G, V16I, D189N, G198S, R223Q, Q255R, and P307L; proteins having the amino acid sequence SEQ ID NOT altered by substitution E262G; proteins having the amino acid sequence SEQ ID NOT altered by substitution E262A; and proteins having the amino acid sequence SEQ ID NOT altered by substitution E262W.
  • the disclosed Cre variants recognize variant lox recombination sites.
  • Preferred variant lox sites are variant lox sites recognized by the Cre variant but not recognized by wild type Cre.
  • Examples of useful variant lox sites include sites having two 13 base pair inverted repeats flanking 8 base pairs, where one of the inverted repeats has the sequence NNNACNNCGTATA (SEQ ID NO:2): sites having the sequence N*N 2 N 3 ACN 4 N 5 CGTATANNNNNNNNTATA CGN 5 'N 4 'GTN 3 'N 2 , N,' (SEQ ID NO:3), where N , N 2 ', N 3 ', N 4 ', and N 5 ' are complementary to Nj, N , N 3 , N 4 .
  • N 5 sites having the sequence N,N 2 N 3 ACN 4 N 5 CGTATANNNNNNTATACGN 5 , N 4 , GN 3 , N 2 'N 1 ' (SEQ ID NO:3), where N 4 N 5 are AA. TC, GT, TG, GG, or CC; and sites having the sequence GATACAACGTATATACCTTTCTATACGTTGTAT (SEQ ID NO:4).
  • DNA sequences comprising first and second lox sites are introduced into cells and contacted with a Cre variant, thereby producing recombination at the lox sites.
  • the location and orientation of the lox sites determines the nature of the recombination.
  • the expression "site-specific recombination” refers to three different types of recombination events:
  • Nucleic acid segment refers to a linear segment of single- or double- stranded nucleic acid, which can be derived from any source. The segment may be a fragment consisting of the segment or a segment within a larger nucleic acid fragment or molecule.
  • the expression "nucleic acid in eukaryotic cells” includes all nucleic acid present in eukaryotic cells.
  • the expression “nucleic acid in yeast” includes all nucleic acid present in yeast cells.
  • DNA segment refers to a linear segment of single- or double-stranded deoxyribonucleic acid (DNA), which can be derived from any source.
  • DNA in eukaryotic cells includes all DNA present in eukaryotic cells.
  • DNA in yeast includes all DNA present in yeast cells.
  • a “gene” is intended to mean a DNA segment which is normally regarded as a gene by those skilled in the art.
  • regulatory molecule refers to a polymer of ribonucleic acid (RNA) or a polypeptide which is capable of enhancing or inhibiting expression of a gene.
  • Regulatory nucleotide sequence refers to a nucleotide sequence located proximate to a gene whose transcription is controlled by the regulatory nucleotide sequence in conjunction with the gene expression apparatus of the cell. Generally, the regulatory nucleotide sequence is located 5' to the gene.
  • the expression “nucleotide sequence” refers to a polymer of DNA or RNA, which can be single- or double-stranded, optionally containing synthetic, non-natural, or altered nucleotides capable of incorporation into DNA or RNA polymers.
  • a “regulatory nucleotide sequence” can include a promoter region, as that term is conventionally employed by those skilled in the art.
  • a promoter region can include an association region recognized by an RNA polymerase, one or more regions which control the effectiveness of transcription initiation in response to physiological conditions, and a transcription initiation sequence.
  • Gene product refers to a polypeptide resulting from transcription, translation, and, optionally, post-translational processing of a selected DNA segment.
  • Recombinases suitable for use in the disclosed method include any enzyme that mediates recombination at specific sites. This includes enzymes identified as recombinases as well as other enzymes that function to produce recombination such as integrases and resolvases. As used herein, recombination at specific sites does not refer only to recombination at completely defined sequences. Rather, a recombinase is considered to mediate recombination at specific sites when the sites of recombination are limited in some way by sequence.
  • wild type Cre recombinase mediates recombination between sites having the sequence N,N 2 N 3 ACTTCGTATANNNNNNNNT ATACGAAGTN 3 'N 2 'N* ⁇ which includes both specific and non-specific sequences.
  • the sequences ACTTCGTATA and TATACGAAGT are recognized by the Cre recombinase.
  • the nonspecific sequences positions with "N" in the recognition sequence, although not limited in sequence, must be compatible with the non-specific sequences of the partner recombination site in order for recombination to be efficient.
  • the recombination sites need not have any particular number of specific nucleotides.
  • recombinases that can be used in the disclosed method include Cre recombinase, FLP recombinase, Beta recombinase of pSM19035 (Diaz et al., J Biol Chem 21 A: 6634-6640 (1999)), Int recombinases (Nunes- Doby et al., Nucleic Acids Res. 26:391-406 (1998)), and resolvases (Hallet et al., FEMS Microbiol Rev. 21 : 157-178 (1997); Oram et al., Curr Biol. 5: 1106- 1109 (1995); Mondragon, Structure 3: 755-758 (1995)).
  • Cre recombinase Cre recombinase
  • FLP recombinase FLP recombinase
  • Beta recombinase of pSM19035 Diaz et al., J Biol Chem 21 A: 6634-6640 (1999
  • Recombination sites are locations within a nucleic acid where recombination mediated by a recombinase takes place.
  • Recombination sites generally include specific sequences, referred to as recognition sequences, through which the recombinase recognizes a given nucleotide sequence as a recombination site.
  • Recognition sequences through which the recombinase recognizes a given nucleotide sequence as a recombination site.
  • Different recombinases generally recognize different recognition sequences. Recombination between two recombination sites requires (1) that the recombinase recognize the sites as recombination sites, and (2) that the sequences of the two sites are sufficiently similar.
  • recombination between two recombination sites can be substantially reduced or prevented by using different compatibility sequences for the recombination sites (the recognition sequences can also differ if the recombinase can recognize different sequences).
  • the variant recombination sites be made incompatible with the recombination sites recognized by non- variant recombinase by using different compatibility sequences for the two sets of recombination sites.
  • Compatibility sequences in a recombination site are those sequences in the recombination site, other than the sequences required for recognition of the site by the recombinase, that must be similar in a pair of recombination sites for recombination to occur between them.
  • recombinases require specific recognition sequences but allow wide variation in compatibility sequences.
  • recombination sites that are recognized by a given recombinase but are incompatible with each other can be readily designed using the disclosed principles.
  • a given base position in the recombination site is a recognition sequence base or a compatibility sequence base may depend on other sequences in the recombination site.
  • a particular base may function as a compatibility sequence base in a recombination site having one sequence while the same base may function as a recognition sequence base in a recombination site having a different sequence.
  • recognition sequences and compatibility sequences do not necessarily occur in blocks within a recombination site. That is, recognition sequence base and compatibility sequence bases may be interspersed in a given recombination site.
  • the disclosed variant recombination sites and the variant recombinases that can act on them allow more freedom in the selection of sites of recombination.
  • the disclosed variant recombinases can allow amino acid changes in a protein of interest while retaining the ability to recombine at a given site.
  • Recognition sequences are regions within a recombination site that must have a specific sequence, or defined range of sequences, for the cognate recombinase to recognize the recombination site. Recognition sequences in a recombination site need not be contiguous. Thus, required nucleotides dispersed in a recombination site are collectively considered recognition sequences. Nucleic acid segments can be said to have a defined range of sequences when every nucleotide position in the nucleic acid segment(s) is limited to one, two, or three nucleotide bases. That is, so long as a nucleotide position cannot have one of the possible nucleotide bases, that position has a defined range of sequence.
  • a nucleotide sequence ATRVB YGC has a defined range of sequences since each nucleotide position has at least one limitation. Standard nomenclature for nucleic acid sequences is used herein.
  • R represents A or G
  • V represents A, C, or G
  • B represents C, G, or T
  • Y represents C or T.
  • Recognition sequences for recombinases are known or can be determined through routine analysis. In general, recognition sequences can be determined by varying the sequence of recombination sites and determining if recombination between the sites can still occur. For this purpose, the pair of sites to be recombined should be identical. That is, the same sequence changes should be made to both sites. This eliminates any incompatibility effect between the recombination sites. If recombination is eliminated or significantly reduced when a specific nucleotide is changed, then that nucleotide is required for recognition of the recombination site by the recombinase.
  • Compatibility sequences are regions in a recombination site that must be similar in a pair of recombination sites for recombination to occur between them. In general, the sequence of recombination sites must be similar for recombination to occur between them. Examples of compatibility sequences are spacer sequences between repeats in recombination sequences. All or some of the nucleotides in the recognition sequences for a recombination site may be involved in compatibility. For example, where some degeneracy of the recognition sequences is allowed, similar recognition sequences may be required in a pair of recombination sites for recombination to occur between them.
  • compatibility between recombination sites can be affected by using different sequences in the compatibility sequences other than the sequences required for recognition of the site by the recombinase (that is, recognition sequences), compatibility sequences that are part of the recognition sequences, or both. It is preferred that compatibility between recombination sites be altered by using different sequences in the compatibility sequences other than the sequences required for recognition of the site by the recombinase.
  • Compatibility sequences for many recombinases are known or can be determined through routine analysis.
  • compatibility sequences can be easily determined by varying the sequence of recombination sites and determining if recombination between the sites can still occur.
  • only one of the sites in the pair of sites to be recombined should be altered. That is, the same sequence changes should not be made to both sites. This isolates incompatibility effect between the recombination sites. Further, only those nucleotide positions that are not a part of the recognition sequence of the site should be altered to avoid recognition problems.
  • nucleotide is required for compatibility of the recombination site. Examples of dissection of the critical sequences in recombination sites of recombinases are described by Hoess et al., Nucleic Acids Res.
  • Cre recombination sites Recognition and compatibility sequences can be further understood using Cre recombination sites as an example. Wild type Cre recombinase mediates recombination between sites having the sequence N ⁇ N 2 N 3 ACTTCGTATANN NNNNNNTATACGAAGTN 3 'N 2 'N , which includes both specific and non-specific sequences (that is, recognition sequences and compatibility sequences, respectively).
  • the sequences ACTTCGTATA and TATACGAAGT (an inverted repeat of the first sequence) are recognized by the Cre recombinase and are the recognition sequences in Cre recombinase sites.
  • Variant Cre recombinases recognize sites having different recognition sequences.
  • the non-specific sequences (positions with "N" in the recognition sequence), although not limited in sequence, must be compatible with the non-specific sequences of the partner recombination site in order for recombination to be efficient.
  • the non-specific sequences are the compatibility sequences of a recombinase site.
  • C. Recombination Constructs Recombination constructs are designed to provide an observable change when recombination between recombination sites occurs. Preferred recombination constructs include two pairs of recombination sites, one pair having a variant sequence and another pair having a sequence recognized by non-mutant recombinase (for example, wild type recombinase).
  • recombination constructs include a first nucleic acid sequence that includes a first reporter gene and first and second recombination sites, where the first and second recombination sites are variant recombination sites, and a second nucleic acid sequence that includes a second reporter gene and third and fourth recombination sites, where the third and fourth recombination sites can be recombined by a non-mutant recombinase.
  • the first and second nucleic acid sequences need not be present on the same vector or on the same nucleic acid molecule (for example, the chromosome), although this is preferred. It is preferred that recombination constructs be embodied in vectors, such as plasmids.
  • the sequence of the recombination sites in the constructs are chosen such that the recognition sequences of the first and second recombination sites differ from the recognition sequences of the third and fourth recombination sites.
  • the sequence of the recombination sites can also be chosen such that the compatibility sequences of the first and second recombination sites differ from the compatibility sequences of the third and fourth recombination sites such that the first and second recombination sites cannot recombine with the third and fourth recombination sites.
  • the sequence of the recombination sites can also be chosen such that the compatibility sequences of the first and second recombination sites are sufficiently similar to allow recombination between the first and second recombination sites, and such that the compatibility sequences of the third and fourth recombination sites are sufficiently similar to allow recombination between the third and fourth recombination sites.
  • Arriving at recombination sites having relationships as described above is preferably accomplished in the following way.
  • a given recombination site sequence (which can be recombined by a non-mutant recombinase)
  • parallel changes are made in the compatibility sequences of the first and second recombination sites.
  • These altered recombination sites should then be tested to make sure that the non-mutant recombinase can still mediate their recombination. This helps insure that compatibility sequence changes have not inadvertently affected the function of the recombination sites. Once this is confirmed, changes can be made to the recognition sequences of the first and second recombination sites.
