WO2024229458A2 - Sites lox et procédés d'ingénierie génomique dans des plantes - Google Patents

Sites lox et procédés d'ingénierie génomique dans des plantes Download PDF

Info

Publication number
WO2024229458A2
WO2024229458A2 PCT/US2024/027932 US2024027932W WO2024229458A2 WO 2024229458 A2 WO2024229458 A2 WO 2024229458A2 US 2024027932 W US2024027932 W US 2024027932W WO 2024229458 A2 WO2024229458 A2 WO 2024229458A2
Authority
WO
WIPO (PCT)
Prior art keywords
cytosine
free
lox
lox site
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2024/027932
Other languages
English (en)
Other versions
WO2024229458A3 (fr
Inventor
Greg GORALOGIA
Steven H. Strauss
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oregon State University
Original Assignee
Oregon State University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oregon State University filed Critical Oregon State University
Publication of WO2024229458A2 publication Critical patent/WO2024229458A2/fr
Publication of WO2024229458A3 publication Critical patent/WO2024229458A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • 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.
  • 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.
  • the present disclosure provides cytosine-free lox sites and methods for their use.
  • 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.
  • the core spacer sequence comprises eight nucleotides and does not include a cytosine.
  • 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.
  • the cytosine-free lox site comprises a core spacer sequence comprising randomly repeating adenine (A) and/or thymine (T) nucleotides.
  • 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
  • the cytosine-free lox site selected from one of as set forth in one of SEQ ID NO: 103 – SEQ ID NO:357.
  • 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.
  • the method comprises the use of a plasmid comprises a transgene flanked by direct repeats of the cytosine-free lox site.
  • the transgene comprises a gene encoding Cas 9 and at least one guide RNA.
  • the plasmid further comprises a Cre coding region.
  • 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.
  • the plasmid comprises a constitutive promoter, an inducible promoter, or a tissue specific promoter controlling Cre expression.
  • the plasmid comprises a reporter gene.
  • 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).
  • FIGURE 2 depicts the strategy used for finding and validating cytosine- free recombination sites.
  • LoxP arm 1 SEQ ID NO:1
  • LoxP arm 2 SEQ ID NO: 2
  • Location of successful excision footprints shown with left pointing arrow or Cre.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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). Amplification of a linear product containing a bacterial origin sequence and an antibiotic resistance gene was undertaken (FIGURE 2).
  • 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.
  • Cytosine Free Lox Sites 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the plasmid further comprises a Cre recombinase coding region. See for example, as specific embodiments FIGURE 5 and FIGURE 7A.
  • 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.
  • 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.
  • 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.
  • the exogenously supplied DNA is either T-DNA, PCR-derived, or plasmid-derived.
  • 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.
  • re-transformation means transformation of cells/tissues/plants which are in themselves transformants.
  • 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.
  • nucleotide sequence refers to a polymer of DNA or RNA, which can be single- or double-stranded, optionally containing synthetic, non-natural, or altered nucleotides capable of incorporation into DNA or RNA polymers.
  • a “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.
  • a “coding region” refers to a DNA segment which encodes a regulatory molecule or any polypeptide.
  • RNA ribonucleic acid
  • 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.
  • 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.
  • “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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a complete single cytosine free lox site comprises the configuration LoxP arm 1 – core spacer sequence – LoxP arm 2.
  • a complete cytosine free lox site flanks each end of the transgene of interest to be deleted.
  • 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).
  • each sequence is the same.
  • 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).
  • 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)).
  • 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 2 ACAACCTCCCTTA (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.
  • 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.
  • 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 ATAACTTCGTATAAA
  • Example 1 Screening Method for Methylation Resistant Lox Sites
  • 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.
  • FIGGURE 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 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.
  • a reporter gene the fluorescent reporter tdTomato
  • 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.
  • GFP green fluorescent protein
  • 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).
  • 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).
  • 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Des modes de réalisation de la présente divulgation proposent une construction et un procédé d'utilisation de la construction pour accroître l'efficacité d'excision de transgène médiée par Cre. Dans certains modes de réalisation, la construction comprend une séquence d'espaceur de recombinaison lox sans cytosine comprenant une séquence nucléotidique telle que présentée dans l'un des SEQ ID NO : 1-256. Dans d'autres modes de réalisation, le procédé vise à accroître l'efficacité d'une excision de transgène médiée par Cre dans une cellule végétale, le procédé consistant à : (a) introduire, dans au moins une cellule végétale, au moins un plasmide comprenant au moins un site lox sans cytosine ; et (b) mettre en contact ledit au moins un site lox sans cytosine avec une recombinase Cre, le transgène étant excisé plus efficacement lorsque ledit au moins un site lox sans cytosine est mis en contact avec la recombinase Cre comparativement à une excision de transgène en l'absence du site lox sans cytosine.
PCT/US2024/027932 2023-05-04 2024-05-06 Sites lox et procédés d'ingénierie génomique dans des plantes Ceased WO2024229458A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363500072P 2023-05-04 2023-05-04
US63/500,072 2023-05-04

