Novel LoxPsym sites for large-scale orthogonal CRE-mediated recombination

Abstract

The present application relates to the fields of genetic engineering and synthetic biology, and more particularly to means and methods for facilitating genetic recombination. The present application discloses novel Cre recombinase-dependent recombination sites that support simultaneous cloning and testing approaches.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present application relates to the fields of genetic engineering and synthetic biology, and more particularly to means and methods for facilitating genetic recombination. The present application discloses novel Cre recombinase-dependent recombination sites that support simultaneous cloning and testing approaches. [Background technology]

[0002] background Site-specific recombination has proven to be a powerful tool in genetic engineering, developmental biology, and systems biology over the past few decades. Such recombination systems are extremely popular in several research fields because they can delete, invert, integrate, and translocate large chunks of genomic DNA in vivo in several host organisms (Meinke et al. 2016 Chem Rev 20:12785-12820). Specifically, Cre( C auses reRecombinase (recombination) is one of the most commonly used recombination systems because it is well understood, has been implemented and characterized in several inducible formats (Guo et al. 2002 Genesis 32:8-18; Wu et al. 2020 Nat Commun 11: 3708; Hochrein et al. 2018 Nat Commun 9: 1931), and is functional in a wide range of host organisms (Adams 1992 J Mol Biol 226:661-73; Sauer 1987 Mol Cell Biol 7: 2087-2096; Hoa et al. 2002 Theor Appl Genet 104: 518-525; Shimshek et al. 2002 Genesis 32: 19-26). Cre recombinase, originally derived from bacteriophage P1, acts through the recognition of LoxP sites, which are 34-bp long sequences consisting of two 13-bp inverted repeats flanking a directional 8-bp spacer (Sternberg and Hoess 1983 Annu Rev Genet 17: 123-154). During recombination, Cre recombinase binds to both inverted repeats of a LoxP site as a dimer, cleaving the spacer region on both strands and initiating strand exchange with another LoxP site (Guo et al. 2000 Genesis 32:8-18). Depending on the orientation and location of the LoxP sites, deletion, inversion, or translocation of DNA fragments can occur.

[0003] To enable non-directional recombination, which allows various recombination events to occur regardless of the orientation of the recombination sites, Hoess et al. (1986 Nucleic Acids Res 14:2287-2300) converted the LoxP site spacer into a palindromic site. This artificial recombination site, called LoxPsym, has recently benefited from a newly discovered interest, as it plays a key role in the Synthetic Yeast Genome project (Sc 2.0). The latter aims to construct the world's first synthetic eukaryotic genome while making it highly evolvable on demand. This was achieved through the introduction of thousands of LoxPsym sites throughout the S. cerevisiae genome, which allows for the rapid introduction of genome rearrangements upon activation of the inducible Cre recombinase (Richardson et al. 2017 Science 355:1040-1044).

[0004] Despite its wide range of applications, the Cre-LoxPsym system is limited to a single recombination system. Therefore, in recent years, several approaches have been undertaken to achieve orthogonal recombination systems within a single organism. Orthogonality allows for simultaneous, large-scale, and independent genetic recombination in various regions of the genome. Orthogonal recombination systems not only enable more sophisticated genome engineering in synthetic biology, but also have wide applications in other fields such as developmental biology (Weng et al., 2022 Trends Cell Biol, 32:324-337), metabolic engineering (Liu et al., 2017 Methods in Molecular Biology, vol. 1642), and environmental monitoring (Akboga et al., 2022 Biosensors, 12: 122).

[0005] Currently, orthogonal recombination is achieved by combining multiple tyrosine recombinases that recognize distinct recombination sites and act without cross-reactivity. Recently, several new tyrosine recombinases that function orthogonally to Cre-LoxP have been discovered. These include Vcre (Liu et al. 2018 Nat Commun 9:1936), SCre (Suzuki et al. 2011 Nucleic Acid Res 39:e49), and Vika (Karimova et al. 2013 Nucleic Acid Res 42:e37). Recently, the development of orthogonal non-directed recombination systems for Vika and Dre has also been published (Wang et al. 2021 iScience 25:103716). However, the number of non-cross-reactive recombinases remains limited, and using several recombination systems in new host organisms requires heterologous expression of various enzymes that are potentially toxic to the host. To address these shortcomings, we have built on existing knowledge and developed an alternative strategy to obtain orthogonal recombination by developing non-cross-reactive LoxPsym recombination sites. Summary of the Invention

[0006] overview In this application, we describe how we first characterized 63 new LoxPsym sites by editing the spacer of the LoxPsym site. Some sites showed higher recombination efficiency compared to commonly used LoxPsym sites. We then performed intensive screening to identify LoxPsym variants that could function without cross-reactivity and found that we could generate several sets of orthogonal LoxPsym variants.

[0007] In a first aspect, the present application provides a compound of the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT wherein the spacer is selected from the list consisting of SEQ ID NOs: 1-63. In one embodiment, the LoxPsym site is cleaved in the presence of recombinase Cre. In another embodiment, the LoxPsym site comprises or consists of a nucleic acid sequence as represented in SEQ ID NOs: 65-127. In one specific embodiment, the LoxPsym site is characterized by a lower recombination efficiency than that of a standard LoxPsym site as represented in SEQ ID NO: 128, wherein the LoxPsym site comprises a spacer selected from the list consisting of SEQ ID NOs: 1-27. In another specific embodiment, the LoxPsym site is characterized by a higher recombination efficiency than that of a standard LoxPsym site as represented in SEQ ID NO: 128, wherein the LoxPsym site comprises a spacer selected from the list consisting of SEQ ID NOs: 28-63. In another specific embodiment, the LoxPsym site is an orthogonal LoxPsym site, meaning that specific DNA recombination cannot occur in the presence of the recombinase Cre between the LoxPsym site and another LoxPsym site that contains different nucleotides at positions 2, 3, 6, and / or 7 of the spacer. In specific embodiments, the LoxPsym site and the other LoxPsym site do not contain the spacers GGGTACCC and AACTAGTT, GGGTACCC and ATATATAT, GAATATTC and AGTTAACT, GAGTACTC and AAATATTT, or GTGTACAC and GCATATGC, respectively. In the most specific embodiment, the LoxPsym site is selected from the list consisting of SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121, and / or 124. Also provided are vectors containing any of the LoxPsym sites described herein, and host cells containing the vectors. The host cell may be a plant cell, a bacterial cell, a yeast cell, an insect cell or a mammalian cell.

[0008] In a second aspect, a set of at least two LoxPsym sites is provided, the LoxPsym sites comprising or consisting of the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT, wherein the spacer is selected from the list consisting of SEQ ID NOs: 1-63, and wherein at least two LoxPsym sites comprise different nucleotides at positions 2, 3, 6, and / or 7 of the spacer, and wherein the set does not include LoxPsym sites represented in SEQ ID NOs: 88 and 89, SEQ ID NOs: 88 and 81, SEQ ID NOs: 90 and 83, SEQ ID NOs: 65 and 92, or SEQ ID NOs: 78 and 103. In specific embodiments, the at least two LoxPsym sites are selected from SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121, and / or 124.

[0009] In a third aspect, there is provided a vector comprising one or more LoxPsym sites according to the first aspect of the invention, hi a specific embodiment, the vector comprises a set of at least two LoxPsym sites according to any embodiment of the second aspect of the invention.

[0010] In another aspect, there is provided a host cell comprising one or more LoxPsym sites according to the first aspect of the invention or a vector according to the third aspect of the invention. In a specific embodiment, the host cell comprises a set of at least two LoxPsym sites according to the second aspect.

[0011] In another aspect, the use of any of the LoxPsym sites or vectors, or sets of LoxPsym sites disclosed herein, is provided for site-specific recombination of one or more nucleic acid sequences. Also provided is the use of these LoxPsym sites, sets of LoxPsym sites, and / or vectors for in vivo cloning and phenotyping. In a specific embodiment, the cloning and phenotyping are performed sequentially in the same cell.

[0012] In yet another aspect, a method for obtaining a recombinant nucleic acid molecule is provided, the method comprising the steps of: i) providing a nucleic acid molecule comprising two or more nucleic acid elements, each flanked by orthogonal LoxPsym sites, or providing first and second nucleic acid molecules, each comprising one or more nucleic acid elements, each of the one or more nucleic acid elements being flanked by an orthogonal LoxPsym site; and ii) reacting the nucleic acid molecule or the first and second nucleic acid molecules with recombinase Cre to obtain a recombinant nucleic acid molecule, wherein the orthogonal LoxPsym sites comprise or consist of any of SEQ ID NOs: 65-127.

[0013] Also provided is a method for shuffling DNA elements within a nucleic acid molecule, the method comprising the steps of: i) providing a nucleic acid molecule comprising at least two nucleic acid elements, each flanked by an orthogonal LoxPsym site; and ii) reacting the nucleic acid molecule with recombinase Cre to obtain a nucleic acid molecule in which the nucleic acid elements have been reshuffled, wherein the orthogonal LoxPsym site comprises or consists of any of SEQ ID NOs: 65-127. In one embodiment of the method, the orthogonal LoxPsym site is a site selected from the list consisting of SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121, and / or 124. In another embodiment, the method further comprises determining the sequence of the recombinant nucleic acid molecule. In yet another embodiment, the method further comprises introducing the recombinant nucleic acid molecule into a cell and / or determining expression of the recombinant nucleic acid molecule in the cell. Also provided is a recombinant nucleic acid molecule obtained by the methods described herein.

[0014] In a final aspect, there is provided a method for optimizing gene expression of one or more genes in a cell, the method comprising the steps of: i) expressing or introducing into the cell one or more vectors comprising one or more genes, each gene under the control of a promoter, the promoter comprising two or more promoter elements, wherein the two or more promoter elements are each flanked by orthogonal LoxPsym sites, and wherein a different LoxPsym site is used for each gene; and ii) optionally, the one or more vectors further comprise a terminator sequence downstream of each gene, the terminator sequence comprising two or more terminator elements, wherein the two or more terminator elements are each flanked by orthogonal LoxPsym sites, and wherein a different LoxPsym site is used for each gene, and wherein any of the LoxPsym sites from step ii) are different from any of the LoxPsym sites used in step i); and iii) expressing the recombinase Cre in the cell; and iv) analyzing the gene expression of one or more genes or analyzing the phenotype of the cell. In one embodiment, the at least one orthogonal LoxPsym site is a LoxPsym site selected from any of the LoxPsym sites disclosed herein, more particularly selected from the list consisting of SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121, and / or 124. In another embodiment, the method further comprises determining the sequence of all or part of the genome of the cell. Also provided are cells, particularly genetically modified cells, obtainable by the method of optimizing gene expression as described herein. In one embodiment, the cell is a plant cell, a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.

[0015] Introducing one or more vectors into a cell is equivalent to genetically transforming the cell with the one or more vectors. Those of skill in the art are familiar with numerous molecular techniques for accomplishing such transformation. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 is a schematic diagram of the gene expression optimization tool disclosed herein, which consists of a promoter (upstream of GOI) and terminator (downstream of GOI) constructs driving expression of a gene of interest (GOI, shown in gray). Orthogonal LoxPsym sites flanking promoter elements (six promoter elements are shown as an example, not intended to be limiting) are visualized by green-colored diamonds, while LoxPsym sites flanking terminator elements (six terminator elements are shown as an example, not intended to be limiting) are visualized by yellow-colored circles. The promoter and terminator elements have different strengths, as indicated by different shades of blue. Upon induction of Cre-mediated recombination, genetic rearrangement of both constructs randomly allows different portions of the constructs to drive expression of the gene of interest, resulting in different expression levels and therefore phenotypic variation within the yeast population (represented by differently colored yeast cells), of which some recombinants will acquire superior phenotypic traits. Expression optimization tools can be applied simultaneously to multiple genes in a pathway.

[0017] [Figure 2] Figure 2 is an illustration of the LoxPsym site or sequence and how variants can be obtained by adapting the first three nucleotides of the spacer, shown in gray, and altering the remainder of the spacer accordingly to obtain a palindromic LoxPsym site. Cre recombinase, illustrated by the green structure, cleaves the LoxPsym site in the spacer region on both strands.

[0018] [Figure 3a-c]Figure 3a shows the gene construct integrated at the CAN1 locus used to determine the frequency of deletions / inversions caused by Cre-LoxPsym recombination. LoxPsym (green diamond) is placed in the TDH3 promoter (yellow arrow) immediately preceding the core promoter. This layout results in yECitrine fluorescence and prevents mCherry fluorescence. Figure 3b shows the gene construct used to determine Cre-LoxPsym recombination efficiency. 64 LoxPsym variants are tested by changing base pairs at positions 1, 2, and 3 of the spacer (blue). LoxPsym sites flank the high-expression cassette pTDH3-yECitrine-tCYC1 integrated at the CAN1 locus. The expression cassette pPGK1-mCherry-tADH1 integrated at the YRO2 locus serves as a control. Figure 3c is a schematic representation of fluorescence in cells after induction of Cre recombinase, resulting in no recombination (NR), deletion (DEL), or inversion (INV) of the fluorescent cassettes shown in panels a (top) and b (bottom). Yellow, red, and white indicate yECitrine, mCherry, or no fluorescence, respectively. [Figure 3d-f] Figure 3d shows the frequency of populations harboring the deleted (diamond), inverted (circle), or original (square) fluorescent cassettes shown in panel a, followed over a 24-hour time course. Figure 3e shows recombination efficiencies grouped by the number of purines / pyrimidines in the spacer sequence. Figure 3f shows recombination efficiencies grouped by the nucleotide at position 1, 2, or 3. [Figure 3g-h]Figures 3g and 3i show the recombination efficiency determined from the cassette shown in panel b between 48 LoxPsym sites after induction of Cre expression for 6 hours. LoxPsym variants are characterized by the nucleotides at positions 1, 2, and 3 of their spacer sequences. Thus, AAA is an abbreviation for the spacer AAATATTT, AAC is an abbreviation for the spacer AACTAGTT, etc. Figure 3h shows the LoxPsym sequences resulting after deletion of the reporter gene. [Figure 3i] Figures 3g and 3i show the recombination efficiency determined from the cassette shown in panel b between 48 LoxPsym sites after induction of Cre expression for 6 hours. LoxPsym variants are characterized by nucleotides at positions 1, 2, and 3 of their spacer sequences. Thus, AAA is an abbreviation for the spacer AAATATTT, AAC is an abbreviation for the spacer AACTAGTT, and so on.

