Lox sites and methods for genome engineering in plants

Abstract

Embodiments of the present disclosure provide a construct and a method of using the construct to increase the efficiency of Cre-mediated transgene excision. In some embodiments, the construct comprises a cytosine-free lox recombination spacer sequence comprising a nucleotide sequence as set forth in one of SEQ ID NOs: 1-256. In other embodiments, the method is directed to increasing the efficiency of a Cre-mediated transgene excision in a plant cell, the method comprising: (a) introducing into at least one plant cell at least one plasmid comprising at least one cytosine-free lox site; and (b) contacting the at least one cytosine-free free lox site with a Cre recombinase, wherein the transgene is excised more efficiently when the at least one cytosine-free lox site is contacted with the Cre recombinase compared to transgene excision in the absence of the cytosine-free lox site.

Description

LOX SITES AND METHODS FOR GENOME ENGINEERING IN PLANTS CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S Provisional Application No.63 / 500072, filed May 4, 2023. STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is 3014-P29WO_Seq_List_20240503.xml. The XML file is 318,282 bytes; was created on May 3, 2024; and is being submitted electronically via Patent Center with the filing of the specification. STATEMENT OF GOVERNMENT LICENSE RIGHTS

[0003] This invention was made with Government support under grant No.2020-33522-32316, awarded by United States Department of Agriculture. The Government has certain rights in the invention. BACKGROUND

[0004] Cre recombinase is a widely used molecular tool for performing DNA manipulations both in vitro and in vivo, with applications in research and biotechnology. This includes deletion, integration, and inversion of target sequences of interest. Cre recombinase acts upon recognition sequences which are called lox sites, the most widely used of which is loxP, which is naturally present in the phage P1 genome.

[0005] Cre recombinase activity in plant somatic cells has been shown to induce gene silencing through methylation of DNA cytosine residues in inserted transgenic regions between added loxP sites. Analysis of these sites with purified DNA and Cre recombinase suggest that methylated cytosines within loxP sites themselves can hamper recombination in vitro. Reducing the plant gene silencing response had positive effects on successful DNA excision in transgenic tobacco plants.

[0006] For vegetatively propagated plants, the removal of gene editing reagents like CRISPR / Cas is difficult. In sexually propagated plant species, segregation of the stably integrated CRISPR / Cas is accomplished through segregation of the transgenic locus via mendelian inheritance while retaining desired edits. Recombinases are commonly used to delete CRISPR / Cas components or other genes useful during genetic transformation (antibiotic resistance genes and / or morphogenic regulators) after they are no longernecessary. However, the deletion of these genes in somatic tissues is currently very inefficient in plants. SUMMARY

[0007] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0008] The present disclosure provides cytosine-free lox sites and methods for their use. In certain embodiments, the disclosure provides a cytosine-free lox site comprising a lox arm 1, and a lox arm 2 separated by a four to twelve nucleotide core spacer sequence. In certain specific embodiments, the core spacer sequence comprises eight nucleotides and does not include a cytosine.

[0009] In certain embodiments the cytosine-free lox site comprises a lox arm one and lox arm two comprising a nucleotide sequence SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:4 through SEQ ID NO:102.

[0010] In certain embodiments, the cytosine-free lox site comprises a core spacer sequence comprising randomly repeating adenine (A) and / or thymine (T) nucleotides. In certain embodiments, the core spacer sequence of the cytosine-free lox site is selected from one of AAAAAAAA (loxW10), AAAAAAAT, AAAAAATA, AAAAAATT, AAAAATAA, AAAAATAT, AAAAATTA, AAAAATTT, AAAATAAA, AAAATAAT, AAAATATA, AAAATATT (loxW9), AAAATTAA, AAAATTAT, AAAATTTA, AAAATTTT, AAATAAAA (loxW18), AAATAAAT, AAATAATA (loxW6), AAATAATT, AAATATAA, AAATATAT, AAATATTA, AAATATTT, AAATTAAA, AAATTAAT, AAATTATA, AAATTATT, AAATTTAA, AAATTTAT (loxW28), AAATTTTA, AAATTTTT, AATAAAAA, AATAAAAT (loxW21), AATAAATA, AATAAATT, AATAATAA, AATAATAT, AATAATTA, AATAATTT, AATATAAA, AATATAAT, AATATATA, AATATATT, AATATTAA, AATATTAT, AATATTTA, AATATTTT, AATTAAAA, AATTAAAT, AATTAATA, AATTAATT, AATTATAA, AATTATAT, AATTATTA, AATTATTT (loxW11), AATTTAAA, AATTTAAT, AATTTATA (loxW4), AATTTATT, AATTTTAA, AATTTTAT, AATTTTTA, AATTTTTT, ATAAAAAA (loxW15), ATAAAAAT, ATAAAATA (loxW22), ATAAAATT, ATAAATAA, ATAAATAT, ATAAATTA, ATAAATTT, ATAATAAA, ATAATAAT, ATAATATA, ATAATATT (loxW26), ATAATTAA (loxW17),ATAATTAT, ATAATTTA, ATAATTTT, ATATAAAA, ATATAAAT, ATATAATA, ATATAATT, ATATATAA, ATTTAATA, ATATATAT, ATATATTA, ATATATTT, ATATTAAA (loxW8), ATATTAAT, ATATTATA, ATATTATT, ATATTTAA, ATATTTAT, ATATTTTA, ATATTTTT, ATTAAAAA, ATTAAAAT, ATTAAATA, ATTAAATT, ATTAATAA, ATTAATAT, ATTAATTA, ATTAATTT, ATTATAAA, ATTATAAT, ATTATATA, ATTATATT, ATTATTAA, ATTATTAT, ATTATTTA, ATTATTTT, ATTTAAAA, ATTTAAAT (loxW25), TAAATTTT, ATTTAATT, ATTTATAA, ATTTATAT (loxW20), ATTTATTA, ATTTATTT, ATTTTAAA, ATTTTAAT, ATTTTATA, ATTTTATT, ATTTTTAA, ATTTTTAT, ATTTTTTA, ATTTTTTT, TAAAAAAA, TAAAAAAT, TAAAAATA, TAAAAATT, TAAAATAA, TAAAATAT, TAAAATTA (loxW12), TAAAATTT, TAAATAAA, TAAATAAT, TAAATATA, TAAATATT, TAAATTAA, TAAATTAT, TAAATTTA, TATATTAA, TATATTAT, TAATAAAA, TAATAAAT, TAATAATA, TAATAATT, TAATATAA, TAATATAT, TAATATTA, TAATATTT, TAATTAAA, TAATTAAT, TAATTATA, TAATTATT, TAATTTAA, TAATTTAT, TAATTTTA, TAATTTTT, TATAAAAA, TATAAAAT, TATAAATA, TATAAATT, TATAATAA, TATAATAT, TATAATTA, TATAATTT, TATATAAA, TATATAAT, TATATATA, TATATATT (loxW7), TTAATAAT, TTAATATA, TATATTTA, TATATTTT, TATTAAAA, TATTAAAT, TATTAATA, TATTAATT, TATTATAA, TATTATAT, TATTATTA, TATTATTT (loxW24), TATTTAAA, TATTTAAT, TATTTATA, TATTTATT, TATTTTAA, TATTTTAT (loxW5), TATTTTTA, TATTTTTT, TTAAAAAA, TTAAAAAT (loxW23), TTAAAATA, TTAAAATT, TTAAATAA, TTAAATAT, TTAAATTA, TTAAATTT, TTAATAAA, TTTAATTA, TTTAATTT, TTTATAAA, TTAATATT, TTAATTAA, TTAATTAT, TTAATTTA (loxW19), TTAATTTT (loxW2), TTATAAAA, TTATAAAT, TTATAATA, TTATAATT, TTATATAA, TTATATAT, TTATATTA, TTATATTT, TTATTAAA, TTATTAAT, TTATTATA, TTATTATT, TTATTTAA (loxW1), TTATTTAT, TTATTTTA, TTATTTTT, TTTAAAAA, TTTAAAAT, TTTAAATA, TTTAAATT, TTTAATAA, TTTAATAT, TTTTAATT, TTTTATAA, TTTTATAT, TTTATAAT, TTTATATA, TTTATATT, TTTATTAA, TTTATTAT, TTTATTTA, TTTATTTT, TTTTAAAA, TTTTAAAT, TTTTAATA, TTTTATTA, TTTTATTT (loxW3), TTTTTAAA, TTTTTAAT, TTTTTATA, TTTTTATT, TTTTTTAA, TTTTTTAT, TTTTTTTA, or TTTTTTTT.