Publications (2)

Publication Number Publication Date
WO2024229458A2 true WO2024229458A2 (fr) 2024-11-07
WO2024229458A3 WO2024229458A3 (fr) 2025-04-03

Family

ID=93333381

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/027932 Ceased WO2024229458A2 (fr) 2023-05-04 2024-05-06 Sites lox et procédés d'ingénierie génomique dans des plantes

Country Status (1)

Country Link
WO (1) WO2024229458A2 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000060091A2 (fr) * 1999-04-06 2000-10-12 Oklahoma Medical Research Foundation Methode de selection de variantes de recombinase a specificite modifiee
WO2007110695A2 (fr) * 2005-10-13 2007-10-04 Bc Cancer Agency genomes modulaires pour biologie synthetique et genie metabolique

Also Published As

Publication number Publication date
WO2024229458A3 (fr) 2025-04-03

Similar Documents

Publication Publication Date Title
CN104293828B (zh) 植物基因组定点修饰方法
EP0959133A1 (fr) Procédé pour l'inhibtion de l' expression de gènes
CN108130342B (zh) 基于Cpf1的植物基因组定点编辑方法
US20030221211A1 (en) Methods of suppressing gene expression
CN110157726A (zh) 植物基因组定点替换的方法
WO2019234132A1 (fr) Édition de bases dans des plantes déficientes en polymérase thêta
Sugimoto et al. Transposition of the maize Ds element from a viral vector to the rice genome
JP2026063247A (ja) 減数分裂及び生殖系列プロモーターを使用する遺伝子編集及び部位特異的組込み事象の増加
JP2025510365A5 (fr)
US20050066386A1 (en) Method of modifying genome in higher plant
Kopertekh et al. Site-specific recombination induced in transgenic plants by PVX virus vector expressing bacteriophage P1 recombinase
CN110582200A (zh) 用于转移细胞质或细胞核性状或组分的组合物和方法
Davies et al. Somatic and germinal inheritance of an FLP-mediated deletion in transgenic tobacco
WO2024229458A2 (fr) Sites lox et procédés d'ingénierie génomique dans des plantes
Sidorenko et al. Functional analysis of two matrix attachment region (MAR) elements in transgenic maize plants
Meynard et al. Thirty years of genome engineering in rice: From gene addition to gene editing
CN108424911B (zh) 种子特异性双向启动子及其应用
JP7518545B2 (ja) 植物細胞のゲノム編集用核酸及びその用途
JP2003510043A (ja) 標的遺伝子除去
US20260071228A1 (en) Guide polynucleotide multiplexing
CN121949571A (zh) 使用Cas12i系统调控植物基因组DNA特定区域甲基化水平的方法
Li et al. The use of hygromycin phosphotransferase gene (hpt) with an artificial intron to obtain marker-off transgenic plants
AU2023254505A1 (en) Compositions and methods for increasing genome editing efficiency
AU2022413848A1 (en) Modified agrobacteria for editing plants
CN111118058A (zh) 用于重组酶介导的特异性位点的基因叠加的籼稻目标系

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24800728

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE