Novel LoxPsym sites for large scale orthogonal CRE mediated recombination

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering and synthetic biology, and more specifically to means and methods for promoting genetic recombination. The present application discloses a novel Cre recombinase-dependent recombination site that supports a method for simultaneous cloning and testing. Background Art

[0002] In the past few decades, site-specific recombination has been shown to be a powerful tool in genetic engineering, developmental biology, and systems biology. This recombination system is very popular in multiple research fields because it can delete, invert, integrate, and translocate large amounts of genomic DNA in vivo in multiple host organisms (Meinke et al., 2016 Chem Rev 20: 12785-12820). Specifically, Cre ( C auses re Cre recombinase is one of the most commonly used recombination systems. As is well known, it has been implemented and characterized in a variety of inducible forms (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 it works 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 was originally derived from bacteriophage P1 and works by recognizing LoxP sites. This is a 34 bp long sequence consisting of two 13 bp inverted repeats flanked by an 8 bp directional spacer (Sternberg and Hoess 1983 Annu Rev Genet 17: 123-154). During the recombination process, the Cre recombinase binds to the two inverted repeats of the LoxP site as a dimer, cuts the spacer on both strands, and initiates strand exchange with another LoxP site (Guo et al., 2000 Genesis 32: 8-18). Depending on the direction and position of the LoxP site deletion, inversion or translocation of the DNA fragment can occur.

[0003] In order to achieve non-directional recombination, that is, to allow different recombination events to occur independently of the direction of the recombination site, the spacer x sequence of the LoxP site has been converted into a palindromic site by Hoess et al. (1986 Nucleic Acids Res 14: 2287-2300). This artificial recombination site, called LoxPsym, has recently attracted new interest because it plays an important role in the synthetic yeast genome project-Sc 2.0. The latter aims to build the world's first synthetic eukaryotic genome while enabling it to be highly evolved as needed. This is achieved by introducing thousands of LoxPsym sites into the genome of Saccharomyces cerevisiae, thereby quickly introducing genomic rearrangements when the inducible Cre recombinase is activated (Richardson et al., 2017 Science 355: 1040-1044).

[0004] Although the Cre-LoxPsym system is widely used, its use is limited to a single recombination system. Therefore, in recent years, a variety of methods have been carried out to obtain an orthogonal recombination system in an organism. Orthogonality allows genetic recombination to be carried out simultaneously, on a large scale and independently in different regions of the genome. Orthogonal recombination systems can not only carry out more complex genome engineering in synthetic biology, but they also have a wide range of 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 ( et al., 2022 Biosensors 12:122).

[0005] At present, orthogonal recombination is obtained by combining multiple tyrosine recombinases, which recognize different recombination sites and therefore work without producing cross-reactivity. In recent years, a variety of novel tyrosine recombinases orthogonal to Cre-LoxP have been found. These novel tyrosine recombinases include Vcre (Liu et al., 2018Nat Commun 9: 1936), SCre (Suzuki et al., 2011Nucleic Acid Res 39: e49) and Vika (Karimova et al., 2013Nucleic Acid Res 42: e37). Recently, the development of orthogonal non-directional recombination systems for Vika and Dre has also been disclosed (Wang et al., 2021iScience 25: 103716). However, the number of non-cross-reactive recombinases is still limited, and the use of multiple recombination systems in new host organisms requires heterologous expression of various enzymes, which may be toxic to the host. To address these deficiencies, we further developed an alternative strategy to obtain orthogonal recombination based on the existing knowledge, namely the development of non-cross-reactive LoxPsym recombination sites. Summary of the invention

[0006] In this application, the inventors disclose how they first characterized 63 new LoxPsym sites by editing the spacers of LoxPsym sites. Compared with the commonly used LoxPsym sites, many sites showed higher recombination efficiency. They then performed intensive screening to identify LoxPsym variants that can work without cross-reactivity and found that multiple sets of orthogonal LoxPsym variants can be prepared.

[0007] In a first aspect, the present application provides a LoxPsym site having the following formula:

[0008] 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT, wherein the spacer is selected from SEQ ID No.1-63. In one embodiment, the LoxPsym site is cleaved in the presence of the recombinase Cre. In another embodiment, the LoxPsym site comprises or consists of the nucleic acid sequence shown in SEQ ID No.65-127. In a specific embodiment, the LoxPsym site is characterized in that its recombination efficiency is lower than the recombination efficiency of the standard LoxPsym site shown in SEQ ID No.128, wherein the LoxPsym site comprises a spacer selected from SEQ ID No.1-27. In another specific embodiment, the LoxPsym site is characterized in that its recombination efficiency is higher than the recombination efficiency of the standard LoxPsym site shown in SEQ ID No.128, wherein the LoxPsym site comprises a spacer selected from SEQ ID No.28-63. In another specific embodiment, the LoxPsym site is an orthogonal LoxPsym site, which means that in the presence of the recombinase Cre, specific DNA recombination cannot occur between the LoxPsym site and another LoxPsym site comprising different nucleotides at positions 2, 3, 6 and / or 7 of the spacer. In a specific embodiment, 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 SEQ ID No. 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and / or 124. A vector comprising any LoxPsym site described herein and a host cell comprising the vector are also provided. The host cell can be a plant cell, a bacterial cell, a yeast cell, an insect cell or a mammalian cell.

[0009] In a second aspect, a set of at least two LoxPsym sites is provided, wherein the LoxPsym site comprises or consists of the following formula:

[0010] 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT, wherein the spacer is selected from SEQ ID NO. 1-63, and wherein the at least two LoxPsym sites comprise different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, and wherein the group does not comprise the LoxPsym sites shown in SEQ ID No. 88 and 89, SEQ ID No. 88 and 81, SEQ ID No. 90 and 83, SEQ ID No. 65 and 92, or SEQ ID No. 78 and 103. In specific embodiments, the at least two LoxPsym sites are selected from SEQ ID No. 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and / or 124.

[0011] In a third aspect, a vector comprising one or more LoxPsym sites according to the first aspect of the invention is provided. In 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.

[0012] In another aspect, 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 is provided. In a specific embodiment, the host cell comprises a set of at least two LoxPsym sites according to the second aspect.

[0013] In another aspect, the use of any of the LoxPsym sites or vectors or multiple sets of LoxPsym sites disclosed herein for site-specific recombination of one or more nucleic acid sequences is provided. Also provided are uses of these LoxPsym sites, multiple sets of LoxPsym sites and / or vectors in in vivo cloning and phenotyping. In a specific embodiment, the cloning and phenotyping are performed sequentially in the same cell.

[0014] On the other hand, a method for obtaining a recombinant nucleic acid molecule is provided, the method comprising the following steps: i) providing a nucleic acid molecule comprising two or more nucleic acid elements, each of which is flanked by an orthogonal LoxPsym site; or providing a first nucleic acid molecule and a second nucleic acid molecule, each of which comprises one or more nucleic acid elements, each of which is flanked by an orthogonal LoxPsym site; and ii) reacting the nucleic acid molecule or the first and second nucleic acid molecules with a recombinase Cre to obtain a recombinant nucleic acid molecule, wherein the orthogonal LoxPsy site comprises or consists of any one of SEQ ID No. 65-127.

[0015] A method for shuffling DNA elements within a nucleic acid molecule is also provided, the method comprising the following steps: i) providing a nucleic acid molecule comprising at least two nucleic acid elements, the nucleic acid elements being flanked by orthogonal LoxPsym sites, respectively; and ii) reacting the nucleic acid molecule with a recombinase Cre to obtain a nucleic acid molecule in which the nucleic acid elements are shuffled, wherein the orthogonal LoxPsym sites comprise or consist of any one of SEQ ID No. 65-127. In one embodiment of the method, the orthogonal LoxPsym sites are selected from SEQ ID No. 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and / or 124. In a further embodiment, the method further comprises the step of determining the sequence of the recombinant nucleic acid molecule. In a further embodiment, the method further comprises 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. A recombinant nucleic acid molecule obtained by the method described herein is also provided.

[0016] In a final aspect, a method for optimizing gene expression of one or more genes in a cell is provided, comprising the following steps: i) expressing or introducing one or more vectors comprising the one or more genes in the cell, each gene being controlled by a promoter, the promoter comprising two or more promoter elements, wherein the two or more promoter elements are respectively flanked by orthogonal LoxPsym sites, wherein each gene uses a different LoxPsym site; 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 respectively flanked by orthogonal LoxPsym sites, wherein each gene uses a different LoxPsym site, and wherein any LoxPsym site from step ii) is different from any LoxPsym site used in step i); and iii) expressing the recombinase Cre in the cell; and iv) analyzing gene expression of the one or more genes or analyzing the phenotype of the cell. In one embodiment, at least one orthogonal LoxPsym site is a LoxPsym site selected from any LoxPsym site disclosed herein, more particularly, selected from SEQ ID No. 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and / or 124. In a further embodiment, the method further comprises the step of determining the sequence of all or part of the cell genome. Also provided is a cell, particularly a genetically engineered cell, obtained by the method for 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.

[0017] Introducing one or more vectors into a cell is the same as genetically transforming the cell with one or more vectors. Those skilled in the art are familiar with a variety of molecular techniques to perform such transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of a genetic expression optimization tool disclosed herein, which is composed of a promoter (located upstream of GOI) and a terminator (located downstream of GOI) construct driving the expression of a target gene (GOI shown in gray). The orthogonal LoxPsym sites flanking the promoter element both sides (6 promoter elements are shown as examples, but not limited thereto) are represented by green diamonds), and the LoxPsym sites flanking the terminator element both sides (6 terminator elements are shown as examples, but not limited thereto) are represented by yellow circles. As shown in different blue tones, promoter and terminator elements have different intensities. After inducing the Cre-mediated recombination, the gene rearrangement of the two constructs will randomly enable different parts of the construct to drive the expression of the target gene, which will result in different expression levels, and therefore result in phenotypic variation in the yeast colony (represented by yeast cells of different colors), in which some recombinants will obtain excellent phenotypic traits. The expression optimization tool can be applied to multiple genes of a pathway simultaneously.

[0019] Figure 2 The LoxPsym site or sequence is shown and how variants can be obtained by adjusting the first three nucleotides of the spacer (shown in grey) and changing the rest of the spacer accordingly to obtain a palindromic LoxPsym site. The Cre recombinase shown in the green structure cleaves the LoxPsym site in the spacer of both strands.

[0020] Figure 3 a shows a gene construct used to determine the frequency of deletion / inversion caused by Cre-LoxPsym recombination, which is integrated at the CAN1 locus. LoxPsym (green diamond) is located in the TDH3 promoter (yellow arrow), just in front of the core promoter. This layout produces yECitrine fluorescence and blocks mCherry fluorescence. Figure 3 b shows the gene construct used to determine the efficiency of Cre-LoxPsym recombination. 64 LoxPsym variants were detected by changing the base pairs at positions 1, 2 and 3 of the spacer (blue). The LoxPsym sites were flanked on both sides of the high expression cassette pTDH3-yECitrine-tCYC1 and integrated into the CAN1 locus. The expression cassette pPGK1-mCherry-tADH1 was integrated into the YRO2 locus for control. Figure 3c is a schematic diagram of fluorescence in cells after induction of Cre recombinase, which results in no recombination (NR), deletion (DEL), or inversion (INV) of the fluorescent boxes shown in panels a (top) and b (bottom). Yellow, red, and white represent yECitrine, mCherry, or no fluorescence, respectively. Figure 3 d shows the frequency of populations with deleted (diamonds), inverted (circles) or original (squares) fluorescent cassettes shown in panel a tracked over a 24 h time course. Figure 3 e shows the recombination efficiency grouped according to the number of purines / pyrimidines in the spacer. Figure 3 f shows the recombination efficiency grouped according to the nucleotide at position 1, 2 or 3. Figure 3 g and 3i show the recombination efficiency between the 48 LoxPsym sites determined by the cassette shown in Figure b 6 h after induction of Cre expression. LoxPsym variants are characterized by the nucleotides at positions 1, 2, and 3 of their spacers. Thus, AAA represents the spacer AAATATTT, AAC represents the spacer AACTAGTT, etc. Figure 3 h shows the LoxPsym sequence generated after reporter gene deletion.

[0021] Figure 4 a shows the comparison of normalized fluorescence of individual SCRaMbLEants between the control group (grey) and the cre-expressing group (yellow), which carries Figure 1 yECitrine genes regulated by the promoter and terminator constructs shown in (N=180). Points represent the normalized fluorescence of individual SCraMbLEants. Fligner-Killeen statistics, p value is 2.20E-16. Green points represent SCraMbLEants that were further analyzed and sequenced, shown in panel b. Figure 4 b shows the normalized fluorescence after recombination of the promoter and terminal elements. The points represent the mean of 3 biological replicates, and the gray error bars represent the standard deviation. The sequences of the recombinant promoter and terminator constructs are depicted on the right side of each figure, and their fluorescence is represented by gray lines.

[0022] Figure 5 a is a schematic diagram of the astaxanthin production pathway in Saccharomyces cerevisiae. Heterologous genes targeted for expression optimization are indicated in orange. Arrows indicate enzymatic conversion of molecules, and dashed lines indicate multiple intermediate steps underlying this conversion. Figure 5 b shows picking of a single colony after induction of SCRaMbLE. The color change is larger in the Cre+ strain compared to the strain without recombinase. Figure 5c shows the quantification of RGB values ​​of 1408 and 1490 single clones from Cre+ and Cre- groups, respectively. Kligner-Killeen statistics, p-values ​​on the x-axis and y-axis are 3.23E-03 and 8.30E-10, respectively. Figure 5 d shows the carotenoid titers obtained by LC-MS analysis of acetone extracts harvested from cultures grown for 72 h in 50 ml YPD2%.Cre+ strains (N=30, right) and strains without recombinase (N=10, left). Figure 5 e shows the ΔΔCT values ​​obtained from qPCR analysis by comparison with the unchanged clone B, showing the gene expression level of each of the six investigated genes (tHMG, CrtE, Crtl, CrtYB, CrtW and CrtZ) of the astaxanthin production pathway. Figure 5 f shows the metabolite concentrations (μg / L) of individual clones.

[0023] Figure 6 Limited cross-reactivity was shown among the 16 LoxPsym variants present simultaneously in Saccharomyces cerevisiae. Figure 6 a shows a total of 18 constructs designed as: 16 test and 2 control constructs. Each construct includes all 16 different LoxPsym variants tested, as well as ADE2 and URA3 expression cassettes. The URA3 cassette was flanked by LoxPsym-TCA on both sides in each case and used as a control to ensure active, functional recombination. The control tested 2 positions upstream of the ADE2 cassette and was surrounded by the LoxPsym-array – no ADE2 deletion would be produced if the 16 LoxPsym variants were operated orthogonally. The 16 test constructs differed in the LoxPsym-NNN variants upstream of ADE2. The different test constructs confirmed cross-reactivity between all sites and recombination activity and specificity between the same LoxPsym-NNN pairs. The LoxPsym variants in the array were separated by 100 bp, allowing recombination between adjacent sites. All constructs were inserted into the CAN1 locus of BY4741ΔADE2. To induce recombination, all strains were transformed with plasmid pSH47-His-Cre or the negative control pSH47-His-Vec. Figure 6 b shows that after 6 h of induction, cells were plated on SC+FOA plates to select for URA3 deletion caused by recombination of the typical LoxPsym sites around the URA3 marker. Red colonies (marked with arrows) indicating ADE2 cassette deletion in the test strain were screened and further studied by PCR and sequencing. Figure 6c shows the recombination efficiency (calculated based on the incidence of ADE2 deletion, red phenotype) of plate counts representing three biological replicates, and the error bars indicate the standard deviation. The control strains showed negligible recombination efficiency (0.4975±0.3518 and 0.5962±0.4268 for controls 1 and 2, respectively). No colonies were observed for the strain carrying pSH47-HisVec. Figure 6 d shows the measured and expected lengths of the recombinant constructs of thirteen randomly picked red colonies, which are represented by dots and crosses, respectively. Three randomly picked samples were analyzed by Sanger sequencing; the different grey colors of the dots represent the sequencing results.

[0024] Figure 7 Recombination and cross-reactivity of LoxPsym variants in maize are shown. Figure 7 a shows the experimental design used to determine the cross-reactivity of LoxPsym variants in maize. A combinatorial library including pairwise combinations of all 256 LoxPsym variants was transfected into maize protoplasts along with a plasmid for constitutive expression of Cre. The presence of recombination was verified using NGS. Figure 7 b shows the design of a combinatorial library transfected into plant protoplasts. Each plasmid encodes two LoxPsym variants (variants are represented by gray diamonds of different shades), separated by a 104bp linker (dashed line) containing restriction enzyme NcoI-HF and PvuI-HF recognition sites (RE1 and RE2). Barcodes are incorporated upstream and downstream of the LoxPsym variants, and each barcode is uniquely connected to a LoxPsym variant. The library contains all 16×16 (=256) combinations between LoxPsym variants. Arrows indicate primer annealing sites for PCR performed after inducing recombination. The size of the PCR products was analyzed on an agarose gel, and short amplicons (indicating that recombination occurred) were analyzed by next-generation sequencing. Figure 7 c shows the recombination efficiency between LoxPsym-NNN variants in maize, calculated from the abundance of sequenced reads. Note that all efficiencies are normalized to the most active recombination site, LoxPsym-GGC, whose efficiency was arbitrarily set to 100%. Data represent the average of three technical replicates of two biological replicates, shown separately in cells divided by diagonal lines.

[0025] Figure 8 Recombination and cross-reactivity of LoxPsym variants in E. coli are shown. Figure 8 a 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 LoxPsym variant (diamonds of different shades), and PCR was used to verify in vivo recombination. Figure 8b shows details about the donor and recipient plasmids. The recipient plasmid encodes the Cre gene controlled by the rhamnose-inducible rhaB promoter and rrnB terminator. If the LoxPsym variant is cross-reactive, induction of Cre expression will lead to recombination. The junction of the recombinant plasmid is amplified using PCR (indicated by small arrows). Recombination was induced by culturing cells in LB supplemented with 2% rhamnose for 4 hours, and the experimental conditions were based on previous reports (e.g., in Sheets 2020, Ceroni 2018). Note that the recombination reaction does not have a final state, as the recombinant plasmid can recombine back into two separate plasmids. Figure 8 c shows the recombination efficiency between LoxPsym-NNN variants in E. coli calculated from densitometric analysis of junction PCR. Data represent the band intensities of PCR performed in technical replicates with template mixtures from three biological replicates.

[0026] Fig. 9 The alternative LoxPsym sites in Yarrowia lipolytica were shown to be orthogonal. Fig. 9 A shows a schematic diagram of the workflow that proves that alternative lox sites in Yarrowia lipolytica have orthogonality. First, a construct with 16 LoxPSym sites (see Table 2) separated by 100bp spacers is integrated into the URA3 locus of Yarrowia lipolytica (strain W29). The bacterial strain carrying the lox site is then transformed with a plasmid carrying Cre recombinase and NAT selection markers to transiently express the recombinase in Yarrowia lipolytica. After transformation, NAT resistance colonies are screened with primers 246-F / 247-R to confirm that different lox sites have orthogonality in Yarrowia lipolytica. Fig. 9 B shows gel electrophoresis after PCR screening of randomly selected transformants with primer pair 246-F / 247-R. A single band (2.3 kb) was amplified from all independent transformants, indicating that no recombination occurred between different lox sites. An additional PCR reaction with genomic DNA from a wild-type untransformed strain (indicated as WT on the gel) was used as a negative control.

[0027] Fig.10 Recombination and cross-reactivity of LoxPsym variants in Yarrowia lipolytica are shown. Fig.10A is a schematic diagram of using orthogonal lox sites to carry out marker excision in Yarrowia lipolytica. The target gene (gene X) has been integrated in the genomic region of Yarrowia lipolytica, which is adjacent to the selectable marker (hph) of two orthogonal sites of flanking joints. The bacterial strain with this construct is then transformed with a plasmid carrying Cre recombinase and selectable marker NAT. The NAT-resistant colony is then transferred to a fresh plate containing NAT or HPH markers. If the site has orthogonality and the marker is successfully excised, growth is not observed on the HPH selection plate. Fig.10 B shows three different LoxPsym sites, and their recombination efficiency after Cre expression is tested, thereby causing marker excision.Removal of hph marker is realized after transformation with the plasmid carrying Cre recombinase.After transformation, 192 NAT resistant bacterium colonies are transferred to the flat board supplemented with hygromycin B, and the growth of transfer bacterium colonies is evaluated after 3 days.The frequency of marker excision is determined by calculating how many bacterium colonies have lost their ability to grow on the flat board containing hygromycin B in 192 bacterium colonies, and the experiment is carried out in triplicate, and the bar graph indicates the SD between repetitions.For comparison, the same experiment is carried out using traditional LoxPsym (LoxP) site. DETAILED DESCRIPTION

[0028] definition

[0029] In order to make it easier to understand this specification, some terms are first defined. Other definitions are set forth throughout the specification. The present invention is described with reference to specific embodiments and with reference to certain drawings, but the present invention is not limited thereto, but is limited only by the claims. Any reference marks in the claims should not be interpreted as limiting the scope. The drawings described are only schematic and non-restrictive. In the drawings, for the purpose of illustration, the sizes of some elements may be enlarged and not drawn to scale. It should be noted that the term "one" or "a kind of" entity refers to one or more of the entities; for example, "a nucleotide sequence" should be understood to represent one or more nucleotide sequences. Therefore, the terms "one" (or "a kind"), "one or more" and "at least one" can be used interchangeably herein. In addition, "and / or" used herein should be understood as specifically disclosing each of the two specified features or components together with or without the other. Therefore, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to cover 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 (alone); B (alone); and C (alone). Unless otherwise specified, when an indefinite or definite article is used when referring to a singular noun, such as "a" or "an", "the", this includes the plural of the noun. In addition, the terms "first", "second", "third", etc. in this specification and in the claims are used to distinguish between similar elements and are not necessarily used to describe a sequential or chronological order. It should be understood that the terms used in this way are interchangeable where appropriate, and that the embodiments of the invention described herein are capable of operating in other sequences than those described or shown herein.

[0030] It should be understood that wherever the language "comprising" is used herein to describe aspects or embodiments, similar aspects or embodiments are also provided as described with the terms "consisting of" and / or "consisting essentially of". Where the term "comprising" is used in this 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 to a person skilled in the art in the field of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in the relevant field of the present disclosure. 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 Edition, 2000, Oxford University Press, provide a general dictionary of many terms used in the present disclosure for those skilled in the art. Practitioners are particularly required to refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 100), John Wiley & Sons, New York (2012) for definitions and terms in the art. The definitions provided herein should not be construed to have a scope less than that understood by one of ordinary skill in the art.

[0032] Units, prefixes and symbols are all expressed in the format accepted by the International System of Units (SI). Numerical ranges include the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction. The headings provided herein are not limitations on the various aspects of the disclosure, and various aspects of the invention can be obtained by reference to the entire specification. Therefore, the terms defined below will be more fully defined by reference to the entire specification.

[0033] 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 of any length, whether deoxyribonucleotides or ribonucleotides, or their analogs. Nucleic acids can have any three-dimensional structure and can perform any known or unknown function. 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 can include promoters, introns, enhancer regions, polyadenylation sites, translation start sites, 5' or 3' untranslated regions, reporter genes, selectable markers, etc. Nucleic acids can include single-stranded or double-stranded DNA or RNA. Nucleic acids can include modified bases or modified backbones. Nucleic acids of no more than about 100 nucleotides are also commonly 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 naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides such as DNA and RNA nucleotides include a ribose moiety, a core base moiety, and one or more phosphate groups (which are not present in nucleosides). Nucleotides without phosphate groups are referred to as "nucleosides", and are therefore a compound comprising a core base moiety and a sugar moiety. As used herein, "core base" refers to a group of atoms that can be connected to a sugar moiety to produce a nucleoside that can be incorporated into an oligonucleotide, and wherein the group of atoms can be combined with a complementary naturally occurring core base of another oligonucleotide or nucleic acid. The core base of naturally occurring RNA or DNA includes purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0034] As used herein, the term "defined by SEQ ID NO.X" or "as shown in SEQ ID NO.X" refers to a biological sequence consisting of the nucleotide sequence given by SEQ ID NO.X. For example, the LoxPsym site defined by SEQ ID NO.X consists of the nucleic acid sequence given by SEQ ID NO.X. Another 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 by SEQ ID No.X but completely comprises the nucleic acid sequence given by SEQ ID No.X, or refers to a nucleic acid sequence consisting of the nucleic acid sequence given by SEQ ID No.X.

[0035] 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 encoding an mRNA and encoding an amino acid sequence, such that the regulatory nucleic acid sequence is capable of regulating the transcription or expression of the associated nucleic acid coding sequence. The regulatory nucleic acid sequence of a chimeric gene is not operably linked to an associated nucleic acid sequence found in nature.

