BR122024004319A2 - FUSION PROTEIN COMPRISING A CAS9 PROTEIN AND A SPO11 PROTEIN, AND HOST CELL COMPRISING THE FUSION PROTEIN - Google Patents

Abstract

The present invention relates to a fusion protein comprising a Cas9 protein and a Spo11 protein, as well as to a host cell comprising said fusion protein.

Description

[001] The present invention is located in the field of eukaryotes, more particularly, in the field of microbiology. It is notably related to a method for improving or modifying a yeast strain by inducing addressed meiotic recombinations.

TECHNOLOGICAL BACKGROUND OF THE INVENTION

[002] Yeasts are used in a wide variety of industries. Due to the harmlessness of a large number of species, yeasts are specifically used in the food industry as a leavening agent in baking, brewing, wine production or distilling, or as extracts for nutritional or flavoring elements. They can also be used in the industrial production of bioethanol or molecules of interest such as vitamins, antibiotics, vaccines, enzymes or steroid hormones, or in cellulosic material degradation processes.

[003] The diversity of industrial applications of yeast means that there is a constant demand for yeast strains that have improved characteristics or at least that are suitable for a new use or new culture conditions.

[004] To obtain a strain that has a specific characteristic, a person skilled in the art can utilize sexual reproduction by crossing two parental strains that have characteristics of interest and selecting a hybrid strain that provides the desired combination of parental characteristics. This method is, however, random and the selection step can be time-consuming.

[005] Alternatively, the strain can also be genetically modified by a recombinant DNA technique. This modification may, however, act to reduce their use, whether for legal, health or environmental reasons.

[006] A third alternative consists of causing a regrouping of paternal and maternal alleles in the genome, during meiotic recombination. Meiotic recombination is an exchange of DNA between homologous chromosomes during meiosis. It is initiated by the formation of double-strand breaks in one or the other homologous chromatid, followed by repair of these breaks, using a chromatid from the homologous chromosome as a matrix. However, meiotic recombinations have the disadvantage of being random and non-uniform. In fact, the double-strand break sites at the origin of these recombinations are not homogeneously distributed throughout the genome. The so-called "hotspot" regions of the chromosome, where the frequency of recombination is high, can then be distinguished from the so-called "cold" regions of the chromosome, where the frequency of recombination can be up to 100 times lower. .

[007] Spo11 is the protein that catalyzes double-strand breaks during meiosis. It acts as a dimer in cooperation with several partners. Currently, the factors that determine the choice of double-strand break sites by Spo11 and its partners remain poorly understood.

[008] Controlling the formation of double strand breaks and, indeed, meiotic recombinations, is crucial for the development of generic engineering techniques. It has recently been shown that it is possible to modify double-strand break formation sites by fusing Spo11 with the DNA-binding domain of the transcriptional activator Gal4 (Pecina et al., 2002 Cell, 111, 173-184). The Gal4-Spo11 fusion protein allows the introduction of double-strand breaks in the so-called "cold" chromosomal regions, at the Gal4 DNA binding sites.

[009] However, in this last approach, the introduction of double-strand breaks is conditioned by the presence of Gal4 binding sites and, therefore, it remains impossible to introduce meiotic recombination phenomena addressed independently of the specific binding sites.

SUMMARY OF THE INVENTION

[0010] The objective of the present invention is to propose a method for inducing meiotic recombinations addressed in eukaryotic cells, preferably in plant or yeast cells, in any region of the genome, independently of any known binding site and, notably, in the so-called "cold" chromosomal regions.

[0011] Therefore, according to a first aspect, the present invention relates to a method for inducing targeted meiotic recombinations in a eukaryotic cell comprising

[0012] - enter in said cell:

[0013] a) a fusion protein comprising a Cas9 domain and a Spo11 domain, or a nucleic acid encoding said fusion protein; It is

[0014] b) one or more guide RNAs or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising an RNA structure for binding to the Cas9 domain of the fusion protein and a sequence complementary to the chromosomal region addressed; It is

[0015] - induce said cell to enter meiotic prophase I.

[0016] The fusion protein may additionally comprise a nuclear localization signal sequence.

[0017] Preferably, the Cas9 domain of the fusion protein is a nuclease-deficient Cas9 protein.

[0018] The nucleic acid encoding said fusion protein can be placed under the control of a constitutive, inducible or meiosis-specific promoter.

[0019] One or more additional guide RNAs that address one or more other chromosomal regions, or nucleic acids that encode said additional guide RNAs, can be introduced into the eukaryotic cell.

[0020] Preferably, the eukaryotic cell is a yeast. In this way, the yeast can then be induced to enter prophase I by transferring it to the sporulation medium.

[0021] Alternatively, the eukaryotic cell is a plant cell.

[0022] Preferably, the introduction of the fusion protein, or the nucleic acid that encodes it, and the gRNA (gRNAs), or the nucleic acid (nucleic acids) that encodes the same, into said cell is simultaneous.

[0023] Alternatively, the introduction of the fusion protein, or the nucleic acid encoding it and the gRNA (gRNAs), or the nucleic acid (nucleic acids) encoding it, into said cell is sequential.

[0024] The introduction of the nucleic acid encoding the fusion protein and the nucleic acid(nucleic acids) encoding the gRNA(gRNAs) into said cell can also be achieved by crossing two cells into which the nucleic acid that encodes the fusion protein and the nucleic acid(nucleic acids) that encodes the gRNA(gRNAs).

[0025] The present invention further relates, according to a second aspect, to a fusion protein as defined in the method above.

[0026] According to a third aspect, the present invention further relates to a nucleic acid encoding the fusion protein defined above.

[0027] The present invention also relates, according to a fourth aspect, to an expression cassette or a vector comprising a nucleic acid as defined above.

[0028] Preferably, the vector is a plasmid comprising a bacterial origin of replication, an expression cassette comprising a nucleic acid as defined above, one or more selection markers and/or one or more sequences that allow targeted insertion of the vector, expression cassette or nucleic acid in the host cell genome. In particular, the plasmid comprises a bacterial origin of replication, preferably the ColE1 origin, an expression cassette comprising a nucleic acid as defined above under the control of a promoter, preferably the ADH1 promoter, a terminator, preferably, the ADH1 terminator, one or more selection markers, preferably resistance markers such as the gene for kanamycin or ampicillin resistance, one or more sequences that allow targeted insertion of the vector, expression cassette or acid nucleic acid in the host cell genome, preferably in the TRP1 locus of the yeast genome. Preferably, the plasmid comprises, or consists of, a nucleotide sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2.

[0029] According to a fifth aspect, the present invention also relates to a host cell comprising a fusion protein, a nucleic acid, a cassette or a vector as defined above.

[0030] Preferably, the host cell is a eukaryotic cell, more preferably, a yeast, plant, fungal or animal cell, and particularly preferably, the host cell is a plant cell or a yeast cell.

[0031] Preferably, the host cell is a yeast cell, more preferably, a yeast selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces castelli, Saccharomyces eubayanus, Saccharomyces kluyveri, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces uvarum, Saccharomyces paradoxus, Saccharomyces pastorianus (also called Saccharomyces carlsbergensis), and hybrids obtained from at least one strain belonging to one of these species and, particularly, preferably, the host cell is Saccharomyces cerevisiae.

[0032] Alternatively, the host cell is a plant cell, more preferably, a plant cell selected from the group consisting of rice, wheat, soybeans, corn, tomatoes, Arabidopsis thaliana, barley, rapeseed, cotton, sugarcane -sugar and beet, and particularly preferably, said host cell is a rice cell.

[0033] The present invention further relates to, in a sixth aspect, a method for generating variants of a eukaryotic organism, other than humans, which comprises:

[0034] - introduce into a cell of said organism:

[0035] a) a fusion protein comprising a Cas9 domain and a Spo11 domain, or a nucleic acid encoding said fusion protein; It is

[0036] b) one or more guide RNAs or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising an RNA structure for binding to the Cas9 domain and a sequence complementary to an addressed chromosomal region ; It is

[0037] - inducing said cell to enter meiotic prophase I;

[0038] - obtain a cell or cells that have the desired recombination (recombinations) in the addressed chromosomal region (addressed chromosomal regions); It is

[0039] - generating a variant of the organism of said recombinant cell.