  • the recombination sites can have a variety of properties and relationships that make them useful for particular purposes.
  • the recombination sites can be designed such that the first and second recombination sites cannot be recombined by non-mutant recombinase to a significant extent. This allows separate assessment of cleavage by mutant and non-mutant recombinase. It is also useful if the first and second recombination sites have identical sequences, and the third and fourth recombination sites have identical sequences.
  • Recombination between the recombination sites can have a variety of effects that allows detection of recombination.
  • the constructs can be designed such that recombination between the first and second recombination sites alters the expression of the first reporter gene, where recombination between the first and second recombination sites is determined by determining if expression of the first reporter gene is altered; recombination between the third and fourth recombination sites alters the expression of the second reporter gene, where recombination between the third and fourth recombination sites is determined by determining if expression of the second reporter gene is altered; recombination between the first and second recombination sites allows the first reporter gene to be expressed;
  • the first nucleic acid sequence includes a spacer sequence flanked by the first and second recombination sites, where the spacer sequence interrupts the first reporter gene such that the first reporter gene is not expressed, and where recombination of the first and second recombination sites excises
  • the constructs can also be designed such that recombination between the first and second recombination sites prevents expression of the first reporter gene; the first reporter gene is flanked by the first and second recombination sites, where recombination of the first and second recombination sites excises the first reporter gene which prevents expression of the first reporter gene; a portion of the first reporter gene is flanked by the first and second recombination sites, where recombination of the first and second recombination sites inverts the flanked portion of the first reporter gene which prevents expression of the first reporter gene: recombination between the third and fourth recombination sites allows the second reporter gene to be expressed; and/or the second nucleic acid sequence includes a spacer sequence flanked by the third and fourth recombination sites, where the spacer sequence interrupts the second reporter gene such that the second reporter gene is not expressed, and where recombination of the third and fourth recombination sites excises the spacer sequence
  • the constructs can also be designed such that a portion of the second reporter gene is inverted, where the inverted portion of the second reporter gene is flanked by the third and fourth recombination sites, and where recombination of the third and fourth recombination sites inverts the inverted portion of the second reporter gene which allows the second reporter gene to be expressed; recombination between the third and fourth recombination sites prevents expression of the second reporter gene to be expressed; the second reporter gene is flanked by the third and fourth recombination sites, where recombination of the third and fourth recombination sites excises the second reporter gene which prevents expression of the second reporter gene; and/or a portion of the second reporter gene is flanked by the third and fourth recombination sites, where recombination of the third and fourth recombination sites inverts the flanked portion of the second reporter gene which prevents expression of the second reporter gene.
  • Expression of a reporter gene can include transcription of the gene, translation of the transcript (if the gene encodes a protein), and/or production of an active protein. As used herein, whether a reporter gene is expressed depends on the context. In general, a gene is considered to be expressed if it produces the expression product to be detected. Such expression products include full or partial transcripts of the gene, full or partial proteins, including active or inactive forms of the proteins, translated from the transcript. Since the goal in using reporter genes in the disclosed method is the detection of expression, any of these forms of expression product can be the object of detection. For example, if the gene's transcript is to be detected, the gene will be considered to be expressed if it produces the transcript, regardless of whether the transcript is translated or whether the resulting protein is active.
  • the gene is not expressed unless active protein is produced—mere transcription of the gene, or even translation to produce an inactive protein, will not be enough in this context.
  • the expression product to be detected will influence the manner in which reporter genes should be interrupted or invented in the disclosed constructs. For example, nearly any interruption of a reporter gene would prevent expression of an active protein encoded by the gene. On the other hand, an interruption of the coding region will usually not prevent production of a transcript.
  • the structure of the disclosed constructs should be designed with these principles in mind.
  • an inactive expression product refers to an expression product that does not have an activity exhibited by the active form of the expression product where that activity is required for detection of expression in the assay scheme being used.
  • the constructs can be designed such that the first nucleic acid sequence is a first nucleic acid construct and the second nucleic acid sequence is on a second nucleic acid construct; the first nucleic acid construct is an extrachromosomal vector and the second nucleic acid construct is in the genome of a host cell; and/or the first and second nucleic acid constructs are on the same nucleic acid construct.
  • Reporter genes are used to monitor whether recombination occurs in the disclosed constructs.
  • Reporter genes can be any gene the expression of which can be detected either directly or indirectly. These include genes encoding enzymes, such as ⁇ -galactosidase, luciferase, and alkaline phosphatase, that can produce specific detectable products, and genes encoding proteins that can be directly detected. Virtually any protein can be directly detected by using, for example, specific antibodies to the protein.
  • a preferred reporter protein that can be directly detected is the green fluorescent protein (GFP). GFP, from the jellyfish Aequorea victoria, produces fluorescence upon exposure to ultraviolet light without the addition of a substrate (Chalfie et al., Science 263:802-5 (1994)).
  • a number of modified GFPs have been created that generate as much as 50-fold greater fluorescence than does wild type GFP under standard conditions (Cormack et al, Gene 173:33-8 (1996); Zolotukhin et al., J. Virol 70:4646-54 (1996)).
  • This level of fluorescence allows the detection of low levels of expression in cells.
  • Reporter genes encoding proteins producing a fluorescent signal are useful since such a signal allows cells to be sorted using FACS.
  • Another way of sorting cells based on expression of the reporter gene involves using the reporter protein as a hook to bind cells.
  • a cell surface protein such as a receptor protein can be bound by a specific antibody. Cells expressing such a protein can be captured by, for example, using antibodies bound to a solid substrate, using antibodies bound to magnetic beads, or capturing antibodies bound to the reporter protein. Many techniques for the use of antibodies as capture agents are known and can be used with the disclosed method.
  • the reporter gene can also encode an expression product that regulates the expression of another gene. This allows detection of expression of the reporter gene by detecting expression of the regulated gene.
  • a repressor protein can be encoded by the reporter gene. Loss of expression of the reporter gene (via recombination) would then result in derepression of the regulated gene.
  • This type of indirect detection allows positive detection of loss of the expression of the reporter gene by the affector RNA molecule.
  • One preferred form of this type of regulation is the use of an antibiotic resistance gene regulated by a repressor protein encoded by the reporter gene. By exposing the host cells to the antibiotic, only those cells in which expression of the reporter gene has been inhibited will grow since expression of the antibiotic resistance gene will be derepressed.
  • the reporter genes can be expressed using any suitable expression sequences. Numerous expression sequences are known and can be used for expression of the reporter genes. Expression sequences can generally be classified as promoters, terminators, and, for use in eukaryotic cells, enhancers. Expression in prokaryotic cells also requires a Shine-Dalgarno sequence just upstream of the coding region for proper translation initiation. Inducible promoters are preferred for use with the first reporter gene since it is preferred that expression of the first reporter gene be adjustable.
  • Promoters suitable for use with prokaryotic hosts illustratively include the ⁇ -lactamase and lactose promoter systems, tetracycline (tet) promoter, alkaline phosphatase promoter, the tryptophan (trp) promoter system and hybrid promoters such as the tac promoter.
  • tet tetracycline
  • trp tryptophan
  • hybrid promoters such as the tac promoter.
  • many other functional bacterial promoters are suitable. Their nucleotide sequences are generally known.
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase, enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosphosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • inducible yeast promoters suitable for use in the disclosed vectors include the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • Yeast enhancers also are advantageously used with yeast promoters.
  • Preferred promoters for use in mammalian host cells include promoters from polymoma virus, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, herpes simplex virus (HSV), Rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters such as the ⁇ actin promoter.
  • Particularly preferred are the early and late promoters of the SV40 virus and the immediate early promoter of the human cytomegalovirus, MMTV LTR, RSV-LTR, and the HSV thymidine kinase promoter.
  • Enhancer sequences are now known from mammalian genes (globin, elastase, albumin, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the disclosed vectors preferably also contain sequences necessary for accurate 3' end formation of both reporter and affector RNAs. In eukaryotic cells, this would be a polyadenylation signal. In prokaryotic cells, this would be a transcription terminator.
  • the disclosed method involves producing mutant recombinases and testing the mutant recombinases with specially designed constructs.
  • the constructs contain variant recombination sites that are not recognized by non- mutant recombinase but will undergo recombination in the presence of a mutant recombinase with altered specificity.
  • the disclosed method also involves determining whether a variant recombinase retains the ability to mediate recombination at recombination sites recognized by non- variant recombinase. This can be accomplished by using constructs containing recombination sites recognized by non-variant recombinase. Recombination at these recombination sites can be monitored or detected by any suitable means.
  • recombination is detected by screening or selection based on the expression or lack of expression of a reporter gene. This can be accomplished by using constructs containing a reporter gene associated with the recombination sites such that the reporter gene is rearranged or deleted, or a spacer sequence interrupting the reporter gene is rearranged or deleted, as a result of recombination at the recombination sites. Recombination of such constructs will result in a loss of expression of the reporter gene, where the construct contained a functional reporter gene, or in a gain in expression of the reporter gene, where the construct contained a non-functional reporter gene. 1. Production of Mutant Recombinases Mutant recombinases can be produced by any suitable technique.
  • a method of generating a variety of recombinase proteins having a variety of amino acid sequences the most preferred way of doing this is to mutagenize or alter nucleic acid encoding the recombinase and then expressing the mutant recombinases.
  • Numerous techniques for introducing alterations into nucleic acid sequences are known and can be used in the disclosed method.
  • alterations can be made by chemical mutagenesis, introduction of degenerate nucleic acid fragments into the base nucleic acid molecule, and low fidelity PCR.
  • the goal of this mutagenesis or alteration will be the generation of a population or set of mutant recombinases having a variety of sequences. The broader the range of variants, the more raw material for the identification process.
  • Variant recombinases that can mediate recombination at variant recombination sites are identified in the disclosed method by selecting for, screening for, or otherwise detecting recombination of specially designed constructs having variant recombination sites.
  • Recombination at variant recombination sites can be monitored or detected by any suitable means. It is preferred that recombination is detected by screening or selection based on the expression or lack of expression of a reporter gene. This can be accomplished by using constructs containing a reporter gene associated with the variant recombination sites such that the reporter gene is rearranged or deleted, or a spacer sequence interrupting the reporter gene is rearranged or deleted, as a result of recombination at the recombination sites.
  • Variant recombinases that can mediate recombination at recombination sites recognized by non-variant recombinase are identified in the disclosed method by selecting for, screening for, or otherwise detecting recombination of specially designed constructs having recombination sites recognized by non-variant recombinase. Recombination at these recombination sites can be monitored or detected by any suitable means. It is preferred that recombination is detected by screening or selection based on the expression or lack of expression of a reporter gene.
  • constructs containing a reporter gene associated with the recombination sites recognized by non-variant recombinase such that the reporter gene is rearranged or deleted, or a spacer sequence interrupting the reporter gene is rearranged or deleted, as a result of recombination at the recombination sites. Recombination of such constructs will result in a loss of expression of the reporter gene, where the construct contained a functional reporter gene, or in a gain in expression of the reporter gene, where the construct contained a non-functional reporter gene.
  • variant recombinase it is preferred that the ability of a variant recombinase to mediate recombination at both variant recombination sites and recombination sites recognized by non-variant recombinase be assessed in the same system (such as a cell strain) either sequentially or simultaneously.
  • variant recombinases are tested for activity on both variant recombination sites and recombination sites recognized by non- variant recombinase in the same system or at the same time, it is preferred that two different reporter genes which can be separately detected or monitored be used. In this case, a first reporter gene can be associated with the variant recombination sites and a second reporter gene can be associated with recombination sites recognized by non-variant recombinase.
  • Variant recombinases produced in the disclosed method can be used for any purpose that unmodified recombinases can be used.
  • the advantage is that the variant recombinases have a different or broader site specificity.
  • the disclosed variant recombinases can be used to mediate recombination of any nucleic acid in any setting, including in vitro, in cell culture, and in vivo. Recombination can be obtained in single celled organisms, such as bacterial cells, fungal cells, yeast cells, prokaryotic cells, and archae bacterial cells, the cells of multicellular organisms, including plants and animals, both in the organism and in culture.
  • the disclosed variant recombinases can also be used in combination with other recombinases (including other variant recombinases) having a different site specificity. Such combinations allow more complex recombination schemes to be used. Examples of such schemes are discussed below.