[0019] [Figure 4] Figure 4a shows a comparison of the normalized fluorescence of single SCRaMbLEants between the control group (gray) and the Cre-expressing group (yellow) with the yECitrine gene regulated by the promoter and terminator constructs shown in Figure 1 (N=180). Dots represent the normalized fluorescence of individual SCRaMbLEants. The Fligner-Killeen statistic had a p-value of 2.20E-16. Green dots represent SCRaMbLEants that were further analyzed and sequenced, as shown in panel b. Figure 4b shows the normalized fluorescence upon recombination of promoter and terminal elements. Dots represent the average of three biological replicates, and gray error bars represent the standard deviation. The sequences of the recombined promoter and terminator constructs are shown to the right of each graph, and gray lines are used to indicate their fluorescence.

[0020] [Figure 5a-c]Figure 5a shows a schematic diagram of the astaxanthin production pathway in S. cerevisiae. Heterologous genes targeted for expression optimization are shown in orange. Arrows indicate enzymatic molecular transformation, and dashed lines indicate multiple intermediate steps underlying this transformation. Figure 5b shows single-picked colonies after SCRaMbLE induction. Larger color variations were obtained in the Cre+ strain compared to the strain without recombinase. Figure 5c shows quantification of the RGB values ​​of 1,408 and 1,490 single clones from the Cre+ and Cre- groups, respectively. Kligner-Killeen statistics had p-values ​​of 3.23E-03 and 8.30E-10 along the x- and y-axes, respectively. [Figure 5d] Figure 5d shows carotenoid titers obtained by LC-MS analysis of acetone extracts taken from cultures grown for 72 hours in 50 mL of YPD 2% for the Cre+ strain (N=30, right) and the recombinase-less strain (N=10, left). [Figure 5e-f] Figure 5e shows the ΔΔCT values ​​obtained from qPCR analysis compared to unmodified clone B, which indicates the gene expression levels of each of the six genes (tHMG, CrtE, Crtl, CrtYB, CrtW, and CrtZ) in the astaxanthin production pathway studied. Figure 5f shows the metabolite concentrations (μg / L) of single clones.

[0021] [Figure 6a]Figure 6 shows the limited cross-reactivity between 16 LoxPsym variants simultaneously in S. cerevisiae. Figure 6a shows a total of 18 constructs designed: 16 test constructs and two control constructs. Each construct contains all 16 different LoxPsym variants tested, as well as the ADE2 and URA3 expression cassettes. The URA3 cassette, flanked by LoxPsym-TCA in each case, served as a control to ensure active and functional recombination. Controls tested two positions of the ADE2 cassette (upstream and surrounded by the LoxPsym array), which should not result in ADE2 deletion if the 16 LoxPsym variants operate orthogonally. The 16 test constructs differed in the LoxPsym-NNN variant upstream of ADE2. The different test constructs verified cross-reactivity between all sites, as well as the activity and specificity of recombination between identical LoxPsym-NNN pairs. The LoxPsym variants in the array were separated by 100 bp, allowing recombination between adjacent sites. All constructs were inserted at the CAN1 locus of BY4741 ΔADE2. To induce recombination, all strains were transformed with the plasmid pSH47-His-Cre or the negative control pSH47-His-Vec. [Figure 6b-d]Figure 6 shows limited cross-reactivity among 16 LoxPsym variants simultaneously in S. cerevisiae. Figure 6b shows that after 6 hours of induction, cells were plated onto SC+FOA plates to select for URA3 deletions caused by recombination of canonical LoxPsym sites surrounding the URA3 marker. Red clones from the test strains showing deletion of the ADE2 cassette were selected for further investigation via PCR and sequencing (marked with arrows). Figure 6c shows the recombination efficiency (ADE2 deletion occurrence, calculated from the red phenotype) represented by plate counts of three biological replicates; error bars indicate standard deviation. The control strains showed negligible recombination efficiencies (0.4975 ± 0.3518 and 0.5962 ± 0.4268 for control strains 1 and 2, respectively). No colonies were observed for strains carrying pSH47-HisVec. Figure 6d shows the measured and predicted lengths of the recombinant constructs of 13 randomly picked red clones from each strain, represented by dots and crosses, respectively. Three randomly picked samples were analyzed by Sanger sequencing; various gray dots indicate the sequencing results.

[0022] [Figure 7A-B]Figure 7 shows the recombination and cross-reactivity of LoxPsym variants in maize (Zea mays). Figure 7a shows the experimental design for determining the cross-reactivity of LoxPsym variants in Z. mays. A combinatorial library encompassing all 256 pairwise combinations of LoxPsym variants was transfected into Z. mays protoplasts along with a plasmid for constitutive Cre expression. The presence of recombination was verified using next-generation sequencing (NGS). Figure 7b shows the design of the combinatorial library transfected into plant protoplasts. Each plasmid encoded two LoxPsym variants (variants represented by different shades of gray diamonds) separated by a 104-bp linker containing recognition sites (RE1 and RE2) for the restriction enzymes NcoI-HF and PvuI-HF (dashed lines). Barcodes were incorporated upstream and downstream of the LoxPsym variants, and each barcode was uniquely linked to one LoxPsym variant. The library contained all 16 × 16 (= 256) combinations between LoxPsym variants. Arrows indicate primer annealing sites for PCR performed after recombination was induced. PCR products were analyzed for size on agarose gels, and short amplicons (indicating recombination had occurred) were analyzed by next-generation sequencing. [Figure 7C] Figure 7 shows the recombination and cross-reactivity of LoxPsym variants in maize (Zea mays). Figure 7c shows the recombination efficiency between LoxPsym-NNN variants in Z. mays, calculated from the abundance of sequenced reads. Note that all efficiencies were normalized to the most active recombination site, LoxPsym-GGC, for which efficiency was arbitrarily set to 100%. Data represent the average of three technical repeats for two biological replicates, each represented by a diagonally divided cell.

[0023] [Figure 8a-b]Figure 8 shows the recombination and cross-reactivity of LoxPsym variants in Escherichia coli. Figure 8a shows the experimental design for determining the cross-reactivity of LoxPsym variants in E. coli. Two plasmids, donor (solid line) and acceptor (dashed line), were co-transformed. Both plasmids carried a single LoxPsym variant (diamonds of different shades), and in vivo recombination was demonstrated using PCR. Figure 8b shows details about the donor and acceptor plasmids. The acceptor plasmid encodes the Cre gene, controlled by the rhamnose-inducible rhaB promoter and rrnB terminator. Induction of Cre expression leads to recombination if the LoxPsym variants are cross-reactive. PCR (indicated by small arrows) was used to amplify the junction of the recombined plasmids. Recombination was induced for 4 hours by growing cells in LB supplemented with 2% rhamnose, experimental conditions based on previous reports (e.g., Sheets 2020, Ceroni 2018). Note that the recombination reaction does not have a final state, as the recombined plasmid can recombine back into two separate plasmids. [Figure 8c] Figure 8 shows the recombination and cross-reactivity of LoxPsym variants in Escherichia coli. Figure 8c shows the recombination efficiency between LoxPsym-NNN variants in E. coli, calculated from densitometric analysis of junction PCR. Data represent band intensities of PCRs performed in paired technical replicates using a mixture of templates from three biological replicates.

[0024] [Figure 9]Figure 9 shows that alternative LoxPsym sites are orthogonal in Yarrowia lipolytica. Figure 9A shows a schematic diagram of the workflow for demonstrating that alternative loxPsym sites are orthogonal in Y. lipolytica. First, a construct containing 16 LoxPsym sites (see Table 2) separated by a 100-bp spacer sequence was integrated into the URA3 locus of Y. lipolytica (strain W29). Then, for transient expression of the recombinase in Y. lipolytica, the strain carrying the loxPsym sites was transformed with a plasmid carrying Cre recombinase and a NAT selection marker. After transformation, NAT-resistant colonies were screened with primer pair 246-F / 247-R to verify that the various loxPsym sites are orthogonal in Y. lipolytica. Figure 9B shows gel electrophoresis of randomly selected transformants after PCR screening with primer pair 246-F / 247-R. A single band (2.3 kb) was amplified from all independent transformants, indicating that no recombination had occurred between the various lox sites. An additional PCR reaction with genomic DNA from a wild-type, untransformed strain (designated WT on the gel) served as a negative control.

[0025] [Figure 10]Figure 10 shows the recombination and cross-reactivity of LoxPsym variants in Y. lipolytica. Figure 10A is a schematic diagram of the use of orthogonal lox sites for marker excision in Y. lipolytica. A gene of interest (gene X) is integrated in the Y. lipolytica genomic region next to a selectable marker (hph), flanked by two putative orthogonal sites. Strains harboring this construct are then transformed with a plasmid carrying Cre recombinase and the selectable marker NAT. NAT-resistant colonies are then transferred to fresh plates containing either the NAT or HPH marker. If the sites are orthogonal and the marker is successfully excised, no growth will be observed on the HPH selection plates. Figure 10B shows three different LoxPsym sites that were tested for their efficiency in recombination following Cre expression, thereby resulting in marker excision. Excision of the hph marker was achieved after transformation with a plasmid carrying Cre recombinase. After transformation, 192 NAT-resistant colonies were transferred to plates supplemented with hygromycin B, and growth of the transferred colonies was assessed after 3 days. The frequency of marker excision was determined by calculating the number of 192 colonies that lost the ability to grow on plates containing hygromycin B. Experiments were performed in triplicate, and bars indicate SD between replicates. For comparison, the same experiment was performed using classical LoxPsym (LoxP) sites. DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description definition In order that this specification may be more readily understood, certain terms are first defined. Additional definitions are provided throughout the detailed description. While the present invention will be described with respect to specific embodiments and with reference to certain drawings, the present invention is not limited thereto and is limited only by the claims. The drawings are merely schematic and non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn to scale for illustrative purposes. It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. Furthermore, when used herein, "and / or" should be interpreted as a specific disclosure of each of two specific features or components, regardless of the presence or absence of the other. Thus, the term "and / or," when used herein in a phrase such as "A and / or B," is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or," when used in a phrase such as "A, B, and / or C," is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single). Where an indefinite or definite article is used when referring to a singular noun (e.g., "a," "an," or "the"), this includes the plural of that noun unless something else is specifically stated. Furthermore, the terms "first," "second," "third," etc. are used in the specification and in the claims to distinguish between like elements and not necessarily to describe an order or chronology.It is to be understood that the terms so used are interchangeable under appropriate circumstances, and that the aspects of the invention described herein are capable of operating in sequences other than those described or illustrated herein.

[0027] When an aspect or embodiment is described herein with the term "comprising," it is understood that similar aspects or embodiments are also provided other than those described with the terms "consisting of" and / or "consisting essentially of." When the term "comprising" is used in the specification and claims, it does not exclude other elements or steps. Unless specifically defined herein, all terms used herein have the same meaning as they would have to one skilled in the art of the invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure pertains.For example, The Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition (2002), CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition (1999), Academic Press; and The Oxford Dictionary of Biochemistry and Molecular Biology, revised (2000), Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.Physicians are particularly referred to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Press, Plainsview, New York (2012); Ausubel et al., Current Protocols in Molecular Biology (Supplement 100), John Wiley & Sons, New York (2012) for the definitions and terms of the relevant technology. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

[0029] Units, prefixes, and symbols are expressed in their Systeme International de Unites (SI) accepted form. Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of this disclosure that may be had by reference to the entire specification. Accordingly, the terms defined immediately below are more fully defined by reference to the entire specification.

[0030] As used herein, the terms "nucleic acid," "nucleic acid sequence," or "nucleic acid molecule" are used interchangeably and refer to a polymeric form of nucleotides (either deoxyribonucleotides or ribonucleotides) of any length, or analogs thereof. Nucleic acids may have any three-dimensional structure and may perform any function, known or unknown. Non-limiting examples of nucleic acids include genes, gene fragments, exons, introns, promoters or fragments thereof, terminators or fragments thereof, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules can be linear or circular. Nucleic acids may contain promoters, introns, enhancer regions, polyadenylation sites, translation initiation sites, 5' or 3' untranslated regions, reporter genes, selectable markers, etc. Nucleic acids may comprise single-stranded or double-stranded DNA or RNA. Nucleic acids may contain modified bases or modified backbones. Nucleic acids up to about 100 nucleotides in length are often referred to as oligonucleotides. As used herein, "nucleotide" refers to the building block of oligonucleotides and polynucleotides, and for the purposes of the present invention, includes both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides, contain a ribose sugar moiety, a nucleobase moiety, and one or more phosphate groups (which are not present in nucleosides). Nucleotides that do not contain phosphate groups are called "nucleosides," and are therefore compounds that contain a nucleobase moiety and a sugar moiety. As used herein, "nucleobase" refers to a group of atoms that can be linked to a sugar moiety to create a nucleoside that can be incorporated into an oligonucleotide, and where the group of atoms can bind to a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid.Naturally occurring nucleobases of RNA or DNA include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0031] The term "defined by SEQ ID NO:X" or "as represented in SEQ ID NO:X," as used herein, refers to a biological sequence consisting of the sequence of nucleotides given in SEQ ID NO:X. For example, a LoxPsym site defined in / by SEQ ID NO:X consists of the nucleic acid sequence given in SEQ ID NO:X. A further example is a nucleic acid sequence comprising SEQ ID NO:X, which refers to a nucleic acid sequence that is longer than the nucleic acid sequence given in SEQ ID NO:X but completely comprises the nucleic acid sequence given in SEQ ID NO:X, or refers to a nucleic acid sequence that consists of the nucleic acid sequence given in SEQ ID NO:X.

[0032] A "chimeric gene," "chimeric gene construct," or "chimeric construct" is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operably linked or associated with a nucleic acid sequence that encodes an mRNA and encodes an amino acid sequence such that the regulatory nucleic acid sequence can regulate the transcription or expression of the associated nucleic acid coding sequence. The regulatory nucleic acid sequence of a chimeric gene is not operably associated with the associated nucleic acid sequence as found in nature.

[0033] A "promoter" is a DNA sequence containing regulatory elements that mediate the expression of a nucleic acid molecule. For expression, a nucleic acid molecule must be operably linked to or contain a suitable promoter that expresses the gene at the correct time and with the required spatial expression pattern. As used herein, the term "operably linked" refers to a functional link between a promoter sequence and a gene of interest such that the promoter sequence can initiate transcription of the gene of interest. A promoter that allows initiation of gene transcription in a eukaryotic cell or host cell is referred to as being "active." To identify a promoter that is active in a eukaryotic cell or host cell, the promoter can be operably linked to a reporter gene, and the level and pattern of expression of the reporter gene can then be assayed. Suitable well-known reporter genes include, for example, beta-glucuronidase, beta-galactosidase, or any fluorescent or luminescent protein. Promoter activity is assayed by measuring the enzymatic activity of beta-glucuronidase or beta-galactosidase. Alternatively, promoter strength can also be assayed by quantifying mRNA levels using methods known in the art, such as Northern blots with densitometric analysis of autoradiograms, quantitative real-time PCR, or RT-PCR, or by comparing the mRNA levels of the nucleic acid to the mRNA levels of housekeeping genes such as 18S rRNA (Heid et al., 1996 Genome Methods 6: 986-994).

[0034] The term "3'-terminal region involved in transcription termination or polyadenylation" refers to a DNA sequence at the end of a transcription unit that signals 3' processing or polyadenylation of the primary transcript and includes regulatory sequences involved in terminating transcription. Regulatory sequences or terminators for transcription termination can be derived from the native gene or from various genes. For expression in yeast, a terminator to be added may be derived, for example, from the TEF or CYC1 gene, or from another yeast gene, or, less preferably, from any other eukaryotic or viral gene.

[0035] The term "vector" refers to any linear or circular DNA construct containing one of the LoxPsym sites of the present application. A vector can refer to an expression cassette or any recombinant expression system intended to express a gene of interest in any cell, including yeast, plant, and mammalian cells, constitutively or inducibly, in vitro or in vivo. A vector can remain episomal or can integrate into the genome of a host cell. A vector can have or not have the ability to autonomously replicate (i.e., drive only transient expression in the cell). The term encompasses recombinant expression cassettes that contain only the minimal elements required for transcription of the recombinant nucleic acid. A vector of the present invention can be a "recombinant vector," which, by definition, is an artificial vector. A vector can also be a viral vector, including lentiviral, retroviral, adenoviral, and adeno-associated viral vectors.

[0036] "Reduction" or "reducing" or "lower", as used herein, refers to a statistically significant reduction, more specifically, said statistically significant reduction is at least a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% reduction compared to the control situation.

[0037] "Increasing" or "increasing" or "enhancing" or "promoting" or "stimulating" as used herein are interchangeable and refer to a statistically significant increase, more specifically, said statistically significant increase being an increase of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% compared to the control situation.

[0038] The term "statistically significantly" different is well known to those skilled in the art. Statistical significance plays a central role in statistical hypothesis testing. It is used to determine whether to reject or retain the null hypothesis. The null hypothesis is the default assumption that nothing has happened or nothing has been changed, and therefore there is no difference in the recombination efficiency of a specific LoxPsym site compared to the recombination efficiency of a standard LoxPsym site, for example, as represented in SEQ ID NO: 128. For the null hypothesis to be rejected, the observed result must be statistically significant, i.e., the observed p-value must be smaller than a pre-specified significance level α. The resulting p-value, p, is the probability of obtaining a certain result, at least as an extreme value, if the null hypothesis is true. In one embodiment, α is 0.05. In a more specific embodiment, α is 0.01. In an even more specific embodiment, α is 0.001.

[0039] Yeast are eukaryotic, unicellular microorganisms classified as members of the fungal kingdom. Like all fungi, yeast can undergo asexual and sexual reproduction cycles. The most common mode of vegetative growth in yeast is asexual reproduction by budding, in which a small bud or daughter cell is formed on a parent cell. The nucleus of the parent cell divides into daughter nuclei and migrates into the daughter cells. The bud continues to grow until it separates from the parent cell, forming a new cell. This reproductive cycle is independent of yeast ploidy; therefore, both haploid and diploid yeast cells can replicate as described above. Haploid cells generally have lower fitness and often die under high-stress conditions, such as nutrient starvation. However, under the same conditions, diploid cells can undergo sporulation and sexual reproduction (meiosis), producing various haploid spores or segregants, which can then proceed to mating (mating) to re-form diploids. A haploid cell contains one set of chromosomes, while a diploid cell contains two sets of chromosomes. A haploid segregant, as used herein, is equivalent to a haploid spore that is the result of sporulation.

[0040] The budding yeast Saccharomyces cerevisiae reproduces as diploid cells by mitosis when nutrients are abundant, but upon starvation, the yeast undergoes meiosis to form haploid spores, which may then reproduce asexually by mitosis.

[0041] As used herein, "engineering" or "engineered" refers to genetic engineering, a technique in which the genome of an organism is modified using biotechnology. This includes, but is not limited to, gene transfer within and across species boundaries, deleting gene fragments or entire genes, and modifying the DNA sequence of an organism by deleting, inserting, or replacing one or more nucleic acid molecules. The means and methods for manipulating microorganisms, especially yeast, are well known to those skilled in the art. The most well-known techniques include classical genetic transformation of yeast and recombinant DNA technology. Today, the most attractive technique for manipulating microorganisms is by using nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and in particular the CRISPR-Cas system as described above.

[0042] Cre / LoxP recombination as a useful genetic engineering tool. DNA recombination is the process by which segments of DNA are broken and recombined to produce new combinations of alleles. While fundamental in generating genetic diversity in all organisms, site-specific recombination has also proven to be a powerful tool in genetic engineering, systems biology, and developmental biology research over the past few decades.

[0043] Cre / LoxP is a widely used site-specific DNA recombination system derived from bacteriophage P1. Cre recombinase catalyzes site-specific recombination between two LoxP sites without the need for accessory factors (Guo et al. Nature 389: 40-46). LoxP sites are 34 base pairs (bp) long and consist of two 13-bp inverted repeats separated by an 8-bp asymmetric spacer sequence. The Cre / LoxP system can be used to generate deletions, inversions, insertions (transpositions), or translocations, depending on the orientation and location of the LoxP sites specified in a particular system (Nagy 2000 Genesis 26:99). The simplicity of the Cre / LoxP system has led to its use in both in vivo and in vitro applications. Previous in vivo applications include targeted gene knockout, gene replacement, etc. (Zou et al. 1994 Curr Biol 4: 1099-1103; Lewandoski & Martin 1997 Nat Genet 17: 223-225), while in vitro applications include high-throughput DNA cloning and adenoviral vector construction (Marsischky & LaBaer 2004 Genome Res 14: 2020-2028; Parks et al. 1999 Gene Ther 10: 2667-2672). The general goal of most existing Cre / LoxP applications is to recover a single recombination event at a defined location. If the LoxP sites encode a symmetric spacer region (LoxPsym), rearrangements should be orientation-independent, and the DNA fragment between the two LoxPsym sites should undergo deletions or inversions with equal frequency (Hoess et al. 1986 Nucleic Acids Res 14: 2287-2300; Shen et al. 2016 Genome Res 26: 36-49).

[0044] A LoxPsym site contains Cre recognition sites or "arms" at the left and right ends, i.e., "LE," and a Cre recognition site or "arm" at the right end, i.e., "RE," sandwiched between the LE and RE arms, i.e., a spacer region. In most wild-type and mutant LoxP sites, the LE and RE arms are each 13 base pairs (bp) in length. In LoxPsym sites, the LE and RE arms are inverted repeat sequences. A non-limiting example of an LE sequence is 5'-ATAACTTCGTATA-3', and a non-limiting example of an RE sequence is 5'-TATACGAAGTTAT-3'. The spacer region is 8 bp in length. Each base in the spacer region is conventionally designated 1, 2, 3, 4, 5, 6, 7, or 8 according to its order in the sequence (5'->3'). Cre-LoxP sites mediate site-specific intra- or inter-strand exchange of DNA molecules catalyzed by Cre recombinase.

[0045] Novel LoxPsym site The efficiency of Cre-Lox recombination events is a major determining factor in setting up genetic engineering experiments. Therefore, the inventors of this application developed 63 variants of the standard LoxPsym site with various recombination efficiencies. Interestingly, 36 new LoxPsym sites appeared to be more efficient than the originally described LoxPsym site, reaching nearly twice the recombination efficiency, while 27 sites showed reduced recombination efficiency compared to the standard LoxPsym site. Since higher or lower recombination efficiency may be desired in certain cases, both groups have value as genetic engineering tools.

[0046] In a first aspect, the present application provides a compound of the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3' wherein the spacer is selected from the list consisting of SEQ ID NOs: 1-63. The LoxPsym site has a nucleotide sequence derived from the loxP site of P1 phage of wild-type E. coli. In one embodiment, the LoxPsym site is cleaved in the presence of recombinase Cre.

[0047] "Cre recombinase" or "recombinase Cre" or "Cre," as used herein, refers to a tyrosine recombinase enzyme derived from P1 bacteriophage (Uniprot ID: Q71TG5), the amino acid sequence of which is represented in SEQ ID NO: 129. SEQ ID NO: 129 (Cre recombinase) MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTL LRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGD

[0048] This enzyme uses a topoisomerase I-like mechanism to carry out site-specific recombination events. This enzyme (38 kDa) is a member of the integrase family of site-specific recombinases and is known to catalyze site-specific recombination events between two DNA recognition sites (LoxP or LoxPsym sites). This 34-nucleotide-long LoxP recognition site consists of two 13-nucleotide palindromic sequences flanked by a short 8-nucleotide spacer region. The product of Cre-mediated recombination at the LoxP sites is dependent on the position and relative orientation of the LoxP sites. Two separate DNA species, both containing LoxP sites, can fuse as a result of Cre-mediated recombination. The DNA sequence found between the two LoxP sites is said to be "floxed." In this case, the product of Cre-mediated recombination depends on the orientation of the LoxP sites. DNA found between two LoxP sites oriented in the same direction will be excised as a circular loop of DNA, while DNA present between two LoxP sites oriented in opposite directions will be inverted (Nagy 2000 Genesis 26:99-109). The enzyme does not require additional cofactors (such as ATP) or accessory proteins for its function (Abremski and Hoess 1984 J Biol Chem 259:1509-1514). When the LoxP sites encode a symmetric spacer region (LoxPsym), rearrangement is orientation-independent, and the DNA fragment between the two LoxPsym sites is deleted or inverted with equal frequency (Hoess et al. 1986 Nucleic Acids Res 14: 2287-2300; Shen et al. 2016 Genome Res 26: 36-49). Table 1. Summary of novel LoxPsym sites and their spacers and recombination efficiencies. [Table 1-1] [Table 1-2] [Table 1-3]

[0049] In one embodiment, the LoxPsym site has a lower recombination efficiency than the standard LoxPsym site described by Hoess et al., 1986, more specifically, the LoxPsym site as represented in SEQ ID NO: 128 (ATAACTTCGTATAATGTACATTATACGAAGTTAT) and includes a spacer as represented in SEQ ID NO: 64 (ATGTACAT). The recombination efficiency of SEQ ID NO: 128 is 47.0%.

[0050] Therefore, the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3' wherein the spacer is selected from the list consisting of SEQ ID NOs: 1-27, or wherein the LoxPsym site is selected from the list consisting of SEQ ID NOs: 65-91. In specific embodiments, the LoxPsym site has a recombination efficiency that is at least 10% lower than that of SEQ ID NO: 128, more specifically, the LoxPsym site comprises a spacer selected from the list consisting of SEQ ID NOs: 1-16. In more specific embodiments, the LoxPsym site has a recombination efficiency that is at least 15% lower than that of SEQ ID NO: 128, more specifically, the LoxPsym site comprises a spacer selected from the list consisting of SEQ ID NOs: 1-12. In even more specific embodiments, the LoxPsym site has a recombination efficiency that is at least 20% lower than that of SEQ ID NO: 128, more specifically, the LoxPsym site comprises a spacer selected from the list consisting of SEQ ID NOs: 1-9.

[0051] In another embodiment, the LoxPsym site has a recombination efficiency higher than that of the standard LoxPsym site described by Hoess et al. 1986, more particularly the LoxPsym site represented in SEQ ID NO: 128, and has a spacer as represented in SEQ ID NO: 64. Thus, the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3' wherein the spacer is selected from the list consisting of SEQ ID NOs: 28-63, or wherein the LoxPsym site is selected from the list consisting of SEQ ID NOs: 92-127. In specific embodiments, the LoxPsym site has a recombination efficiency that is at least 10% higher than the recombination efficiency of SEQ ID NO: 128, more specifically, the LoxPsym site comprises a spacer selected from the list consisting of SEQ ID NOs: 32-63. In more specific embodiments, the LoxPsym site has a recombination efficiency that is at least 15% higher than the recombination efficiency of SEQ ID NO: 128, more specifically, the LoxPsym site comprises a spacer selected from the list consisting of SEQ ID NOs: 34-63. In even more specific embodiments, the LoxPsym site has a recombination efficiency that is at least 20% higher than the recombination efficiency of SEQ ID NO: 128. low It has recombination efficiency, and more specifically, the LoxPsym site comprises a spacer selected from the list consisting of SEQ ID NOs: 35 to 63.