[0011] In certain embodiments the cytosine-free lox site selected from one of as set forth in one of SEQ ID NO: 103 – SEQ ID NO:357.

[0012] The present disclosure also provides a method to increase the efficiency of Cre-mediated transgene excision in a plant cell, the method comprising: (a) introducing into at least one plant cell at least one plasmid comprising at least one cytosine-free lox site according to any of the above embodiments; and (b) contacting the at least one cytosine- free free lox site with a Cre recombinase; wherein the transgene is excised more efficiently when the at least one cytosine-free lox site is contacted with the Cre recombinase compared to transgene excision in the absence of the cytosine-free lox site.

[0013] In certain embodiments the method comprises the use of a plasmid comprises a transgene flanked by direct repeats of the cytosine-free lox site. In certain embodiments the transgene comprises a gene encoding Cas 9 and at least one guide RNA. In certain embodiments, the plasmid further comprises a Cre coding region. In certain embodiments the cytosine-free lox site is selected from a sequence as set forth in any one of SEQ ID NO:103 – SEQ ID NO:357.

[0014] In certain embodiments, the plasmid comprises a constitutive promoter, an inducible promoter, or a tissue specific promoter controlling Cre expression. In certain embodiments, the plasmid comprises a reporter gene. In certain more specific embodiments the reporter is a fluorescent or DNA methylation sensitive reporter. DESCRIPTION OF THE DRAWINGS

[0015] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

[0016] FIGURE 1 depicts how steric hinderance of methylated cytosine residues in the wild-type loxP sequence can impair Cre recombinase activity in vitro (Liu et al., DNA methylation occurring in Cre-expressing cells inhibits loxP recombination and silences loxP-sandwiched genes. New Phytol. 231(1):210-224, 2021). The present disclosure demonstrates the discovery and use of recombination sites whose core spacer sequences lack cytosines that improve excision efficiency in plants.

[0017] FIGURE 2 depicts the strategy used for finding and validating cytosine- free recombination sites. LoxP arm 1 (SEQ ID NO:1) and LoxP arm 2 (SEQ ID NO: 2) aredemonstrated. Location of successful excision footprints shown with left pointing arrow or Cre.

[0018] FIGURE 3 shows that cytosine-free lox sites were more efficient in excision than wild-type loxP sequences. Leaves from tobacco were imaged 4 days after agroinfiltration. Strong reductions of tdTomato (an exceptionally bright red fluorescent protein) expression can be seen in Cre-containing constructs (bottom panels) relative to a no-Cre control (top panels), which could be due to excision of the tdTomato fragment or silencing without excision. However, use of the disclosed fluorescent-switch-reporter constructs (that enable green fluorescent protein (GFP) expression after excision) verified that excision was occurring, and that the cytosine-free LoxP core site of LoxW7 (TATATATT)-containing leaves were more effective than native LoxP in supporting excision (Bottom panel).

[0019] FIGURES 4A and 4B demonstrate that cytosine-free lox sites were more efficient at inducing successful excision in stable poplar transgenics. Figure 4A shows LoxP and LoxW7-containing transgenes with constitutive Cre and a methylation-sensitive excision reporter exhibit reduced shoot regeneration rates vs. no Cre containing controls in poplar, but LoxW7 transgenes allow (approximately 10%) excised shoot recovery. Figure 4B shows LoxP and LoxW7-containing transgenes with constitutive Cre and methylation- resistant excision reporter (26% GC content proximal to Lox sites) substantially reduce LoxW7 negative-regeneration effects but not LoxP. Using this reporter, LoxW7 sites resulted in greater recovery of excised shoots versus LoxP.

[0020] FIGURE 5 shows a plant transformation vector example utilizing cytosine-free lox sites to enable CRISPR and / or morphogenic gene excision after transformation. Cytosine-free lox sites (as an example, the loxW7 site is designated) were added in direct repeats in the flanking arms of the T-DNA, 100-300 base pairs inboard of the T-DNA left and right borders. After excision, only a small piece of residual inserted DNA remains in the genome comprising a lox site and the left and right border regions. (bottom construct diagram).

[0021] FIGURE 6 shows LoxW7-containing transgenes with heat-shock inducible Cre and a methylation-resistant excision reporter do not pre-excise early in transgenesis, allowing for dynamic control of excision via heat shock during transformation.

[0022] FIGURES 7A through 7C show LoxW7-containing transgenes with heat- shock inducible Cre, gene editing components and DEV genes result in efficient transgene excision in poplar. FIGURE 7A provides a map of the vector construct. FIGURE 7B depicts regenerated shoots that demonstrate excision of the transgene and examples of developing plants with the RUBY transgene and with the RUBY transgene excised. FIGURE 7C shows the percent of excision events with and without heat shock and the transgenic efficiency of excision at the DNA level. Forty eight percent (48%) of events yielded visually excised propagules after two weeks of heat shock treatment and one month on SIM (shoot induction media), and 50% of those propagules were verified to be transgene-free by PCR (FIGURE 7C). DETAILED DESCRIPTION

[0023] The process of transgene excision would be greatly aided by improved efficiency of recombinases for their target sites in plants. The present disclosure provides recombination sites for the most widely used recombinase which facilitate increased plant activity by making them resistant to cytosine methylation which occurs in unique contexts in plants.

[0024] In embodiments, the present disclosure describes cytosine-free lox sequences, and a method for generating them, which were designed to increase Cre- mediated excision rates on their respective lox sites specifically in plant cells. Because plants have different methods and contexts of DNA methylation compared with animals, cytosine methylation prevents efficient recombination of target sites (FIGURE 1). Discovery and implementation of lox sites which are resistant to cytosine methylation improves their ability to be acted on successfully by Cre recombinase when Cre is expressed. Although many alternative lox sites have been discovered through previous screening strategies, none are cytosine-free in the spacer sequence nor are any optimized for use as excision tools in plants. Method of Identifying Methylation Resistant Lox Sites Lacking Cytosine

[0025] The present disclosure provides a unique screening method to identify lox sites located in the recombination core spacer sequence and that lack cytosines and are therefore less or non-susceptible to methylation in plants.

[0026] In embodiments, this method leverages flanking degenerate primer sequences (both 3’ and 5’) whose overhangs include lox sites with spacers which only contain the DNA bases A or T for use in PCR (polymerase chain reaction). Amplificationof a linear product containing a bacterial origin sequence and an antibiotic resistance gene was undertaken (FIGURE 2). These PCR products were treated in vitro with purified Cre recombinase, and those with compatible lox sites with cytosine-free spacer sequences circularized into plasmids capable of replication and selection in E. coli. Colonies produced through this method were sequenced. The resulting lox sites with cytosine-free core spacer regions were screened for activity through cloning two direct repeats in plasmids and treating with purified Cre recombinase for excision products. Increased Recombination Efficiency in Plants

[0027] To implement these lox sequences and test for increased recombination efficiency in plants, well-performing sites were cloned in direct repeats, the first placed in the 5’ UTR of a fluorescent reporter gene, and the second placed after the terminator of the same fluorescent gene. A second fluorescent reporter was placed downstream of this site without a promoter such that excision via Cre of the first product turns on expression of the second reporter (FIGURE 3). Testing of these vectors with a constitutively expressed Cre in tobacco agroinfiltration assays (Schöb et al. Mol. Genet. Genomics 256:581-585, 1997; Norkunas et al. Plant Methods 14:71, 2018; both incorporated herein by reference) showed significantly increased transgene excision with cytosine-free spacer lox sites relative to the same vector with traditional loxP sites. The same result was found in stable transgenic calli and shoots in the hybrid poplar P. tremula x alba using constitutive Cre recombinase (FIGURES 4A and 4B). Uses of the Cytosine Free Lox Sites