  • first, second, and fourth DNA sequences comprising a first recombination site, a second recombination site, and a third recombination site, respectively, are introduced into cells.
  • recombination site means a nucleotide sequence at which a recombinase or variant recombinase can catalyze a site-specific recombination.
  • Methods for introducing a DNA sequence into cells are known in the art. These methods typically include the use of a DNA vector to introduce the sequence into the DNA of a single or limited number of eukaryotic cells and then growing such cell or cells to generate a suitable population of cells.
  • the term "vector” includes plasmids, viruses, and viral vectors.
  • the DNA sequences are introduced by a plasmid capable of transforming a selected cell while carrying a DNA sequence.
  • the particular vector which is employed to introduce the DNA sequence into a selected cell is not critical.
  • the recombination sites are contacted with a variant recombinase, thereby producing the site specific recombination.
  • a preferred means of contacting the DNA to be recombined with a variant recombinase is to place the DNA to be recombined into a cell expressing nucleic acid encoding the variant recombinase.
  • expression of the variant recombinase is under the control of a regulatory nucleotide sequence. Suitable regulatory nucleotide sequences are known in the art. The regulatory nucleotide sequence which is employed with a selected eukaryotic cell is not critical to the method.
  • a partial list of suitable regulatory nucleotide sequences includes the long terminal repeat of Moloney sarcoma virus described by Blochlinger and Diggelmann, Mol. Cell Bio., 4:2929-2931 (1984); the mouse metallothionein-I promoter described by Pavlakis and Hamer, Proc. Natl. Acad. Sci USA, 80:397-401 (1983); the long terminal repeat of Rous sarcoma virus described by Gorman et al., Proc. Natl. Acad. Sci USA, 19:6111-61 ! (1982); and the early region promoter of SV40 described by Southern and Berg, J. Mol. Appl. Genet., 1 :327-341 (1982).
  • suitable regulatory nucleotide sequences include GALl, GAL10. ADH1, CYC1, and TRP5 promoters.
  • GALl and GAL10 promoters are present on plasmid pBM150 which is described by Johnston and Davis. Molec. Cell. Biol, 4:1440 (1984).
  • the ADH1 promoter, also called ADC1 is present on plasmid pAAH5 which is described by Ammer, Methods Enzymol, 101 :192 (1983).
  • the CYC1 promoter is described by Stiles et al., Cell, 25:277 (1981).
  • the TRP5 promoter is described by Zalkin and Yanofsky, J. Biol. Chem., 257:1491 (1982).
  • the regulatory nucleotide sequence is a GALl promoter.
  • the first, second, and optionally, third and fourth DNA sequences are introduced into one strain of yeast.
  • the DNA sequences are introduced into two different strains of yeast of opposite mating types which are subsequently mated to form a single strain having all three or four DNA sequences.
  • the plasmid contains either (1) a nucleotide sequence of DNA homologous to a resident yeast sequence to permit integration into the yeast DNA by the yeast's recombination system or (2) a nucleotide sequence of DNA which permits autonomous replication in yeast.
  • One nucleotide sequence which permits autonomous replication in yeast is an ARS sequence described by Stinchcomb et al., Nature, 282:39 (1979).
  • a partial list of plasmids capable of transforming yeast includes YIP5, YRP17 and YEP24. These plasmids are disclosed and described by Botstein and Davis, The Molecular Biology of the Yeast Saccharomyces, Metabolism and Gene Expression (ed. Strathern et al), (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982), at page 607.
  • two recombination sites on the same DNA molecule can have the same or opposite orientations with respect to each other.
  • Recombinations between recombination sites in the same orientation result in a deletion of the DNA segment located between the two recombination sites and a connection between the resulting ends of the original DNA molecule.
  • the deleted DNA segment forms a circular molecule of DNA.
  • the original DNA molecule and the resulting circular molecule each contain a single recombination site (see Figure 2).
  • Recombination between recombination sites in opposite orientations on the same DNA molecule result in an inversion of the nucleotide sequence of the DNA segment located between the two recombination sites (see Figure 2).
  • recombinases including the disclosed variants and wild type recombinases.
  • Recombination using the disclosed variant recombinases can be used in vitro to produce site-specific recombination of nucleic acid molecules. This is useful for a wide variety of manipulations that currently employ wild type recombinases or involve traditional restriction enzyme cleavage followed by ligation. Examples include recombination of libraries of DNA fragments into vectors or in desired structures, and labeling of DNA via recombination.
  • Recombined DNA formed by in vitro recombination can then be introduced into cells.
  • constructs formed in vitro can be introduced into cells to resolve the structures formed in vitro or to select active structures.
  • large concatemers of subject DNA and spacer/vector fragments can be made, introduced into cells, and circularized into vector units in the cells. Such recombination could also be performed in vitro if desired.
  • the disclosed variant recombinases can be used to label DNA by recombining a DNA molecule of interest with a labeled DNA molecule.
  • Use of a recombinase for labeling is advantageous since it involves fewer steps than traditional labeling via DNA synthesis or ligation. These considerations are particularly important when large DNA molecules (over 20 kb) are to be labeled since such large molecules will fragment more the more they are manipulated.
  • Recombination mediated by the disclosed variant recombinases and variant recombination sites can be used to manipulate a host cell genome as desired and simultaneously introduce a marker gene flanked by the recognition sites of a second recombinase. After selection, leading to an accumulation of cells carrying the desired genomic alteration, one could simply remove the marker gene by expression of the second site-specific recombinase.
  • a large number of recombinases suitable for this purpose exists in nature, including ⁇ Integrase (Int), yeast Flp, etc. (Nunes-Doby et al., Nucl. Acids Res., 26:391-406 (1998)).
  • Variant recombinases having different site specificity can also be used.
  • wild type recombinases recognize both wild type recombination sites and variant recombination sites that are not recognized by wild type recombinase
  • wild type recombinase and variant recombinases can be used to mediate sequential recombination between nucleic acids containing a combination of wild type recombination sites and variant recombination sites.
  • generation of knockout animals and plants can be made more efficient by using a structure wild type recombination site-selectable marker- wild type recombination site-endogenous gene-variant recombination site (rather than the conventional wild type recombination site-selectable marker- wild type recombination site-endogenous gene-wild type recombination site).
  • a structure allows the selectable marker to be removed by the action of wild type recombinase without disturbing the gene since wild type recombinase will not recognize the variant recombination site to any significant degree.
  • the endogenous gene can then be removed later by the action of a variant recombinase since the disclosed variant recombinases recognize both wild type and variant recombination sites.
  • the first and second DNA sequences are introduced into cells connected by a pre-selected DNA segment.
  • the segment can be a gene or any other sequence of deoxyribonucleotides of homologous, heterologous or synthetic origin.
  • the pre-selected DNA segment is a gene for a structural protein, an enzyme, or a regulatory molecule. If the first and second recombination sites have the same orientation, activation of the regulatory nucleotide sequence produces a deletion of the pre-selected DNA segment. If the first and second recombination sites have opposite orientation, activation of the regulatory nucleotide sequence produces an inversion of the nucleotide sequence of the pre-selected DNA segment.
  • a fourth DNA sequence (containing the third recombination site) is also introduced into cells, it is preferred that the second and fourth DNA sequences be introduced into cells connected by a second pre-selected DNA segment.
  • the second segment can be a gene or any other sequence of deoxyribonucleotides of homologous, heterologous or synthetic origin.
  • the second pre-selected DNA segment is a gene for a structural protein, an enzyme, or a regulatory molecule. If the second and third recombination sites have the same orientation, activation of the regulatory nucleotide sequence produces a deletion of the second pre-selected DNA segment. If the second and third recombination sites have opposite orientation, activation of the regulatory nucleotide sequence produces an inversion of the nucleotide sequence of the second pre-selected DNA segment.
  • Combinations of wild type and variant recombination sites, and combinations of different orientations of the recombination sites, in DNA introduced into cells can multiply recombination options. For example, if the first and second recombination sites are wild type recombination sites and the third recombination site is a variant recombination site (all in the same orientation) then wild type recombinase can produce a deletion of the first preselected DNA segment (but not the second) and a variant recombinase can produce a deletion of the first, second, or both pre-selected DNA segments.
  • This arrangement allows sequential deletion of the first and second pre-selected DNA segments.
  • first and second recombination sites are wild type recombination sites and the third recombination site is a variant recombination site, and the first recombination site has the opposite orientation from the second and third recombination sites (which, of course, have the same orientation) then wild type recombinase can produce an inversion of the first pre-selected DNA segment and a variant recombinase can produce a deletion of the second pre-selected DNA segment (and/or produce an inversion of the first pre-selected DNA segment or the entire section spanning the first, second, and third recombination sites).
  • first and third recombination sites are wild type recombination sites and the second recombination site is a variant recombination site, and the second recombination site has the opposite orientation from the first and third recombination sites (which, of course, have the same orientation) then wild type recombinase can produce a deletion of the entire section spanning the first, second, and third recombination sites, and a Cre variant can produce an inversion of the first, second, or both pre-selected DNA segments.
  • first and third recombination sites are wild type recombination sites and the second recombination site is a variant recombination site, and the first recombination site has the opposite orientation from the second and third recombination sites (which, of course, have the same orientation) then wild type recombinase can produce an inversion of the entire section spanning the first, second, and third recombination sites, and a variant recombinase can produce a deletion of the second pre-selected DNA segments and an inversion of the first pre-selected DNA segment.
  • variant recombinase can also be used with a different variant recombinase having a different site specificity rather than wild type recombinase.
  • the above examples illustrate the general principles involved in designing specific recombinations that may be desired. It should be understood that the above combinations of recombination sites can be extended to the use of more recombination sites (that is more than three) and more intervening, pre-selected DNA segments.
  • lox site means a nucleotide sequence at which the gene product of the cre gene, referred to herein as Cre, and/or the disclosed Cre variants, can catalyze a site- specific recombination.
  • LoxP site is a 34 base pair nucleotide sequence ( Figure 1) which can be directly synthesized or isolated from bacteriophage PI by methods known in the art.
  • the Lox P site is an example of a wild type lox site.
  • LoxP site consists of two 13 base pair inverted repeats separated by an 8 base pair spacer region.
  • the nucleotide sequences of the insert repeats and the spacer region are as follows.
  • lox sites include LoxB, LoxL and LoxR sites which are nucleotide sequences isolated from E. coli. These sequences are disclosed and described by Hoess et al., Proc. Natl. Acad. Sci. USA, 79:3398-3402 (1982). Preferred wild type lox sites are LoxP or LoxC2. Lox sites can also be produced by a variety of synthetic techniques which are known in the art. For example, synthetic techniques for producing lox sites are disclosed by Ito et al., Nuc. Acid Res., 10:1755 (1982) and Ogilvie et al., Science, 214:270 (1981).
  • the gene product of the cre gene is a recombinase herein designated "Cre" which effects site-specific recombination of DNA at lox sites.
  • Cre a recombinase herein designated "Cre” which effects site-specific recombination of DNA at lox sites.
  • the expression "cre gene” means a nucleotide sequence which codes for a gene product which effects site-specific recombination of DNA in cells at lox sites.
  • One cre gene (the wild type cre gene) can be isolated from bacteriophage PI by methods known in the art. One method for isolating a cre gene from bacteriophage PI is disclosed by Abremski et al., Cell, 32:1301-1311 (1983). Genes engineered into cells for producing a foreign protein are often placed under the control of a highly active promoter.
  • the activity of the promoter can result in an overproduction of the protein which interferes with the growth of the engineered cell.
  • This overproduction of the protein can make it difficult to grow the engineered cell in sufficient quantity to make protein production economically feasible.
  • the present invention provides a method whereby engineered cells can be grown to a desired density prior to expressing the engineered gene.
  • the engineered gene is expressed, as desired, by activating a regulatory nucleotide sequence responsible for controlling expression of DNA encoding a variant recombinase.
  • Methods of controlling the expression of an engineered gene include the following: (1 ) A DNA segment flanked by recombination sites in the same orientation is introduced into DNA in a cell between a promoter and an engineered gene to render the promoter incapable of expressing the gene.
  • a second DNA sequence comprising a regulatory nucleotide sequence and DNA encoding a variant recombinase is also introduced in the DNA. After the engineered cells are grown to a desired density, the regulatory nucleotide sequence is activated thereby effecting expression of the variant recombinase and producing a deletion of the DNA segment. The engineered gene would then be expressed.