[0052] In a third aspect, a vector is provided that comprises any of the LoxPsym sites disclosed herein. In a specific embodiment, the vector comprises a nucleic acid sequence flanked by LoxPsym sites at both the 5' and 3' ends. In a specific embodiment, the nucleic acid sequence is flanked by LoxPsym sites. In another specific embodiment, the LoxPsym sites flanking the nucleic acid sequence are different. In another specific embodiment, the nucleic acid sequence is a gene of interest or a fragment thereof, its promoter or a fragment thereof, a terminator or a fragment thereof, or any coding, non-coding, or regulatory nucleic acid sequence (e.g., a 3'-terminal region involved in transcription termination or polyadenylation).

[0053] In yet another embodiment, a host cell is provided that contains any of the vectors described above or any of the LoxPsym sites disclosed herein. In specific embodiments, the host cell is a microorganism, a plant cell, an insect cell, a mammalian cell, or a yeast cell. In specific embodiments, one or more LoxPsym sites according to any embodiment of the first aspect are amplified, e.g., by PCR, and the resulting product, preferably a linear product, is directly transformed into a host cell. In another specific embodiment, one or more LoxPsym sites according to any embodiment of the first aspect are incorporated by a vector according to any embodiment of the third aspect. It should be understood that any method known to those skilled in the art can be used to incorporate one or more LoxPsym sites into a host cell as disclosed herein without departing from the scope of the present application. In even more specific embodiments, the host cell is a bacterium, such as Escherichia coli, or a yeast cell, even more specifically a yeast of the genus Saccharomyces, Yarrowia, or Pichia. In another specific embodiment, the host cell is a cell of a species selected from Saccharomyces cerevisiae, Escherichia coli, Zea mays, and Yarrowia lipolytica.

[0054] Novel orthogonal LoxPsym sites Microbial biotechnology, or microbial engineering, explores the power of bacteria and yeast to obtain economically valuable products or activities on an industrial scale. Synthetic biology and recombinant DNA techniques enable the expression of heterologous pathways in host cells, which are not limited to microorganisms but also extend to plant, insect, and mammalian cells. When cells are used as factories, the entire biosynthetic pathway is preferably inserted into the cell's genome. Because high expression of all biosynthetic genes often does not result in maximum product yield (e.g., intermediates may accumulate, causing feedback on the system, be toxic, or result in suboptimal growth), the expression levels of several modules or genes must be optimized and coordinated with each other. Currently available approaches, such as those based on simply trying many different combinations, directed evolution, computational prediction, and self-regulating systems with feedback inhibition, are laborious, expensive, and time-consuming.

[0055] The present inventors developed a simultaneous in vivo cloning and testing approach based on the well-known Cre-Lox system. Some of the 63 variants of the LoxPsym site described above were tested for cross-reactivity. Specifically, 1056 interactions between LoxPsym variants were tested in a fluorescence-based assay to identify orthogonal LoxPsym variants. From this interaction matrix, as shown in Figure 3g, several sets of orthogonal LoxPsym sites can be selected, and the largest set can be found to include 16 variants that are all cleavable by Cre but do not recombine with each other. Examples of such sets are a set consisting of LoxPsym sites selected from the list consisting of SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and 124, or a set consisting of LoxPsym sites comprising a spacer selected from the list consisting of SEQ ID NOs: 1, 5-7, 10, 13, 18, 20, 36, 41, 43-45, 48, 57 and 60. Other non-limiting examples of sets of orthogonal LoxPsym sites consist of SEQ ID NOs: 70, 77, 82, 101-102, 105, 107, 109, 112, 114, 117-118, 123-124, 126-127, or consist of SEQ ID NOs: 65, 71-74, 84, 86, 88, 90, 103, 106-108, 111, 121, 124, or LoxPsym sites selected from the list consisting of SEQ ID NOs: 72, 74, 77-78, 80, 82, 84, 86, 88, 102, 106, 109, 114, 118, 123, 127, or SEQ ID NOs: 65, 70-71, 73, 80, 90, 101, 103, 105, 108, 111-112, 117, 121, 124, 126. Smaller sets of LoxPsym sites are also provided. A non-limiting example is a set consisting of LoxPsym sites selected from the list consisting of SEQ ID NOs: 69, 70, 74, 77, 84, 86, 88, 100, 108, 109, 114, and 124.

[0056] The orthogonal LoxPsym sites disclosed herein can be used, for example, as follows: cells are transformed with one or more constructs containing genes A, B, C, ... Upstream of the genes, several promoter elements are present, separated by LoxPsym sites, where all promoter elements preceding gene A are separated by the same LoxPsym site, but different LoxPsym sites are used for all other genes (Figure 1). The same approach can be taken to optimize terminator sequences downstream of genes: several terminator elements are separated by LoxPsym sites, i.e., the same for all terminator elements of one gene, but different LoxPsym sites from the LoxPsym sites of the promoter elements of that gene and different from the LoxPsym sites of other genes (Figure 1). By inducing expression of Cre recombinase in cells, different combinations of promoter and terminator elements are made, either separately or simultaneously for multiple genes (orthogonal recombination). The genetic diversity of the resulting cell population leads to phenotypic diversity for traits of interest, such as production of valuable compounds (Figure 1). The best-performing cells are then sequenced to reveal optimized promoter / terminator combinations for each gene and, optionally, re-engineered in a clean manner.

[0057] In a second aspect, the present invention provides orthogonal LoxPsym sites that may be used in the assembly of nucleic acid constructs. The term "orthogonal" or "orthogonality" in (synthetic) biology describes the inability of two or more molecules of similar composition and / or function to interact with each other or affect their respective substrates; thus, "orthogonal," as used herein, refers to "acting independently" or "not cross-reacting."

[0058] Thus, if, in the presence of Cre recombinase, a LoxPsym site can recombine only with LoxPsym sites having the same nucleic acid sequence, but not with LoxPsym sites having different nucleic acid sequences, then the LoxPsym site is "orthogonal," which is a major advantage because specific recombination events can be initiated simultaneously without affecting each other.

[0059] Orthogonal recombination is currently achieved by using multiple recombinases that recognize distinct recombination sites (Wang et al. 2022 iScience 25:103716). However, the number of such non-cross-reactive recombinases is still limited, and in cellulo expression can potentially result in a dragging effect. The developed orthogonal LoxPsym variants disclosed herein overcome these disadvantages.

[0060] The present application provides a selection of orthogonal or non-cross-reactive LoxPsym sites. More specifically, mutant LoxPsym sites or sequences that can be cleaved in the presence of the recombinase Cre, and in which specific DNA recombination can occur between LoxPsym sites with identical nucleotide sequences, but not between LoxPsym sites with different nucleotide sequences. In a specific embodiment, the LoxPsym site comprises or consists of the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', where the spacer is selected from the list consisting of SEQ ID NOs: 1-63.

[0061] In one embodiment, a compound of the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3' wherein the spacer is selected from the list consisting of SEQ ID NOs: 1-63, and wherein the LoxPsym site in the presence of recombinase Cre can be cleaved, and wherein specific DNA recombination can occur only between LoxPsym sites having identical nucleotide sequences or containing the same nucleotides at positions 2 to 7 of the spacer. In a specific embodiment, recombination between said LoxPsym sites will not occur if said sites contain different nucleotides at positions 2, 3, 4, 5, 6 and / or 7 of the spacer.

[0062] In specific embodiments, the LoxPsym site, mutant LoxPsym site, or orthogonal LoxPsym site has the following properties: -the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3' wherein the spacer consists of 8 bases and is selected from the list consisting of SEQ ID NOs: 1 to 63; and In the presence of the recombinase Cre, a LoxPsym site can only recombine with a LoxPsym site having an identical nucleotide sequence or comprising or consisting of the formula 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from the list consisting of SEQ ID NOs: 1 to 63, and positions 2 to 7 of the spacer comprise an identical nucleotide sequence. "Spacer positions 2 through 7" means that both positions 2 and 7 are included.

[0063] In the presence of Cre recombinase, an orthogonal LoxPsym site cannot recombine with a LoxPsym site that has a different nucleotide at positions 2, 3, 4, 5, 6, or 7 of the spacer.

[0064] In another aspect, a set of LoxPsym sites is provided, comprising at least a first and a second LoxPsym site, wherein the LoxPsym site is selected from the list consisting of SEQ ID NOs: 65-127 or has the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', where the spacer is selected from the list consisting of SEQ ID NOs: 1-63. In one embodiment, in the presence of recombinase Cre, specific DNA recombination between the first and second LoxPsym site can occur only if the first and second LoxPsym site have the same nucleic acid sequence or share the same nucleotide at positions 2-7 of the spacer. In another embodiment, in the presence of recombinase Cre, specific DNA recombination between any LoxPsym site from the set and any other LoxPsym site from the set can occur if the LoxPsym sites share the same nucleotide at positions 2-7 of the spacer. In specific embodiments, the set of LoxPsym sites does not include SEQ ID NOs:88 and 89, SEQ ID NOs:88 and 81, SEQ ID NOs:90 and 83, or SEQ ID NOs:78 and 103.

[0065] In another embodiment, specific DNA recombination between the first and second LoxPsym sites of the set in the presence of recombinase Cre cannot occur if the spacers of the first and second LoxPsym sites are different and / or if the spacer of the first LoxPsym site contains different nucleotides at positions 2, 3, 4, 5, 6 and / or 7, more particularly at positions 2, 3, 6 and / or 7 of the spacer, compared to the spacer of the second LoxPsym site, provided that the first and second LoxPsym sites are different from each other in the set of spacers. Except when the first and second LoxPsym sites contain the combinations GGGTACCC-AACTAGTT, GGGTACCC-ATATATAT, GAATATTC-AGTTAACT, GAGTACTC-AAATATTT, or GTGTACAC-GCATATGC, or alternatively, when the first and second LoxPsym sites contain the spacers GGGTACCC and AACTAGTT, GGGTACCC and ATATATAT, GAATATTC and AGTTAACT, GAGTACTC and AAATATTT, or GTGTACAC and GCATATGC, respectively.

[0066] In another embodiment, in the presence of recombinase Cre, specific DNA recombination between the first and second LoxPsym sites of the set cannot occur if the first and second LoxPsym sites contain different nucleotides at positions 2, 3, 4, 5, 6 and / or 7 of the spacer, except when the first and second LoxPsym sites contain the spacer combinations GGGTACCC-AACTAGTT, GGGTACCC-ATATATAT, GAATATTC-AGTTAACT, GAGTACTC-AAATATTT, or GTGTACAC-GCATATGC.

[0067] In another embodiment, a set of at least two LoxPsym sites is provided, wherein the LoxPsym sites comprise or consist of the formula 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from the list consisting of SEQ ID NOs: 1 to 63, and wherein the at least two LoxPsym sites comprise different nucleotides at positions 2, 3, 4, 5, 6 and / or 7 of the spacer, and wherein the set does not include LoxPsym sites represented in SEQ ID NOs: 88 and 89, SEQ ID NOs: 88 and 81, SEQ ID NOs: 90 and 83, SEQ ID NOs: 65 and 92, or SEQ ID NOs: 78 and 103.

[0068] In one embodiment, a set of LoxPsym sites is provided, comprising at least a first and a second LoxPsym site, wherein the LoxPsym sites have the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from the list consisting of SEQ ID NOs: 1-63, and i) wherein, in the presence of the recombinase Cre, specific DNA recombination between the first and second LoxPsym sites can occur if the first and second LoxPsym sites have the same nucleic acid sequence, or alternatively, if the first and second LoxPsym sites are identical; and ii) wherein, in the presence of recombinase Cre, specific DNA recombination between the first and second LoxPsym sites cannot occur if the first and second LoxPsym sites contain different nucleotides at positions 2, 3, 4, 5, 6 and / or 7 of the spacer, except for the spacer combinations GGGTACCC-AACTAGTT, GGGTACCC-ATATATAT, GAATATTC-AGTTAACT, GAGTACTC-AAATATTT, or GTGTACAC-GCATATGC.

[0069] In another embodiment, a set of at least two LoxPsym sites is provided, wherein the LoxPsym sites have the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', where the spacer is selected from the list consisting of SEQ ID NOs: 1-63, and wherein the at least two LoxPsym sites differ from each other in at least one nucleotide residue at positions 2-7 of the spacer. In a specific embodiment, the at least two LoxPsym sites do not comprise the spacers GGGTACCC and AACTAGTT, GGGTACCC and ATATATAT, GAATATTC and AGTTAACT, GAGTACTC and AAATATTT, or GTGTACAC and GCATATGC. In another specific embodiment, the at least two LoxPsym sites do not comprise SEQ ID NOs: 88 and 89, 88 and 81, 90 and 83, 65 and 92, or 78 and 103.

[0070] In specific embodiments, the set includes at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 LoxPsym sites. In more specific embodiments, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 LoxPsym sites are selected from the list consisting of SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121, and / or 124.

[0071] In another aspect, a chimeric gene construct is provided, comprising one or more LoxPsym sites as described herein.In one embodiment, the chimeric gene construct comprises at least two LoxPsym sites.In a specific embodiment, the chimeric gene construct comprises a gene of interest or a fragment thereof, a promoter or a fragment thereof, a terminator or a fragment thereof, or any other coding, non-coding or regulatory nucleic acid sequence, flanked at the 5' end and / or 3' end by one or more LoxPsym sites as disclosed herein.

[0072] In another aspect, a vector is provided that includes one or more LoxPsym sites described herein or that includes any of the chimeric gene constructs described above.

[0073] Also provided are host cells containing the vector or any of the LoxPsym sites disclosed herein. In certain embodiments, the host cell is a microorganism, a plant cell, an insect cell, or a mammalian cell. In certain embodiments, the host cell is a bacterial cell or a yeast cell. In more specific embodiments, the host cell is a yeast, even more specifically a yeast of the genus Saccharomyces, Yarrowia, or Pichia, most specifically S. cerevisiae. In another specific embodiment, the host cell is a cell of a species selected from Saccharomyces cerevisiae, Escherichia coli, Zea mays, and Yarrowia lipolytica.