[0028] Possible uses for the disclosed methods and lox sites include excision of morphogenic genes used in plant transformation and gene editing, excision of gene editing genes, site-specific integration of transgenes, development of biosensors or inducible switches using Cre recombinase in plants, and fate-mapping tools for use in plants. Use of these cytosine free lox sites is conventional for one skilled in the art with existing strategies using loxP, such as providing direct repeats of loxP sites flanking desired segments to be deleted after transformation, or during the induction of a genetic switch. Conversely, Cre recombinase will act upon a single site at a previously inserted locus should gene integration be desired. Cre recombinase expression control is conventional with cytosine- free sites, however due to the increased rates of Cre activity (e.g., excision), additional control of baseline Cre recombinase expression may be desirable based on the somatic or germline cell type of interest where a recombination event is to be triggered.Vectors Containing Lox Sequences

[0029] For the deletion of gene editing and morphogenic genes after the initial transformation step using Cre recombinase and cytosine-free lox sites, vectors which are flanked by direct repeats of the lox sites, between the T-DNA left and right borders (FIGURE 5). These are placed 100-300 base pairs inboard of these locations to allow successful excision in cases where T-DNA insertion fidelity is imperfect and is truncated relative to the T-DNA left or right border. The remaining “footprint” of DNA remaining in the genome is then generally less than 500 base pairs and contains only non-coding DNA elements including an intact cytosine-fee lox site unless a transgene is desired to be inserted at the same time as gene editing is to commence. Cytosine-free lox sites can be added to existing T-DNA vectors in a conventional PCR-based approach via long primers, Gibson assembly, oligo annealing, or other common cloning techniques to those skilled in the art. (Han et al. Plant Cell Rep. 19:315-320, 2000; Birch Nuc. Acids Res. 26(11):2729-2734, 1998; both incorporated herein in their entirety).

[0030] In accordance with the foregoing, in one aspect, the disclosure provides for a cytosine-free lox recombination core spacer sequence comprising a nucleotide sequence as set forth in one of the nucleotide sequences in Table 2.

[0031] In another aspect, the disclosure provides for a method to increase the efficiency of Cre recombinase-mediated transgene excision in a plant cell, the method comprising: (a) introducing into at least one plant cell at least one plasmid comprising at least one cytosine-free lox site; and (b) contacting the at least one cytosine-free free lox site with a Cre recombinase, wherein the transgene is excised more efficiently when the at least one cytosine-free lox site is contacted with the Cre recombinase compared to transgene excision in the absence of the cytosine-free lox site.

[0032] In some embodiments, the plasmid comprises a transgene flanked by direct repeats of the cytosine-free lox site. See for example, as specific embodiments FIGURE 4A and FIGURE 4B which provide examples of molecular switches.

[0033] In other embodiments, the plasmid further comprises a Cre recombinase coding region. See for example, as specific embodiments FIGURE 5 and FIGURE 7A.

[0034] In some embodiments, the cytosine-free lox site comprises a Cre recognition sequence, and its palindrome selected from a sequence as set forth in any one of SEQ ID NOs: 1, 2, and 4 through 102 separated by a 4 to 12 base pair cytosine-free core spacer sequence.

[0035] In other embodiments, the cytosine-free lox site comprises an eight base pair core spacer sequence comprising randomly repeating adenine (A) and / or thymine (T) nucleotides. In some embodiments, the core sequence comprises between 4 to 12 randomly repeated A and / or T nucleotides.

[0036] In certain embodiments, the cytosine-free lox site comprises a loxP recognition sequence and its palindrome separated by an eight base pair cytosine-free nucleotide core spacer sequence wherein the loxP cytosine-free site is selected from one of the nucleotide sequences SEQ ID NO:103-357. Additional definitions

[0037] As used herein, “transformation” means processes by which cells / tissues / plants acquire properties encoded on a nucleic acid molecule that has been transferred to the cell / tissue / plant. “Transferring” refers to methods to transfer DNA into cells including, but not limited to, microinjection, permeabilizing the cell membrane with various physical (e.g., electroporation) or chemical (e.g., polyethylene glycol, PEG) treatments, high-velocity microprojectile bombardment also termed biolistics, or infection with Agrobacterium tumefaciens or A. rhizogenes. In some embodiments, the exogenously supplied DNA is either T-DNA, PCR-derived, or plasmid-derived.

[0038] As used herein, “transformant” means a plant which has acquired properties encoded on a nucleic acid molecule that has been transferred to cells during the process known as transformation. As used herein, “re-transformation” means transformation of cells / tissues / plants which are in themselves transformants.

[0039] As used herein, Cre recombinase-mediated transgene excision refers to but is not limited to: (i) deletion of a pre-selected DNA segment flanked by lox sites; (ii) inversion of the nucleotide sequence of a pre-selected DNA segment flanked by lox sites; and (iii) reciprocal exchange of DNA segments proximate to lox sites on different DNA molecules. Subsequent to excision a cytosine-free lox site remains after excision of the selected transgene.

[0040] The expression “nucleotide sequence,” as used herein refers to a polymer of DNA or RNA, which can be single- or double-stranded, optionally containing synthetic, non-natural, or altered nucleotides capable of incorporation into DNA or RNA polymers. A “DNA segment” refers to a linear fragment of single- or double-stranded DNA, which can be derived from any source. The expression “DNA in plant cells” includes all DNA present in plant cells.

[0041] A “coding region” refers to a DNA segment which encodes a regulatory molecule or any polypeptide. The expression “regulatory molecule” refers to a polymer of ribonucleic acid (RNA), such as antisense RNA or a ribozyme, or a polypeptide which is capable of enhancing or inhibiting expression of a gene product. The term “expression” refers to the synthesis of gene product from a gene coding for the sequence of the gene product. The gene product can be a RNA or a protein.

[0042] As used herein, the term “promoter region” refers to a sequence of DNA, usually upstream (5’) of the coding sequence, which controls the expression of the coding region by providing the recognition for RNA polymerase and / or other factors required for transcription to start at the correct site. A “promoter fragment” constitutes a DNA sequence consisting of the promoter region. A promoter region can include one or more regions which control the effectiveness of transcription initiation in response to physiological conditions (e.g., an inducible promoter), and a transcription initiation sequence. A “tissue specific promoter” as referred to herein is one that directs gene expression primarily in specific tissues such as roots, leaves, stems, pistils, anthers, flower petals, seed coat, seed nucellus or epidermal layers. Transcription stimulators, enhancers or activators may be integrated into tissue specific promoters to create a promoter with a high level of activity that retains tissue specificity.

[0043] “Introducing” as used herein is intended to mean presenting to the organism, such as a plant, or the cell a polynucleotide or polypeptide in such a manner that the polynucleotide or polypeptide comprising a sequence of nucleotides or amino acids gains access to the interior of a cell of the organism or to the cell itself. The methods and compositions do not depend on a particular method for introducing a polynucleotide or polypeptide into an organism or cell, only that the polynucleotide or polypeptide gains access to the interior of at least one cell of the organism. Methods for introducing polynucleotides or polypeptides into plants are well known in the art and include, but not limited to, stable transformation methods, transient transformation methods, virus- mediated methods, and sexual breeding.

[0044] “Stable transformation” as used herein is intended to mean that the nucleotide construct introduced into a plant integrates into a genome of the plant and is capable of being inherited by the progeny thereof. “Transient transformation” as used herein is intended to mean that a polynucleotide is introduced into the plant and does not integrate into a genome of the plant, or a polypeptide is introduced into a plant.

[0045] As used herein a “transgene” is a gene which is introduced into the genome of a plant cell either artificially or naturally. A “transgene of interest” is a gene that is selected for a particular purpose to be introduced into the plant cell. The method of introduction can include any number of genetic engineering techniques well known in the art.

[0046] The wild-type recognition site on the bacteriophage P1 recognized by Cre is a 34 base pair (bp) double stranded DNA sequence known as LoxP. The LoxP site is palindromic with the exception of its eight innermost base pairs, which impart directionality to the site. As used herein, a complete single cytosine free lox site comprises the following configuration: lox arm 1 – core spacer sequence - lox arm 2; wherein lox arm 1 is the 5’ Cre recognition sequence and lox arm 2 is the Cre 3’ recognition sequence. The wild-type core spacer sequence is the nucleotide sequence GCATACAT. In some particular embodiments, a complete single cytosine free lox site comprises the configuration LoxP arm 1 – core spacer sequence – LoxP arm 2. In some embodiments, a complete cytosine free lox site flanks each end of the transgene of interest to be deleted. In some embodiments, the complete cytosine free lox site flanking each end of a transgene of interest to be deleted comprises identical sequences (i.e., each sequence is the same). In some embodiments, the complete cytosine free lox site flanking each end of the transgene of interest to be deleted comprises different sequences (i.e, each sequence is different). One skilled in the art would understand that for some applications, it can be advantageous if sites had very low promiscuity with divergent sequences, for example, if there were more than two complete single cytosine free lox sites in the construct and they were to operate in pairs.

[0047] The Cre recombinase has been shown to be active on various nucleotide sequences that vary considerably from the bacteriophage recognition sequence LoxP (LoxP arm 1 – ATAACTTCGTATA (SEQ ID NO: 1); LoxP arm 2 – TATACGAAGTTAT (SEQ ID NO: 2)). The nucleotide sequences of the alternative recognition sites are provided below and derivation of these sequences to form full length cytosine-free lox sites is considered part of the presently disclosed compositions and methods. The alternative lox recognition sequences (lox arm 1 and lox arm 2 are provided in Table 1 and are as follows:Table 1- Alternative Cre recognition sequences. Lox arm 1 Lox arm 2ACAACCTCCCTTA (SEQ ID NO:63) TAAGGGAGGTTGT (SEQ ID NO:64)Nucleotide sequences for cytosine-free recombination

[0048] The following nucleotide sequences can be useful in practicing the various embodiments and broader concepts of the present disclosure and envisioned herein.

[0049] As above, the structure of the cytosine-free Cre recombinase recognition sites have the basic structure of lox arm 1 – core spacer sequence - lox arm 2. Lox arm 2 is the reverse nucleotide sequence lox arm 1. In specific embodiments of the cytosine-free Cre recombinase recognition the nucleotide sequences comprise LoxP arm 1 – core spacer sequence – LoxP arm 2. The nucleotide sequences of the cytosine-free core spacer sequences are provided below in Table 2.Table 2 - Cytosine-free recombination core sequences AAAAAAAA (loxW10) AAATTTAA AAAAAAAT AAATTTAT (loxW28) AAAAAATA AAATTTTA AAAAAATT AAATTTTT AAAAATAA AATAAAAA AAAAATAT AATAAAAT (loxW21) AAAAATTA AATAAATA AAAAATTT AATAAATT AAAATAAA AATAATAA AAAATAAT AATAATAT AAAATATA AATAATTA AAAATATT (loxW9) AATAATTT AAAATTAA AATATAAA AAAATTAT AATATAAT AAAATTTA AATATATA AAAATTTT AATATATT AAATAAAA (loxW18) AATATTAA AAATAAAT AATATTAT AAATAATA (loxW6) AATATTTA AAATAATT AATATTTT AAATATAA AATTAAAA AAATATAT AATTAAAT AAATATTA AATTAATA AAATATTT AATTAATT AAATTAAA AATTATAA AAATTAAT AATTATAT AAATTATA AATTATTA AAATTATT AATTATTT (loxW11) AATTTAAA ATATATAT AATTTAAT ATATATTA AATTTATA (loxW4) ATATATTT AATTTATT ATATTAAA (loxW8)AATTTTAA ATATTAAT AATTTTAT ATATTATA AATTTTTA ATATTATT AATTTTTT ATATTTAA ATAAAAAA (loxW15) ATATTTAT ATAAAAAT ATATTTTA ATAAAATA (loxW22) ATATTTTT ATAAAATT ATTAAAAA ATAAATAA ATTAAAAT ATAAATAT ATTAAATA ATAAATTA ATTAAATT ATAAATTT ATTAATAA ATAATAAA ATTAATAT ATAATAAT ATTAATTA ATAATATA ATTAATTT ATAATATT (loxW26) ATTATAAA ATAATTAA (loxW17) ATTATAAT ATAATTAT ATTATATA ATAATTTA ATTATATT ATAATTTT ATTATTAA ATATAAAA ATTATTAT ATATAAAT ATTATTTA ATATAATA ATTATTTT ATATAATT ATTTAAAA ATATATAA ATTTAAAT (loxW25) ATTTAATA TAAATTTT ATTTAATT TAATAAAA ATTTATAA TAATAAAT ATTTATAT (loxW20) TAATAATA ATTTATTA TAATAATT ATTTATTT TAATATAA ATTTTAAA TAATATAT ATTTTAAT TAATATTAATTTTATA TAATATTT ATTTTATT TAATTAAA ATTTTTAA TAATTAAT ATTTTTAT TAATTATA ATTTTTTA TAATTATT ATTTTTTT TAATTTAA TAAAAAAA TAATTTAT TAAAAAAT TAATTTTA TAAAAATA TAATTTTT TAAAAATT TATAAAAA TAAAATAA TATAAAAT TAAAATAT TATAAATA TAAAATTA (loxW12) TATAAATT TAAAATTT TATAATAA TAAATAAA TATAATAT TAAATAAT TATAATTA TAAATATA TATAATTT TAAATATT TATATAAA TAAATTAA TATATAAT TAAATTAT TATATATA TAAATTTA TATATATT (loxW7) TATATTAA TTAATAAT TATATTAT TTAATATA TATATTTA TTAATATT TATATTTT TTAATTAA TATTAAAA TTAATTAT TATTAAAT TTAATTTA (loxW19) TATTAATA TTAATTTT (loxW2) TATTAATT TTATAAAA TATTATAA TTATAAAT TATTATAT TTATAATA TATTATTA TTATAATT TATTATTT (loxW24) TTATATAATATTTAAA TTATATAT TATTTAAT TTATATTA TATTTATA TTATATTT TATTTATT TTATTAAA TATTTTAA TTATTAAT TATTTTAT (loxW5) TTATTATA TATTTTTA TTATTATT TATTTTTT TTATTTAA (loxW1) TTAAAAAA TTATTTAT TTAAAAAT (loxW23) TTATTTTA TTAAAATA TTATTTTT TTAAAATT TTTAAAAA TTAAATAA TTTAAAAT TTAAATAT TTTAAATA TTAAATTA TTTAAATT TTAAATTT TTTAATAA TTAATAAA TTTAATAT TTTAATTA TTTTAATT TTTAATTT TTTTATAA TTTATAAA TTTTATAT TTTATAAT TTTTATTA TTTATATA TTTTATTT (loxW3) TTTATATT TTTTTAAA TTTATTAA TTTTTAAT TTTATTAT TTTTTATA TTTATTTA TTTTTATT TTTATTTT TTTTTTAA TTTTAAAA TTTTTTAT TTTTAAAT TTTTTTTA TTTTAATA TTTTTTTT

[0050] Representative combined LoxP arm 1 – cysteine-free core sequence - LoxP arm 2 nucleotide sequences are as follows: ATAACTTCGTATAAAAAAAAATATACGAAGTTAT (SEQ ID NO:103) designated loxW10; comprising LoxP arm 1 (SEQ ID NO:1) the core spacer sequence AAAAAAAA and LoxP arm 2 (SEQ ID NO:2); and ATAACTTCGTATA TATATATTT ATACGAAGTTAT (SEQ ID NO:273) designated loxW7, comprising LoxP arm 1 (SEQ ID NO:1) the core sequence TATATATT, and LoxP arm 2 (SEQ ID NO:2). Table 3. Combined lox recognition sequences and cytosine free core spacer sequences Combined loxP - cytosine free core spacer sequence (full length) SEQ ID NO: ATAACTTCGTATAAAAAAAAATATACGAAGTTAT 103 ATAACTTCGTATAAAAAAAATTATACGAAGTTAT 104 ATAACTTCGTATAAAAAAATATATACGAAGTTAT 105 ATAACTTCGTATAAAAAAATTTATACGAAGTTAT 106 ATAACTTCGTATAAAAAATAATATACGAAGTTAT 107 ATAACTTCGTATAAAAAATATTATACGAAGTTAT 108 ATAACTTCGTATAAAAAATTATATACGAAGTTAT 109 ATAACTTCGTATAAAAAATTTTATACGAAGTTAT 110 ATAACTTCGTATAAAAATAAATATACGAAGTTAT 111 ATAACTTCGTATAAAAATAATTATACGAAGTTAT 112 ATAACTTCGTATAAAAATATATATACGAAGTTAT 113 ATAACTTCGTATAAAAATTAATATACGAAGTTAT 114 ATAACTTCGTATAAAAATTATTATACGAAGTTAT 115 ATAACTTCGTATAAAAATTTATATACGAAGTTAT 116 ATAACTTCGTATAAAAATTTTTATACGAAGTTAT 117 ATAACTTCGTATAAAATAAAATATACGAAGTTAT 118 ATAACTTCGTATAAAATAAATTATACGAAGTTAT 119 ATAACTTCGTATAAAATAATATATACGAAGTTAT 120 ATAACTTCGTATAAAATAATTTATACGAAGTTAT 121 ATAACTTCGTATAAAATATAATATACGAAGTTAT 122 ATAACTTCGTATAAAATATATTATACGAAGTTAT 123 ATAACTTCGTATAAAATATTATATACGAAGTTAT 124 ATAACTTCGTATAAAATATTTTATACGAAGTTAT 125 ATAACTTCGTATAAAATTAAATATACGAAGTTAT 126ATAACTTCGTATAAAATTAATTATACGAAGTTAT 127 ATAACTTCGTATAAAAAAAAATATACGAAGTTAT 128 ATAACTTCGTATAAAATTATTTATACGAAGTTAT 129 ATAACTTCGTATAAAATTTAATATACGAAGTTAT 130 ATAACTTCGTATAAAATTTATTATACGAAGTTAT 131 ATAACTTCGTATAAAATTTTATATACGAAGTTAT 132 ATAACTTCGTATAAAATTTTTTATACGAAGTTAT 133 ATAACTTCGTATAAATAAAAATATACGAAGTTAT 134 ATAACTTCGTATAAATAAAATTATACGAAGTTAT 135 ATAACTTCGTATAAATAAATATATACGAAGTTAT 136 ATAACTTCGTATAAATAAATTTATACGAAGTTAT 137 ATAACTTCGTATAAATAATAATATACGAAGTTAT 138 ATAACTTCGTATAAATAATATTATACGAAGTTAT 139 ATAACTTCGTATAAATAATTATATACGAAGTTAT 140 ATAACTTCGTATAAATAATTTTATACGAAGTTAT 141 ATAACTTCGTATAAATATAAATATACGAAGTTAT 142 ATAACTTCGTATAAATATAATTATACGAAGTTAT 143 ATAACTTCGTATAAATATATATATACGAAGTTAT 144 ATAACTTCGTATAAATATATTTATACGAAGTTAT 145 ATAACTTCGTATAAATATTAATATACGAAGTTAT 146 ATAACTTCGTATAAATATTATTATACGAAGTTAT 147 ATAACTTCGTATAAATATTTATATACGAAGTTAT 148 ATAACTTCGTATAAATATTTTTATACGAAGTTAT 149 ATAACTTCGTATAAATTAAAATATACGAAGTTAT 150 ATAACTTCGTATAAATTAAATTATACGAAGTTAT 151 ATAACTTCGTATAAATTAATATATACGAAGTTAT 152 ATAACTTCGTATAAATTAATTTATACGAAGTTAT 153 ATAACTTCGTATAAATTATAATATACGAAGTTAT 154 ATAACTTCGTATAAATTATATTATACGAAGTTAT 155 ATAACTTCGTATAAATTATTATATACGAAGTTAT 156 ATAACTTCGTATAAATTATTTTATACGAAGTTAT 157 ATAACTTCGTATAAATTTAAATATACGAAGTTAT 158 ATAACTTCGTATAAATTTAATTATACGAAGTTAT 159ATAACTTCGTATAAATTTATATATACGAAGTTAT 160 ATAACTTCGTATAAATTTATTTATACGAAGTTAT 161 ATAACTTCGTATAAATTTTAATATACGAAGTTAT 162 ATAACTTCGTATAAATTTTATTATACGAAGTTAT 163 ATAACTTCGTATAAATTTTTATATACGAAGTTAT 164 ATAACTTCGTATAAATTTTTTTATACGAAGTTAT 165 ATAACTTCGTATAATAAAAAATATACGAAGTTAT 166 ATAACTTCGTATAATAAAAATTATACGAAGTTAT 167 ATAACTTCGTATAATAAAATATATACGAAGTTAT 168 ATAACTTCGTATAATAAAATTTATACGAAGTTAT 169 ATAACTTCGTATAATAAATAATATACGAAGTTAT 170 ATAACTTCGTATAATAAATATTATACGAAGTTAT 171 ATAACTTCGTATAATAAATTATATACGAAGTTAT 172 ATAACTTCGTATAATAAATTTTATACGAAGTTAT 173 ATAACTTCGTATAATAATAAATATACGAAGTTAT 174 ATAACTTCGTATAATAATAATTATACGAAGTTAT 175 ATAACTTCGTATAATAATATATATACGAAGTTAT 176 ATAACTTCGTATAATAATATTTATACGAAGTTAT 177 ATAACTTCGTATAATAATTAATATACGAAGTTAT 178 ATAACTTCGTATAATAATTATTATACGAAGTTAT 179 ATAACTTCGTATAATAATTTATATACGAAGTTAT 180 ATAACTTCGTATAATAATTTTTATACGAAGTTAT 181 ATAACTTCGTATAATATAAAATATACGAAGTTAT 182 ATAACTTCGTATAATATAAATTATACGAAGTTAT 183 ATAACTTCGTATAATATAATATATACGAAGTTAT 184 ATAACTTCGTATAATATAATTTATACGAAGTTAT 185 ATAACTTCGTATAATATATAATATACGAAGTTAT 186 ATAACTTCGTATAATATATATTATACGAAGTTAT 187 ATAACTTCGTATAATATATTATATACGAAGTTAT 188 ATAACTTCGTATAATATATTTTATACGAAGTTAT 189 ATAACTTCGTATAATATTAAATATACGAAGTTAT 190 ATAACTTCGTATAATATTAATTATACGAAGTTAT 191 ATAACTTCGTATAATATTATATATACGAAGTTAT 192ATAACTTCGTATAATATTATTTATACGAAGTTAT 193 ATAACTTCGTATAATATTTAATATACGAAGTTAT 194 ATAACTTCGTATAATATTTATTATACGAAGTTAT 195 ATAACTTCGTATAATATTTTATATACGAAGTTAT 196 ATAACTTCGTATAATATTTTTTATACGAAGTTAT 197 ATAACTTCGTATAATTAAAAATATACGAAGTTAT 198 ATAACTTCGTATAATTAAAATTATACGAAGTTAT 199 ATAACTTCGTATAATTAAATATATACGAAGTTAT 200 ATAACTTCGTATAATTAAATTTATACGAAGTTAT 201 ATAACTTCGTATAATTAATAATATACGAAGTTAT 202 ATAACTTCGTATAATTAATATTATACGAAGTTAT 203 ATAACTTCGTATAATTAATTATATACGAAGTTAT 204 ATAACTTCGTATAATTAATTTTATACGAAGTTAT 205 ATAACTTCGTATAATTATAAATATACGAAGTTAT 206 ATAACTTCGTATAATTATAATTATACGAAGTTAT 207 ATAACTTCGTATAATTATATATATACGAAGTTAT 208 ATAACTTCGTATAATTATATTTATACGAAGTTAT 209 ATAACTTCGTATAATTATTAATATACGAAGTTAT 210 ATAACTTCGTATAATTATTATTATACGAAGTTAT 211 ATAACTTCGTATAATTATTTATATACGAAGTTAT 212 ATAACTTCGTATAATTATTTTTATACGAAGTTAT 213 ATAACTTCGTATAATTTAAAATATACGAAGTTAT 214 ATAACTTCGTATAATTTAAATTATACGAAGTTAT 215 ATAACTTCGTATAATTTAATATATACGAAGTTAT 216 ATAACTTCGTATAATTTAATTTATACGAAGTTAT 217 ATAACTTCGTATAATTTATAATATACGAAGTTAT 218 ATAACTTCGTATAATTTATATTATACGAAGTTAT 219 ATAACTTCGTATAATTTATTATATACGAAGTTAT 220 ATAACTTCGTATAATTTATTTTATACGAAGTTAT 221 ATAACTTCGTATAATTTTAAATATACGAAGTTAT 222 ATAACTTCGTATAATTTTAATTATACGAAGTTAT 223 ATAACTTCGTATAATTTTATATATACGAAGTTAT 224 ATAACTTCGTATAATTTTATTTATACGAAGTTAT 225ATAACTTCGTATAATTTTTAATATACGAAGTTAT 226 ATAACTTCGTATAATTTTTATTATACGAAGTTAT 227 ATAACTTCGTATAATTTTTTATATACGAAGTTAT 228 ATAACTTCGTATAATTTTTTTTATACGAAGTTAT 229 ATAACTTCGTATATAAAAAAATATACGAAGTTAT 230 ATAACTTCGTATATAAAAAATTATACGAAGTTAT 231 ATAACTTCGTATATAAAAATATATACGAAGTTAT 232 ATAACTTCGTATATAAAAATTTATACGAAGTTAT 233 ATAACTTCGTATATAAAATAATATACGAAGTTAT 234 ATAACTTCGTATATAAAATATTATACGAAGTTAT 235 ATAACTTCGTATATAAAATTATATACGAAGTTAT 236 ATAACTTCGTATATAAAATTTTATACGAAGTTAT 237 ATAACTTCGTATATAAATAAATATACGAAGTTAT 238 ATAACTTCGTATATAAATAATTATACGAAGTTAT 239 ATAACTTCGTATATAAATATATATACGAAGTTAT 240 ATAACTTCGTATATAAATATTTATACGAAGTTAT 241 ATAACTTCGTATATAAATTAATATACGAAGTTAT 242 ATAACTTCGTATATAAATTATTATACGAAGTTAT 243 ATAACTTCGTATATAAATTTATATACGAAGTTAT 244 ATAACTTCGTATATAAATTTTTATACGAAGTTAT 245 ATAACTTCGTATATAATAAAATATACGAAGTTAT 246 ATAACTTCGTATATAATAAATTATACGAAGTTAT 247 ATAACTTCGTATATAATAATATATACGAAGTTAT 248 ATAACTTCGTATATAATAATTTATACGAAGTTAT 249 ATAACTTCGTATATAATATAATATACGAAGTTAT 250 ATAACTTCGTATATAATATATTATACGAAGTTAT 251 ATAACTTCGTATATAATATTATATACGAAGTTAT 252 ATAACTTCGTATATAATATTTTATACGAAGTTAT 253 ATAACTTCGTATATAATTAAATATACGAAGTTAT 254 ATAACTTCGTATATAATTAATTATACGAAGTTAT 255 ATAACTTCGTATATAATTATATATACGAAGTTAT 256 ATAACTTCGTATATAATTATTTATACGAAGTTAT 257 ATAACTTCGTATATAATTTAATATACGAAGTTAT 258ATAACTTCGTATATAATTTATTATACGAAGTTAT 259 ATAACTTCGTATATAATTTTATATACGAAGTTAT 260 ATAACTTCGTATATAATTTTTTATACGAAGTTAT 261 ATAACTTCGTATATATAAAAATATACGAAGTTAT 262 ATAACTTCGTATATATAAAATTATACGAAGTTAT 263 ATAACTTCGTATATATAAATATATACGAAGTTAT 264 ATAACTTCGTATATATAAATTTATACGAAGTTAT 265 ATAACTTCGTATATATAATAATATACGAAGTTAT 266 ATAACTTCGTATATATAATATTATACGAAGTTAT 267 ATAACTTCGTATATATAATTATATACGAAGTTAT 268 ATAACTTCGTATATATAATTTTATACGAAGTTAT 269 ATAACTTCGTATATATATAAATATACGAAGTTAT 270 ATAACTTCGTATATATATAATTATACGAAGTTAT 271 ATAACTTCGTATATATATATATATACGAAGTTAT 272 ATAACTTCGTATATATATATTTATACGAAGTTAT 273 ATAACTTCGTATATATATTAATATACGAAGTTAT 274 ATAACTTCGTATATATATTATTATACGAAGTTAT 275 ATAACTTCGTATATATATTTATATACGAAGTTAT 276 ATAACTTCGTATATATATTTTTATACGAAGTTAT 277 ATAACTTCGTATATATTAAAATATACGAAGTTAT 278 ATAACTTCGTATATATTAAATTATACGAAGTTAT 279 ATAACTTCGTATATATTAATATATACGAAGTTAT 280 ATAACTTCGTATATATTAATTTATACGAAGTTAT 281 ATAACTTCGTATATATTATAATATACGAAGTTAT 282 ATAACTTCGTATATATTATATTATACGAAGTTAT 283 ATAACTTCGTATATATTATTATATACGAAGTTAT 284 ATAACTTCGTATATATTATTTTATACGAAGTTAT 285 ATAACTTCGTATATATTTAAATATACGAAGTTAT 286 ATAACTTCGTATATATTTAATTATACGAAGTTAT 287 ATAACTTCGTATATATTTATATATACGAAGTTAT 288 ATAACTTCGTATATATTTATTTATACGAAGTTAT 289 ATAACTTCGTATATATTTTAATATACGAAGTTAT 290 ATAACTTCGTATATATTTTATTATACGAAGTTAT 291ATAACTTCGTATATATTTTTATATACGAAGTTAT 292 ATAACTTCGTATATATTTTTTTATACGAAGTTAT 293 ATAACTTCGTATATTAAAAAATATACGAAGTTAT 294 ATAACTTCGTATATTAAAAATTATACGAAGTTAT 295 ATAACTTCGTATATTAAAATATATACGAAGTTAT 296 ATAACTTCGTATATTAAAATTTATACGAAGTTAT 297 ATAACTTCGTATATTAAATAATATACGAAGTTAT 298 ATAACTTCGTATATTAAATATTATACGAAGTTAT 299 ATAACTTCGTATATTAAATTATATACGAAGTTAT 300 ATAACTTCGTATATTAAATTTTATACGAAGTTAT 301 ATAACTTCGTATATTAATAAATATACGAAGTTAT 302 ATAACTTCGTATATTAATAATTATACGAAGTTAT 303 ATAACTTCGTATATTAATATATATACGAAGTTAT 304 ATAACTTCGTATATTAATATTTATACGAAGTTAT 305 ATAACTTCGTATATTAATTAATATACGAAGTTAT 306 ATAACTTCGTATATTAATTATTATACGAAGTTAT 307 ATAACTTCGTATATTAATTTATATACGAAGTTAT 308 ATAACTTCGTATATTAATTTTTATACGAAGTTAT 309 ATAACTTCGTATATTATAAAATATACGAAGTTAT 310 ATAACTTCGTATATTATAAATTATACGAAGTTAT 311 ATAACTTCGTATATTATAATATATACGAAGTTAT 312 ATAACTTCGTATATTATAATTTATACGAAGTTAT 313 ATAACTTCGTATATTATATAATATACGAAGTTAT 314 ATAACTTCGTATATTATATATTATACGAAGTTAT 315 ATAACTTCGTATATTATATTATATACGAAGTTAT 316 ATAACTTCGTATATTATATTTTATACGAAGTTAT 317 ATAACTTCGTATATTATTAAATATACGAAGTTAT 318 ATAACTTCGTATATTATTAATTATACGAAGTTAT 319 ATAACTTCGTATATTATTATATATACGAAGTTAT 320 ATAACTTCGTATATTATTATTTATACGAAGTTAT 321 ATAACTTCGTATATTATTTAATATACGAAGTTAT 322 ATAACTTCGTATATTATTTATTATACGAAGTTAT 323 ATAACTTCGTATATTATTTTATATACGAAGTTAT 324ATAACTTCGTATATTATTTTTTATACGAAGTTAT 325 ATAACTTCGTATATTTAAAAATATACGAAGTTAT 326 ATAACTTCGTATATTTAAAATTATACGAAGTTAT 327 ATAACTTCGTATATTTAAATATATACGAAGTTAT 328 ATAACTTCGTATATTTAAATTTATACGAAGTTAT 329 ATAACTTCGTATATTTAATAATATACGAAGTTAT 330 ATAACTTCGTATATTTAATATTATACGAAGTTAT 331 ATAACTTCGTATATTTAATTATATACGAAGTTAT 332 ATAACTTCGTATATTTAATTTTATACGAAGTTAT 333 ATAACTTCGTATATTTATAAATATACGAAGTTAT 334 ATAACTTCGTATATTTATAATTATACGAAGTTAT 335 ATAACTTCGTATATTTATATATATACGAAGTTAT 336 ATAACTTCGTATATTTATATTTATACGAAGTTAT 337 ATAACTTCGTATATTTATTAATATACGAAGTTAT 338 ATAACTTCGTATATTTATTATTATACGAAGTTAT 339 ATAACTTCGTATATTTATTTATATACGAAGTTAT 340 ATAACTTCGTATATTTATTTTTATACGAAGTTAT 341 ATAACTTCGTATATTTTAAAATATACGAAGTTAT 342 ATAACTTCGTATATTTTAAATTATACGAAGTTAT 343 ATAACTTCGTATATTTTAATATATACGAAGTTAT 344 ATAACTTCGTATATTTTAATTTATACGAAGTTAT 345 ATAACTTCGTATATTTTATAATATACGAAGTTAT 346 ATAACTTCGTATATTTTATATTATACGAAGTTAT 347 ATAACTTCGTATATTTTATTATATACGAAGTTAT 348 ATAACTTCGTATATTTTATTTTATACGAAGTTAT 349 ATAACTTCGTATATTTTTAAATATACGAAGTTAT 350 ATAACTTCGTATATTTTTAATTATACGAAGTTAT 351 ATAACTTCGTATATTTTTATATATACGAAGTTAT 352 ATAACTTCGTATATTTTTATTTATACGAAGTTAT 353 ATAACTTCGTATATTTTTTAATATACGAAGTTAT 354 ATAACTTCGTATATTTTTTATTATACGAAGTTAT 355 ATAACTTCGTATATTTTTTTATATACGAAGTTAT 356 ATAACTTCGTATATTTTTTTTTATACGAAGTTAT 357

[0051] In certain specific embodiments that LoxP arm 1 (SEQ ID NO:1) and LoxParm 2 (SEQ ID NO:2) have been combined with the core spacer sequences designated as loxW1 to loxW12, loxW15 to loxW26, and loxW28 above and have validated in the screening methods provided herein. EXAMPLES Example 1 – Screening Method for Methylation Resistant Lox Sites

[0052] Example one describes a screening method used for determining the nucleotide sequences for methylation resistant lox sites. Vectors were constructed having flanking degenerate primer sequences (both 3’ and 5’) whose overhangs included LoxP sites with loxP arm 1 (SEQ ID NO:1), a core spacer sequence which only contained the DNA bases A and T, and loxP arm 2 (SEQ ID NO:2) for use in PCR amplification of the linear product containing a bacterial origin of replication sequence and an antibiotic resistance gene (AmpR). (FIGURE 2). The PCR products were treated in vitro with purified Cre recombinase, and those vectors with compatible lox sites with cytosine free core spacer sequences were circularized into plasmids capable of replication and selection in E. coli. (FIGURE 2, left panel). Colonies produced through this method were sequenced. (FIGURE 2, upper right panel). The resulting lox sites with cytosine-free core spacer sequences were screened for activity through cloning two direct repeats in plasmids and treating with purified Cre recombinase for excision products. (FIGURE 2, lower right panel). Lox core spacer sequences loxW1 to loxW12, loxW15 to loxW26, and loxW28 described and disclosed above and have been fully validated using this method. Example 2 – Test for Increased Recombination in Plant Cells

[0053] To implement the discovered lox sites and to test for increased recombination in plant cells, well-performing sites were cloned by standard methods into a vector in direct repeats, the first site was placed in the 5’ UTR of a reporter gene (the fluorescent reporter tdTomato), and the second site was placed after the terminator of the same fluorescent protein gene. A second fluorescent reporter (green fluorescent protein, GFP) was placed downstream of this second lox site without a promoter such that excision via Cre recombinase of the first product turns on expression of the second reporter. Testing of these vectors with a constitutively expressed Cre recombinase in tobacco in well-known agrofiltration assays showed significantly increased transgene excision with loxPcytosine-free core spacer sequences relative to the same vector with traditional wild-type loxP sites as shown in FIGURE 3.

[0054] In a second test, the vectors were transformed into hybrid poplar (Populus tremula x alba) by standard treatment with Agrobacterium. Vectors with LoxP and LoxW7-containing transgenes with constitutive Cre expression and a methylation-sensitive excision reporter (FIGURE 4A) exhibited reduced shoot regeneration rates versus no Cre containing control vectors in poplar. Vectors with the LoxW7 transgene allow for approximately 10% excised shoot recovery. (FIGURE 4A).

[0055] A second vector configuration was also tested in poplar. In the second vector configuration, a fluorescent reporter coding sequence (mScarlet3) was separated into two exons with a third exon in the middle of the gene encoding an inflexible linker. The inflexible linker prevented proper folding of the fluorescent protein. (FIGURE 4B). Because the sequence contexts of each of the two vector constructs around the lox sites have very different GC content and important gene features, the first promoter-less switch reporter was anticipated to be DNA-methylation sensitive, and the latter intron-linker excision reporter (which has 26% GC content in introns around lox sequences) to be DNA-methylation resistant. Testing of the DNA methylation-sensitive reporter with a constitutively expressed Cre in tobacco agroinfiltration assays showed significantly increased transgene excision with cytosine-free spacer lox sites relative to the same vector with traditional wild-type loxP sites (FIGURE 3). The same result was found in stable transgenic calli and shoots in the hybrid poplar P. tremula x alba using the DNA methylation-sensitive reporter and constitutive Cre recombinase (FIGURE 4B). Using the DNA methylation-resistant reporter and constitutive Cre recombinase, cytosine-free core spacer sequences resulted in greater excised plant recovery compared with loxP and reduced negative impacts on plant regeneration (FIGURE 4A). With a heat-shock inducible Cre recombinase coupled with a methylation-resistant reporter, loxP containing transgenic poplars excised uncontrollably without heat-shock stimulus, whereas in the lox site with a cytosine-free core spacer sequence containing transgenic poplars, shoots remained unexcised (FIGURE 6). This result additionally suggested that lox sites with cytosine-free core spacer sequences have enhanced dynamic control of excision compared with conventional loxP sites.Example 3 – Use of Lox Sites with a Cystosine-free Core Spacer Sequence for Excision of a Morphogenic Gene and CRISPR / Cas9 Transgenes

[0056] Example 3 provides an example of the use of lox sites with a cytosine-free core spacer sequence. T-DNA vectors which contain morphogenic root inducing genes (rol genes from Agrobacterium rhizogenes strain A4), morphogenic shoot-inducing genes (WUSCHEL (WUS) from Arabidopsis thaliana and ipt from Agrobacterium tumefasciens), as well as fluorescent markers, a red betalain pigment gene (RUBY), Cre recombinase, Cas9, and gRNAs was assembled and flanked by cytosine-free lox sites (Figure 7A). This assembly was produced using cloning techniques common to those skilled in the art. After transformation and recovery of transgenic roots, shoot formation and transgene excision were induced by heat shock for two weeks at 38 °C for four hours daily, and many shoots lacking transgenic markers were found and propagated successfully (Figure 7B). When analyzed in detail, 48% of recovered initial transgenic root events could form excised shoots lacking marker gene expression, and 50% of surveyed product shoots were fully excised of transgene components at the DNA level (Figure 7C). These vectors show highly efficient generation of transgene-excised plants using lox sites having cytosine-free core spacer sequences.