  • a gene for a regulatory molecule flanked by recombination sites in the same orientation is introduced into DNA in a cell. The regulatory molecule inhibits expression of an engineered gene.
  • a second DNA sequence comprising a regulatory nucleotide sequence and DNA encoding a variant recombinase is also introduced into the DNA. After the engineered cells are grown to a desired density, the regulatory nucleotide sequence is activated thereby effecting expression of the variant recombinase and producing a deletion of the gene for the regulatory molecule. The engineered gene would then be expressed.
  • An engineered gene lacking a promoter and flanked by two recombination sites in opposite orientations is introduced into DNA in a cell such that the 3' end of the gene lies adjacent to the transcription start site of a regulatory nucleotide sequence.
  • a second DNA sequence comprising a regulatory nucleotide sequence and DNA encoding a variant recombinase is also introduced into the DNA. Since the engineered gene would be transcribed in the antisense direction, no engineered protein would be produced. After the engineered cell is grown to a desired density, the regulatory nucleotide sequence is activated thereby effecting expression of the variant recombinase and producing an inversion of the desired gene. The engineered gene could then be transcribed in the proper direction and expressed.
  • Cre recombinase and other, similar recombinases such as FLP.
  • the disclosed variant recombinases can also be used in any of these methods. Adaptation of these methods to the use of the disclosed variant recombinases is straightforward. Generally, all that is required is substitution of a variant recombinase (or a gene expressing a variant recombinase) for the original recombinase (or recombinase gene) and, if appropriate, substitution of variant (or wild type) recombination sites for the original recombination sites used in the method.
  • Examples of methods involving wild type recombinases and wild type recombination sites that can be adapted for use with the disclosed variant recombinases and recombination sites include recombination of DNA in phage packaging systems, recombination of DNA to form phage display libraries (for example, Fisch et al, Proc Natl Acad Sci USA 93(15):7761-6 (1996), and
  • Nucleic acids can generally be introduced into plant protoplasts, with or without the aid of electroporation, polyethylene glycol, or other processes known to alter membrane permeability. Nucleic acid constructs can also be introduced into plants using vectors comprising part of the Ti- or Ri-plasmid, a plant virus, or an autonomously replicating sequence. Nucleic acid constructs can also be introduced into plants by microinjection or by high- velocity microprojectiles, also termed "particle bombardment” or "biolistics” (Sanford, J. C, Tibtech 6: 299 (1988)), directly into various plant parts.
  • the preferred means of introducing a nucleic acid fragment into plant cells involves the use of A. lumefaciens containing the nucleic acid fragment between T-DNA borders either on a disarmed Ti-plasmid (that is, a Ti-plasmid from which the genes for tumorigenicity have been deleted) or in a binary vector in trans to a disarmed Ti-plasmid.
  • the Agrobacterium can be used to transform plants by inoculation of tissue explants, such as stems, roots, or leaf discs, by co-cultivation with plant protoplasts, or by inoculation of seeds or wounded plant parts.
  • Foreign genes can be introduced into a wide range of crop species.
  • the disclosed variant recombinases and method are applicable to a broad range of agronomically or horticulturally useful plants.
  • the particular method which is employed to introduce the DNA sequence into a selected plant cell is not critical.
  • DNA sequences are introduced into plant cells by co-cultivation of leaf discs with tumefaciens essentially as described by Horsch et al., Science. 227: 1229-1231 (1985) omitting the nurse cultures.
  • the recombination sites are contacted with a variant recombinase, thereby producing the site specific recombination.
  • a variant recombinase, or messenger RNA encoding a variant recombinase is introduced into the cells directly by micro-injection, biolistics, or other protein or RNA introduction procedure.
  • DNA encoding the variant recombinase is introduced into the plant cell under the control of a promoter that is active in plant cells. Suitable regulatory nucleotide sequences are known in the art.
  • the promoter which is employed with a selected plant cell is not critical to the method of the invention.
  • a partial list of suitable promoters include the 35S promoter of cauliflower mosaic virus described by Odell et al, Nature, 313: 810-812 (1985); the promoter from the nopaline synthase gene of A.
  • Variant recombinases can be expressed throughout the plant generally in all cells at all stages of development, or expression of variant recombinases can be more specifically controlled through the use of promoters or regulatory nucleotide sequences having limited expression characteristics. Variant recombinases can be expressed in a tissue specific manner, for example only in roots, leaves, or certain flower parts.
  • Variant recombinases can be expressed in a developmentally specific time period, for example only during seed formation or during reproductive cell formation. Expression of variant recombinases can also be placed under the control of a promoter that can be regulated by application of an inducer. In this case expression is off or very low until the external inducer is applied. Promoters active in plant cells have been described that are inducible by heat shock (Gurley et al., Mol. Cell. Biol 6: 559- 565 (1986)), ethylene (Bfoglie et al., Plant Cell 1 : 599-607 (1989)), auxin (Hagan and Guilfoyle, Mol. Cell. Biol.
  • control of expression of variant recombinases by the safener- inducible promoter 2-2, or its derivatives, allows the expression to be turned on only when the inducing chemical is applied and not in response to environmental or phytohormonal stimuli. Thus expression can be initiated at any desired time in the plant life cycle.
  • the regulatory nucleotide sequence is a 35S promoter or a 2-2 promoter.
  • Hybridization of a crop involves the crossing of two different lines to produce hybrid seed from which the crop plants are grown.
  • Hybrid crops are superior in that more of the desired traits can be introduced into the production plants.
  • quality traits such as oil content, herbicide resistance, disease resistance, adaptability to environmental conditions, and the like, can be hybridized in offspring so that the latter are invested with the most desirable traits of its parents.
  • progeny from a hybrid cross may possess new qualities resulting from the combination of the two parental types, such as yield enhancement resulting from the phenomenon known as heterosis. Controlled cross-fertilization to produce hybrid seeds has been difficult to achieve commercially due to competing self-fertilization, which occurs in most crop plants.
  • Hybrid seed production is typically performed by one of the following means: (a) mechanically removing or covering the male organs to prevent self- fertilization followed by exposing the male-disabled plants to plants with male organs that contain the trait(s) desired for crossing; (b) growing genetically male-sterile plants in the presence of plants with fertile male organs that contain the trait that is desired for crossing: or (c) treating plants with chemical hybridizing agents (CHA) that selectively sterilize male organs followed by exposing the male-disabled plants to plants with fertile male organs that contain the trait that is desired for crossing.
  • CHA chemical hybridizing agents
  • Some disadvantages to each of these methods include: (a) applicability only to a few crops, such as corn, where the male and female organs are well separated; and it is labor intensive and costly; (b) genetically male sterile lines are cumbersome to maintain, requiring crosses with restorer lines; (c) all CHAs exhibit some degree of general phytotoxicity and female fertility reduction. Also CHAs often show different degrees of effectiveness toward different crop species, or even toward different varieties within the same species.
  • a molecular genetic approach to hybrid crop production applicable to a wide range of crops and involves genetic male sterility is described in EPA 89- 344029.
  • This system involves the introduction of a cell disruption gene that is expressed only in the tapetal tissue of anthers thereby destroying the developing pollen.
  • the resulting genetically male sterile plants serve as the female parents in the cross to produce hybrid seed.
  • This system could be highly effective and desirable.
  • one disadvantage is that since the male sterile parent is heterozygous for the sterility gene which acts as a dominant trait, only 50% of the plants grown from the hybrid seed are fertile, the rest retain the sterility gene. This situation will result in reduced pollen shed in the production field which may lead to reduced seed set and yield.
  • seedless watermelon is sold that actually contains some developed seed and a large number of immature seed that varies in size up to that of fully mature seed.
  • To produce these watermelon first a hybrid cross is made between a tetraploid maternal parent and a diploid pollinator. The resulting triploid seed produces self-infertile plants that are crossed with a diploid pollinator to produce seedless fruit (Kihara, Proc. Soc. Hort. Sci., 58: 217-230, (1951)).
  • This production scheme suffers the following problems: (i) Creating a tetraploid plant, which is accomplished by a chromosome duplication method, is difficult. Also the number of seeds per fruit on this tetraploid plant must be low since this has a positive correlation with seed number in the final product (Andrus, Production of Seedless Watermelons, USDA Tech. Bull. No. 1425 (1971)); (ii) good combining ability of the diploid pollinator and the tetraploid plant is difficult to achieve (Henderson, J. Amer. Soc. Hort. Sci., 102: 293-297 (1977)); (iii) the triploid seeds are much inferior to regular diploid seeds in vigor and germinability (Maynard, Hort.
  • a recombination site/poly A- inactivated cell disruption gene regulated by a seed-specific promoter is introduced into a plant. When this plant is crossed to a plant expressing a variant recombinase. the disruption gene is activated and expressed in the seed, thereby disrupting seed development. The certainty of endosperm failure
  • the seed-specific promoter used can be selected from the group of promoters known to direct expression in the embryo and/or the endosperm of the developing seed, most desirably in the endosperm.
  • Examples of seed- specific promoters include but are not limited to the promoters of seed storage proteins. The seed storage proteins are strictly regulated, being expressed almost exclusively in seeds in a highly tissue-specific and stage-specific manner (Higgins et al, Ann. Rev. Plant Physiol.
  • seed storage proteins may be expressed at different stages of seed development and in different parts of the seed.
  • seed-specific expression of seed storage protein genes in transgenic dicotyledonous plants include genes from dicotyledonous plants for bean ⁇ -phaseolin (Sengupta-Goplalan et al., Proc. Natl. Acad. Sci. USA 82: 3320-3324 (1985) and Hoffman et al., Plant Mol. Biol.
  • soybean kunltz trypsin inhibitor (Perez-Grau and Goldberg Plant Cell 1 : 1095-1109 (1989)), soybean ⁇ -conglycinin (Beachy et al., EMBO J 4: 3047-3053 (1985), Barker et al., Proc. Natl. Acad. Sci. 85: 458-462 (1988), Chen et al., EMBO J1: 297-302 (1988), Chen et al., Dev. Genet, 10: 112-122 (1989), Naito et al., Plant Mol. Biol 11 : 683-695 (1988)), pea vicillin (Higgins et al., Plant Mol. Biol.
  • promoters of seed-specific genes operably linked to heterologous coding regions in chimeric gene constructions also maintain their temporal and spatial expression pattern in transgenic plants.
  • Such examples include Arabidopsis thaliana 2S seed storage protein gene promoter to express enkephalin peptides in Arabidopsis and Brassica napus seeds (Vandekerckhove et al., Bio/Technology 7: 929-932 (1989)), bean lectin and bean ⁇ -phaseolin promoters to express luciferase (Riggs et al., Plant Sci.
  • the cell disruption gene used can be selected from a group of genes encoding products that disrupt normal functioning of cells. There are many proteins that are toxic to cells when expressed in an unnatural situation. Examples include the genes for the restriction enzyme EcoRI (Barnes and Rine, Proc. Natl. Acad. Sci. USA 82: 1354-1358 (1985)), diphtheria toxin A (Yamaizumi et al., Cell 15: 245-250 (1987)), streptavidin (Sano and Cantor, Proc. Natl Acad. Sci. USA 87: 142-146 (1990)), and barnase (Paddon and
  • a highly desirable seedless system is one in which fully fertile FI seed develops, that can then be grown into plants that produce only seedless fruit.
  • This system is economically favorable in that for each cross pollination, a large number of seedless fruits result: the number of FI seed from one cross X the number of fruits produced on an F 1 plant.
  • Also incorporated in this scheme are the advantages of growing a hybrid crop, including the combining of more valuable traits and hybrid vigor. This is accomplished in the same manner as described above except that the recombination site/poly A-inactivated disruption gene is expressed from a seed maternal tissue (seed coat or nucellus)-specific promoter. For example, the seed coat is the outgrowth of the integuments, a strictly maternal tissue. Therefore the hybrid cross that brings the recombination site/poly A-inactivated disruption gene together with the recombinase gene does not involve this seed coat tissue.
  • the seed coat of the FI seed has either recombination sites or recombinase, depending on which is used as the female parent, and thus FI seed develop normally. After the FI seed gives rise to a fruit-bearing FI plant, all vegetative cells (including seed coat cells) inherit both recombination sites and recombinase from the embryo. Thus the seed coat of the FI plant has an activated cell disruption gene.
  • the seed coat is an essential tissue for seed development and viability.
  • the seed coat When the seed is fully matured, the seed coat serves as a protective layer to inner parts of the seed.
  • the seed coat is a vital nutrient-importing tissue for the developing embryo.
  • the seed is nutritionally "parasitic" to the mother plant. All raw materials necessary for seed growth must be imported.
  • the vascular tissue enters the seed through the funiculus and then anastamoses in the seed coat tissue. There is no vascular tissue connection or plasmodesmata linkage between the seed coat and the embryo. Therefore, all nutrient solutes delivered into the developing seed must be unloaded inside the seed coat and then move by diffusion to the embryo.
  • the disclosed tissue-specific and site-directed DNA recombination can be used to obtain seedless fruit production. This method is useful for the production of seedless watermelon, for example.
  • a combination of gene expression specific for maternally inherited seed tissue and the disclosed recombinase system can be used for the production of seedless watermelon.
  • the system can be universally applied to any horticultural crop in which the presence of seeds is undesirable and difficult to be eliminated through conventional breeding methods.
  • the system also allows the normal production of FI seeds.
  • the ability to maintain heterosis is an advantage of producing F2 seedless fruits.
  • the seed coat (including the integumentary tapetum) and nucellus (the tissue encompassing the embryo sac) are the remaining seed tissues that are maternally inherited.
  • the seed coat and nucellus also play an important role in importing nutrients into the developing embryo and endosperm. Seed development will be aborted if this vital nutrient- importing mechanism of the seed coat/nucellus is debilitated. This will be accomplished by using the recombinase system to activate a cell-damaging gene only in these tissues. Controlling the gene activation in a maternal tissue- specific manner allows production of normal FI seed, but abortion of F2 seed.
  • a seed coat or nucellus promoter is coupled to a tissue-destructive (lethal) gene in order to prevent seeds from forming.
  • the destructive gene is inactive in the seed parent due to the presence of a blocking transcription terminator.
  • the terminator is flanked by recombination sites for subsequent excision by a recombinase-mediated recombination event.
  • Expression of the recombinase is also controlled by the seed coat/nucellus-specific promoter.
  • the self-pollinated or out-crossed plants will produce seedless fruits or vegetables, since in seed coat/nucellus tissues recombinase and recombination sites are combined, and the lethal gene is activated.
  • the disclosed variant recombinases can also be used to aid in phage packaging.
  • the cloning system described herein utilizes a headful in vitro packaging system to clone foreign DNA fragments as large as 95 kb which permits the isolation of DNA fragments that are at least twice the size of those that can be obtained by lambda cosmid cloning.
  • This increased cloning capacity has the following utility: (1) Genes in the 45-95 kb size range and, more particularly, in the 70-95 kb size range can now be directly cloned and genes in the 25-45 kb size range can be cloned more easily.
  • the cloning system of the invention is useful as a means for the delivery of DNA efficiently to bacteria which otherwise do not take up DNA from solution well.
  • the headful packaging system of this invention for cloning foreign DNA fragments as large as 95 kb comprises:
  • step (a) modifying vector DNA by inserting a stuffer fragment into a blunt end producing site which is proximal to a pac site; (b) digesting the product of step (a) to produce two vector arms each of which contains (i) a blunt end, (ii) another end which is compatible with the foreign DNA fragment which is to be cloned, and (iii) a recombination site;
  • step (c) ligating the foreign DNA to the product of step (b) without generating concatemers
  • step (d) reacting the product of step (c) with pac cleavage proficient extract and head-tail proficient extract wherein the ratio of large heads to small heads in the head-tail extract is at least 5:1 ;
  • step (e) infecting a bacterial strain expressing a variant recombinase with the product of step (d);
  • pac is a generic name which refers to the site needed to initiate packaging of DNA.
  • the pac cleavage proficient extract contains the recognition proteins necessary to cleave the pac site and, thus, initiate packaging.
  • the head-tail proficient extract contains the heads and tails needed to package the cloned DNA into a virus particle.
  • concatemer means a DNA molecule consisting of repeating units arranged in a head-to-tail configuration.
  • stuffer fragment refers to a DNA fragment which is inserted into the vector DNA at a unique site, and within which headful packaging is terminated.
  • bacteriophage and phage are used interchangeably herein.
  • Bacteriophages which are suitable to practice the invention must have a large head capacity and the elements necessary for packaging DNA must be defined.
  • the necessary packaging elements are defined.
  • P22 and Tl do not have a very large head capacity.
  • the necessary packaging elements have not been defined.
  • the elements necessary for packaging DNA are the following: (1 ) a unique site, pac, which is cleaved by recognition proteins; it is the pac cleavage proficient extract which contains the recognition proteins necessary to cleave the pac site; and
  • initiation of packaging is site-specific (cleavage of pac site initiates packaging)
  • termination of packaging is not site-specific. In other words, no unique site is recognized, as packaging will terminate at whatever point the head has been filled.
  • the DNA substrate used in the packaging reaction during the viral life cycle is a concatemer consisting of individual units of the PI chromosome arranged in a head-to-tail manner.
  • Headful packaging using either PI phage or any other phage, is a four step process: (1) In the first step a unique site, pac, is recognized and cleaved by the pac recognition proteins (PRPs); (2) DNA on one side of the cleavage is packaged into an empty phage head until the head has been completely filled; (3) a second cleavage event is then triggered (the "headful" cut) that separates the packaged DNA away from the rest of the concatemer; and (4) initiation of a second round of DNA packaging from the free end generated by the previous "headful" cut—hence the term processive headful cutting.
  • PRPs pac recognition proteins
  • the ends of the packaged PI DNA do not contain complementary single-stranded sequences, as do the ends of packaged bacteriophage lambda DNA, and consequently after PI DNA is injected into a bacterium its cyclization does not occur by strand annealing but rather by recombination between homologous sequences present at the ends of the molecule. Because of this circumstance, any vector that uses PI packaging, or for that matter any headful packaging mechanism, must devise a means of cyclizing the linear packaged DNA by recombination. Cyclizing is accomplished by incorporating recombination sites into the vector and using a disclosed variant recombinase to cyclize the DNA after injection into gram-negative bacterial strains expressing the variant recombinase.
  • PI produces two head sizes, a big head that can accommodate 105-110 kb of DNA, and a small head that can accommodate no more than 45 kb of DNA. Normally the ratio of big to small heads in a PI wild-type infection is
  • the radio of head sizes is 1 :1.
  • the head-tail packaging lysate prepared from the cm mutant of PI contained the usual ratio of big to small heads which is about 10:1. This is the preferred lysate for preparing head- tail packaging extract.
  • the DNA must be bigger than that which can be accommodated by the small heads. It is generally desired that there be a large excess of big heads. However, the ratio of large heads to small heads should not fall below a ratio of about 5:1. Illustration
  • the method preferably uses the following components: 1. An in vitro mutagenesis system;
  • a recombinase expression plasmid that allows varying levels of expression by a simple environmental control (for example, by the presence of varying amounts of an inducer substance in the growth media, by temperature, or by osmolarity); 3.
  • An indicator/selector bacterial strain The strain carries both an indicator recombination substrate for detection of recombination at the wild type recombination site and a second recombination substrate that allows selection for recombinase mutants that have gained the ability to recognize and perform recombination at a target mutant recombination site (that is, a variant recombination site).
  • the wild type and target mutant sites are designed so that recombination between the mutant and wild type sites is blocked even with a mutant recombinase that can recognize both the wildtype site and also the target mutant site.
  • This design prevents unwanted recombination between the wildtype and target mutant recombination sites that could interfere with either selection or detection of desired recombinational outcomes.
  • the block is imposed by designing the wildtype and mutant sites to have different spacer regions (that is, different compatibility sequences), for example, the normal "wt" spacer for the wildtype recombination site, and an alternative spacer "Al" for the other recombination substrate.
  • DNA recombination proceeds efficiently both for recombination sites having the wt spacer (that is, a recombination between two wt sites) and also for sites having the Al spacer (that is, a recombination between two Al sites). Yet, recombination between the Al site and the wt site is blocked (that is, recombination between a wt site and an Al does not occur).
  • This strategy is applicable to all recombinases that have a recombination target site displaying one or more recombinase binding sites (repeat elements) on each side of a spacer region in which recombination occurs (Nunes-D ⁇ by et al., Nucl. Acids Res., 26:391-406 (1998)).
  • Such sites display a requirement for homology in the spacer elements for optimal recombination activity and has been shown to be the case for members of the Int family of recombinases, including Cre, lambda Int, and FLP (Craig, Ann. Rev. Genet. 22:77-105 (1988)).
  • the preferred in vitro mutagenesis system is that of Stemmer (Stemmer,
  • the expression plasmid to be used can be any of the "inducible" expression plasmids available in bacteria.
  • one of the pBAD plasmids for E. coli was chosen that allows expression of recombinase by growth on arabinose (Guzman et al., J. Bacteriol. 177:4121-4130 (1995)), and which can be turned off by growth on glucose (and no arabinose).
  • the expression plasmid carries the replication origin of pAC YC , and the FLP recombinase gene is under the control of the E. coli ara promoter region.
  • the plasmid carries the selectable marker Cm r which confers resistance to the antibiotic chloramphenicol. Because the pACYC replicon is low copy, its use may be advantageous in preventing excessive expression of FLP. Alternatively, a higher copy replicon could be used, such as that of Col ⁇ l. In that case the expression level of FLP must also be carefully controlled using the inducer substance arabinose.
  • the indicator/selector bacteria carries two different reporter constructs for FLP-mediated recombination.
  • the first reporter construct consists of two FRT sites (FLP recombination target; that is, the recombination site recognized by FLP recombinase) in direct orientation (an excision substrate) and resides on a low copy replicon that is compatible with the FLP expression construct.
  • FLP recombination target that is, the recombination site recognized by FLP recombinase
  • an excision substrate resides on a low copy replicon that is compatible with the FLP expression construct.
  • the first substrate is integrated into the E. coli genome. This can be done by incorporating the FRT substrate onto phage lambda and then constructing a lambda lysogen.
  • the FRT substrate could reside on a low-copy replicon that is compatible with that of the FLP expression vector and which has an additional selectable marker, for example resistance to bleomycin.
  • This FRT substrate carries two FRT sites in direct orientation flanking a gene whose presence can be easily monitored.
  • a constitutively expressing lacZ gene was used whose presence can be determined simply by growing colonies plates containing X-gal, upon which they will become blue in color. Loss of the lacZ gene by FLP-mediated recombination results in white colony formation on X-gal plates.
  • the indicator/selector bacterial strain also carries a second FRT-like substrate.
  • This is a plasmid element having two FRT-M sites in direct orientation flanking DNA sequences (STOP) that disallow expression of a downstream selectable marker.
  • STOP direct orientation flanking DNA sequences
  • nonexpression of the selectable marker is achieved by placing genetic elements in the following order: constitutive promoter - FRT-M - STOP - FRT-M - 'neo, where 'neo indicates the promoterless neo gene of Tn5.
  • this cassette cannot express neo and cells are sensitive to the antibiotic kanamycin. Excision of STOP by recombination at FRT-M is designed to permit expression of neo so that cells now become resistant to kanamycin.
  • the plasmid carries an additional selectable marker, Ap r conferring resistance to ampicillin, to maintain presence of the plasmid in E. coli.
  • the STOP sequence here is the strong transcriptional terminator ⁇ BTlT2 (Liebke et al.. Nucleic Acids Res. 13:5515-5525 (1985)).
  • Figure 17 shows wt FRT, FRT-A1, and FRT-M sites used in this illustration.
  • wildtype FRT site displays three inverted repeat elements, recombination proceeds efficiently with sites carrying two of these repeats in the inverted configuration shown (Jayaram, Proc. Natl. Acad. Sci. USA 82:5875-5879 (1985)).
  • Either the full or minimal site can be used since both are recombinationally functional.
  • the FRT-A1 site is designed to have an altered spacer but which is still functional for self X self recombination (Senecoff et al, J. Biol. Chem. 261 :7380-7386 (1986)).
  • the target FRT-M site is designed to carry symmetrical mutations in the repeat elements that disallow efficient FLP-mediated recombination (Senecoff et al., J. Mol. Biol. 201 :405- 421 (1988)), and also the spacer mutation of FRT-A1.
  • the FRT-M site differs from the FRT site in two ways.