[0074] In another embodiment, a set or combination or selection of vectors is provided, wherein each vector comprises a nucleic acid sequence flanked at its 5' end by one of the LoxPsym sites disclosed herein and at its 3' end by the same LoxPsym site, wherein all vectors of the set of vectors comprise different LoxPsym sites. The nucleic acid sequence may be a promoter or promoter element, a terminator or terminator element, an exon or exon fragment, an intron or intron fragment, or any other regulatory, coding or non-coding DNA sequence.

[0075] in vivo and / or in vitro recombination methods As described herein, LoxPsym sites according to the present invention can be used in combination with Cre recombinase for site-specific recombination events of one or more nucleic acid sequences.

[0076] In another aspect, there is provided the use of any of the LoxPsym sites disclosed herein for in vivo and / or in vitro cloning.

[0077] More specifically, there is provided an in vivo or in vitro method for obtaining a recombinant nucleic acid sequence or molecule, said method comprising: - combining a nucleic acid sequence or molecule comprising at least two or more nucleic acid segments or elements and one or more site-specific recombinase recognition sites capable of being recognized by a recombinase with a recombinase that recognizes the site-specific recombinase recognition sites, such that the nucleic acid molecules or sequences recombine to provide a recombinant nucleic acid molecule; and - optionally determining the sequence of the recombinant nucleic acid molecule or sequence after introducing the recombinant nucleic acid molecule or sequence into a cell and / or determining its expression.

[0078] In one embodiment, the site-specific recombinase recognition site(s) are LoxPsym sites, and the recombinase is Cre recombinase, which recognizes the LoxPsym sites. In a specific embodiment, the LoxPsym sites are selected for the LoxPsym sites disclosed herein, more specifically, LoxPsym sites comprising or consisting of SEQ ID NOs: 65-127. In another specific embodiment, the DNA segment can be a nucleic acid sequence encoding a protein or non-coding RNA, a promoter element, a terminator element, or any other regulatory nucleic acid sequence.

[0079] Also provided is a method for replacing a DNA element A with a different DNA element B, the method comprising the steps of: providing a nucleic acid molecule A' comprising, in this 5'-3' order, a LoxP site, a DNA element A and a LoxP site, and a nucleic acid molecule B' comprising, in this 5'-3' order, a LoxP site, a DNA element B and a LoxP site, wherein at least one LoxP site is a LoxPsym site according to the invention, more particularly a LoxPsym site comprising a nucleic acid sequence as represented in SEQ ID NOs: 65 to 127; - reacting nucleic acid molecules A' and B' in the presence of recombinase Cre to obtain a nucleic acid molecule in which DNA element A has been replaced by DNA element B.

[0080] In one embodiment, at least two, at least three, at least four, or all of the LoxPsym sites are selected from the LoxPsym sites disclosed herein. In a specific embodiment, the LoxPsym sites are selected from the list consisting of SEQ ID NOs: 65-91, 65-80, 65-76, 65-73, 92-127, 96-127, 98-127, and 99-127, and / or selected from the list consisting of SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121, and 124.

[0081] Methods for optimizing gene expression A particular application of the orthogonal LoxPsym sites disclosed herein is simultaneous in vivo cloning and phenotyping, preferably performed in the same cell.

[0082] More specifically, a method of optimizing gene expression of one or more genes in a cell is provided, the method comprising the steps of: a. expressing or introducing into a cell one or more vectors comprising one or more genes of interest under the control of a promoter, the promoter comprising two or more promoter elements, wherein the two or more promoter elements are individually or each flanked by orthogonal LoxPsym sites, wherein a different LoxPsym site is used for each gene or promoter; b. optionally, the one or more vectors comprise a terminator sequence downstream of the one or more genes of interest, the terminator sequence comprising two or more terminator elements, wherein the two or more terminator elements are individually or each flanked by orthogonal LoxPsym sites, wherein a different LoxPsym site is used for each gene, and wherein any of the LoxPsym sites from step b) is different from any of the LoxPsym sites used in step a); c. expressing the recombinase Cre in said cells; d. Analyzing the gene expression level of said gene of interest or analyzing the phenotype of the cell.

[0083] Also provided is a method of optimizing gene expression of one or more genes in a cell, the method comprising the steps of: a. expressing or introducing into a cell one or more vectors comprising one or more genes of interest under the control of a promoter, wherein the one or more vectors comprise a terminator sequence downstream of the one or more genes of interest, the terminator sequence comprising two or more terminator elements, wherein the two or more terminator elements are individually or each flanked by orthogonal LoxPsym sites, and wherein a different LoxPsym site is used for each gene or for each terminator; b. optionally, the promoter comprises two or more promoter elements, wherein the two or more promoter elements are individually or each flanked by orthogonal LoxPsym sites, wherein a different LoxPsym site is used for each gene, and wherein any of the LoxPsym sites from step b) is different from any of the LoxPsym sites used in step a); c. expressing the recombinase Cre in said cells; d. Analyzing the gene expression level of said gene of interest or analyzing the phenotype of the cell.

[0084] In one embodiment, the orthogonal LoxPsym site is any of the LoxPsym sites disclosed herein, more particularly, a site of the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3' a LoxPsym site selected from the group consisting of: wherein the spacer is selected from the list consisting of SEQ ID NOs: 1 to 63, and wherein, in the presence of recombinase Cre, specific DNA recombination between a LoxPsym site and another LoxPsym site comprising a spacer having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3' and selected from the list consisting of SEQ ID NOs: 1 to 63 cannot occur if the LoxPsym site contains different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, except when the LoxPsym site contains the spacer combination GGGTACCC-AACTAGTT, GGGTACCC-ATATATAT, GAATATTC-AGTTAACT, GAATATTC-AGTTAACT or GTGTACAC-GCATATGC.

[0085] In another embodiment, the orthogonal LoxPsym site is a LoxPsym site having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from the list consisting of SEQ ID NOs: 1-63, and wherein, in the presence of recombinase Cre, specific DNA recombination between a LoxPsym site and another LoxPsym site having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT and comprising a spacer selected from the list consisting of SEQ ID NOs: 1-63 can occur only if the spacers from both LoxPsym sites contain the same nucleotide sequence between positions 2 and 7 of the spacer, and cannot occur if the LoxPsym sites contain different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, with the exception of the spacer combinations GGGTACCC-AACTAGTT, GGGTACCC-ATATATAT, GAATATTC-AGTTAACT, GTGTACAC-GCATATGC.

[0086] In another embodiment, the orthogonal LoxPsym site is selected from any of the sets of LoxPsym sites disclosed herein, more specifically, a set of LoxPsym sites comprising at least two LoxPsym sites having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from the list consisting of SEQ ID NOs: 1 to 63, and wherein at least two LoxPsym sites comprise different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, and wherein the set does not include LoxPsym sites represented in SEQ ID NOs: 88 and 89, SEQ ID NOs: 88 and 81, SEQ ID NOs: 90 and 83, SEQ ID NOs: 65 and 92, or SEQ ID NOs: 78 and 103.

[0087] In specific embodiments, the orthogonal LoxPsym site is selected from SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and / or 124, or is selected from the list consisting of SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and 124.

[0088] In one aspect, the method further provides for sequencing the cells or determining the sequence of the cells, more particularly determining the sequence of the recombined nucleic acid molecules responsible for the phenotype.

[0089] "Phenotype," as used herein, includes, but is not limited to, cell proliferation, reproductive fitness, synthesis of one or more compounds, detectable markers, or any other observable characteristic.

[0090] "Individually flanking" or "each flanking," as used herein, means that a nucleic acid (e.g., a promoter or terminator element) contains additional elements, such as LoxPsym sites, at both the 5' and 3' ends.

[0091] Also provided is a method of shuffling DNA elements, the method comprising the steps of: providing a nucleic acid molecule comprising at least two DNA elements each flanked by orthogonal LoxP sites; - reacting the nucleic acid molecule with the recombinase Cre to obtain a nucleic acid molecule in which at least two DNA elements have been reshuffled.

[0092] In one embodiment, the nucleic acid molecule is a gene promoter, and at least two DNA elements are promoter elements. In another embodiment, the nucleic acid molecule is a terminator sequence, and at least two DNA elements are terminator elements. In yet another embodiment, the nucleic acid molecule is a protein-coding or non-coding gene, and at least two DNA elements are introns and / or exons.

[0093] In another embodiment, the orthogonal LoxP site is any of the LoxPsym sites disclosed herein, more particularly, the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT and a LoxPsym site selected from the group consisting of: wherein the spacer is selected from the list consisting of SEQ ID NOs: 1 to 63, and wherein, in the presence of recombinase Cre, specific DNA recombination between a LoxPsym site and another LoxPsym site comprising a spacer having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT and selected from the list consisting of SEQ ID NOs: 1 to 63 cannot occur if the LoxPsym site contains different nucleotides at positions 2, 3, 6, and / or 7 of the spacer, except when the LoxPsym site contains the spacer combinations GGGTACCC and AACTAGTT, GGGTACCC and ATATATAT, GAATATTC and AGTTAACT, GAGTACTC and AAATATTT, and / or GTGTACAC and GCATATGC.

[0094] In another embodiment, the orthogonal LoxPsym site is a LoxPsym site having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT, wherein the spacer is selected from the list consisting of SEQ ID NOs: 1-63, and wherein, in the presence of recombinase Cre, specific DNA recombination between a LoxPsym site and another LoxPsym site having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT and comprising a spacer selected from the list consisting of SEQ ID NOs: 1-63 can occur only if the spacers from both LoxPsym sites contain the same nucleotide sequence between positions 2 and 7 of the spacer, and cannot occur if the LoxPsym sites contain different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, with the exception of the spacer combinations GGGTACCC and AACTAGTT, GGGTACCC and ATATATAT, GAATATTC and AGTTAACT, GAGTACTC and AAATATTT, and / or GTGTACAC and GCATATGC.

[0095] In another embodiment, the orthogonal LoxPsym site is selected from any of the sets of LoxPsym sites disclosed herein, more specifically, a set of LoxPsym sites comprising at least two LoxPsym sites having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from the list consisting of SEQ ID NOs: 1 to 63, and wherein at least two LoxPsym sites comprise different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, and wherein the set does not include LoxPsym sites represented in SEQ ID NOs: 88 and 89, SEQ ID NOs: 88 and 81, SEQ ID NOs: 90 and 83, SEQ ID NOs: 65 and 92, or SEQ ID NOs: 78 and 103.

[0096] In specific embodiments, the orthogonal LoxPsym site is selected from SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and / or 124, or is selected from the list consisting of SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and 124.

[0097] Also provided is a method for modifying expression of a gene, the method comprising the steps of: - providing a nucleic acid molecule comprising a gene operably fused to a promoter, the promoter comprising two or more promoter elements each flanked by orthogonal LoxPsym sites; - Optionally, the nucleic acid molecule comprises a terminator sequence downstream of the gene, wherein the terminal sequence comprises two or more terminator elements each flanked by an orthogonal LoxPsym site; - Reshuffling the promoter elements, and optionally the terminator elements, by reacting the nucleic acid molecule with the recombinase Cre.

[0098] In one embodiment, the orthogonal LoxPsym site is any of the LoxPsym sites disclosed herein, more particularly, the site of the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3' a LoxPsym site selected from the group consisting of: wherein the spacer is selected from the list consisting of SEQ ID NOs: 1 to 63, and wherein, in the presence of recombinase Cre, specific DNA recombination between a LoxPsym site and another LoxPsym site comprising a spacer having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3' and selected from the list consisting of SEQ ID NOs: 1 to 63 cannot occur if the LoxPsym site contains different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, except when the LoxPsym site contains the spacer combination GGGTACCC-AACTAGTT, GGGTACCC-ATATATAT, GAATATTC-AGTTAACT, GAATATTC-AGTTAACT or GTGTACAC-GCATATGC.

[0099] In another embodiment, the orthogonal LoxPsym site is a LoxPsym site having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from the list consisting of SEQ ID NOs: 1-63, and wherein, in the presence of recombinase Cre, specific DNA recombination between a LoxPsym site and another LoxPsym site having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT and comprising a spacer selected from the list consisting of SEQ ID NOs: 1-63 can occur only if the spacers from both LoxPsym sites contain the same nucleotide sequence between positions 2 and 7 of the spacer, and cannot occur if the LoxPsym sites contain different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, with the exception of the spacer combinations GGGTACCC-AACTAGTT, GGGTACCC-ATATATAT, GAATATTC-AGTTAACT, GTGTACAC-GCATATGC.

[0100] In another embodiment, the orthogonal LoxPsym site is selected from any of the sets of LoxPsym sites disclosed herein, more particularly, a set of LoxPsym sites comprising at least two LoxPsym sites having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from the list consisting of SEQ ID NOs: 1 to 63, and wherein at least two LoxPsym sites comprise different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, and wherein the set does not include LoxPsym sites represented in SEQ ID NOs: 88 and 89, SEQ ID NOs: 88 and 81, SEQ ID NOs: 90 and 83, SEQ ID NOs: 65 and 92, or SEQ ID NOs: 78 and 103.

[0101] In specific embodiments, the orthogonal LoxPsym site is selected from SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and / or 124, or is selected from the list consisting of SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and 124.

[0102] In some embodiments of the methods described herein, the activity of the recombinase is stopped by manipulating the in vitro reaction by applying heat or a denaturant or chelator. In certain embodiments of the present invention, the methods provided herein include the additional step of introducing a recombinant polynucleotide into a cell, more specifically a plant cell, an insect cell, a mammalian cell, or a microorganism, to obtain a genetically modified cell, and determining the function of the recombinant polynucleotide by analyzing the genetically modified cell. In certain embodiments, the modified cell is a microorganism, even more specifically a bacterium or yeast, most specifically a yeast of the genus Saccharomyces, Yarrowia, or Pichia. In another specific embodiment, the modified cell is a cell of a species selected from Saccharomyces cerevisiae, Escherichia coli, Zea mays, and Yarrowia lipolytica. In specific embodiments, the methods provided herein include the additional step of determining the sequence of the recombinant polynucleotide and / or one or more functions of the protein or functional RNA encoded by the recombinant polynucleotide.