[0040] Preferably, the eukaryotic organism is a yeast or a plant, more preferably, a yeast, notably a strain of yeast of industrial interest.

[0041] In a seventh aspect, the present invention also relates to a method for identifying or locating genetic information encoding a characteristic of interest in a eukaryotic cell genome comprising:

[0042] - introduce into the eukaryotic cell:

[0043] a) a fusion protein comprising a Cas9 domain and a Spo11 domain, or a nucleic acid encoding said fusion protein; It is

[0044] b) one or more guide RNAs or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising an RNA structure for binding to the Cas9 domain and a sequence complementary to an addressed chromosomal region ; It is

[0045] - inducing said cell to enter meiotic prophase I;

[0046] - obtain a cell or cells that have the desired recombination (recombinations) in the addressed chromosomal region (addressed chromosomal regions); It is

[0047] - analyze the genotypes and phenotypes of recombinant cells in order to identify or locate the genetic information that encodes the characteristic of interest.

[0048] Preferably, the eukaryotic cell is a yeast or a plant, more preferably, a yeast, notably a strain of yeast of industrial interest.

[0049] Preferably, the characteristic of interest is a quantitative tract of interest (QTL).

[0050] The present invention further relates to, in an eighth aspect, a kit comprising a fusion protein, a nucleic acid, a cassette, a vector or a host cell as defined above.

[0051] Finally, in a ninth aspect, the present invention relates to the use of a kit as defined above to implement a method as defined above, in particular, to (i) induce targeted meiotic recombinations in a eukaryotic cell, (ii ) generate variants of a eukaryotic organism, and/or (iii) identify or locate the genetic information that encodes a characteristic of interest in a eukaryotic cell genome.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1: Diagram representing plasmids P1 (SEQ ID NO: 1) and P2 (SEQ ID NO: 2) of the SpCas9-Spo11 or SpCas9*-Spo11 fusion. P1 and P2 respectively encode the expression of NLS-SpCas9-Spo11 and NLS-SpCas9*- Spo11 in yeast. Black blocks represent the constitutive ADH1 promoter (pADH1) and the ADH1 terminator (tADH1). The arrow indicates the direction of transcription under the control of the ADH1 promoter.

[0053] Figure 2: Diagram representing gRNA expression plasmids in yeast cells. (A) Diagram of the plasmid containing a single gRNA expression cassette to address a single region of the yeast genome. RE indicates the restriction site into which the sequence that determines the specificity (SDS) of the gRNA is inserted by the Gibson method. The promoter and terminator (Term) are dependent on RNA polymerase III. The arrow on the promoter indicates the direction of transcription of the sequence. A gRNA expression cassette contains a gRNA flanked by a promoter and a terminator. (B) Diagram of the plasmid containing several gRNA expression cassettes to address multiple regions of the yeast genome (multiplexed addressing). The various gRNA cassettes are distinguished by their specificity-determining sequence (SDS). They were successively introduced into the multiple cloning site (MCS) by conventional ligation/cloning techniques.

[0054] Figure 3: Diagram representing the P1 plasmid (SEQ ID NO: 1).

[0055] Figure 4: Diagram representing the P2 plasmid (SEQ ID NO: 2).

[0056] Figure 5: Viability of spores derived from the sporulation of strains that do or do not express the SpCas9*-Spo11 fusion protein. Growth of meiosis-derived spores of the diploid strains SPO11/SPO11 (ORD7339), spo11/spo11 (AND2822), spoll/spoll dCAS9-SPO11/0 (AND2820), and spoll/spoll dCAS9-SPOll/dCAS9-SPOll (AND2823).