[0036] "Promoter" is a DNA sequence comprising a regulatory element that mediates the expression of a nucleic acid molecule. In order to express, a nucleic acid molecule must be operably connected to or comprise a suitable promoter that expresses the gene at the correct time point and in a desired spatial expression pattern. As used herein, the term "operably linked" refers to a functional connection between a promoter sequence and a target gene, such that the promoter sequence can initiate transcription of the target gene. A promoter that can initiate transcription of a gene in a eukaryotic or host cell is referred to as "active". In order to identify a promoter that is active in a eukaryotic or host cell, a promoter can be operably connected to a reporter gene, after which the expression level and pattern of the reporter gene can be determined. Suitable well-known reporter genes include, for example, β-glucuronidase, β-galactosidase, or any fluorescent protein or luminescent protein. Promoter activity is determined by measuring the enzymatic activity of β-glucuronidase or β-galactosidase. Alternatively, promoter strength can be determined by quantifying mRNA levels or by comparing mRNA levels of a nucleic acid to mRNA levels of a housekeeping gene such as 18S rRNA using methods known in the art, such as Northern blotting with autoradiographic density analysis, quantitative real-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6:986-994).

[0037] The term "3' terminal region involved in transcriptional termination or polyadenylation" encompasses control sequences, which are DNA sequences at the end of the transcription unit that signal 3' processing or polyadenylation of the primary transcript and participate in the termination of transcription. Control sequences or terminators for transcriptional termination can be derived from natural genes or from a variety of genes. For expression in yeast, the terminator to be added can be derived from, for example, the TEF or CYC1 genes, or alternatively from another yeast gene, or less preferably from any other eukaryotic or viral gene.

[0038] The term "vector" refers to any linear or circular DNA construct comprising one of the LoxPsym sites of the present application. A vector may refer to an expression cassette or any recombinant expression system for expressing a target gene in vivo or in vitro in any cell (including yeast, plant and mammalian cells) in a constitutive or inducible manner. The vector may still be an episome or integrated into the host cell genome. The vector may have self-replication capability or may not have self-replication capability (i.e., only driving transient expression in the cell). The term includes a recombinant expression cassette containing only the minimum elements required for transcribing a recombinant nucleic acid. The vector of the present invention may be a "recombinant vector" defined by an artificial vector. The vector may also be a viral vector, including lentivirus, retrovirus, adenovirus and adeno-associated virus vectors.

[0039] As used herein, "reduction," "reducing," or "lower" refers to a statistically significant decrease, more particularly a statistically significant decrease of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to a control.

[0040] As used herein, "increasing," "increase," "enhancing," "promoting," or "stimulating" are used interchangeably and refer to a statistically significant increase, more particularly, the statistically significant increase is an increase of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to a control.

[0041] The term "statistically significant" difference is well known to those skilled in the art. Statistical significance plays a key role in statistical hypothesis testing. It is used to determine whether the null hypothesis should be rejected or retained. The null hypothesis is the default assumption that nothing has happened or nothing has changed, so, for example, the recombination efficiency of a specific LoxPsym site is no different from the recombination efficiency of the standard LoxPsym site shown 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 is less than a pre-specified significance level α. In the case where the null hypothesis is true, the p-value of the result p is the probability of obtaining at least the same extreme result. 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.

[0042] Yeast is a eukaryotic unicellular microorganism, classified as a member of the kingdom Fungi, and like all fungi, yeast can have asexual and sexual reproduction cycles. The most common mode of vegetative growth of yeast is asexual reproduction by budding. At this point, a small bud or daughter cell is formed on the parent cell. The nucleus of the mother cell splits into daughter nuclei and migrates into the daughter cell. The bud continues to grow until it separates from the mother cell to form a new cell. This reproduction cycle does not depend on the ploidy of yeast, so both haploid and diploid yeast cells can be replicated as described above. Haploid cells usually have lower adaptability, and they often die under high stress conditions such as nutrient deficiency, while under the same conditions, diploid cells can undergo sporulation, enter sexual reproduction (meiosis) and produce a variety of haploid spores or haploid segregants, which can continue to mate (join), and re-form diploid. Haploid cells contain one set of chromosomes, and diploid cells contain two sets of chromosomes. As used herein, haploid segregants are equivalent to haploid spores, which are the result of sporulation.

[0043] When nutrients are plentiful, the budding yeast Saccharomyces cerevisiae reproduces as diploid cells by mitosis, but when starved, the yeast undergoes meiosis to form haploid spores. The haploid cells can then reproduce asexually by mitosis.

[0044] As used herein, "engineering" or "engineered" refers to genetic engineering, a technique that uses biotechnology to modify the genome of an organism. This includes, but is not limited to, transferring genes within and across species boundaries, deleting gene fragments or deleting 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 of engineering microorganisms, especially yeast, are well known to those skilled in the art. The most known techniques include traditional gene transformation of yeast and recombinant DNA techniques. Today, the most attractive techniques for engineering microorganisms are through the use of nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), large-range nucleases, but especially the CRISPR-Cas system as described above.

[0045] Cre / LoxP recombination as a valuable genetic engineering tool

[0046] DNA recombination is a process by which DNA fragments break and recombine to produce new allele combinations. Site-specific recombination is the basis for the generation of genetic diversity in all organisms and has been proven to be a powerful tool in genetic engineering, systems biology, and developmental biology research over the past few decades.

[0047] Cre / LoxP is a widely used site-specific DNA recombination system derived from bacteriophage P1. Cre recombinase catalyzes the site-specific recombination reaction between two LoxP sites, and does not require auxiliary factors (Guo et al., Nature 389: 40-46). The length of the LoxP site is 34 base pairs (bp), consisting of two 13bp inverted repeats, which are separated by an 8bp asymmetric spacer. The Cre / LoxP system can be used to produce deletions, inversions, insertions (transpositions) or translocations, depending on the direction and position of the LoxP site specified in a given system (Nagy 2000 Genesis 26: 99). The simplicity of the Cre / LoxP system makes it possible to be used for 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), and 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 restore a single recombination event at a defined position. If the LoxP sites encode symmetric spacers (LoxPsym), the rearrangement is independent of orientation, and the DNA fragment between the two LoxPsym sites should be deleted or inverted at the same frequency (Hoess et al., 1986 Nucleic Acids Res 14:2287-2300; Shen et al., 2016 Genome Res 26:36-49).

[0048] The LoxPsym site includes a left end and a right end, i.e., a "LE" Cre recognition site or "arm"; a right end, i.e., a "RE" Cre recognition site or "arm", and a sandwich between the LE and RE arms, i.e., a spacer. In most wild-type and mutant LoxP sites, the length of the LE arm and the RE arm are each 13 base pairs (bp). In the LoxPsym site, the LE arm and the RE arm are inverted repeats. A non-limiting example of the LE sequence is 5'-ATAACTTCGTATA-3', and a non-limiting example of the RE sequence is 5'-TATACGAAGTTAT-3'. The length of the spacer is 8bp. Each base in the spacer is usually named 1, 2, 3, 4, 5, 6, 7 or 8 according to its order in the sequence (5'→3'). The Cre-LoxP site mediates site-specific intrachain exchange or interchain exchange of DNA molecules catalyzed by the Cre recombinase.

[0049] Novel LoxPsym sites

[0050] The efficiency of Cre-Lox recombination events is a major determinant in establishing genetic engineering operations. Therefore, the inventors of the present application developed 63 variants of standard LoxPsym sites with variable recombination efficiencies. Interestingly, 36 new LoxPsym sites appear to be more efficient than the originally described LoxPsym sites, achieving almost twice as high recombination efficiencies, while 27 sites exhibit reduced recombination efficiencies compared to standard LoxPsym sites. Both sets of sites have value as genetic engineering tools because high or low recombination efficiencies may be required in specific circumstances.

[0051] In a first aspect, the present application provides a LoxPsym site or alternatively expressed as a LoxPsym sequence or a LoxPsym oligonucleotide having the following formula:

[0052] 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from SEQ ID No. 1 to 63. The LoxPsym site has a nucleotide sequence from the wild-type Escherichia coli P1 phage loxP site. In one embodiment, the LoxPsym site is cleaved in the presence of the recombinase Cre.

[0053] As used herein, “Cre recombinase” or “recombinase Cre” or “Cre” refers to the tyrosine recombinase from P1 phage (Uniprot ID: Q71TG5), and its amino acid sequence is shown in SEQ ID NO.129.

[0054] SEQ ID No.129 (Cre recombinase)

[0055] MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPA

[0056] EPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAK

[0057] QALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIG

[0058] RTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGI

[0059] FEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDS

[0060] ETGAMVRLLEDGD

[0061] The enzyme utilizes a topoisomerase I-like mechanism to carry out site-specific recombination events. The 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 is composed of two palindromic sequences of 13 nucleotides flanked by a short spacer of 8 nucleotides. The recombination product of the LoxP site mediated by Cre depends on the position and relative orientation of the LoxP site. As a result of the recombination mediated by Cre, two independent DNA species containing LoxP sites can be fused. The DNA sequence between the two LoxP sites is referred to as "floxed". In this case, the recombination product mediated by Cre depends on the direction of the LoxP site. The DNA between the two identical LoxP sites in the two directions will be excised in the form of circular DNA, and the intervening DNA between the two opposite LoxP sites in the two directions will be inverted (Nagy 2000 Genesis 26: 99-109). The enzyme does not require additional cofactors (such as ATP) or auxiliary proteins to perform its function (Abremski and Hoess 1984 J Biol Chem 259: 1509-1514). If the LoxP site encodes a symmetric spacer (LoxPsym), the rearrangement is independent of direction, and the DNA fragment between the two LoxPsym sites is deleted or inverted at the same frequency (Hoess et al., 1986 Nucleic Acids Res 14: 2287-2300; Shen et al., 2016 Genome Res 26: 36-49).

[0062] Table 1. Overview of novel LoxPsym sites, their spacers and recombination efficiencies.

[0063]

[0064]

[0065]

[0066] In one embodiment, the recombination efficiency of the LoxPsym site is lower than the recombination efficiency of the standard LoxPsym site described by Hoess et al. 1986, more specifically, lower than the recombination efficiency of the LoxPsym site as shown in SEQ ID No. 128 (ATAACTTCGTATAATGTACATTATACGAAGTTAT) comprising the spacer as shown in SEQ ID No. 64 (ATGTACAT). The recombination efficiency of SEQ ID No. 128 is 47.0%.

[0067] Thus, a LoxPsym site having the formula:

[0068] 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from SEQ ID No. 1-27 or wherein the LoxPsym site is selected from SEQ ID No. 65-91. In a specific embodiment, the recombination efficiency of the LoxPsym site is at least 10% lower than the recombination efficiency of SEQ ID No. 128, more specifically, the LoxPsym site comprises a spacer selected from SEQ ID No. 1-16. In a more specific embodiment, the recombination efficiency of the LoxPsym site is at least 15% lower than the recombination efficiency of SEQ ID No. 128, more specifically, the LoxPsym site comprises a spacer selected from SEQ ID No. 1-12. In an even more specific embodiment, the recombination efficiency of the LoxPsym site is at least 20% lower than the recombination efficiency of SEQ ID No. 128, more specifically, the LoxPsym site comprises a spacer selected from SEQ ID No. 1-9.

[0069] In another embodiment, the recombination efficiency of the LoxPsym site is higher than the recombination efficiency of the standard LoxPsym site described by Hoess et al. 1986, more specifically, higher than the recombination efficiency of the LoxPsym site as shown in SEQ ID No. 128, having the spacer shown in SEQ ID No. 64. Thus, a LoxPsym site having the following formula is provided:

[0070] 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from SEQ ID No. 28-63 or wherein the LoxPsym site is selected from SEQ ID No. 92-127. In a specific embodiment, the recombination efficiency of the LoxPsym site is at least 10% higher than the recombination efficiency of SEQ ID No. 128, more specifically, the LoxPsym site comprises a spacer selected from SEQ ID No. 32-63. In a more specific embodiment, the recombination efficiency of the LoxPsym site is at least 15% higher than the recombination efficiency of SEQ ID No. 128, more specifically, the LoxPsym site comprises a spacer selected from SEQ ID No. 34-63. In an even more specific embodiment, the recombination efficiency of the LoxPsym site is at least 20% lower than the recombination efficiency of SEQ ID No. 128, more specifically, the LoxPsym site comprises a spacer selected from SEQ ID No. 35-63.

[0071] In a third aspect, a vector comprising any of the LoxPsym sites disclosed herein is provided. In a specific embodiment, the vector comprises a nucleic acid sequence flanked by LoxPsym sites at 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 target gene or a fragment thereof, a 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).

[0072] In another aspect, a host cell comprising any vector as described above or comprising any LoxPsym site disclosed herein is provided. In a specific embodiment, the host cell is a microorganism, a plant cell, an insect cell, a mammalian cell or a yeast cell. In a specific embodiment, one or more LoxPsym sites according to any embodiment of the first aspect are amplified, for example, 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 of 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 disclosed herein without departing from the scope of the present application. In an even more specific embodiment, the host cell is a bacterium, such as Escherichia coli, or a yeast cell, even more specifically a yeast of the genus Saccharomyces, the genus Yarrowia or the genus Pichia. In another specific embodiment, the host cell is a cell of a species selected from the group consisting of Saccharomyces cerevisiae, Escherichia coli, Zea mays and Yarrowia lipolytica.

[0073] Novel orthogonal LoxPsym sites

[0074] Microbial biotechnology or microbial engineering explores the ability of bacteria and yeast to obtain products or activities with economic value on an industrial scale. Synthetic biology and recombinant DNA technology enable heterologous pathways to be expressed in host cells, which are not limited to microorganisms, but extend to plant cells, insect cells and mammalian cells. When cells are used as factories, preferably the entire biosynthetic pathway is inserted into the genome of the cell. Since the high expression of all biosynthetic genes does not usually bring maximum product yield (for example, intermediates will accumulate, feedback will be generated to the system, toxicity will be generated, or growth will be undesirable), the expression level of multiple modules or genes should be optimized and adjusted. Currently available methods are based on, for example, simply trying many different combinations, directed evolution, computational prediction, self-adjusting systems with feedback inhibition, etc., which are laborious, expensive and time-consuming.

[0075] The inventors of the present application developed a method for simultaneous in vivo cloning and testing based on the well-known Cre-Lox system. The cross-reactions of many of the 63 variants of the above-mentioned LoxPsym sites were tested. Specifically, 1056 interactions between LoxPsym variants were tested in a fluorescence-based assay to identify orthogonal LoxPsym variants. Figure 3In this interaction matrix shown in g, it can be found that multiple groups of orthogonal LoxPsym sites can be selected, the largest group including 16 variants, all of which can be cut by Cre but do not recombine with each other. Examples of such groups are groups consisting of LoxPsym sites selected from SEQ ID No. 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and 124, or groups consisting of LoxPsym sites containing spacers selected from SEQ ID No. 1, 5-7, 10, 13, 18, 20, 36, 41, 43-45, 48, 57 and 60. Other non-limiting examples of orthogonal LoxPsym site groups consist of LoxPsym sites selected from the group consisting of SEQ ID No. 70, 77, 82, 101-102, 105, 107, 109, 112, 114, 117-118, 123-124, 126-127, or SEQ ID No. 65, 71-74, 84, 86, 88, 90, 103, 106-108, 111, 121, 124, or SEQ ID No. 72, 74, 77-78, 80, 82, 84, 86, 88, 102, 106, 109, 114, 117-118, 123-124, 126-127, or SEQ ID No. ID No. 65, 70-71, 73, 80, 90, 101, 103, 105, 108, 111-112, 117, 121, 124, 126. In addition, smaller groups of LoxPsym sites are also provided. A non-limiting example is a group consisting of LoxPsym sites selected from SEQ ID No. 69, 70, 74, 77, 84, 86, 88, 100, 108, 109, 114 and 124.

[0076] The orthogonal LoxPsym sites disclosed herein can be used, for example, as follows: a cell is transformed with one or more constructs comprising genes A, B, C, ...; multiple promoter elements separated by LoxPsym sites are present upstream of the genes, in such a way that all promoter elements in front of gene A are separated by the same LoxPsym site, but for each of the other genes, a different LoxPsym site is used ( Figure 1 The same method can be used to optimize the terminator sequence downstream of the gene: multiple terminator elements are separated by LoxPsym sites, that is, the LoxPsym sites of all terminator elements of a gene are the same, but different from the LoxPsym sites of the promoter element of the gene and the LoxPsym sites of other genes ( Figure 1By inducing the expression of Cre recombinase in cells, different combinations are made between promoter elements and terminator elements, and different combinations of multiple genes are made simultaneously (orthogonal recombination). The genetic diversity of the resulting cell population leads to phenotypic diversity of target traits, such as the production of high-value compounds ( Figure 1 ). The best performing cells are then sequenced to reveal the optimized promoter / terminator combination for each gene and optionally re-engineered in a clean fashion.

[0077] In a second aspect, the invention provides orthogonal LoxPsym sites that can be used to assemble nucleic acid constructs. The term "orthogonal" or "orthogonality" in (synthetic) biology describes that two or more molecules of similar composition and / or function cannot interact with each other or affect their respective substrates, so "orthogonal" as used herein refers to "independently acting" or "non-cross reacting".

[0078] Therefore, when Cre recombinase is present, LoxPsym sites can only recombine with LoxPsym sites with the same nucleic acid sequence, but not with LoxPsym sites with different nucleic acid sequences, and the LoxPsym sites are "orthogonal". This is a huge advantage because specific recombination events can be initiated simultaneously without affecting each other.

[0079] Orthogonal recombination is currently obtained by using multiple recombinases that recognize different recombination sites (Wang et al., 2022iScience25:103716). However, the number of such non-cross-reacting recombinases is still limited, and expression in cells may potentially have a yield dragging effect. The orthogonal LoxPsym variants disclosed herein overcome these shortcomings.

[0080] The present application provides the selection of orthogonal or non-cross-reactive LoxPsym sites. More specifically, the mutant LoxPsym site or sequence can be cut in the presence of the recombinase Cre, and specific DNA recombination can occur between LoxPsym sites with the same nucleotide sequence, but recombination cannot occur between LoxPsym sites with different nucleotide sequences. In a specific embodiment, the LoxPsym site comprises or consists of the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from SEQ ID No.1-63.

[0081] In one embodiment, the orthogonal LoxPsym site comprises or consists of the formula:

[0082] Provided is 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from SEQ ID No. 1-63, and wherein the LoxPsym site can be cleaved in the presence of the recombinase Cre, and wherein specific DNA recombination occurs only between LoxPsym sites having the same nucleotide sequence, or between LoxPsym sites containing the same nucleotide at positions 2-7 of the spacer. In a specific embodiment, when the site contains different nucleotides at positions 2, 3, 4, 5, 6 and / or 7 of the spacer, recombination does not occur between the LoxPsym sites.

[0083] In specific embodiments, the LoxPsym site, mutant LoxPsym site or orthogonal LoxPsym site has the following properties:

[0084] - A nucleic acid sequence comprising or consisting of the following formula:

[0085] 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3',

[0086] wherein the spacer consists of 8 bases and is selected from SEQ ID No. 1-63; and

[0087] - In the presence of the recombinase Cre, the LoxPsym site can only recombine with a LoxPsym site with the same nucleotide sequence or with a site containing the formula 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3'

[0088] or LoxPsym site recombination consisting of the same, wherein the spacer is selected from SEQ ID No. 1-63, and comprises the same nucleotide sequence from position 2 to position 7 of the spacer.

[0089] "From position 2 to position 7 of the spacer" means that positions 2 and 7 are both included.

[0090] In the presence of Cre recombinase, the orthogonal LoxPsym site cannot recombine with a LoxPsym site having a different nucleotide at position 2, 3, 4, 5, 6 or 7 of the spacer.

[0091] In a further aspect, a set of LoxPsym sites is provided, comprising at least a first LoxPsym site and a second LoxPsym site, the LoxPsym site being selected from SEQ ID No. 65-127 or having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from SEQ ID No. 1-63. In one embodiment, in the presence of the recombinase Cre, specific DNA recombination occurs between the first LoxPsym site and the second LoxPsym site only when the first LoxPsym site and the second LoxPsym site have the same nucleic acid sequence or share the same nucleotides at positions 2-7 of the spacer. In a further embodiment, in the presence of the recombinase Cre, specific DNA recombination can occur between any LoxPsym site from the set and any other LoxPsym site from the set when the LoxPsym sites share the same nucleotides 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.

[0092] In another embodiment, in the presence of the recombinase Cre, when the spacers of the first LoxPsym site and the second LoxPsym site are different and / or when the spacer of the first LoxPsym site and the spacer of the second LoxPsym site comprise different nucleotides at positions 2, 3, 4, 5, 6 and / or 7 of the spacer, more specifically including positions 2, 3, 6 and / or 7 of the spacer, specific DNA recombination cannot occur between the first LoxPsym site and the second LoxPsym site of the group, unless the first LoxPsym site and the second LoxPsym site respectively comprise a spacer combination GGGTACCC-AACTAGTT, GGGTACCC-ATATATAT, GAATATTC-AGTTAACT, GAGTACTC-AAATATTT or GTGTACAC-GCATATGC or in other words, or unless when the first LoxPsym site and the second LoxPsym site comprise the spacers GGGTACCC and AACTAGTT, GGGTACCC and ATATATAT, GAATATTC and AGTTAACT, GAGTACTC and AAATATTT or GTGTACAC and GCATATGC, respectively.

[0093] In another embodiment, in the presence of the recombinase Cre, when the first LoxPsym site and the second LoxPsym site comprise different nucleotides at positions 2, 3, 4, 5, 6 and / or 7 of the spacer, specific DNA recombination cannot occur between the first LoxPsym site and the second LoxPsym site of the group unless the first LoxPsym site and the second LoxPsym site comprise the spacer combination GGGTACCC-AACTAGTT, GGGTACCC-ATATATAT, GAATATTC-AGTTAACT, GAGTACTC-AAATATTT or GTGTACAC-GCATATGC.

[0094] In a further embodiment, a set of at least two LoxPsym sites is provided, the LoxPsym sites comprising or consisting of the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from SEQ ID NOs. 1-63, 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 comprise the LoxPsym sites shown 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.

[0095] In one embodiment, a set of LoxPsym sites is provided, comprising at least a first LoxPsym site and a second LoxPsym site, wherein the LoxPsym site has the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from SEQ ID No. 1-63, and

[0096] i) wherein in the presence of the recombinase Cre, when the first LoxPsym site and the second LoxPsym site have the same nucleic acid sequence, or in other words, when the first LoxPsym site and the second LoxPsym site are identical, specific DNA recombination can occur between the first LoxPsym site and the second LoxPsym site; and

[0097] ii) wherein in the presence of the recombinase Cre, when the first LoxPsym site and the second LoxPsym site comprise different nucleotides at positions 2, 3, 4, 5, 6 and / or 7 of the spacer, specific DNA recombination cannot occur between the first LoxPsym site and the second LoxPsym site, except for the spacer combinations GGGTACCC-AACTAGTT, GGGTACCC-ATATATAT,

[0098] GAATATTC-AGTTAACT, GAGTACTC-AAATATTT, or GTGTACAC-GCATATGC.

[0099] In a further embodiment, a set of at least two LoxPsym sites is provided, the LoxPsym sites having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from SEQ ID No. 1-63, 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 a further specific embodiment, the at least two LoxPsym sites do not comprise SEQ ID No. 88 and 89, SEQ ID No. 88 and 81, SEQ ID No. 90 and 83, SEQ ID No. 65 and 92 or SEQ ID No. 78 and 103.

[0100] In a specific embodiment, the group comprises 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 a more specific embodiment, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 LoxPsym sites are selected from SEQ ID No. 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and / or 124.

[0101] In a further aspect, a chimeric gene construct comprising one or more LoxPsym sites as described herein is provided. 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' and / or 3' end by one or more LoxPsym sites disclosed herein.

[0102] In a further aspect, a vector comprising one or more LoxPsym sites described herein or comprising any of the chimeric gene constructs described above is provided.

[0103] A host cell is also provided, comprising the vector or comprising any LoxPsym site disclosed herein. In a specific embodiment, the host cell is a microorganism, a plant cell, an insect cell or a mammalian cell. In a specific embodiment, the host cell is a bacterium or a yeast cell. In a more specific embodiment, the host cell is a yeast, even more particularly a yeast of the genus Saccharomyces, the genus Yarrowia or the genus Pichia, most particularly Saccharomyces cerevisiae. In another specific embodiment, the host cell is a cell of a species selected from the group consisting of Saccharomyces cerevisiae, Escherichia coli, Zea mays and Yarrowia lipolytica.

[0104] In a further embodiment, a set or combination or selection of vectors is provided, wherein each vector comprises a nucleic acid sequence flanked by one of the LoxPsym sites disclosed herein at the 5' end of the nucleic acid sequence and flanked by the same LoxPsym site at the 3' end of the nucleic acid sequence, wherein each vector of the set of vectors comprises a different LoxPsym site. 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.

[0105] In vivo and / or in vitro recombinant methods

[0106] As described herein, the LoxPsym sites according to the present invention can be used for site-specific recombination events in combination with one or more nucleic acid sequences and the Cre recombinase.

[0107] In further aspects, there is provided the use of any of the LoxPsym sites disclosed herein for in vivo and / or in vitro cloning.

[0108] More specifically, an in vivo or in vitro method for obtaining a recombinant nucleic acid sequence or molecule is provided, comprising:

[0109] - 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 are recombined to provide a recombinant nucleic acid molecule; and

[0110] - optionally after introduction of said recombinant nucleic acid molecule or sequence into a cell, determining said sequence and / or determining the expression of said recombinant nucleic acid molecule or sequence.

[0111] In one embodiment, the site-specific recombinase recognition site is a LoxPsym site, and the recombinase is a Cre recombinase that recognizes the LoxPsym site. In a specific embodiment, the LoxPsym site is selected from the LoxPsym sites disclosed herein, more specifically, the LoxPsym site comprises or consists of SEQ ID No.65-127. In another specific embodiment, the DNA segment can be a nucleic acid sequence encoding a protein or a non-coding RNA, a promoter element, a terminator element, or any other regulatory nucleic acid sequence.

[0112] Also provided is a method for replacing a DNA element A with a different DNA element B, the method comprising the steps of:

[0113] - providing a nucleic acid molecule A' comprising a LoxP site, a DNA element A and a LoxP site in a 5'-3' sequence, and a nucleic acid molecule B' comprising a LoxP site, a DNA element B and a LoxP site in a 5'-3' sequence, wherein at least one LoxP site is a LoxPsym site according to the present invention, more specifically, the LoxPsym site comprises a nucleic acid sequence shown in SEQ ID No. 65-127;

[0114] - Nucleic acid molecules A' and B' are reacted in the presence of the recombinase Cre to obtain a nucleic acid molecule in which DNA element A is replaced by DNA element B.

[0115] In one embodiment, at least two, at least three, at least four or all LoxP sites are selected from the LoxPsym sites disclosed herein. In a specific embodiment, the LoxPsym site is selected from SEQ ID No. 65-91, SEQ ID No. 65-80, SEQ ID No. 65-76, SEQ ID No. 65-73, SEQ ID No. 92-127, SEQ ID No. 96-127, SEQ ID No. 98-127, SEQ ID No. 99-127 and / or selected from SEQ ID No. 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and 124.

[0116] Methods for optimizing gene expression

[0117] The specific use of the orthogonal LoxPsym sites disclosed herein is to perform in vivo cloning and phenotyping simultaneously. Preferably, the in vivo cloning and phenotyping are performed in the same cell.

[0118] More specifically, a method for optimizing gene expression of one or more genes in a cell is provided, comprising the steps of:

[0119] a. expressing or introducing into a cell one or more vectors comprising one or more genes of interest controlled by a promoter, wherein the promoter comprises two or more promoter elements, wherein the two or more promoter elements are flanked by orthogonal LoxPsym sites, respectively, wherein each gene or each promoter uses a different LoxPsym site;

[0120] b. Optionally, the one or more vectors comprise a terminator sequence located 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 respectively flanked by orthogonal LoxPsym sites, wherein each gene uses a different LoxPsym site, and wherein any LoxPsym site from step b) is contiguous with the LoxPsym site from step a).

[0121] Any LoxPsym sites used in are different;

[0122] c. expressing the recombinase Cre in the cell;

[0123] d. Analyze the gene expression level of the target gene or analyze the phenotype of the cell.

[0124] Also provided is a method of optimizing gene expression of one or more genes in a cell, comprising the steps of:

[0125] a. expressing or introducing into a cell one or more vectors comprising one or more target genes under the control of a promoter, the one or more vectors comprising a terminator sequence located downstream of the one or more target genes, the terminator sequence comprising two or more terminator elements, wherein the two or more terminator elements are respectively flanked by orthogonal LoxPsym sites, wherein each gene or each terminator uses a different LoxPsym site;

[0126] b. Optionally, the promoter comprises two or more promoter elements, wherein the two or more promoter elements are respectively or each flanked by orthogonal LoxPsym sites, wherein each gene uses a different LoxPsym site, and wherein any LoxPsym site from step b) is different from any LoxPsym site used in step a);

[0127] c. expressing the recombinase Cre in the cell;

[0128] d. Analyze the gene expression level of the target gene or analyze the phenotype of the cell.

[0129] In one embodiment, the orthogonal LoxPsym site is selected from any LoxPsym site disclosed herein, more specifically, from a LoxPsym site having the formula:

[0130] 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3',

[0131] wherein the spacer is selected from SEQ ID No. 1-63, and wherein in the presence of the recombinase Cre, when the LoxPsym site comprises different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, specific DNA recombination cannot occur between the LoxPsym site and another LoxPsym site, the other LoxPsym site having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3' and comprising a spacer selected from SEQ ID No. 1-63, unless the LoxPsym site comprises the spacer combination GGGTACCC-AACTAGTT, GGGTACCC-ATATATAT, GAATATTC-AGTTAACT, GAATATTC-AGTTAACT or GTGTACAC-GCATATGC.

[0132] In another embodiment, the orthogonal LoxPsym site is a LoxPsym site having the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from SEQ ID No. 1-63, and wherein in the presence of the recombinase Cre, specific DNA recombination can occur between the LoxPsym site and another LoxPsym site only when the spacers of the two LoxPsym sites comprise the same nucleotide sequence between positions 2 and 7 of the spacer, wherein the other LoxPsym site has the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT and comprises a spacer selected from SEQ ID NO. 1-63, while specific DNA recombination cannot occur when the LoxPsym site comprises different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, unless the spacer combination is GGGTACCC-AACTAGTT, GGGTACCC-ATATATAT, GAATATTC-AGTTAACT, GTGTACAC-GCATATGC.

[0133] In a further embodiment, the orthogonal LoxPsym sites are selected from any group of LoxPsym sites disclosed herein, more specifically, from a group of LoxPsym sites comprising at least two LoxPsym sites having the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from SEQ ID NOs. 1-63, and wherein the at least two LoxPsym sites comprise different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, and wherein the group does not comprise the LoxPsym sites shown 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.

[0134] In specific embodiments, the orthogonal LoxPsym sites are selected from SEQ ID No. 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and / or 124, or are selected from SEQ ID No. 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and 124.

[0135] In one embodiment, the method further provides a step of sequencing or determining the sequence of the cell, more specifically a step of determining the sequence of the recombinant nucleic acid molecule responsible for the phenotype.

[0136] As used herein, "phenotype" includes, but is not limited to, cell growth, reproductive fitness, synthesis of one or more compounds, detectable markers, or any other observable characteristic.

[0137] As used herein, "individually flanked" or "each flanked" means that a nucleic acid (eg, a promoter or terminator element) comprises additional elements, such as LoxPsym sites, at both the 5' and 3' ends.

[0138] Also provided is a method of shuffling DNA elements, comprising the steps of:

[0139] - providing a nucleic acid molecule comprising at least two DNA elements, each flanked by an orthogonal LoxP site;

[0140] - reacting the nucleic acid molecule with the recombinase Cre to obtain a nucleic acid molecule in which at least two DNA elements are shuffled.

[0141] In one embodiment, the nucleic acid molecule is a gene promoter, and at least two DNA elements are promoter elements. In other embodiments, the nucleic acid molecule is a terminator sequence, and at least two DNA elements are terminator elements. In another embodiment, the nucleic acid molecule is a protein encoding gene or a non-coding gene, and at least two DNA elements are introns and / or exons.

[0142] In further embodiments, the orthogonal LoxP site is selected from any LoxPsym site disclosed herein, more specifically, from a LoxPsym site having the formula:

[0143] 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT,

[0144] wherein the spacer is selected from SEQ ID No. 1-63, and wherein in the presence of the recombinase Cre, when the LoxPsym site comprises different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, specific DNA recombination cannot occur between the LoxPsym site and another LoxPsym site, the other LoxPsym site having the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT, and comprising a spacer selected from SEQ ID No. 1-63, unless the LoxPsym site comprises the spacer combination GGGTACCC and AACTAGTT, GGGTACCC and ATATATAT, GAATATTC and AGTTAACT, GAGTACTC and AAATATTT and / or GTGTACAC and GCATATGC.

[0145] In a further embodiment, the orthogonal LoxPsym site is a LoxPsym site having the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT, wherein the spacer is selected from SEQ ID No. 1-63, and wherein in the presence of the recombinase Cre, specific DNA recombination can occur between the LoxPsym site and another LoxPsym site only when the spacers of the two LoxPsym sites comprise the same nucleotide sequence between positions 2 and 7 of the spacers, the other LoxPsym site having the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT, and comprising a nucleotide sequence selected from SEQ ID 1-63, and specific DNA recombination cannot occur when the LoxPsym site contains different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, unless the spacer combination is GGGTACCC and AACTAGTT, GGGTACCC and ATATATAT, GAATATTC and AGTTAACT, GAGTACTC and AAATATTT and / or GTGTACAC and GCATATGC.

[0146] In a further embodiment, the orthogonal LoxPsym sites are selected from any group of LoxPsym sites disclosed herein, more specifically, from a group of LoxPsym sites comprising at least two LoxPsym sites having the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from SEQ ID NOs. 1-63, and wherein the at least two LoxPsym sites comprise different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, and wherein the group does not comprise the LoxPsym sites shown 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.

[0147] In specific embodiments, the orthogonal LoxPsym sites are selected from SEQ ID No. 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and / or 124, or are selected from SEQ ID No. 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and 124.

[0148] Also provided is a method for modifying gene expression, comprising the steps of:

[0149] - providing a nucleic acid molecule comprising a gene operably fused to a promoter comprising two or more promoter elements each flanked by an orthogonal LoxPsym site;

[0150] - Optionally, the nucleic acid molecule comprises a terminator sequence located downstream of the gene, wherein the terminator sequence comprises two or more terminator elements, each of which is flanked by an orthogonal LoxPsym site;

[0151] - Reshuffling the promoter element and optionally the terminator element by reacting the nucleic acid molecule with the recombinase Cre.

[0152] In one embodiment, the orthogonal LoxPsym site is selected from any LoxPsym site disclosed herein, more specifically, from a LoxPsym site having the formula:

[0153] 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from SEQ ID No. 1-63, and wherein in the presence of the recombinase Cre, when the LoxPsym site comprises different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, specific DNA recombination cannot occur between the LoxPsym site and another LoxPsym site, wherein the other LoxPsym site has the following formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3' and comprises a spacer selected from SEQ ID No. 1-63, unless the LoxPsym site comprises the spacer combination GGGTACCC-AACTAGTT, GGGTACCC-ATATATAT, GAATATTC-AGTTAACT, GAATATTC-AGTTAACT or GTGTACAC-GCATATGC.

[0154] In another embodiment, the orthogonal LoxPsym site is a LoxPsym site having the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from SEQ ID No. 1-63, and wherein in the presence of the recombinase Cre, specific DNA recombination can occur between the LoxPsym site and another LoxPsym site only when the spacers of the two LoxPsym sites comprise the same nucleotide sequence between positions 2 and 7 of the spacer, wherein the other LoxPsym site has the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT and comprises a spacer selected from SEQ ID NO. 1-63, while specific DNA recombination cannot occur when the LoxPsym site comprises different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, unless the spacer combination is GGGTACCC-AACTAGTT, GGGTACCC-ATATATAT, GAATATTC-AGTTAACT, GTGTACAC-GCATATGC.

[0155] In a further embodiment, the orthogonal LoxPsym sites are selected from any group of LoxPsym sites disclosed herein, more specifically, from a group of LoxPsym sites comprising at least two LoxPsym sites having the formula: 5'-ATAACTTCGTATA-spacer-TATACGAAGTTAT-3', wherein the spacer is selected from SEQ ID NOs. 1-63, and wherein the at least two LoxPsym sites comprise different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, and wherein the group does not comprise the LoxPsym sites shown 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.

[0156] In specific embodiments, the orthogonal LoxPsym sites are selected from SEQ ID No. 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and / or 124, or are selected from SEQ ID No. 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and 124.