  • both of the 13 bp inverted repeat elements that is the recognition sequences
  • the spacer region that is, the compatibility sequence; the 8 bp region between the 13 bp inverted repeats
  • the alternate spacer when present in an otherwise wt FRT site which we will call FRT-A1 , is permissive for FLP-mediated recombination between two FRT-A1 sites, but does not permit recombination between FRT-A1 and FRT.
  • Use of the FRT-M site which contains the heterologous spacer prevents FLP-mediated recombination between FRT and FRT-M by FLP or a mutant FLP protein that might otherwise catalyze recombination between the wt FRT substrate and the mutant target FRT-M substrate.
  • Unwanted recombination between the wildtype and target mutant recombination sites would decrease efficiency of the selection procedure by (a) not limiting recombination at the target mutant site specifically to these sites and thus compromising the selection (at FRT-M sites) for mutant FLP recombinases, (b) affecting the accuracy of the specific indication of activity at the wt FRT sites, and (c) decreasing either the plasmid stability of the FRT-M selector substrate or the integrity of bacterial chromosome (or compatible plasmid) carrying the wt FRT sites.
  • the pool of mutagenized FLP genes is transformed into the FRT indicator/selector strain which is pre-induced with arabinose and/or induced with arabinose during DNA transformation.
  • Bacterial colonies are then selected to be simultaneously resistant to chloramphenicol (to retain the FLP expression plasmid), ampicillin or carbenicillin (to retain the selector plasmid) and kanamycin (to select for cells in which FRT-M X FRT-M recombination has occurred) on agar plates containing either arabinose (for continued FLP expression) or glucose (to prevent prolonged FLP expression).
  • chloramphenicol to retain the FLP expression plasmid
  • ampicillin or carbenicillin to retain the selector plasmid
  • kanamycin to select for cells in which FRT-M X FRT-M recombination has occurred
  • Example 1 Selection of Variant Cre Recombinases
  • Cre mutants characterized by a wider substrate recognition were created, applying a technique called directed molecular evolution: Multiple rounds of a random mutagenesis procedure (DNA shuffling; Stemmer, W. P. C, Proc. Natl. Acad. Sci. USA, 91:10747- 10751 (1994)) and a sensitive selection for the desired phenotypes allow to accumulate candidate mutants within the generated pools of mutated sequences.
  • the Cre mutants created in this example showed wt-like activity on lox? sites.
  • Tris-HCl (1 M, pH 7.5 and 8.0) came from Quality Biological, Inc. (Gaithersburg, MD), as well as autoclaved LB (Luria- Bertani) and SOC broth.
  • L-(+)-arabinose >99% was ordered from Sigma- Aldrich Fine Chemicals (St. Louis. MO) and anyhdrous D-glucose from Mallinckrodt Laboratory Chemicals (Phillipsburg, NJ).
  • agarose TBE gels 0.8% agarose TBE gels were used (GTG Sea Kem Agarose (FMC, Rockland. ME)). Gels were prestained with 0.25 ⁇ g/ml EtBr (Ethidium Bromide, 10 mg/ml (Life Technologies, Inc., Grand Island, NY)).
  • the used electrophoresis apparatus was a DNA SUB CELLTM (BioRad, Hercules, CA) with an OSP 105 (OWL, Woburn, MA) powersupply. Gels were run at 60 V (5 V/cm) as recommended by Sambrook et al., Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press (Second Edition) (1989).
  • Plasmids for diagnostics, cloning, and sequence analysis were prepared using the WizardTM Minipreps Plus Kit (Promega, WizardTM Minipres Plus DNA Purification System. Instruction Manual (Madison, WI) (1/96)). Useful ones were assigned a pBS number and stored in TE pH 8.0 at +4°C. Oligonucleotides
  • oligonucleotides used as PCR primers, for plasmid construction, or in the mutagenesis procedure were ordered from Midland, Inc. (Midland, TX) in gel filtration (GF) quality.
  • the lyophilized oligonucleotides were assigned a BSB number, suspended in HPLC grad water (Sigma-Aldrich) at a final concentration of 300 ⁇ M, and stored at -20°C.
  • electroporation was preferred over chemical protocols.
  • Electorcompetent cells were made and used for electroporation as described by Smith et al., Focus, 12:38-40 (1990).
  • the appropriate cell porator and cuvettes were from Life Technologies.
  • the time in SOC medium Smith et al., Focus, 12:38-40 (1990) at 37°C under agitation (Lab-Line ® Orbit Environ-Shaker, Lab Line Instruments, Inc., Melrose Park, IL) prior to plating on selection medium was 1 h for ampicillin (Ap) and 2 h or more h for kanamycin (Kan) selection.
  • Table 1 List of reagents used for selection and screening of E. coli cultures. Reagent Concentration Stock Solution
  • Z-ara 0.006% 2% in DMF (w/v) The concentration of stock solutions, stored at -20°C and their dilutions in liquid LB medium or LB-agar plates is given.
  • X-gal stands for 5-bromo-4- chloro-3-indolyl- ⁇ -D-galactopyranoside
  • Z-ara for 5-bromo-3-indolyl- ⁇ -L- arabinofuranoside. All reagents, except Z-ara, were purchased from Life Technologies and aliquoted in the desired stock concentration for storage.
  • Z-ara (Berlin and Sauer, Anal. Biochem., 243:171-175 (1996)) was a generous gift from W. Berlin.
  • Standard PCR reactions were carried out in 50 ⁇ l total volume with the following reagents (all, except noted, from Perkin Elmer, Foster City, CA): lx PE buffer II (without MgCl 2 ), 2 mM MgCl 2 , 250 ⁇ M of each dNTP, 0.8 ⁇ M of each primer, ca. 50 ng of template DNA, qsp. H O (HPLC grade, Sigma-
  • PCR products When finished, all PCR products immediately were loaded on an agarose gel, or separated from enzyme, nucleotides and primers by applying the WizardTM PCR Preps Kit (Promega, WizardTM Minipres Plus DNA Purification System. Instruction Manual (Madison, WI) (1/96)). PCR products were recovered using deionized water and stored frozen at -20°C. Sequence Analysis
  • cre gene for the following DNase I shuffling reaction was amplified by PCR using 5' forward primer BSB436 (5'
  • AAATAATCTAGACTGAGTGTGAAATGTCC 3' AAATAATCTAGACTGAGTGTGAAATGTCC 3'
  • 3' reverse primer BSB376 (5' ATATATAAGCTTATCATTTACGCGTTAATGG 3'), introducing an Xba I and Hind III cloning site, respectively (underlined).
  • Mutagenic and non-mutagenic PCR * s were carried out: (94°C, 30 s; 52°C, 30 s; 72°C, 90 s) 45 or 30 times, respectively.
  • the 5' primer was designed to include the endogenous Shine-Dalgarno (SD) of cre, whereas its three endogenous promoters were excluded (position -17, 6; positions refer to the adenine of the start codon of the cre coding sequence as position 1).
  • a 60 cycle non-mutagenic PCR (as described above) was carried out without added primers, allowing the fragments to prime themselves and thereby to undergo shuffling while reassembling.
  • Conditions for PCR were: 94°C, 90 s; (94°C, 30 s; 45°C, 30 s; 72°C, 90 s) 60 times; 72°C, 10 min.
  • the self-priming step never yielded a single size product but rather a range of fragments between 300 bp to 2000 bp ( Figure 5), the self-priming PCR mixture was diluted 1/40 in a non-mutagenic PCR mix with primers BSB376 and BSB436 (see above), and subjected to an additional 20 cycles
  • pBAD33 contains the promoter of the arabiniose operon (pBAD), as well as expresses the regulatory protein AraC.
  • cre expression therefore will be under complete control of the pBAD promoter. This is important for the selection procedure (see below) that was intended for few Cre molecules acting on different lox sites. High concentrations or long term background expression of cre could eventually defeat the selection since wt Cre also catalyzes at very low frequencies recombination events between altered lox sites.
  • Plasmids and E. coli Strains Used for Selection and Screen Mutant lox sites Figure 1 compares the original lox? site to the two mutant sites, loxKl and /oxK2, used during the described experiments.
  • the lox sites with 5' Sal I and Xho I compatible, and 3' Xba I and Nhe I compatible ends were received as single stranded oligonucleotides from Midland and annealed by heating the appropriate ones together at 70°C, followed by a gradual cool down. Plasmids for Selection and Screening
  • Plasmid pBS561 was constructed using three fragments: (i) the 5' modified neo gene derived from pBS398 (Sauer et al., Methods, 4:143-149 (1992), (ii) the RSVneo (Gorman et al., Science, 221 :551-553 (1983) backbone without the neo gene, and (iii) the oligonucleotide-derived MCS ( Figure 7).
  • FIG. 8 summarizes the procedures used to construct plasmids pBS583 and pBS584.
  • the loxK cassettes containing MIu J/Kpn I-fragments from pBS568 and pBS569 were ligated into the RSV neo backbone that contains MIu I and Bgl II sites.
  • the Bgl II - Kpn I junction was achieved by filling the 3 ' recessed end of Bgl II with Klenow ( ⁇ EB) followed by a blunt-end ligation to the Kpn I end. This junction also was checked by sequencing and found to be correct.
  • a transcriptional terminator, rr «BTjT derived from pBAD33 by non-mutagenic PCR with primers BSB425 (5' ATAAGCGGCCGCTGAGCTTGGCTGTT TTGGCGG 3') and BSB426 (5' GCCGTCTCGAGAGAGTTTGTAG AAACGCAAAAAGGC 3'), was inserted into the loxK 2 cassette 3' of the EGFP gene after digest of the its Not I and Xho I linkers (underlined).
  • lox? 2 cassette selection plasmid also was designed (pBS613, Figure 9), to be used as a control.
  • Cre mutants the frequency of lox? recombination by Cre mutants could be determined in the same manner as used to evaluate /oxKl or /oxK2 recombination by pBS583 and pBS584.
  • Figure 10 summarizes the construction process for pBS601 (loxKl ) and pBS602 (loxKl ): In a first step, the neo resistance marker of pBS581 and pBS582 (intermediates in the construction of pBS583 and pBS584) was removed by deleting the Pvu II fragment and thus restoring the possibility to use neo for a different selection procedure.
  • the newly generated polylinker between the two lox sites permitted insertion of the Xba I - BamU I fragment of pBS481 (that carries the ⁇ b/A.st marker gene) into its Xba I and Bgl II sites.
  • E. coli strains expressing a recombinant ⁇ -L- arabinofuranosidase gene from Streptomyces lividans can be detected by eye on LB plates containing 5-bromo-3-indolyl- ⁇ -L-arabinofuranoside (Z- ara) Berlin and Sauer, Anal. Biochem., 243:171-175 (1996).
  • the E. coli strain BS583, DH5 ⁇ lac ( ⁇ D69 ZoxP 2 [/ ⁇ cZ L ⁇ U2]), was chosen as the bacterial background for the selection procedure for Kl + or K2 + Cre mutants by plasmids pBS583 or pBS584. Due to the lox? 2 [lacZ] containing ⁇ prophage, Cre activity on lox? can be evaluated simply by using X-gal plates.
  • the selection strains BS1493 and BS1494 were made by introducing the selection plasmids pBS583 and pBS584 into BS583 (Table 2).
  • BS1493 or BS1494 electrocompetent cells were transformed with 2 ⁇ l of the microdialyzed reaction mixture (VS membrane, MilliporeTM' Bedford, MA). To induce expression of the cre pool, the transformed cells were incubated at 37°C in induction medium for 2.5 h and/or 4 h under agitation (as mentioned before). Cultures were diluted 1/500 or 1/5000 and grown on LB plates with the following formulation: Ap, Cm, glucose, and X-gal for determining the transformation efficiency, referred to as non-selection plates.
  • Dilutions of 1/5 and occasionally 1/50 were grown on plates with addition of Kan, used to select for Kl + or K2 + mutants and called selection-plates.
  • the formulation of the plates served the following purposes: (i) Ap and Cm were added to assure that all clones contained both, selection and expression plasmid, (ii) X-gal to distinguish between P + and P " clones (Table 2). After overnight incubation at 37°C, blue and white colonies were counted and pools prepared for the next round of DNA shuffling. Alternatively, certain mutants were chosen for further analysis (see below).
  • the cre pool obtained was cloned into pBAD33 and transformed into BS583 cells. After 2 h of cre expression, the transformed cells were grown on X-gal plates. After overnight incubation at 37°C, two white colonies (indicating lox? recombination) were picked for plasmid preparation and complete sequencing. No point mutation was found in either one, so that each could be used as a control plasmid for wt Cre expression. One of the two was selected for further use and named pBS606.