[0103] The present application also provides recombinant polynucleotides produced by any of the methods described herein, and cells, more particularly microorganisms such as yeast or bacteria, comprising any of the polynucleotides produced by the methods described herein.

[0104] In one approach, the present disclosure includes the use of the Cre-LoxPsym system described herein in combination with one or more other recombination systems selected from the list consisting of: Flp recombinase, which functions in the Flp / FRT system; Dre recombinase, which functions in the Dre-rox system; Vika recombinase, which functions in the Vika / vox system; Bxb1 recombinase, which functions with attP and attB sites; long terminal repeat (LTR) site-specific recombinase (Tre); and other serine recombinases, such as phiC31 integrase, which mediates recombination between two 34 base pair sequences called attachment sites (att); Hin recombinase, which recognizes 26 bp imperfect inverted repeats; or int2-13, which each recognize distinct target sites of 39-66 bp.

[0105] The present application also provides cell lines containing multiple landing pads integrated into the genomic DNA of a parent cell line. The parent cell line may be a wild-type cell line or a cell line with existing genomic modifications. In the latter case, the cell line will serve as the "parent" of a cell line generated from further modification of its genomic DNA. A "landing pad" is a foreign DNA sequence integrated into a location in the host genome that contains a LoxPsym site. In some cases, the foreign DNA sequence includes a LoxPsym site, a constitutive promoter operably linked to a nucleotide sequence encoding a detectable marker, followed by a nucleotide sequence encoding a first selectable marker. In one type of landing pad, the LoxPsym site is located between the promoter and the nucleotide sequence encoding the detectable protein. When more than one landing pad is used in a given cell, it is preferred that the LoxPsym site of one landing pad be orthogonal to the LoxPsym site of any other landing pad. The landing pad is used for further genetic manipulation and integration of a nucleic acid molecule of interest via site-specific recombination. Landing pads can be integrated into the parent genome using any method known in the art, for example, by using zinc finger nucleases, TALEN, or CRISPR-Cas systems. In some embodiments, the number of landing pads integrated into the cell line is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the LoxPsym site in the landing pad is selected from SEQ ID NOs: 65-127, and most particularly selected from SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121, and / or 124. In some embodiments, the detectable marker in the landing pad is a fluorescent protein, such as eGFP, eYFP, eCFP, mKate2, mCherry, mPlum, mGrape2, mRaspberry, mGrapel, mStrawberry, mTangerine, mBanana, and mHoneydew, luciferase, or LacZ.In some embodiments, the selectable marker hydrolyzes a drug such as puromycin, hygromycin, G418, neomycin, or bleomycin.

[0106] Also provided herein is a method for integrating a gene circuit or multiple gene circuits into a cell containing multiple landing pads. In some embodiments, one gene circuit is integrated into a cell line. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more circuits may be integrated into the cell line, provided that the number of landing pads in the cell line is sufficient to accommodate the number of gene circuits to be integrated into the cell line. In some cases, it may be preferable that the number of landing pads is at least the number of gene sequences or circuits to be integrated. In other cases, a single landing pad can encompass multiple circuits under the control of different promoters. As used herein, a "gene circuit" refers to a rationally designed artificial gene regulatory network with robust function, comprising primary genetic elements or building blocks. Non-limiting examples of primary genetic elements include promoters, ribosome binding sites, transcriptional activators and repressors, gene coding sequences, 5'UTRs, 3'UTRs, polyA signals, and terminators. Independent modules of genetic circuits can be constructed using primary genetic elements. Methods for constructing these genetic circuits are known to those skilled in the art. In some embodiments, multiple landing pads may be integrated into different locations of the genome, allowing modification of multiple loci of the genome via site-specific recombination.

[0107] In addition to the above detailed description of the invention, the following experimental details will further enable one skilled in the art to practice all details of the present invention.

[0108] example Example 1. New LoxPsym recombination sites strongly affect recombination efficiency in Saccharomyces cerevisiae. Similar to the development of the original LoxPsym site by Hoess et al. (1986 J Cell Biochem), we edited the spacer of the LoxP site to obtain new non-directional recombination sites. In doing so, we focused on altering the first three nucleotides of the spacer (Figure 2). This is because it was previously shown that changing the nucleotides at positions 4 and 5 of the spacer (T and A, respectively) prevents recombination (Hoess et al. 1986). The last three nucleotides were adapted accordingly to ensure that the spacer maintained its palindromic nature. Thus, we were able to design up to 63 new potential LoxPsym sites. To test whether the Cre enzyme could still recombine the new LoxPsym variants, we performed a fluorescence-based assay in yeast via genomic integration of a fluorescent cassette flanked by identical LoxPsym variants at both sites (Figure 3a, c). Recombination of the functional LoxPsym variants allows deletion of the fluorescent cassette. The proportion of non-fluorescent cells in the population after induction of Cre-mediated recombination was used to determine the recombination efficiency of the new LoxPsym site. To verify whether this setup is effective for estimating recombination efficiency, we assessed the frequency of inversions / deletions over time using the dual-fluorescence reporter system shown in Figure 3a. Measurements after 0, 6, 10, and 24 hours of induction showed that the frequency of inversions was negligible compared to deletions over time, thus demonstrating the validity of the reporter system for determining recombination efficiency (Figure 3d). Recombination efficiencies varied significantly among the different LoxPsym variants (Table 1). Sixty-three new LoxPsym variants were obtained, with recombination efficiencies ranging from 13% to 89%. Interestingly, the 36 new LoxPsym sites exhibited higher recombination efficiency than the original LoxPsym sites currently used by the research community (Hoess et al. 1986, Richardson et al. 2017), with a 1.9-fold increase in observed recombination events (Table 1).

[0109] Finally, we investigated whether the purine / pyrimidine content of the spacer or the distribution of purines / pyrimidines within the spacer region could explain the large variability in recombination efficiency among LoxPsym variants, but no clear relationship was observed (Fig. 3e).

[0110] Example 2. A large set of orthogonal recombination sites could be identified. After confirming that the newly developed LoxPsym site allows recombination in yeast, we next sought to identify orthogonal LoxPsym sites. Because the spacer is the target for cleavage and strand exchange during Cre-mediated recombination, we reasoned that a non-homologous spacer would prevent recombination. Therefore, we performed a similar assay to that described previously, except that in this case, the LoxPsym sites flanking the fluorescent cassette were different, upstream and downstream of the fluorescent marker (Figure 3b-c). To enable the identification of a large number of orthogonal sites, we evaluated the interactions between 48 LoxPsym variants. Specifically, this set contains all variants with a nucleotide T, A, or G at the first position of the spacer. Thus, an additional 1,008 S. cerevisiae strains were constructed to evaluate the interactions between different LoxPsym variants, and the recombination efficiencies are shown in Figure 3g. The high recombination efficiency, indicated by the diagonal lines in the matrix, indicates that recombination occurs not only between identical sites, but also between non-identical sites that differ only in the first and last nucleotides of the spacer (due to the palindromic nature of the LoxPsym site). Interestingly, these nucleotides still play a role in generating cross-reactivity between variants that differ at other positions in the spacer. For example, the recombination efficiency of LoxPsym variants and the spacer sequence AGT TAACG- GAA When recombination occurs between TATTC and LoxPsym spacer AGT TAACG- TAARecombination between TATTA and TATTA was prevented, thus indicating that the first nucleotide of the spacer cannot simply be ignored in the screen. Most importantly, by combining all the data, we were able to identify a large set of 16 orthogonal LoxPsym variants that were still functional but did not interact with each other. Finally, to verify the interactions between non-identical LoxPsym variants, we analyzed some strains in which the fluorescent cassette had been deleted via Sanger sequencing (Fig. 3h). Sequences of the remaining LoxPsym sites show that mismatches at the first and last nucleotides of both spacers consistently result in hybrid LoxPsym variants that contain traces from both parental sites and are therefore no longer fully symmetric. Mismatches between LoxPsym variants in the middle of the spacer do not result in a hybrid scar; rather, only the sequence of one of the two original LoxPsym sites can be found.

[0111] Example 3. Applying expression optimization tools to the astaxanthin production pathway in yeast can enhance strain performance. As one application of the orthogonal LoxPsym sites developed in this study, the new variants were used to shuffle promoter and terminator elements of genes. First, we evaluated whether combinations of several repeats of two orthogonal LoxPsym sites (in either the promoter or terminator construct) could diversify gene expression upon Cre recombinase induction. This was done by targeting a fluorescent reporter (yECitrine) and analyzing several single clones after recombination (Figure 4a - violin plot). We observed that shuffling both the promoter and the terminator could alter expression. Sequencing confirmed that this was achieved without cross-reactivity between the two orthogonal LoxPsym variants used (specifically, the spacers SEQ ID NOs: 6 and 20, and SEQ ID NOs: 70 and 84) (Figure 4b - sequencing results).

[0112] Next, we used a combination of 12 orthogonal LoxPsym variants (SEQ ID NOs: 69, 70, 74, 77, 84, 86, 88, 100, 108, 109, 114, and 124) to alter the expression of six genes in the astaxanthin production pathway in S. cerevisiae (the genes are indicated by the upper arrows in Figure 5A). Induction of recombination resulted in variations in the color of the strains due to changes in the production of intermediate carotenoids (e.g., lycopene, beta-carotene, and zeaxanthin) (Figures 5B-C). Several single clones were further analyzed for carotenoid content (Figures 5D and 5F) and gene expression levels of the six genes of interest (Figure 5E). Thus, this application demonstrates that multiple orthogonal sites can be simultaneously applied to simultaneously optimize the expression of multiple genes in vivo. This allows for the optimization of a desired phenotype, in this particular example, resulting in a >2-fold increase in astaxanthin production.

[0113] Example 4. A set of orthogonal LoxPsym variants can be applied for multiplex genome engineering.We further investigated the ability to simultaneously use all 16 orthogonal LoxPsym variants identified in previous assays. More specifically, we analyzed the application of all 16 sites present in the same genome while maintaining orthogonality. This is because this is an absolute requirement for applying these recombination sites to facilitate complex metabolic engineering efforts, for example, when several genomic loci are typically modified simultaneously or sequentially for gene insertion or deletion. Our study relied on 18 constructs, including 16 test constructs, each evaluating the function of one LoxPsym variant in the presence of all other variants, and two controls (Fig. 6a). To select for recombination in all cases, all constructs utilized a deletion of URA3 (resulting in resistance to 5-fluorouracil; FOA) by surrounding the marker with two LoxPsym-TCA sites and plating on SC+FOA. In addition, each construct encoded an ADE2 marker to determine the efficiency of each specific LoxPsym site in the presence of all other sites. Deletion of this marker resulted in red-colored yeast colonies, allowing for the detection of secondary recombination events. Two control constructs (different in the location of the ADE2 marker) displayed only one copy of each LoxPsym site (with the exception of LoxPsym-TCA, which allowed for the selection of recombination-positive clones), and no deletion of ADE2 was predicted. In contrast, each test construct contained an extra copy of one specific LoxPsym variant upstream of the ADE2 marker, and deletion of ADE2 was predicted and used as a readout for the recombination efficiency of that site (Figure 6b-c). LoxPsym-CAC displayed a very low recombination rate (0.9116 ± 0.6606%), indicating that recombination of this LoxPsym variant was reduced by the presence of the 15 other sites. Other LoxPsym variants showed higher activity, but recombination efficiencies were consistently lower and did not correlate well with those previously calculated, which may be a result of differences in the experimental setup.Notably, our results suggest that the genomic context of the site plays a major role, since the two sites showing the highest efficiency (LoxPsym-TTA and -TCA) were located at both ends of the LoxPsym array. Moreover, there was a negative correlation (R ) between efficiency and the distance to the end of the LoxPsym array. 2 =0.37, p-value=0.013), indicating that more LoxPsym variants prevent the desired recombination site from binding or finding its correct interaction partner, resulting in decreased efficiency. We speculate that the underlying cause of this observation is a combination of the correlation between recombination efficiency and the distance between interacting recombination sites (Hoess et al. 1985 Gene 40:325-329, Zheng et al. 2000 Molec. Cell. Biol. 20:648-55), a reduced ratio of Cre enzyme to its target site, and the formation of unproductive synapses between incompatible recombination sites, which may block recombination sites from recombining with functional interaction partners (Lee and Saito 1998 Gene 216:55-65, Fan 2012 Nucleic Acids Res. 40:6208-6222).

[0114] Additionally, because recombination between different LoxPsym sites results in reporters with different lengths, the design of the constructs allows for frequencies to be calculated (Figure 6). The measured PCR fragment lengths deviated from the expected size in only 9 of 208 (13 per test construct) randomly picked red colonies, indicating some level of cross-reactivity. To further ensure that recombination events corresponded to the expected pattern, we sequenced three PCR fragments per test construct and observed the expected result in all but one case.

[0115] Example 5. LoxPsym variants are functional and orthogonal in prokaryotes and higher eukaryotes. In addition to identifying and multiplexing a set of 16 orthogonal LoxPsym variants in yeast, we further analyzed the potential of these novel recombination sites as functional and orthogonal tools in other species, particularly Escherichia coli and Zea mays. To assess the function and cross-reactivity of LoxPsym variants in E. coli, we set up a plasmid-based assay to test pairwise combinations between 16 donor and acceptor plasmids, each carrying a single LoxPsym variant (Figure 8a-b). After inducing recombination, we detected cross-reactivity between LoxPsym variants via PCR amplification of the junctions spanning the recombined recombination sites. All tested LoxPsym variants exhibited recombination activity in bacterial cells, but no correlation was observed with the activity of the respective sites in yeast and plants (Figure 8c). In contrast to the data obtained for S. cerevisiae, we observed cross-reactivity in some cases. Sequencing of these recombined scars revealed up to three mutations in the LoxPsym site resulting from recombination between cross-reactive partners. Indeed, it has previously been observed that results demonstrating orthogonal recombination are not always transferable between prokaryotes and eukaryotes. This may be caused by slight modifications in the protein structure or activity of recombinases in different host organisms or by differences in native cellular processes (such as the DNA mismatch repair pathway involved in repairing mismatches that appear after recombination). Additionally, differences in the experimental setup may also explain the observed differences: for experiments in yeast, recombination (deletion) was irreversible, whereas in bacteria, recombination of the two plasmids was reversible. Importantly, cross-reactivity was much lower than the recombination activity observed between identical LoxPsym sites.

[0116] To characterize cross-reactivity in higher eukaryotes, we used Zea mays, one of the most important cereal crops with a wide range of applications in food and feed, as well as industrial applications. We performed a plasmid-based assay using maize mesophyll protoplasts. Briefly, we constructed a library of plasmids containing two LoxPsym sites separated from each other by a short linker incorporating two restriction sites for digestion of the plasmid later in the workflow (Figure 7a-b). Each LoxPsym variant was labeled with a unique barcode, and sequencing the barcodes surrounding the recombined LoxPsym sites allowed us to identify which LoxPsym pairs were involved in the recombination process. Using a combinatorial cloning scheme, all 256 combinations of the 16 LoxPsym variants were present in the final plasmid pool and confirmed by NGS sequencing. Plasmid pools and plasmids constitutively expressing Cre recombinase or empty backbones were co-transfected into maize mesophyll protoplasts, and regions spanning the LoxPsym site(s) were amplified by PCR 48 hours posttransfection. Recombination was detected only in the presence of Cre recombinase, and the reactions were sent for NGS sequencing to assess the efficiency of recombination for each LoxPsym pair by identifying barcode frequencies in the pool (normalized to abundance in the starting pool) (Figure 7c). Results confirmed the activity of recombination in the higher eukaryote Z. mays and demonstrated the absence of cross-reactivity among selected LoxPsym variants. Moreover, despite the weak correlation to efficiency we observed in yeast (R 2 = 0.02, p value = 0.6056), a wide range of recombination efficiencies associated with various LoxPsym were detected.

[0117] In conclusion, we used pairwise interaction assays to assess cross-reactivity among a selection of LoxPsym variants and identified a set of 16 orthogonal LoxPsym variants that can be used simultaneously with no or minimal cross-reactivity. We showed that the sites described herein can also be used in other species, including E. coli and Z. mays. Together, these findings dramatically expand the potential for using Cre-LoxPsym as a gene editing technique, for example, in strain construction in metabolic engineering efforts, particularly when recursive and / or multiplexed recombination is desired.

[0118] Example 6. LoxPsym variants are also functional and orthogonal in Y. lipolytica . To demonstrate that the alternative LoxPsym sites (Table 2) are orthogonal in Y. lipolytica, we transformed the wild-type strain W29 with a construct consisting of 16 LoxPsym sites separated by spacer sequences (100 bp). The construct had a total length of 2284 bp and was targeted at the URA3 locus (Figure 9A). After precise integration of this construct, the resulting strain was subsequently transformed with a Y. lipolytica replicable plasmid containing an expression cassette for Cre recombinase (codon-optimized for S. cerevisiae) and a NAT selectable marker conferring resistance to neomycin (Figure 9A). Resistant colonies were then screened by PCR for Cre-mediated recombination. The primers used were designated 246-F (TGGTTTAGTGTATGTTGCGC) and 247-R (CTAAGTCTGTGCTCCTTCC), which flank a construct with 16 LoxPsym sites. If the sites are orthogonal, Cre-mediated recombination will not occur, and the primers will amplify a single product of 2284. If some of the LoxPsym sites cross-react, smaller bands of various sizes will appear after PCR screening, depending on the specific loxPsym sites that were recombined. A total of 192 transformants were screened after transient expression of Cre recombinase. In all cases, a single band of approximately 2.3 kb was amplified (Figure 9B), indicating that the alternative loxPsym sites are orthogonal in Y. lipolytica.

[0119] One of the most common uses of the Cre / lox system is marker recycling. In this case, a selectable marker is flanked by LoxP sites, and after transient expression of Cre recombinase, the selectable marker can be looped out and used for the next round of transformation. This Cre-mediated recombination leaves behind LoxP sites that can cross-react with newly inserted LoxP sites in the next round, resulting in gene rearrangements (Steensels et al. 2018, Nat Commun 9: 1937). To further test whether the new LoxPsym sites are orthogonal and can be used for multiple rounds of marker curation without the risk of genome scrambling (Steensels et al., 2018, Nat Commun 9: 1937), we checked the efficiency of looping out the selectable marker hph, which confers resistance to hygromycin, using four different LoxPsym sites (loxP, LoxPSym0, LoxPSym2, and LoxPSym4; Figure 10A; Table 2). We repeated these experiments three times, and each time, we confirmed 192 colonies for resistance to hygromycin (by growing them on plates supplemented with 100 μg / ml hygromycin B). The average frequencies of marker excision were 81.9% for LoxPSym0, 80.7% for LoxPSym2, 80.6% for LoxPSym4, and 79.3% for loxP (Figure 10B). The results indicate that alternative LoxPSyms are orthogonal, have high frequencies of Cre-mediated recombination, and can therefore be combined without risk of cross-reactivity when multiple rounds of genetic engineering and subsequent marker recycling are required.

[0120] Table 2. Orthogonal LoxPsym sites in Yarrowia lipolytica . [Table 2]

[0121] material and method Molecular methods. DNA amplification was performed by PCR using SapphireAmp Fast PCR Mix (Takara Bio) and Phusion (NEB) or GXL (Takara Bio) DNA polymerase. DNA oligonucleotides were obtained from Integrated DNA Technologies (IDT). Synthesis of longer DNA constructs was ordered from Qinglan Biotech, BGI. The pV1382 backbone (Addgene Plasmid #111436) was used to express sgRNAs, which were ligated into the BsmBI-digested backbone after oligonucleotide annealing as previously described (Vyas et al. 2018 mSphere 3:e00154-1). Plasmids reported in this study were constructed using Gibson Assembly (NEBuilder HiFi DNA Assembly Master Mix) for plasmids used in E. coli and S. cerevisiae, and Golden Gate cloning (GreenGate cloning standard reported by Lampropoulos et al. 2013 PLoS One 8: e83043) for plasmids used in Z. mays. Plasmids required for experiments in yeast and bacteria were purified using the QIAprep Spin Miniprep Kit (Qiagen). Plasmids required for experiments in plant cells were purified using the ZymoPURE II Plasmid Midiprep Kit (Zymo Research). Sanger sequencing was performed by Eurofins Genomics.

[0122] Strains and growth conditions. E. coli strains were constructed from the laboratory strain DH5α (NEB). Cells were grown in Luria Bertani (LB) medium (10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract) at 37°C with shaking at 200 rpm. Antibiotics (chloramphenicol, carbinicillin, and kanamycin) were added at 50 μg / mL. The inducer L-rhamnose was added at 2%. S. cerevisiae strains were constructed from the laboratory strain BY4741, a derivative of S288C with the genotype MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0. Cells were grown in synthetic complete (SC) medium (0.79 g / L SCM, 6.7 g / L YNB) or SC-histidine medium. Carbon sources (glucose, raffinose, and galactose) were added at 2%. Z. mays protoplasts (cv. B104) were isolated as previously described and suspended in W5 solution; see below (Gaillochet et al. 2023 Genome Biol. 24:6).

[0123] S. cerevisiae transformation protocol. One mL of overnight (ON) culture in 2x YPD (20 g / L yeast extract, 40 g / L peptone, 4 g / L glucose) was inoculated into 50 mL of 2x YPD for 3 hours. The cells were centrifuged (3 min, 3000 rpm) and washed successively with 10 mL and 1 mL of 0.1 M lithium acetate (LiOAc). The cells were resuspended in 100 μL of 0.1 M LiOAc. PCR-amplified donor DNA (50 μL) and / or plasmid DNA (200 ng) was added. CRISPR / Cas9 was used for genomic DNA insertion using pV1382, into which the gRNA of interest had been inserted. A mixture containing 620 μL of 50% PEG3350, 4 μL of salmon sperm DNA, and 90 μL of 1 M LiOAc was added and mixed by vortexing. The cells were incubated at 30°C, 400 rpm, for 30 minutes. 100 μL of DMSO was added, followed by a 15-minute heat shock at 42°C. Cells were harvested by centrifugation (3 minutes, 3000 rpm) and washed with 5 mM CaCl2. Cells were incubated for a 3-hour recovery period at 30°C and 400 rpm, and finally plated onto selective medium. Positive transformants were identified using colony PCR (SapphireAmp Fast PCR Master Mix, TaKaRa) to amplify the desired insert junction using templates prepared by boiling clones in 50 μL of NaOH (0.02 M) at 99°C for 10 minutes.

[0124] Fluorescence assay and recombination induction in S. cerevisiae. The strain was derived from BY4741, which has constitutive expression of the fluorescent reporter mCherry (Smukalla, S. et al. 2008 Cell 135:726-737). To test LoxPsym variants, the strain carried an overexpressed yECitrine reporter gene, controlled by the TDH3 promoter and CYC1 terminator, flanked by two LoxPsym variants (inserted via LoxPsym-tailed primers) and integrated into the genome at the CAN1 locus. A single colony was inoculated into 100 μL of SC-His 2% glucose for ON growth. Cells were washed and diluted to a final OD of 0.05 in SC-His 2% raffinose and grown ON. Cells were washed and diluted in SC-His 2% raffinose, 2% galactose for induction of Cre expression from a control backbone (no Cre) and a plasmid carrying the pGAL1-Cre expression cassette. Cells were induced for 6 hours unless otherwise indicated. Cells were washed and diluted (1 / 20 dilution) against SC 2% glucose for recovery in ON, after which cells were plated onto YPD and / or used for flow cytometry analysis.

[0125] Fluorescence analysis. Flow cytometry was performed using an Attune NxT flow cytometer and autosampler. Cultured yeast cells were diluted in focusing fluid and measured at a flow rate of 200 μL / min. Cytometry data were gated based on the FSC-H to FSC-A map to select for single cells. For determination of recombination efficiency, additional gating was performed using the control fluorescent reporter mCherry (mCherry+ cells were selected for further analysis). yECitrine and mCherry were measured using channels BL1-A (excitation at 488 nm and emission at 574 nm, 20 nm bandwidth) and YL2-A (excitation at 561 nm and emission at 610 nm, 20 nm bandwidth), respectively. Analysis and gating steps were performed using FlowJo software with a (non-)fluorescent control strain as a reference. To remove noise from the data, recombination efficiencies lower than the reference were set to zero. To determine the yECitrine fluorescence of single clones, single colonies were inoculated into SC 2% glucose and fluorescence was measured using a plate reader (TECAN Infinite 200 Pro) with excitation at 498 nm with a 9 nm bandwidth and emission at 535 nm with a 20 nm bandwidth. Data were acquired after normalization by absorbance at 600 nm. Fluorescent / non-fluorescent groups were determined by comparison with values ​​obtained for the control strain.

[0126] Multiplexed LoxPsym assay and recombination induction in S. cerevisiae. Strains derived from BY4741 carrying the pADE2-ADE2-tADE2 deletion were constructed using sgRNA3, with test and control (Figure 6a) inserted at the CAN1 locus and either P1 (Cre) or P2 (control). Similar recombination induction was performed as described above. After ON recovery in SC 2% glucose, cells were plated onto SC and SC + FOA and incubated at 30°C for 48 hours, after which colonies were counted for each plate. Red colonies were selected for PCR amplification of the recombined construct. Amplicon length was determined using capillary electrophoresis (QIAxcel Advanced instrument, QIAxcel DNA Screening Cartridge, QX Size Marker 250 bp - 4 kb v2.0) to visualize slight differences in band length.

[0127] E. coli transformation protocol. For heat shock transformation, chemically competent E. coli cells were thawed on ice for 30 minutes. Plasmid DNA (50–100 ng) or 2 μL of the Gibson / Golden Gate reaction was mixed with 25 μL of competent cells in an ice-cold 1.5 mL Eppendorf tube. After 30 minutes of incubation on ice, the reaction was heat-shocked at 42°C for 30 seconds and cooled on ice for 5 minutes. A volume of 300 μL of SOC medium was added, and the tube was incubated at 37°C in a shaking incubator for 60 minutes. Finally, 100 μL of cells were plated onto prewarmed (37°C) LB medium containing the appropriate antibiotic and incubated at 37°C for growth in ON. For electroporation, we used 2 × 10 10Commercially available NEB 10β cells (NEB) with a transformation efficiency of 100 cfu / μg were used. 2 μL of the assembly reaction mixture was mixed with 50 μL of competent cells and placed inside a chilled electroporation cuvette (0.2 cm gap, BioRad). Electroporation was performed in a GenePulser (BioRad) according to the manufacturer's conditions, after which 900 μL of SOC medium was immediately added to the cells. The cells were incubated at 37°C in a shaking incubator for 60 minutes. Finally, 100 μL of cells were plated per preheated (37°C) LB plate containing the appropriate antibiotic.

[0128] Recombination assay in E. coli. The bacterial strain was derived from DH5α after cotransformation of the acceptor and donor plasmids using a double-selective medium of LB + kanamycin (Kan) + chloramphenicol (Cm). A single colony was inoculated into 100 μL of LB + Kan + Cm for growth in ON. The cells were washed and diluted (1 / 20) in LB 2% rhamnose + Kan + Cm for induction of Cre expression from the acceptor plasmid (under the control of the rhaB promoter). After 4 hours of induction, the cells were washed and grown in LB + Kan + Cm in ON. The harvested cells were collected by centrifugation (3500 rpm, 5 minutes) and suspended in dH2O. The cells were boiled at 99°C for 10 minutes, and the remaining mixture was used as a template for PCR to amplify the junction of the recombined donor and acceptor plasmids. We reasoned that amplifying one of the two possible recombined junctions (the donor plasmid can insert into the acceptor plasmid in two orientations) was sufficient because recombination between symmetric sites should not favor one of both options, and the combination of two independent plasmids avoided the accumulation of single recombination outcomes. Amplicons were subjected to densitometric analysis using Image J software to extract peak areas (from lane plots). Junction peak areas were normalized by division by the area extracted from the most abundant control amplicon (derived from PCR performed on separate donor and acceptor plasmids).

[0129] Combinatorial LoxPsym library construction for assays in Z. mays. For the construction of the LoxPsym combinatorial library, we applied Golden Gate cloning using the GreenGate cloning standard (Lampropoulos et al. 2013 PLoS One 8: e83043) to assemble five entry clones. Entries A and E were constructed by ligating annealed oligonucleotides into BsaI-digested entry vectors pGGA000 (Addgene #48856) and pGGE000 (Addgene #48860), respectively. Entries for the 16 barcode-LoxPsym combinations at positions B and D were generated in the same manner using oligonucleotides. The linker at position C was PCR amplified from the pUC19 plasmid (Addgene #50005). After gel purification using the Zymoclean Gel DNA Recovery Kit, the purified product was combined with pGGC000 (Addgene #48858) in a Gibson assembly reaction using the NEBuilder Master Mix (NEB). For the final Golden Gate reaction of the LoxPsym combinatorial library, all entries were pooled, and entries B and D contained a mixture of all LoxPsym variant plasmids at equal concentrations (16 plasmids / position; quantified by Qubit™ dsDNA HS) to create a combinatorial library of plasmids containing 256 different LoxPsym combinations, which were then transformed into DH10B cells. After overnight incubation, colonies (>50,000 colonies) from nine different plates were scraped and suspended in LB medium. Plasmid DNA was extracted with the ZymoPURE II Plasmid Midiprep Kit (Zymo Research). Plasmids were diluted to 1 μg / μL. A plasmid expressing Cre recombinase was also constructed using Golden Gate, starting from available parts ( https: / / gatewayvectors.vib.be / ), and similarly purified and diluted.

[0130] Isolation and transfection of Z. mays protoplasts. Maize protoplast isolation and transfection were performed as previously described (Gaillochet et al., 2023 Genome Biol. 24:6). Transfections were performed by transfecting 100 μL of protoplasts (10 5 Transfections were performed using 100 μL of 10 ...

[0131] Z. mays DNA extraction. A modified Edwards extraction protocol was used to isolate protoplast DNA (Edwards et al., 1991, Nucleic Acids Res. 19:1349). The extraction buffer consisted of 100 mM Tris-HCl (pH 8), 500 mM NaCl, 50 mM EDTA, and 0.7% SDS. The protoplasts were transferred to a 1.5 mL Eppendorf tube and spun down at 12,000 rcf for 5 minutes, after which the supernatant was removed. A volume of 200 μL of extraction buffer was added to the Eppendorf tube, and the tube was manually shaken to dissolve the pellet. After incubation at 60°C for 15 minutes, the tube was cooled to room temperature. A volume of 200 μL of 100% isopropanol was added, and the tube was spun down at 12,000 rcf for 10 minutes. The supernatant was removed, and the pellet was washed with 200 μL of 80% ethanol. After air-drying for 15 min, the pellet was dissolved in 20 μL of 10 mM Tris-HCl, pH 8 (preheated to 60° C.). After incubation of the tubes in a 60° C. thermoblock for 10 min, the tubes were stored at −20° C. until further processing.

[0132] Next-generation sequencing. To sequence the input plasmid library for Z. mays transfection, we set up a 40 μL PCR reaction using the Phire Plant Direct PCR Kit (Thermo Scientific) with 4 μL of diluted miniprep (100 ng / μL) as template and primer OF / R82. The following PCR conditions were used: 98°C / 2 min + 10× (98°C / 5 s + 62°C / 5 s + 72°C / 10 s) + 72°C / 2 min + 23°C / ∞. Fragments of the correct size (approximately 270 bp) were purified using the Zymoclean Gel DNA Recovery Kit according to the manufacturer's instructions. A similar setup was used for sequencing the protoplast assay fragments, using 4 μL of protoplast DNA as template and primers with different demultiplexing tags for each sample in a total reaction volume of 40 μL. The PCR conditions used were as follows: 98°C / 2 min + 25×(98°C / 5 s + 62°C / 5 s + 72°C / 10 s) + 72°C / 2 min + 23°C / ∞. We were unable to detect any evidence of recombination in our agarose gel electrophoresis results, and reasoned that this could be due to the vast amount of transfected plasmid DNA (approximately 32 million plasmid copies per protoplast). Therefore, we used restriction enzyme digestion of extracted DNA to specifically cleave the C-linker-D module of unrecombined plasmids and bias against amplification of these DNA species. Protoplast DNA was digested with NcoI-HF (NEB) and PvuI-HF (NEB) in CutSmart buffer at 37°C for 12 h. Amplicons were constructed using primers OF / R83-88 purified with the GeneJET PCR Purification Kit (Thermo Fisher) according to the manufacturer's instructions. Samples were sent to Eurofins (Germany) for adapter ligation and NGS sequencing (5 million paired reads, 2 × 150 bp).For each plasmid, the number of reads detected for protoplast DNA was normalized by the number of reads present in the input library.

[0133] Yarrowia lipolytica strains and culture conditions. The reference strain Y. lipolytica W29 was used in all experiments mentioned. Yeast was grown in yeast extract-peptone-dextrose (YPD) at 30°C / 220 rpm (if liquid culture was used) for 2-3 days. Selection reagents were added at the following concentrations: norseothricin (CloNAT), 220 μg / ml; hygromycin B, 100 μg / ml.

[0134] Transformation of yeast (Y. lipolytica). Transformation of Y. lipolytica was performed according to a previously described protocol (Abdel-Mawgoud and Stephanopoulos 2020, Metab Eng 62: 106-115) with minor modifications.

[0135] Colony picking and PCR screening. Transformed colonies were picked from the transformation plates and pinned onto selection plates using a PIXL Precision Microbial Colony Picker (Singer Instruments). Colony PCR screening was performed using the primer pair 246-F / 247-R with SapphireAmp fast PCR polymerase (Takara Bio).

Claims

1. A LoxPsym site comprising the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT, wherein the spacer is selected from the list consisting of SEQ ID NOs: 1-63.

2. The LoxPsym site of claim 1, wherein the LoxPsym site is cleaved in the presence of the recombinase Cre.

3. 3. The LoxPsym site of claim 1, comprising a nucleic acid sequence selected from the list consisting of SEQ ID NOs: 65 to 127.

4. A LoxPsym site according to any one of claims 1 to 3, characterized by a lower recombination efficiency than a standard LoxPsym site as represented in SEQ ID NO: 128, and wherein the LoxPsym site comprises a spacer selected from the list consisting of SEQ ID NOs: 1 to 27.

5. A LoxPsym site according to any one of claims 1 to 3, characterized by a higher recombination efficiency than a standard LoxPsym site as represented in SEQ ID NO: 128, and wherein the LoxPsym site comprises a spacer selected from the list consisting of SEQ ID NOs: 28 to 63.

6. A LoxPsym site as described in claim 1, wherein in the presence of recombinase Cre, specific DNA recombination cannot occur between the LoxPsym site and a second LoxPsym site as described in claim 1, and wherein the LoxPsym site and the second LoxPsym site contain different nucleotides at positions 2, 3, 6 and / or 7 of the spacer.

7. 7. The LoxPsym site of claim 6, wherein the LoxPsym site and the second LoxPsym site do not contain the spacers GGGTACCC and AACTAGTT, GGGTACCC and ATATATAT, GAATATTC and AGTTAACT, GAGTACTC and AAATATTT, or GTGTACAC and GCATATGC.

8. the LoxPsym site is selected from the list consisting of SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and / or 124, and / or selected from the list consisting of SEQ ID NOs: 70, 77, 82, 101-102, 105, 107, 109, 112, 114, 117-118, 123-124, 126 and / or 127, and / or selected from the list consisting of SEQ ID NOs: 65, 71-74, 84, 86, 88, 90, 103, 106-108, 111, 121 and / or 124, and / or 8. The LoxPsym site of claim 6 or 7, selected from the list consisting of sequence numbers 72, 74, 77-78, 80, 82, 84, 86, 88, 102, 106, 109, 114, 118, 123 and / or 127, and / or selected from the list consisting of SEQ ID NOs: 65, 70-71, 73, 80, 90, 101, 103, 105, 108, 111-112, 117, 121, 124 and / or 126, and / or selected from the list consisting of SEQ ID NOs: 69, 70, 74, 77, 84, 86, 88, 100, 108, 109, 114 and / or 124.

9. A vector comprising the LoxPsym site according to any one of claims 1 to 8.

10. A host cell comprising a LoxPsym site according to any one of claims 1 to 8 or a vector according to claim 9.

11. The host cell of claim 10 , wherein the host cell is a plant cell, a microorganism, an insect cell, or a mammalian cell.

12. The host cell of claim 11 , wherein the host cell is a yeast cell or a bacterial cell.

13. The host cell of claim 11, wherein the host cell is a cell of a species selected from the group consisting of Saccharomyces cerevisiae, Escherichia coli, Zea mays, and Yarrowia lipolytica.

14. A set of at least two LoxPsym sites according to claim 1, wherein at least two LoxPsym sites contain different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, and wherein the set does not include LoxPsym sites represented in SEQ ID NOs: 88 and 89, SEQ ID NOs: 88 and 81, SEQ ID NOs: 90 and 83, SEQ ID NOs: 65 and 92, or SEQ ID NOs: 78 and 103.

15. at least two LoxPsym sites are selected from the list consisting of SEQ ID NOs: 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and / or 124, and / or selected from the list consisting of SEQ ID NOs: 70, 77, 82, 101-102, 105, 107, 109, 112, 114, 117-118, 123-124, 126 and / or 127, and / or selected from the list consisting of SEQ ID NOs: 65, 71-74, 84, 86, 88, 90, 103, 106-108, 111, 121 and / or 124; and / or selected from the list consisting of SEQ ID NOs: 72, 74, 77-78, 80, 82, 84, 86, 88, 102, 106, 109, 114, 118, 123 and / or 127, and / or selected from the list consisting of SEQ ID NOs: 65, 70-71, 73, 80, 90, 101, 103, 105, 108, 111-112, 117, 121, 124 and / or 126, and / or selected from the list consisting of SEQ ID NOs: 69, 70, 74, 77, 84, 86, 88, 100, 108, 109, 114 and / or 124.

16. Use of a LoxPsym site according to any one of claims 1 to 8, or of a vector according to claim 9, or of a set of at least two LoxPsym sites according to claim 14 or 15, for site-specific recombination of one or more nucleic acid sequences.

17. Use of a LoxPsym site according to any one of claims 1 to 8, or of a vector according to claim 9, or of a set of at least two LoxPsym sites according to claim 14 or 15, for in vivo cloning and phenotyping.

18. 18. The use according to claim 17, wherein cloning and phenotyping are carried out sequentially in the same cell.

19. 1. A method for obtaining a recombinant nucleic acid molecule, comprising the steps of: a. providing a nucleic acid molecule comprising two or more nucleic acid elements, each flanked by orthogonal LoxPsym sites, or providing first and second nucleic acid molecules, each comprising one or more nucleic acid elements, each flanked by orthogonal LoxPsym sites; b. reacting the nucleic acid molecule or the first and second nucleic acid molecules with recombinase Cre to obtain a recombinant nucleic acid molecule; wherein the orthogonal LoxPsym site is selected from the list consisting of SEQ ID NOs: 65-127. The method comprising:

20. 1. A method for shuffling DNA elements within a nucleic acid molecule, comprising the steps of: a. providing a nucleic acid molecule comprising at least two nucleic acid elements, each flanked by orthogonal LoxPsym sites; b. reacting the nucleic acid molecule with recombinase Cre to obtain a nucleic acid molecule in which the nucleic acid elements have been reshuffled; wherein the orthogonal LoxPsym site is selected from the list consisting of SEQ ID NOs: 65-127. The method comprising:

21. The method of claim 19 or 20, wherein the orthogonal LoxPsym site is a LoxPsym site according to any one of claims 6 to 8.

22. The method of any one of claims 19 to 21, further comprising the step of sequencing the recombinant nucleic acid molecule.

23. The method of any one of claims 19 to 22, further comprising the step of introducing the recombinant nucleic acid molecule into a cell and / or determining the expression of the recombinant nucleic acid molecule in the cell.

24. A recombinant nucleic acid molecule obtainable by the method according to any one of claims 19 to 22.

25. 1. A method for optimizing gene expression of one or more genes in a cell, comprising the steps of: a. introducing into a cell one or more vectors comprising one or more genes, each gene under the control of a promoter, the promoter comprising two or more promoter elements, each flanked by orthogonal LoxPsym sites, wherein a different LoxPsym site is used for each gene; b. Optionally, the one or more vectors further comprise a terminator sequence downstream of each gene, the terminator sequence comprising two or more terminator elements, each flanked by orthogonal LoxPsym sites, wherein a different LoxPsym site is used for each gene, and wherein any of the LoxPsym sites from step b) is different from any of the LoxPsym sites used in step a); c. expressing the recombinase Cre in said cells; d. Analyzing gene expression of one or more genes or analyzing the phenotype of a cell The method comprising:

26. 26. The method of claim 25, wherein the at least one orthogonal LoxPsym site is selected from the list consisting of SEQ ID NOs: 65-127.

27. The method according to claim 25 or 26, characterized in that at least one orthogonal LoxPsym site is a LoxPsym site according to any one of claims 6 to 8.

28. 26. The method of claim 25, wherein all orthogonal LoxPsym sites are LoxPsym sites according to any one of claims 6 to 8.

29. The method of any one of claims 25 to 28, further comprising determining the sequence of all or part of the genome of the cell.

30. 30. The method of any one of claims 25 to 29, wherein the cell is a plant cell, a microorganism, an insect cell or a mammalian cell.

31. 30. The method of any one of claims 25 to 29, wherein the cell is a yeast cell or a bacterial cell.

32. A genetically modified cell obtainable by the method according to any one of claims 25 to 28.

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