[0157] In some embodiments of the methods described herein, the in vitro reaction is manipulated by applying heating or a denaturant or chelating agent to terminate the activity of the recombinase. In a specific embodiment of the present invention, the method provided herein includes the following additional steps: introducing the recombinant polynucleotide into a cell, more specifically into 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 a specific embodiment, the modified cell is a microorganism, even more particularly a bacterium or yeast, most particularly a yeast genus (Saccharomyces), a genus Yarrowia (Yarrowia) or a genus Pichia yeast. In another specific embodiment, the modified cell is a cell of a species selected from the group consisting of: Saccharomyces cerevisiae, Escherichia coli (Escherichia coli), Zea mays and Yarrowia lipolytica (Yarrowia lipolytica). In a specific embodiment, the method provided herein includes an additional step of determining the sequence of the recombinant polynucleotide and / or one or more functions of a protein or functional RNA encoded by the recombinant polynucleotide.

[0158] The present application also provides recombinant polynucleotides prepared by any of the methods described herein, and cells, more specifically microorganisms such as yeast or bacteria, comprising any of the polynucleotides prepared by the methods described herein.

[0159] In certain methods, the invention comprises the use of the Cre-LoxPsym system described herein in combination with one or more other recombination systems selected from the following: Flp recombinase that functions in the Flp / FRT system, Dre recombinase that functions in the Dre-Rox system, Vika recombinase that functions in the Vika / Vox system, Bxb1 recombinase that functions at attP and attB sites, long terminal repeat (LTR) site-specific recombinase (Tre), and other serine recombinases, such as phiC31 integrase that mediates recombination between two 34 base pair sequences called attachment sites (att), Hin recombinase that recognizes a 26 bp imperfect inverted repeat sequence, or int2-13 that each recognizes different target sites of 39-66 bp.

[0160] The application also provides a cell line comprising multiple landing pads integrated into the genomic DNA of the parental cell line. The parental cell line can be a wild-type cell line, or a cell line with existing genome modification. In the latter case, the cell line is the "parent" of the cell line produced by the further modification of its genomic DNA." Landing pad" is an exogenous DNA sequence integrated into the position of the host genome including the LoxPsym site. In some cases, the exogenous DNA sequence includes a LoxPsym site, a constitutive promoter effectively connected to the nucleotide sequence encoding a detectable marker, followed by a nucleotide sequence encoding a first selectable marker. In certain types of landing pads, the LoxPsym site is located between the promoter and the nucleotide sequence encoding a detectable protein. When more than one landing pad is used in a given cell, it is preferred that the LoxPsym site of a landing pad is orthogonal to the LoxPsym site in any other landing pad. The landing pad is used for further genetic engineering and integration of target nucleic acid molecules by site-specific recombination. The landing zone can be integrated into the parental genome using any method known in the art, such as by using zinc finger nucleases, TALENs, or CRISPR-Cas systems. In some embodiments, the number of landing zones 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 zone is selected from SEQ ID No. 65-127, most specifically selected from SEQ ID No. 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 zone 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.

[0161] Also provided herein is a method for integrating one or more genetic circuits into a cell comprising a plurality of landing zones. In some embodiments, a genetic 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 a cell line, as long as the number of landing zones in the cell line is sufficient to accommodate the number of genetic circuits to be integrated into the cell line. In some cases, it may be preferred that the number of landing zones is at least the number of genetic sequences or circuits to be integrated. In other cases, a single landing zone may include multiple circuits controlled by different promoters. As used herein, "genetic circuit" is a rationally designed artificial gene regulatory network with powerful functions, which comprises a primary genetic element or building block. Non-limiting examples of primary genetic elements are promoters, ribosome binding sites, transcriptional activators and repressors, gene coding sequences, 5'UTR, 3'UTR, polyA signals and terminators. Independent modules of genetic circuits can be constructed using primary genetic elements. Methods for constructing these genetic circuits are well known to those skilled in the art. In some embodiments, multiple landing pads can be integrated into different locations in the genome, allowing modification at multiple loci of the genome through site-specific recombination.

[0162] In addition to the above detailed description of the present invention, the following experimental details further enable those skilled in the art to put all the details of the present invention into practice.

[0163] Example

[0164] Example 1. The novel LoxPsym recombination site strongly affects the recombination efficiency in Saccharomyces cerevisiae.

[0165] 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 a new non-targeted recombination site. In this case, we focused on changing the first three nucleotides of the spacer ( Figure 2 ), as previously shown, 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 adjusted accordingly to ensure that the spacer remained palindromic. Thus, we were able to design up to 63 new potential LoxPsym sites. To test whether the Cre enzyme was still able to recombine new LoxPsym variants, we performed a fluorescence-based assay in yeast by genomically integrating a fluorescent cassette that flanked the same LoxPsym variant at both sites ( Figure 3a, c). Recombination of functional LoxPsym variants can delete the fluorescent cassette. After induction of Cre-mediated recombination, the proportion of non-fluorescent cells in the population was used to determine the recombination efficiency of the novel LoxPsym site. To verify whether this setup is effective for evaluating recombination efficiency, the fluorescent cassette was used to determine the recombination efficiency of the novel LoxPsym site. Figure 3 The dual fluorescent reporter system shown in a was used to assess the frequency of inversions / deletions over time. Measurements at 0, 6, 10, and 24 hours after induction demonstrated that the frequency of inversions was negligible compared to deletions over time, thus indicating that the reporter system used to determine recombination efficiency is effective ( Figure 3 d). The recombination efficiency varied widely between different LoxPsym variants (Table 1). 63 new LoxPsym variants were obtained, and their recombination efficiency ranged from 13% to 89%. Interestingly, 36 new LoxPsym sites showed 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).

[0166] Finally, we investigated whether the purine / pyrimidine content of the spacer or the distribution of purines / pyrimidines within the spacer could explain the large differences in recombination efficiency between LoxPsym variants, but no clear relationship was observed ( Figure 3 e).

[0167] Example 2. A large set of orthogonal recombination sites can be identified.

[0168] After demonstrating that the newly developed LoxPsym sites were capable of recombination in yeast, our next goal was to identify orthogonal LoxPsym sites. Since spacers are targets for cleavage and strand exchange during Cre-mediated recombination, we reasoned that non-homologous spacers would prevent recombination. Therefore, similar experiments were performed as described above, but in this case, the LoxPsym sites flanking the fluorescent cassette were different, located upstream and downstream of the fluorescent marker ( Figure 3 bc). In order to be able to identify a large number of orthogonal sites, we evaluated the interactions between 48 LoxPsym variants. Specifically, this group contains all variants with a nucleotide T, A or G in the first position of the spacer. In this way, an additional 1008 S. cerevisiae strains were constructed to evaluate the interactions between the different LoxPsym variants. The recombination efficiency was calculated as follows: Figure 3g. The high recombination efficiency shown on the diagonal in the matrix indicates that recombination occurs between identical sites and between non-identical sites that differ only at the first and last nucleotides of the spacer (due to the palindromic nature of the LoxPsym site). Interestingly, it can be seen that these nucleotides still play a role in the cross-reactivity between variants that differ at other positions in the spacer. For example, when the LoxPsym variant is AGT TAACG- GAA When recombination occurs between TATTC, the LoxPsym spacer AGT TAACG- TAA Recombination between TATTA was prevented, thus indicating that the first nucleotide of the spacer cannot simply be ignored in the screen. Most importantly, combining all the data allowed us 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 interaction between non-identical LoxPsym variants, some strains in which the fluorescent cassette was deleted were analyzed by Sanger sequencing ( Figure 3 h). The sequences of the remaining LoxPsym sites show that mismatches at the first and last nucleotides of the two spacers always result in a heterozygous LoxPsym variant that contains traces from both parental sites and is therefore no longer completely symmetrical. Mismatches between LoxPsym variants in the middle of the spacer do not result in a heterozygous scar, but rather only the sequence of one of the two original LoxPsym sites can be found.

[0169] Example 3. Application of expression optimization tools to the astaxanthin production pathway in yeast can enhance strain performance.

[0170] As an application of the orthogonal LoxPsym sites developed in this study, the new variants were used to reorganize genetic promoter and terminator elements. First, it was evaluated whether the combination of multiple repeats of 2 orthogonal LoxPsym sites (in the promoter or terminator construct) allowed for diversification of gene expression after Cre recombinase induction. This was done by targeting a fluorescent reporter (yECitrine) and analyzing multiple single clones after recombination ( Figure 4 a - violin plot). It was observed that expression could be altered by shuffling both the promoter and the terminator. Sequencing confirmed that this was done without cross-reactions with the two orthogonal LoxPsym variants used (particularly the spacers SEQ ID No. 6 and 20 and SEQ ID No. 70 and 84) ( Figure 4 b-sequencing results).

[0171] Next, a combination of 12 orthogonal LoxPsym variants (SEQ ID Nos. 69, 70, 74, 77, 84, 86, 88, 100, 108, 109, 114, and 124) was used to alter the expression of 6 genes in the astaxanthin production pathway in Saccharomyces cerevisiae ( Figure 5 The induction of recombination results in a color change in the strain due to altered production of intermediate carotenoids such as lycopene, β-carotene, and zeaxanthin ( Figure 5 BC). The carotenoid content of multiple monoclonal clones was further analyzed ( Figure 5 D, 5F) and gene expression levels of 6 target genes ( Figure 5 E). Thus, the present application demonstrates that multiple orthogonal sites can be simultaneously applied to optimize the in vivo expression of multiple genes simultaneously. This allows optimization of the desired phenotype, in this particular example an increase in astaxanthin production of >2 fold.

[0172] Example 4. A set of orthogonal LoxPsym variants can be applied to multiplex genome engineering.

[0173] The ability to use all 16 orthogonal LoxPsym variants identified in previous experiments simultaneously was further investigated. In more detail, the application of all 16 sites present in the same genome while maintaining orthogonality was analyzed, as this is an absolute requirement for the application of these recombination sites, for example, to facilitate complex metabolic engineering efforts, where multiple genomic loci are often altered simultaneously or sequentially for gene insertions or deletions. Our tests relied on 18 constructs, including 16 test constructs and 2 controls, each of which evaluated the functionality of one LoxPsym variant in the presence of all the other variants ( Figure 6 a). To select for recombination in all cases, all constructs used the URA3 deletion (leading to tolerance to 5-fluoroorotic acid FOA) by surrounding the marker with two LoxPsym-TCA sites and plating on SC+FOA. In addition, to determine the efficiency of each specific LoxPsym site when all other sites were present, each construct encoded the ADE2 marker. Deletion of this marker resulted in a red yeast colony and allowed detection of a second recombination event. The two control constructs (with different positions of the ADE2 marker) showed only one copy of each LoxPsym site (except LoxPsym-TCA, which allowed selection of recombinant positive clones), and ADE2 was not expected to be deleted. Instead, each test construct included an additional copy of a specific LoxPsym variant upstream of the ADE2 marker, and ADE2 was expected to be deleted and used as a readout of the recombination efficiency of this site ( Figure 6bc). LoxPsym-CAC showed a very low recombination rate (0.9116 ± 0.6606%), indicating that the presence of 15 other sites induced the recombination of this LoxPsym variant. The other LoxPsym variants showed higher activity, although the recombination efficiency was always lower and did not correlate well with the previously calculated recombination efficiency, which may be the result of differences in the experimental settings. Specifically, our results indicate that since the two sites showing the highest efficiency (LoxPsym-TTA and LoxPsym-TCA) are located at the two edges of the LoxPsym array, the genomic background of these sites plays a major role. In addition, a negative correlation can be observed between the efficiency and the distance to the edge of the LoxPsym array (R 2 =0.37, p value = 0.013), indicating that the efficiency is reduced because more LoxPsym variants hinder the target recombination site to be combined or find its correct interaction partner. We infer that the reason behind 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 non-productive synapses between incompatible recombination sites, which may prevent the recombination site from recombination with a functional interaction partner (Lee and Saito 1998 Gene 216: 55-65, Fan 2012 Nucleic Acids Res. 40: 6208-6222).

[0174] Furthermore, the design of the construct allows calculation of the frequency of cross-reactivity by PCR, since recombination between different LoxPsym sites will result in reporter molecules with different lengths ( Figure 6 ). Of 208 randomly picked red colonies (13 per test construct), only 9 measured PCR fragment lengths deviated from the expected size, indicating a certain level of cross-reactivity. To further ensure that the recombination events were consistent with the expected pattern, we sequenced three PCR fragments for each test construct and observed the expected results in all but one case.

[0175] Example 5. LoxPsym variants are also functional and orthogonal in prokaryotes and higher eukaryotes.

[0176] In addition to identifying and multiplexing a set of 16 orthogonal LoxPsym variants in yeast, we also analyzed the potential of these novel recombination sites as functionalized and orthogonal tools in other species, specifically Escherichia coli and maize. To evaluate the functionality and cross-reactivity of the LoxPsym variants in E. coli, we established a plasmid-based assay testing pairwise combinations between 16 different donor and recipient plasmids, each carrying a LoxPsym variant ( Figure 8 ab). After induction of recombination, cross-reactivity between LoxPsym variants was tested by PCR amplification of the junction spanning the recombination site. All tested LoxPsym variants showed recombination activity in bacterial cells, although no correlation with activity at the corresponding sites in yeast and plants was observed ( Figure 8 c). In contrast to the data obtained for Saccharomyces cerevisiae, we did observe cross-reactivity in some cases. Sequencing of these recombination scars revealed up to three mutations in the LoxPsym sites, resulting from recombination between the cross-reacting partners. Indeed, it has been previously observed that the results of orthogonal recombination are not always transferable between prokaryotes and eukaryotes, which could be caused by slightly altered protein structure or activity of the recombinases in the different host organisms, or by differences in natural cellular processes, such as the DNA mismatch repair pathways involved in restoring mismatches that arise after recombination. Additionally, differences in the experimental setup could also be an explanation for the observed differences: the recombination (deletion) in the yeast experiments was irreversible, whereas the recombination of the two plasmids in bacteria was reversible. Importantly, the cross-reactivity was much lower than the recombination activity observed between the same LoxPsym sites.

[0177] To characterize cross-reactivity in higher eukaryotes, we used maize, one of the most important cereal crops, widely used for food and feed as well as industrial applications. We performed plasmid-based experiments using maize mesophyll protoplasts. Briefly, we constructed a plasmid library containing two LoxPsym sites separated from each other by a short linker that incorporated two restriction sites to digest the plasmid later in the workflow ( Figure 7ab). Each LoxPsym variant is attached with a unique barcode, and by sequencing the barcodes around the recombinant LoxPsym site, it is possible to identify which LoxPsym pair is involved in the recombination process. A combinatorial cloning scheme was used, and all 256 combinations of the 16 LoxPsym variants were present in the final plasmid pool and confirmed by NGS sequencing. The plasmid pool and a plasmid constitutively expressing Cre recombinase or an empty backbone were co-transfected into maize mesophyll protoplasts, and the region spanning the LoxPsym site was amplified by PCR 48 hours after transfection. Recombination was detected only in the presence of Cre recombinase, and the reaction was sent for NGS sequencing to assess the recombination efficiency of each LoxPsym pair by identifying the barcode frequency in the pool (normalized to the abundance in the starting pool). Figure 7 c). The results confirmed the recombination activity in higher eukaryotic organisms such as maize and showed that there was no cross-reactivity between the selected LoxPsym variants. In addition, a wide range of recombination efficiencies associated with different LoxPsym sites was detected, although the correlation with the efficiencies we observed in yeast was weak (R 2 =0.02, p-value=0.6056).

[0178] In summary, we evaluated cross-reactivity between selected LoxPsym variants by pairwise interaction assays and identified a set of 16 orthogonal LoxPsym variants that can be used simultaneously with no or only minimal cross-reactivity. We demonstrated that the loci described herein can also be used in species other than yeast, including Escherichia coli and maize. Together, these findings greatly expand the potential of using Cre-LoxPsym as a gene editing technology, especially in situations where repetitive and / or multiplexed recombination is required, such as during strain construction in metabolic engineering.

[0179] Example 6. LoxPsym variants are also functional and orthogonal in Yarrowia lipolytica.

[0180] To demonstrate that the alternative LoxPsym sites (Table 2) are orthogonal in Yarrowia lipolytica, we transformed wild-type strain W29 with a construct consisting of 16 LoxPsym sites separated by spacers (100 bp). The total length of the construct was 2284 bp and targeted the URA3 locus ( Fig. 9 A). After correct integration of the construct, the resulting strain was then transformed with a Yarrowia lipolytica replicative plasmid containing an expression cassette for the Cre recombinase (codons optimized for S. cerevisiae) and a NAT selectable marker conferring neomycin resistance ( Fig. 9A). Resistant colonies were then screened for Cre-mediated recombination using PCR. The primers used were called 246-F (TGGTTTAGTGTATGTTGCGC) and 247-R (CTAAGTCTGTGCTCCTTCC) and they flanked 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 bp. If some of the LoxPsym sites cross-react, then after PCR screening, smaller bands of varying sizes will appear depending on the specific lox sites that were recombined. A total of 192 transformants were screened after transient expression of the Cre recombinase. In all cases, a single band of ~2.3 kb was amplified ( Fig. 9 B), Demonstrating orthogonality of alternative lox sites in Yarrowia lipolytica.

[0181] One of the most common applications of the CRE / LOX system is marker recycling. In this case, the selectable marker is flanked by LoxP sites, and after transient expression of the Cre recombinase, the selectable marker is looped and can be used for the next round of transformation. This Cre-mediated recombination leaves behind a LoxP site that can cross-react with the newly inserted LoxP site in the next round, leading to genetic rearrangement (Steensels et al. 2018, Nat Commun 9:1937). To further test whether the new LoxPsym site is orthogonal and can be used for multiple rounds of marker recycling without the risk of genomic SCRAMBLE (Steensls et al., Nat Commun 9:1937), we used 4 different LoxPsym sites (loxP, LoxPsym0, LoxPsym2, LoxPsym4; Fig.10 A; Table 2) examined the efficiency of looping of the selectable marker hph, which confers hygromycin resistance. We repeated these experiments three times, each time examining 192 colonies for hygromycin resistance (by growing them on plates supplemented with 100 μg / ml hygromycin B). The average frequency of marker excision was 81.9% for LoxPsym0, 80.7% for LoxPsym2, 80.6% for LoxPsym4, and 79.3% for loxP ( Fig.10 B) The results show that the alternative LoxPSYMs are orthogonal, have high Cre-mediated recombination frequencies, and can therefore be combined without the risk of cross-reactivity when multiple rounds of genetic engineering and subsequent marker recycling are required.

[0182] Table 2. Orthogonal LoxPsym sites in Yarrowia lipolytica.

[0183]

[0184]

[0185] Materials and methods

[0186] Molecular methods.

[0187] DNA amplification was performed by PCR using SapphireAmp Fast PCR Mix (Takara Bio), Phusion (NEB) or GXL (TakaraBio) DNA polymerase. DNA oligonucleotides were obtained from Integrated DNA Technologies (IDT). The synthesis of longer DNA constructs was ordered from Qinglan Biotech (BGI), a subsidiary of BGI. The pV1382 backbone (Addgene plasmid #111436) was used to express sgRNA, and sgRNA was connected to the backbone digested with BsmBI after oligonucleotide annealing, as previously described (Vyas et al., 2018mSphere 3: e00154-1). The plasmid reported in this study was constructed using Gibson Assembly (NEBuilder HiFiDNA Assembly Master Mix) for plasmids used in Escherichia coli and Saccharomyces cerevisiae, and Golden Gate cloning (Lampropoulos et al., 2013PLoS One 8: GreenGate cloning standards reported in e83043) for plasmids used in corn. Plasmid purification for experiments in yeast and bacteria was performed using the QIAprep Spin Miniprep Kit (Qiagen). Plasmid purification for experiments in plant cells was performed using the ZymoPURE II Plasmid Midiprep Kit (Zymo Research). Sanger sequencing was performed by Eurofins Genomics.

[0188] Strains and growth conditions.

[0189] Escherichia coli strains were constructed from laboratory strain DH5α (NEB) and 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. 50 μg / mL antibiotics (chloramphenicol, carbinicilin and kanamycin) were added. 2% of the inducer L-rhamnose was added. Saccharomyces cerevisiae strains were constructed from laboratory strain BY4741, which is a laboratory strain of S288Cl origin with a genotype of MATa his3Δ1leu2Δ0met15Δ0ura3Δ0. Cells were grown in synthetic complete (SC) medium (0.79 g / L SCM, 6.7 g / L YNB) or SC-histidine medium. 2% of carbon sources (glucose, raffinose and galactose) were added. Maize protoplasts (cv. B104) were isolated and suspended in W5 solution as described previously, see below (Gaillochet et al., 2023 Genome Biol. 24:6).

[0190] Saccharomyces cerevisiae transformation protocol.

[0191] 1mL of culture grown overnight (ON) in 2xYPD (20g / L yeast extract, 40g / L peptone, 4g / L glucose) was inoculated into 50mL 2xYPD and cultured for 3 hours. The cells were centrifuged (3min, 3000rpm) and washed with 10mL and 1mL 0.1M lithium acetate (LiOAc) continuously. The cells were resuspended in 100μL of 0.1M LiOAc. PCR amplified donor DNA (50μL) and / or plasmid DNA (200ng) were added. Genomic DNA was inserted using CRISPR / Cas9 using pV1382 with inserted target gRNA. A mixture containing 620μL 50% PEG 3350, 4μL salmon sperm DNA and 90μL 1M LiOAc was added and mixed by vortexing. The cells were incubated at 30°C, 400rpm for 30 minutes. 100 μL DMSO was added, followed by heat shock at 42°C for 15 minutes. Cells were harvested by centrifugation (3 min, 3000 rpm) and washed with 5 mM CaCl2. Cells were incubated for a 3-hour recovery period at 30°C, 400 rpm, and finally plated on selective medium. Colony PCR (Sapphire Amp Fast PCR Master Mix, TaKaRa) was performed using a template to identify positive transformants, wherein the template was prepared by boiling the clone in 50 μL NaOH (0.02 M) (99°C, 10 minutes) to amplify the desired insertion point.

[0192] Fluorescence assay and recombination induction in Saccharomyces cerevisiae.

[0193] The strain is derived from BY4741 (Smukalla, S. et al., 2008 Cell 135: 726-737) with constitutive expression of the fluorescent reporter mCherry. To test the LoxPsy variant, the strain carries an overexpressed yECitrine reporter gene, which is regulated by the TDH3 promoter and the CYC1 terminator, flanked by two LoxPsy variants (inserted by LoxPsy-tailing primers) and integrated into the genome at the CAN1 locus. Single colonies were inoculated in 100 μL SC-His 2% glucose for overnight (ON) growth. The cells were washed and diluted to a final OD of 0.05 in SC-His 2% raffinose and cultured overnight (ON). The cells were washed and diluted in SC-His 2% raffinose 2% galactose to induce Cre expression from a control backbone (without Cre) and a plasmid containing a pGAL1-Cre expression cassette. Unless otherwise stated, the cells were induced for 6 hours. Cells were washed and diluted into SC 2% glucose for overnight (ON) recovery (1 / 20 dilution) before plating cells on YPD and / or using for flow cytometry analysis.

[0194] Fluorescence measurement.

[0195] Flow cytometry was performed using an Attune NxT flow cytometer and an autosampler. The cultured yeast cells were diluted in a focusing liquid and measured at a flow rate of 200 μL / min. Cell counting data were gated according to the FSC-H to FSC-A profile to select single cells. In order to determine the recombination efficiency, additional gating was performed using the control fluorescent reporter molecule mCherry (mCherry+ cells were selected for further analysis). yECitrine and mCherry were measured using channels BL1-A (excitation wavelength of 488 nm, emission wavelength of 574 nm, bandwidth of 20 nm) and YL2-A (excitation wavelength of 561 nm, emission wavelength of 610 nm, bandwidth of 20 nm), respectively. FlowJo software was used to perform analysis and gating steps using a (non) fluorescent control strain as a reference. The recombination efficiency below the reference value was set to 0 to remove noise from the data. To determine the yECitrine fluorescence of individual clones, individual colonies were inoculated in SC 2% glucose and fluorescence was measured using a microplate reader (TECAN Infinite200Pro) with an excitation wavelength of 498 nm, a bandwidth of 9 nm, and an emission wavelength of 535 nm, a bandwidth of 20 nm. Data were obtained after normalization by absorbance at 600 nm. The values ​​obtained were compared with those obtained for the control strain and divided into fluorescent / non-fluorescent groups.

[0196] Multiplex LoxPsym analysis and recombination induction in Saccharomyces cerevisiae.

[0197] The strain was derived from BY4741, pADE2-ADE2-tADE2 was deleted, constructed with sgRNA3, and inserted into the CAN1 locus for test and control ( Figure 6 a) and P1 (Cre) or P2 (control). Recombination was induced similarly to the above method. After overnight recovery in SC 2% glucose, cells were plated on SC and SC + FOA and incubated at 30 ° C for 48 hours, after which the colonies on each plate were counted. Red colonies were selected for PCR amplification of the recombinant construct. The length of the amplicon was determined using capillary electrophoresis (QIAxcel Advanced instrument, QIAxcel DNA Screening Cartridge, QX Size Marker 250bp–4kb v2.0) to observe slight differences in band length.

[0198] Escherichia coli transformation protocol.

[0199] For heat shock transformation, chemically competent E. coli cells were thawed on ice for 30 minutes. Plasmid DNA (50-100ng) or 2μL Gibson / Golden Gate reactions were mixed with 25μL competent cells in an ice-cold 1.5mL Eppendorf tube. After incubation on ice for 30 minutes, the reactions were heat shocked at 42°C for 30 seconds and frozen on ice for 5 minutes. 300μL SOC medium was added and the tubes were incubated at 37°C in a shaking incubator for 60 minutes. Finally, 100μL of cells were plated on preheated (37°C) LB medium containing appropriate antibiotics and incubated at 37°C for overnight growth. For electroporation, we used commercial NEB 10β cells (NEB) with a transformation efficiency of 2×10 10 cfu / μg. 2 μL of assembly reaction was mixed with 50 μL competent cells and placed in a cold electroporation tube (0.2 cm gap, BioRad). Electroporation was performed in a GenePulser (BioRad) according to the manufacturer's conditions, and 900 μL 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 on each preheated (37°C) LB plate containing the appropriate antibiotic.

[0200] Recombination assay in Escherichia coli.

[0201] After co-transformation of the recipient and donor plasmids using the dual selection medium LB + kanamycin (Kan) + chloramphenicol (Cm), bacterial strains were obtained from DH5α. Single colonies were inoculated in 100 μL LB + Kan + Cm for overnight growth. The cells were washed and diluted (1 / 20) in LB 2% rhamnose + Kan + Cm to induce Cre expression of the recipient plasmid (controlled by the rhaB promoter). After 4 hours of induction, the cells were washed and grown in LB + Kan + Cm. The recovered cells were harvested by centrifugation (3500rpm, 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 connection points of the recombinant donor and recipient plasmids. We infer that it is sufficient to amplify one of the two recombinant connection possibilities (the donor plasmid can be inserted into the recipient plasmid in two directions) because the recombination between the symmetric sites should not be biased towards one of the two options, and the combination of two independent plasmids avoids the accumulation of a species group result. Densitometric analysis of the amplicons was performed to extract peak areas (from lane plots) using Image J software. Peak areas at junctions were normalized by dividing by the area extracted from the most abundant control amplicon (derived from PCR performed on separate donor and recipient plasmids).

[0202] Construction of a combinatorial LoxPsym library for assays in maize.

[0203] To construct the LoxPsym combinatorial library, we applied Golden Gate cloning to assemble 5 entry clones using the GreenGate cloning standard (Lampropoulos et al2013 PLoS One8:e83043). Entries A and E were constructed by ligating the annealed oligonucleotides into the entry vectors pGGA000 (Addgene#48856) and pGGE000 (Addgene#48860) digested with BsaI, respectively. Entries for 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 a 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 NEBuilder master mix (NEB). For the final GoldenGate 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; PCR reaction was performed using Qubit TMdsDNA HS assay for quantification) to form a plasmid combinatorial library containing 256 different LoxPsym combinations, which were then transformed into DH10B cells. After overnight incubation, colonies from nine different plates (>50,000 colonies) were scraped and suspended in LB medium. Plasmid DNA was extracted using the ZymoPURE II Plasmid Midiprep Kit (Zymo Research). The plasmid was diluted to 1 μg / μL. A plasmid expressing Cre recombinase was also constructed using Golden Gate, starting from the available part ( https: / / gatewayvectors.vib.be / ) and were purified and diluted similarly.

[0204] Isolation and transfection of maize protoplasts.

[0205] Isolation and transfection of maize protoplasts were performed as previously described (Gaillochet et al., 2023 Genome Biol. 24:6). 100 μL of protoplasts (10 5 Cells), 110 μL PEG solution (0.2M mannitol, 100mM CaCl2) and 40% PEG 4000 and 20 μg plasmid DNA (10 μg combined loxP plasmid library and 10 μg control or Cre expression plasmid), transfected in 1 mL test tube (TN0946-08B, National Scientific Supply Co). Each transfection was performed in triplicate. Protoplasts were suspended in W5 solution and incubated in the dark in a 24-well plate on a shaking platform (20 rpm) at 25°C. Samples were collected two days later and stored at -20°C until further processing.

[0206] Extraction of maize DNA.

[0207] Protoplast DNA was isolated using a modified Edwards extraction protocol (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, rotated at 12000 rcf for 5 minutes, and then the supernatant was removed. 200 μL of extraction buffer was added to the Eppendorf tube, and the tube was manually shaken to dissolve the precipitate. After incubation at 60° C. for 15 minutes, the test tube was cooled to room temperature. 200 μL of 100% isopropanol was added, and the test tube was rotated at 12000 rcf for 10 minutes. The supernatant was removed and the precipitate was washed with 200 μL of 80% ethanol. After air drying for 15 minutes, the pellet was dissolved in 20 μL of 10 mM Tris-HCl pH 8 (pre-warmed at 60° C.) After incubating the tubes in a 60° C. heat block for 10 minutes, the tubes were stored at −20° C. until further processing.

[0208] Next generation sequencing.

[0209] To sequence the input plasmid library for maize transfection, we set up a 40 μL PCR reaction using the Phire Plant DirectPCR Kit (Thermo Scientific), using 4 μL of diluted midiprep (100 ng / μL) as template and primer OF / R82. The PCR conditions used were as follows: 98°C / 2 min + 10x (98°C / 5 s + 62°C / 5 s + 72°C / 10 s) + 72°C / 2 min + 23°C / ∞. The correct size fragment (approximately 270 bp) was 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, primers with different demultiplexing tags for each sample, and a total reaction volume of 40 μL. The PCR conditions used were as follows: 98°C / 2min+25x(98°C / 5s+62°C / 5s+72°C / 10s)+72°C / 2min+23°C / ∞. We did not detect any evidence of recombination in the agarose electrophoresis results and inferred that this may be due to the large amount of transfected plasmid DNA (~32 million plasmid copies / protoplast). Therefore, we restricted the extracted DNA to specifically cut the C-linker-D module of the non-recombinant plasmid to bias the 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 hours, and amplicons were constructed using primers OF / R83-88 purified using 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 from protoplast DNA was normalized by the number of reads present in the input library.

[0210] Yarrowia lipolytica strains and culture conditions.

[0211] 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 (when liquid culture was used) for 2-3 days. Selective agents were added at the following concentrations: nourseothricin (CloNAT), 220 μg / ml; hygromycin B 100 μg / ml.

[0212] Yeast (Yarrowia lipolytica) transformation.

[0213] Transformation of Yarrowia lipolytica followed a protocol already described (Abdel-Mawgoud and Stephanopoulos 2020, Metab Eng 62:106–115) with slight modifications.

[0214] Colony picking and PCR screening.

[0215] Transformed colonies were picked from the transformation plates using a PIXL Precision Microbial Colony Picker (Singer Instruments) and fixed on selection plates. Colony PCR screening was performed using 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 SEQ ID No. 1-63. 2 . The LoxPsym site according to claim 1 , wherein the LoxPsym site is cleaved in the presence of recombinase Cre.

3. The LoxPsym site according to any one of claims 1 or 2, comprising a nucleic acid sequence selected from SEQ ID No. 65-127.

4. The LoxPsym site according to any one of claims 1 to 3, characterized in that the recombinant The efficiency is lower than the recombination efficiency of the standard LoxPsym site shown in SEQ ID No. 128, and wherein the LoxPsym site comprises a spacer selected from SEQ ID No. 1-27.

5. The LoxPsym site according to any one of claims 1 to 3, characterized in that the recombinant The efficiency is higher than the recombination efficiency of the standard LoxPsym site shown in SEQ ID No. 128, and wherein the LoxPsym site comprises a spacer selected from SEQ ID No. 28-63.

6. The LoxPsym site according to claim 1, wherein in the presence of the recombinase Cre, specific DNA recombination cannot occur between the LoxPsym site and the second LoxPsym site according to claim 1, and wherein the LoxPsym site and the second LoxPsym site comprise different nucleotides at positions 2, 3, 6 and / or 7 of the spacer.

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

8. The LoxPsym site according to any one of claims 6 or 7, wherein the LoxPsym site is selected from SEQ ID No. 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and / or 124, and / or is selected from SEQ ID No. 70, 77, 82, 101-102, 105, 107, 109, 112, 114, 117-118, 123-124, 126 and / or 127, and / or is selected from SEQ ID No. 65, 71-74, 84, 86, 88, 90, 103, 106-108, 111, 121 and / or 124, and / or is selected from SEQ ID No. ID No.72, 74, 77-78, 80, 82, 84, 86, 88, 102, 106, 109, 114, 118, 123 and / or 127, and / or is selected from SEQ ID No.65, 70-71, 73, 80, 90, 101, 103, 105, 108, 111-112, 117, 121, 124 and / or 126, and / or is selected from SEQ ID No. SEQ ID No.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 the preceding claims. 10 . A host cell comprising the LoxPsym site according to any one of claims 1 to 8 or the vector according to claim 9 . The host cell according to claim 10 , wherein the host cell is a plant cell, a microorganism, an insect cell or a mammalian cell.

12. The host cell according to 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: The at least two LoxPsym sites comprise different nucleotides at positions 2, 3, 6 and / or 7 of the spacer, and wherein the group does not comprise the LoxPsym sites shown 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. The panel according to claim 14, wherein the at least two LoxPsym sites are selected from SEQ ID No. 65, 69-71, 74, 77, 82, 84, 100, 105, 107-109, 112, 121 and / or 124, and / or selected from SEQ ID No.70, 77, 82, 101-102, 105, 107, 109, 112, 114, 117-118, 123-124, 126 and / or 127, and / or selected from SEQ ID No.65, 71-74, 84, 86, 88, 90, 103, 106-108, 111, 121 and / or 124, and / or selected from SEQ ID No.72, 74, 77-78, 80, 82, 84, 86, 88, 102, 106, 109, 114, 118, 123 and / or 127, and / or is selected from SEQ ID No.65, 70-71, 73, 80, 90, 101, 103, 105, 108, 111-112, 117, 121, 124 and / or 126, and / or is selected from SEQ ID No.SEQ ID No.69, 70, 74, 77, 84, 86, 88, 100, 108, 109, 114 and / or 124.

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

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

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

19. A method for obtaining a recombinant nucleic acid molecule, the method comprising the following steps: a. Providing a nucleic acid molecule comprising two or more nucleic acid elements, each of which is flanked by an orthogonal LoxPsym sites, or providing a first nucleic acid molecule and a second nucleic acid molecule, each of which comprises one or more nucleic acid elements, each of which is flanked by an orthogonal LoxPsym site; b. reacting the nucleic acid molecule or the first and second nucleic acid molecules with a recombinase Cre to obtain a recombinant nucleic acid molecule, The orthogonal LoxPsym sites are selected from SEQ ID No. 65-127.

20. A method for shuffling DNA elements within a nucleic acid molecule, the method comprising the steps of: a. Providing a nucleic acid molecule comprising at least two nucleic acid elements, each of which is flanked by an orthogonal LoxPsym site; b. reacting the nucleic acid molecule with a recombinase Cre to obtain a nucleic acid molecule in which the nucleic acid elements are shuffled, wherein the orthogonal LoxPsym site is selected from SEQ ID No. 65-127.

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

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

23. The method according to 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 a cell.

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

25. 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 the one or more genes, each gene being controlled by a promoter comprising two or more promoter elements, wherein the two or more promoter elements are respectively flanked by orthogonal LoxPsym sites, wherein each gene uses a different LoxPsym site; b. Optionally, the one or more vectors further comprise a terminator sequence located downstream of each gene, the terminator sequence comprising two or more terminator elements, wherein the two or more terminator elements are flanked by orthogonal LoxPsym sites, wherein each gene uses a different LoxPsym site, and wherein any of the The LoxPsym sites are all different from any LoxPsym sites used in step a); c. expressing the recombinase Cre in the cell; d. analyzing the gene expression of the one or more genes or analyzing the phenotype of the cells.

26. The method of claim 25, wherein at least one orthogonal LoxPsym site is selected from SEQ ID No. 65-127.

27. The method according to any one of claims 25 or 26, wherein the at least one orthogonal LoxPsym site is a LoxPsym site according to any one of claims 6 to 8.

28. 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 according to any one of claims 25 to 28, further comprising the step of determining the sequence of all or part of the genome of the cell.

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

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

32. Genetically engineered cells obtained by the method according to any one of claims 25 to 28.

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