  • minipreps were digested with the restriction enzyme Aat II which only cuts the selection plasmid.
  • Aat II restriction enzyme Aat II which only cuts the selection plasmid.
  • different dilutions were grown on LB agar plates supplemented with Cm and X-gal to select for pBAD33. Plates with Ap plus Cm were used to determine the background of contamination with uncut selection plasmid.
  • the Stratagene QuickChangeTM Site-Directed Mutagenesis Kit (Stratagene Cloning Systems, La Jolla, CA) was used to create cre mutants with mutations at the determined location, only.
  • the Stratagene QuickChangeTM Site-Directed Mutagenesis Kit (Stratagene Cloning Systems, La Jolla, CA) was used to create cre mutants with mutations at the determined location, only.
  • BSB 465 to BSB 470, Table 4 three different mutant primer sets (BSB 465 to BSB 470, Table 4), all steps were carried out as detailed in the manufacturer's instruction manual, except that electrocompetent BS1494 cells were used for transformation and mutant selection, replacing the provided XL1 blue cells.
  • the made mutant candidates were subjected to functional testing and sequencing as detailed before.
  • control cell line BSl 541 (Table 2) permits the combined P + selection and P " screen.
  • the frequency of P " Cre mutants obtained after non-mutagenic and after error-prone PCR was determined by the following experiment: After one mutagenic or one non-mutagenic PCR on the wt Cre expression plasmid pBSl 85, the resulting cre pools were inserted into the expression vector pBAD33 and transformed into BS583 cells. After 2.5 h of arabinose-mediated induction or glucose-mediated repression (by SOC medium) or cre expression, dilutions were transferred to LB plates with Ap, Cm, glucose and X-gal. The results are presented in Table 5: Under glucose repression, exclusively blue colonies could be identified (first line in Table 5), indicating that cre expression is insufficient for lox?
  • the observed discrepancy between 11% and 20% of phenotypical mutants may be due to a variety of reasons, among which: (i) the size difference between the two genes (633 bp vs. 1020 bp), (ii) different elongation times during PCR, and (iii) different sensibility of the two proteins for disabling point mutations.
  • the level of loxKl and / ⁇ xK2 recombination due to wt cre expression was determined using the wt Cre control plasmid pBS606. After transformation of the cell lines BSl 493 and BSl 494 with pBS606 and 2.5 h and 4 h of cre expression, cells were grown on selection and non-selection plates (as described previously). The recombination frequency between the altered lox sites was considered equal to the observed frequency of Kan R phenotype: for /oxKl, it was about 10 "5 after 2.5 h and 2 X 10 "5 after 4 h of wt cre expression, for / ⁇ xK2, it changed from about 2 X 10 "5 to 2 X 10 "3 .
  • cre mutants E262G, E262A, and E262W were excised by Hind III and Xba I and reinserted into the MCA of fresh pBAD33.
  • the three defined Cre mutants for the amino acid position 262 were subjected to a functional test for / ⁇ xK2, oxKl and lox? recombination activity, as mentioned before.
  • the results are summarized in Table 9.
  • the results described previously for the E262G and E262A mutations were confirmed: As indicated in column three, the / ⁇ xK2 recombination frequency increased 10 fold with the E262G mutant compared to the wt enzyme, whereas the E262A mutant shows only an increase of 200 fold.
  • the E262W mutant also achieved a similar activity on / ⁇ xK2 as seen with the E262G mutant.
  • lox Sites lox? and /oxK2 differ at several locations as illustrated in Figure 1 : First, the three outermost bp of the inverted repeats are altered, facilitating the construction of the various plasmids used for selection.
  • loxKl could interfere with wt Cre binding at two distinct DNA-protein interaction sites as compared to / ⁇ xK2, where only one location of incompatibility is available. For this reason, /oxK2 was chosen for the initial set of experiments, described in this work.
  • Cre Mutants after Three Rounds of Directed Evolution Three iterations of the in vitro evolution procedure were necessary to identify 36 candidates, expressing Cre mutants that could process / ⁇ xK2 (based on the applied selection procedure in E. coli).
  • Tests showed that five out of six selected ones had 10 3 and 10 4 fold increased activity on / ⁇ xK2 when compared to wt Cre.
  • On lox? and loxKl there was almost no difference between wt and the mutant enzymes.
  • the mutants therefore had developed an increased tolerance from transversions at positions 1 1 and 12 (/oxK2) of the lox sequence, but not for other positions like 10 and 14 (/oxKl). To indicate this phenotype they were referred to as K2 + /P + .
  • glutamate at position 262 located in the J helix of the enzyme, may be a DNA contacting residue and permit the formation of a hydrogen bond between the carboxyl group of its side chain and an amino group of one of the two adenines at positions 11 ' or 12' in the lox? sequence ( Figure 1).
  • changing these two bases to thymines in the / ⁇ xK2 sequence should lead to an electrostatic repulsion between their oxygens and the acidic side chain of glutamate. This could explain the observation that wt Cre is unable to catalyze a recombination between two /oxK2 sites.
  • R258 is a DNA contacting residue that forms hydrogen bonds with the guanine at position 10' of lox? and may also interact with the bp at position 11. There is yet no confirmatory experimental evidence for this proposal.
  • results from three initially isolated mutants indicated about 50%) of recombination frequency on / ⁇ xK2. This is about ten fold higher than that obtained with the E262G mutation alone. It is therefore likely that some of the additional point mutations identified in these three mutants account for this increase in activity.
  • Table 8 lists all point mutations that were found. If silent and conservative mutations are considered not to influence specificity, only a limited number of candidates to account for the phenotype remains. Among these, S254G and Q255R of mxoxox 2 and 3, because of their location close to the amino-terminus of the J helix, could be expected to influence DNA contacts with positions 11 or 12 of the lox site.
  • the other mutations are scattered in the N- and C-terminal domain of Cre. All, except Rl 01 Q of mxoxox 5, affect aa that are not located within the proximity of DNA contacting areas. Some appear independently in two mutants, e.g. D29A or Dl 89N, that could influence protein folding or the interactions among the four Cre enzymes necessary for recombination. Such alterations could influence for example the orientation of the J helix and thereby reduce remaining interference between the ⁇ xK2 site and the enzyme. Alternatively, some mutations, also silent ones, could influence protein expression, leading to a faster accumulation of enzymes and consequently to higher recombination frequency. This possibility should however also influence lox? recombination.
  • the aliphatic side chain (a methyl group) of A262 could be the reason for slight sterical interference. This would explain the observed reduced frequency of / ⁇ xK2 activity with E262A. lox? recognition, however, could not be found to be affected compared to E262G. The lowered / ⁇ xK2 activity explains why no E262A mutation was identified in the small pool of six analyzed mutants: With a ten fold decrease in activity, one would expect to encounter the corresponding mutation ten times less often during selections as well.
  • Kl + or K2 + Cre mutant capable for /oxKl or /oxK2 recombination
  • Kan Kan R , Kan S
  • Kanamycin resistant, sensitive
  • Vectors pBS606, 614, 626, 627, 628 and 650 pBAD33 with wt, E262G, E262G/D29A, E262G/D189N, E262G/T316S, and R3M3 cre insertion used for expression of the corresponding Cre proteins in DH5 ⁇ for in vivo testing.
  • pBS632 to pBS641 pUC 19 based plasmids for in vivo tests of different
  • pRH200 wt Cre expression plasmid (a generous gift from Ron Hoess, DuPont, Wilmington, DE) used to overexpress wt Cre in BL21(DE3) (Novagen, Madison, WI) strain.
  • pBS654 topBS658 wt cre of pRH200 was replaced with different cre mutants (E262G, E262G/D29A, E262G/D189N, E262G/T316S, and R3M3) using Age I and Ml ⁇ I restriction sites.
  • BS583 The E. coli strain BS583, DH5 ⁇ lac ( ⁇ D69 loxP 2 [lacZ LEU2]), was chosen as the bacterial background for the selection procedure using plasmid pBS584. Due to the loxP [lacZ] containing ⁇ prophage, Cre activity on loxP can be evaluated simply by using X-gal plates.
  • BSl 494 The E coli strain for selection was established by introducing the selection plasmid pBS584 into BS583.
  • BS1494 allows a kanamycin- selection for loxK2 and in parallel a blue/white-screen for loxP recombination with 5-bromo-4chloro-3-indolyl- ⁇ -D-galactopyranoside (X-gal).
  • X-gal 5-bromo-4chloro-3-indolyl- ⁇ -D-galactopyranoside
  • FASl spacer region of the loxK2 site
  • BSl 576 to BSl 581 For the in vivo recombination experiments wt and mutant Cre expressing strains were generated by introducing plasmids pBS606, 614, 626, 627, 628, and 650 into DH5 ⁇ . Transformation of E. coli
  • electroporation was preferred over chemical protocols. ⁇ lectrocompetent cells were made and used for electroporation as described by Smith et al, Focus, 12:38-40 (1990). The appropriate cell porator and cuvettes were from Life Technologies (Bethesda, MD).
  • Plasmids pBS632 to pBS641 were transformed into Cre- expressing E. coli strains BSl 576 (wt), BSl 577 (E262G), BSl 578 (E262G/D29A), BSl 579 (E262G/D189N), BSl 580 (E262G/T316S), and BSl 581 (R3M3). After 1 hour of induction of cre expression with 0.2% L-(+)- arabinose, 10° dilutions were plated on non-selection medium containing 0.2%) D-glucose.
  • plasmids pBS632 to pBS641 served as substrates, whereas for the DNA binding reactions ⁇ [ 33 P]- dATP (Amersham Pharmacia Biotech, Piscataway, NJ) end-labeled 35 bp oligonucleotides were used, each encoding a /ox-halfsite and one half of the FASl spacer.
  • recombination assays with different lox sites were carried out with the following Cre enzymes: the wt enzyme, one of the originally sequenced third round mutants (R3M3), and the generated single and double mutants. All lox sites used for the in vivo tests were designed to have the same 8 bp spacer region (FASl) so that recombinational specificity was completely dependent on Cre's recognition of the symmetrical inverted repeats of the lox sites.
  • Cre enzymes the wt enzyme, one of the originally sequenced third round mutants (R3M3), and the generated single and double mutants. All lox sites used for the in vivo tests were designed to have the same 8 bp spacer region (FASl) so that recombinational specificity was completely dependent on Cre's recognition of the symmetrical inverted repeats of the lox sites.
  • Figure 14 presents the recombination frequencies of various mutant lox " substrates and combination of sites (loxP with loxK2 and as control with loxKl) from the marker excision assay. Mutant lox sites with symmetric nucleotide substitutions at positions 11 and 12 of the loxP sequence were tested with the wt enzyme and the five variant Cre mutants, including the multiple mutant R3M3(A). All enzymes showed a maximum in recombination (close to 100%)
  • Figure 14B shows the observed recombination frequencies on mixed substrates (e.g. loxP with loxK2).
  • loxKl recombination with loxP by wt enzyme was substantially less than for loxP' recombination.
  • This recombination frequency was increased dramatically with all of the mutant Cre protein.
  • E262G-Cre activity on the loxKl control site in vitro but not in vivo probably derives from differences in ionic strength and/or enzyme concentration between the assays.
  • Incompatible spacers (original loxP and FASl) formed the basis for the simultaneous selection for loxK2 recombination and screen for loxP recombination with E. coli strain BSl 494 which led to the disclosed variant Cre recombinases.
  • LoxKl the other lox sequence used in this study, bears two critical bp exchanges per arm as well, however at different positions: 10 and 14. It was used as a control lox site, addressing the question whether the generated Cre mutants with novel specificity for loxK2 can also tolerate adjacent alterations within the lox sequence. Cre Mutants after Three Rounds of Directed Evolution
  • the in vivo and in vitro recombination assays showed a similar pattern in recombination frequencies for the different enzymes on the different sites tested.
  • recombination frequencies on mutant substrates were distinctively the highest with R3M3.
  • the double mutant E262G/D29A was about half as effective as R3M3, whereas the other double mutants and the single mutant E262G showed slightly further decreased recombination frequencies on the altered sites.
  • the wt enzyme did not recombine any of the mutant substrates presented here, neither in vivo nor in vitro.