[0057] While representative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the compositions and methods provided and claimed.

Claims

CLAIMS The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A cytosine-free lox site comprising a lox arm 1, and a lox arm 2 separated by a four to twelve nucleotide core spacer sequence.

2. The cytosine-free lox site according to Claim 1, wherein the core spacer sequence comprises eight nucleotides and does not include a cytosine.

3. The cytosine-free lox site according to any one of Claims 1 or 2, wherein lox arm one and lox arm two comprise a nucleotide sequence SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:4 through SEQ ID NO:

102.

4. The cytosine-free lox site according to any one of Claims 1-3, wherein the cytosine-free lox site comprises a core spacer sequence comprising randomly repeating adenine (A) and / or thymine (T) nucleotides.

5. The cytosine-free lox site according to any one of Claims 1-3, wherein the core spacer sequence is selected from one of AAAAAAAA (loxW10), AAAAAAAT, AAAAAATA, AAAAAATT, AAAAATAA, AAAAATAT, AAAAATTA, AAAAATTT, AAAATAAA, AAAATAAT, AAAATATA, AAAATATT (loxW9), AAAATTAA, AAAATTAT, AAAATTTA, AAAATTTT, AAATAAAA (loxW18), AAATAAAT, AAATAATA (loxW6), AAATAATT, AAATATAA, AAATATAT, AAATATTA, AAATATTT, AAATTAAA, AAATTAAT, AAATTATA, AAATTATT, AAATTTAA, AAATTTAT (loxW28), AAATTTTA, AAATTTTT, AATAAAAA, AATAAAAT (loxW21), AATAAATA, AATAAATT, AATAATAA, AATAATAT, AATAATTA, AATAATTT, AATATAAA, AATATAAT, AATATATA, AATATATT, AATATTAA, AATATTAT, AATATTTA, AATATTTT, AATTAAAA, AATTAAAT, AATTAATA, AATTAATT, AATTATAA, AATTATAT, AATTATTA, AATTATTT (loxW11), AATTTAAA, AATTTAAT, AATTTATA (loxW4), AATTTATT, AATTTTAA,AATTTTAT, AATTTTTA, AATTTTTT, ATAAAAAA (loxW15), ATAAAAAT, ATAAAATA (loxW22), ATAAAATT, ATAAATAA, ATAAATAT, ATAAATTA, ATAAATTT, ATAATAAA, ATAATAAT, ATAATATA, ATAATATT (loxW26), ATAATTAA (loxW17), ATAATTAT, ATAATTTA, ATAATTTT, ATATAAAA, ATATAAAT, ATATAATA, ATATAATT, ATATATAA, ATTTAATA, ATATATAT, ATATATTA, ATATATTT, ATATTAAA (loxW8), ATATTAAT, ATATTATA, ATATTATT, ATATTTAA, ATATTTAT, ATATTTTA, ATATTTTT, ATTAAAAA, ATTAAAAT, ATTAAATA, ATTAAATT, ATTAATAA, ATTAATAT, ATTAATTA, ATTAATTT, ATTATAAA, ATTATAAT, ATTATATA, ATTATATT, ATTATTAA, ATTATTAT, ATTATTTA, ATTATTTT, ATTTAAAA, ATTTAAAT (loxW25), TAAATTTT, ATTTAATT, ATTTATAA, ATTTATAT (loxW20), ATTTATTA, ATTTATTT, ATTTTAAA, ATTTTAAT, ATTTTATA, ATTTTATT, ATTTTTAA, ATTTTTAT, ATTTTTTA, ATTTTTTT, TAAAAAAA, TAAAAAAT, TAAAAATA, TAAAAATT, TAAAATAA, TAAAATAT, TAAAATTA (loxW12), TAAAATTT, TAAATAAA, TAAATAAT, TAAATATA, TAAATATT, TAAATTAA, TAAATTAT, TAAATTTA, TATATTAA, TATATTAT, TAATAAAA, TAATAAAT, TAATAATA, TAATAATT, TAATATAA, TAATATAT, TAATATTA, TAATATTT, TAATTAAA, TAATTAAT, TAATTATA, TAATTATT, TAATTTAA, TAATTTAT, TAATTTTA, TAATTTTT, TATAAAAA, TATAAAAT, TATAAATA, TATAAATT, TATAATAA, TATAATAT, TATAATTA, TATAATTT, TATATAAA, TATATAAT, TATATATA, TATATATT (loxW7), TTAATAAT, TTAATATA, TATATTTA, TATATTTT, TATTAAAA, TATTAAAT, TATTAATA, TATTAATT, TATTATAA, TATTATAT, TATTATTA, TATTATTT (loxW24), TATTTAAA, TATTTAAT, TATTTATA, TATTTATT, TATTTTAA, TATTTTAT (loxW5), TATTTTTA, TATTTTTT, TTAAAAAA, TTAAAAAT (loxW23), TTAAAATA, TTAAAATT, TTAAATAA, TTAAATAT, TTAAATTA, TTAAATTT, TTAATAAA, TTTAATTA, TTTAATTT, TTTATAAA, TTAATATT, TTAATTAA, TTAATTAT, TTAATTTA (loxW19), TTAATTTT (loxW2), TTATAAAA, TTATAAAT, TTATAATA, TTATAATT,TTATATAA, TTATATAT, TTATATTA, TTATATTT, TTATTAAA, TTATTAAT, TTATTATA, TTATTATT, TTATTTAA (loxW1), TTATTTAT, TTATTTTA, TTATTTTT, TTTAAAAA, TTTAAAAT, TTTAAATA, TTTAAATT, TTTAATAA, TTTAATAT, TTTTAATT, TTTTATAA, TTTTATAT, TTTATAAT, TTTATATA, TTTATATT, TTTATTAA, TTTATTAT, TTTATTTA, TTTATTTT, TTTTAAAA, TTTTAAAT, TTTTAATA, TTTTATTA, TTTTATTT (loxW3), TTTTTAAA, TTTTTAAT, TTTTTATA, TTTTTATT, TTTTTTAA, TTTTTTAT, TTTTTTTA, or TTTTTTTT. The cytosine-free lox site according to any one of Claims 1-5, wherein the cytosine-free lox site is selected from one of as set forth in one of SEQ ID NO: 103 – SEQ ID NO:

357.

7. A method to increase the efficiency of Cre-mediated transgene excision in a plant cell, the method comprising: (a) introducing into at least one plant cell at least one plasmid comprising at least one cytosine-free lox site according to any one of Claims 1-6; and (b) contacting the at least one cytosine-free free lox site with a Cre recombinase, wherein the transgene is excised more efficiently when the at least one cytosine-free lox site is contacted with the Cre recombinase compared to transgene excision in the absence of the cytosine-free lox site.

8. The method according to Claim 7, wherein the plasmid comprises a transgene flanked by direct repeats of the cytosine-free lox site.

9. The method according to Claim 8, wherein the transgene comprises Cas 9 and at least one guide RNA.

10. The method according to any one of Claims 7-9, wherein the plasmid further comprises a Cre coding region.

11. The method according to any one of Claims 7 to 10, wherein the cytosine-free lox site is selected from a sequence as set forth in any one of SEQ ID NO:103 – SEQ ID NO:

357.

12. The method according to any one of Claims 7 to 10, wherein the plasmid comprises a constitutive promoter, an inducible promoter, or a tissue specific promoter controlling Cre expression.

13. The method according to Claims 7 to 12, wherein the plasmid comprises a reporter gene.

14. The method according to Claim 13, wherein the reporter is a fluorescent or DNA methylation sensitive reporter.

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