  • the Q255R mutation of R3M3 could be expected to influence DNA contacts. Other mutations may influence protein folding or protein- protein interactions which could result in a higher flexibility within the Cre-/ ⁇ x interface and thus allowing a better tolerance of alterations of the lox sequence. This hypothesis is also supported by the observation that R3M3 recognizes the loxKl site at frequencies similar to E262G recognizing loxK2. The double and single Cre mutants, on the other hand, did not show activity on loxKl.
  • Cre double-mutants E262G with D29A, D189N, or T316S
  • table 16 the observed mean percentages of binding to loxP, loxK2, and loxKl halfsites with the five different Cre mutants and the wt enzyme are given.
  • all mutants - in contrast to the wt enzyme - do bind to loxK2 with similar frequencies, except R3M3 which shows surprisingly low retardation.
  • this phenomenon may be explained with an increased tolerance, i.e., flexibility, of R3M3 for altered lox sites.
  • the E262G mutation confers a generally elevated level of recombination at a number of variant lox sites, those having any of a large number of alterations at positions 11 and 12 (and mirroring 11 ' and 12' alterations).
  • the D 189N mutation in conjunction with the E262G mutation appears to fine tune the broadened specificity of the E262G mutation by reducing recombination at the loxK2 variants 'GG', 'TC and 'CC without decreasing recombination at loxK2 and the 'GT " variant. This mutation is thus useful to limit the broadened specificity of E262G. 3.
  • the T316S mutation when in conjunction with E262G provides a slight boost in recombination frequency with the loxK2 variants 'GT " and 'CC, and has no deleterious effect on recombination at the other variant loxK2 sites.
  • D29A mutation together with E262G boosts recombination at loxK2 and the variants 'CC and 'GG'.
  • D29A does not reduce recombination at the other variant lox sites or at loxP.
  • R3M3 must account for the further increased recombination frequency on any of the tested mutant lox sites, including loxKl but do not compromise loxP recognition.
  • the disclosed variant recombinases have a number of useful features and applications. By recognizing an altered, user-defined target site, they were designed to allow both genetic targeting events in prokaryotes and eukaryotes like wt Cre but on different sites and in vitro recombination strategies. With a wider variety of possible target sequences being now accessible, multiple and defined genomic alterations will now become feasible. This opens more possibilities in designing genomic manipulations in all DNA-based organisms by site-specific recombination.
  • Table 14 Presented are the frequencies of Kan R of six chosen candidate mutants from round three of the mutagenesis procedure (R3M1 to 6) when retesting them in the indicator strain. Also, they were tested for their performance on loxP sites in identical fashion. The obtained Kan R frequencies indicate the percentages of recombination within the allotted induction time of 2.5 hours.
  • mutant oligonucleotides used in the QuickChangeTM Site-Directed Mutagenesis Kit (Stratagene) to introduce single point mutations (mismatches with the wt sequence are highlighted by bold letters).
  • BSB465/466 a E262G mutation
  • BSB467/468 a E262A mutation is introduced in Cre.
  • the last set of primers (BSB469/470) represent an equimolar mixture of all possible bases at the assigned positions. This mixture results in all possible aa combinations at positions 261 to 263 of Cre.
  • the indicated positions refer to 5' and 3' end of the oligonucleotides in the cre coding sequence.
  • the three following columns show the calculated percentages of Kan R when subjecting the different Cre mutants and wt Cre to the three selection strains.
  • the frequency of the observed Kan R phenotype is considered as an indicator for the frequency of lox recombination: On loxK2, all mutants, except mxoxox 4, show remarkably increased Kan R frequencies compared to wt Cre. On lox?, all (including mxoxox 4), show very similar results as wt Cre, indicating, that lox? recombination is at best slightly affected On loxKl neither mutant nor wt Cre show more than background activity, even after four hours of cre expression. The obtained mutants are therefore characterized by a broader substrate recognition compared to the wt enzyme.
  • the frequency of the observed Kan R phenotype is considered as an indicator for the frequency of lox 2 recombination
  • E262G and E262W show approximately an 10 ⁇ fold increased Kan R frequency compared to wt Cre, whereas E262A results in a 200 fold increase.
  • On lox? all show very similar results as wt Cre, indicating, that lox? recombination is at best slightly affected
  • On loxKl neither mutant nor wt Cre show more than background activity This indicates, that E262G and E262W alone are sufficient to remarkably increase Cre's activity on /O K2, that these mutations do not increase loxKl recognition, and that lox? activity may be slightly affected
  • % of recombination is calculated from the obtained fluorescence intensity of substrate and product on an agarose gel.
  • % of gelretardation is calculated from the obtained intensity of the shifted band (Cre bound to lox halfsite) and free lox halfsites (labeled oligonucleotides). The values given here were all obtained with 0.2 pmol of lox halfsites. Without enzyme added, no retardation of either halfsite could be observed. The obtained data was normalized for better comparability to 100% retardation on loxP halfsite, for each enzyme tested.

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Abstract

L'invention concerne des variantes de recombinase Cre augmentant la spécificité du site de recombinaison. Plus particulièrement, l'invention concerne des variantes servant de médiation dans la recombinaison entre des séquences autres que la séquence loxP et autres séquences du site lox sur lesquelles la recombinase Cre de type sauvage est active. En général, les variantes Cre de l'invention servent de médiation dans la recombinaison efficace entre les sites lox sur lesquels peut agir la Cre de type sauvage (désignés sous le nom de sites lox de type sauvage), entre des sites lox variants n'étant pas utilisés de manière efficace par la Cre de type sauvage (désignés sous le nom de sites lox variants), et entre un site lox sauvage et un site lox variant. L'invention concerne également des méthodes de recombinaison d'acides nucléiques à l'aide des variantes Cre de l'invention. Par exemple, ces variantes Cre peuvent être utilisées dans toute méthode ou technique dans lesquelles peut être utilisée la recombinase Cre (ou d'autres recombinases semblables telles que la FLP). De plus, les variantes Cre permettent d'effectuer des recombinaisons diverses. En effet, les variantes Cre permettent une recombinaison beaucoup plus efficace entre les sites lox de type sauvage et les sites lox variants. La commande d'une recombinaison de ce type permet de réaliser des recombinaisons séquentielles plus sophistiquées et d'obtenir des résultats impossibles à atteindre avec la recombinase Cre de type sauvage.
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WO2001005961A1 (fr) * 1999-07-14 2001-01-25 Clontech Laboratories, Inc. Procedes faisant intervenir la recombinase pour la production de vecteurs d'expression et compositions permettant d'atteindre ce resultat
US6270969B1 (en) 1995-06-07 2001-08-07 Invitrogen Corporation Recombinational cloning using engineered recombination sites
US6277608B1 (en) 1997-10-24 2001-08-21 Invitrogen Corporation Recombinational cloning using nucleic acids having recombination sites
WO2002044409A2 (fr) * 2000-12-01 2002-06-06 European Molecular Biology Laboratory Evolution dirigee liee a un substrat (slide)
WO2002083910A2 (fr) * 2001-01-18 2002-10-24 Clontech Laboratories, Inc. Methodes basees sur une recombinase specifique de sequence permettant de produire des vecteurs contenant un intron et compositions destinees a la mise en pratique de ces methodes
JP2003528594A (ja) * 2000-02-18 2003-09-30 ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー ゲノム修飾のための改変リコンビナーゼ
US6632980B1 (en) 1997-10-24 2003-10-14 E. I. Du Pont De Nemours And Company Binary viral expression system in plants
US6746870B1 (en) 1999-07-23 2004-06-08 The Regents Of The University Of California DNA recombination in eukaryotic cells by the bacteriophage PHIC31 recombination system
US6936747B2 (en) 2000-07-21 2005-08-30 The United States Of America As Represented By The Secretary Of Agriculture Methods for the replacement, translocation and stacking of DNA in eukaryotic genomes
US7521240B2 (en) 2001-05-30 2009-04-21 Smithkline Beecham Corporation Chromosome-based platforms
EP2067858A1 (fr) 2007-12-07 2009-06-10 Universidad de Sevilla Modèles d'animaux pour maladies neurodégénératives
WO2009138544A1 (fr) 2008-05-16 2009-11-19 Proyecto De Biomedicina Cima, S.L. Adénovirus auxiliaires auto-inactivants pour la production d'adénovirus de recombinaison de capacité élevée
US8883988B2 (en) 1999-03-02 2014-11-11 Life Technologies Corporation Compositions for use in recombinational cloning of nucleic acids
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US7198924B2 (en) 2000-12-11 2007-04-03 Invitrogen Corporation Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites
EP1697534B1 (fr) 2003-12-01 2010-06-02 Life Technologies Corporation Molecule d'acide nucleique contenant des sites de recombinaison et leurs procedes d'utilisation

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Title
SAUER, METHODS, vol. 14, 1998, pages 381 - 392

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US6270969B1 (en) 1995-06-07 2001-08-07 Invitrogen Corporation Recombinational cloning using engineered recombination sites
US6277608B1 (en) 1997-10-24 2001-08-21 Invitrogen Corporation Recombinational cloning using nucleic acids having recombination sites
US6632980B1 (en) 1997-10-24 2003-10-14 E. I. Du Pont De Nemours And Company Binary viral expression system in plants
US8883988B2 (en) 1999-03-02 2014-11-11 Life Technologies Corporation Compositions for use in recombinational cloning of nucleic acids
WO2001005961A1 (fr) * 1999-07-14 2001-01-25 Clontech Laboratories, Inc. Procedes faisant intervenir la recombinase pour la production de vecteurs d'expression et compositions permettant d'atteindre ce resultat
US8129598B2 (en) 1999-07-23 2012-03-06 The Regents Of The University Of California DNA recombination in eukaryotic cells by the bacteriophage PHIC31 recombination system
US6746870B1 (en) 1999-07-23 2004-06-08 The Regents Of The University Of California DNA recombination in eukaryotic cells by the bacteriophage PHIC31 recombination system
US7115798B1 (en) 1999-11-17 2006-10-03 E. I. Du Pont De Nemours And Company Methods for regulated expression of triats in plants using multiple site-specific recombination systems
JP2003528594A (ja) * 2000-02-18 2003-09-30 ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー ゲノム修飾のための改変リコンビナーゼ
US7972857B2 (en) 2000-07-21 2011-07-05 The United States Of America As Represented By The Secretary Of Agriculture Methods for the replacement, translocation and stacking of DNA in eukaryotic genomes
US6936747B2 (en) 2000-07-21 2005-08-30 The United States Of America As Represented By The Secretary Of Agriculture Methods for the replacement, translocation and stacking of DNA in eukaryotic genomes
WO2002044409A2 (fr) * 2000-12-01 2002-06-06 European Molecular Biology Laboratory Evolution dirigee liee a un substrat (slide)
WO2002044409A3 (fr) * 2000-12-01 2003-03-13 European Molecular Biology Lab Embl Evolution dirigee liee a un substrat (slide)
WO2002083910A3 (fr) * 2001-01-18 2003-02-20 Clontech Lab Inc Methodes basees sur une recombinase specifique de sequence permettant de produire des vecteurs contenant un intron et compositions destinees a la mise en pratique de ces methodes
US6977165B2 (en) 2001-01-18 2005-12-20 Clontech Laboratories, Inc. Sequence specific recombinase-based methods for producing intron containing vectors and compositions for use in practicing the same
WO2002083910A2 (fr) * 2001-01-18 2002-10-24 Clontech Laboratories, Inc. Methodes basees sur une recombinase specifique de sequence permettant de produire des vecteurs contenant un intron et compositions destinees a la mise en pratique de ces methodes
US7521240B2 (en) 2001-05-30 2009-04-21 Smithkline Beecham Corporation Chromosome-based platforms
EP2067858A1 (fr) 2007-12-07 2009-06-10 Universidad de Sevilla Modèles d'animaux pour maladies neurodégénératives
WO2009138544A1 (fr) 2008-05-16 2009-11-19 Proyecto De Biomedicina Cima, S.L. Adénovirus auxiliaires auto-inactivants pour la production d'adénovirus de recombinaison de capacité élevée
WO2014188042A1 (fr) 2013-05-20 2014-11-27 3P Biopharmaceuticals Vecteurs alpha viraux et lignées cellulaires permettant la production de protéines recombinantes
US10011847B2 (en) 2013-05-20 2018-07-03 3P Biopharmaceuticals, S.L. Alphaviral vectors and cell lines for producing recombinant proteins

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