[0057] Figure 6: Addressing meiotic DSBs by the SpCas9*-Spo11 fusion protein and a guide RNA specific for the YCR048W region. To the right of the gel, a graph indicates the position of the genes (coding regions, gray arrows) and the position of the probe. Black squares indicate natural DSB sites. The black triangle indicates the DSB sites addressed by the UAS1-YCR048W guide RNA. The percentage of DSBs corresponds to the ratio between the signal intensity of the fragment considered and the total signal of the band.

[0058] Figure 7: Addressing meiotic DSBs by the SpCas9*-Spo11 fusion protein and two guide RNAs specific to the YCR048W region. To the right of the gel, a graph indicates the position of the genes (coding regions, gray arrows) and the position of the probe. Black squares indicate natural DSB sites. The black triangle indicates the DSB sites addressed by the UAS1-YCR048W guide RNA. The black circle indicates the DSB sites addressed by the UAS2-YCR048W guide RNA. The percentage of DSBs corresponds to the ratio between the signal intensity of the fragment considered and the total signal of the band.

[0059] Figure 8: Addressing meiotic DSBs by the SpCas9*-Spo11 fusion protein and a guide RNA specific for the GAL2 region. To the right of the gel, a graph indicates the position of the genes (coding regions, gray arrows) and the position of the probe. The percentage of DSBs corresponds to the ratio between the signal intensity of the fragment considered and the total signal of the band.

[0060] Figure 9: Addressing meiotic DSBs by the SpCas9*-Spo11 fusion protein and a guide RNA specific for the SWC3 region. To the right of the gel, a graph indicates the position of the genes (coding regions, gray arrows) and the position of the probe (hatched triangle). The percentage of DSBs corresponds to the ratio between the signal intensity of the fragment considered and the total signal of the band.

[0061] Figure 10: Multiplexed addressing of meiotic DSBs by the SpCas9*-Spo11 fusion protein and several guide RNAs specific to the GAL2 region. To the right of the gel, a graph indicates the position of the genes (coding regions, gray arrows) and the position of the probe (hatched triangle). The percentage of DSBs corresponds to the ratio between the signal intensity of the fragment considered and the total signal of the band.

[0062] Figure 11: Stimulation of meiotic recombination by the SpCas9*-SPO11 protein and a guide RNA in the GAL2 target region. (A) Diagram of the genetic test to detect recombinants at the GAL2 site. (B) Test for genetic recombination at the GAL2 locus.

[0063] Figure 12: The addressing of meiotic DSBs by the SpCas9*-Spo11 protein and a guide RNA specific for the PUT4 gene coding sequence. To the right of the gel, a graph indicates the position of the genes (coding regions, gray arrows) and the position of the probe (hatched triangle). The percentage of DSBs corresponds to the ratio between the signal intensity of the fragment considered and the total signal of the band.

[0064] Figure 13: Sequences of the gRNAs used. The upper and lower case characters respectively indicate the sequence that determines the specificity of the gRNA (20 nucleotides long) and the sequence that constitutes the structure of the gRNA (“handle”, 82 nucleotides long).

[0065] Figure 14: Diagram of the construct to express the dCas9-Spo11 fusion protein in rice. pZmUbi and tNOS correspond, respectively, to the promoter and terminator used in this construct.

DETAILED DESCRIPTION OF THE INVENTION

[0066] The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is a bacterial defense system against exogenous DNA. This system is essentially found in the association of a Cas9 protein and a “guide” RNA (gRNA or sgRNA) responsible for the specificity of the cleavage site. It can be used to create DNA double-strand breaks (DSBs) at sites addressed by the CRISPR/Cas9 system. This system has already been used for targeted genome engineering in eukaryotic cells (see, for example, patent application EP2764103), notably human cells (Cong L et al., 2013, Science 339(6121):819-823; Mali P et al., 2013, Science, 339(6121):823-826; Cho SW et al., 2013, Nature Biotechnology 31(3):230-232), rat cells (Li D, et al., 2013, Nature Biotechnology, 31(8):681-683; WO 2014/089290), mouse cells (Wang H et al., 2013, Cell, 153(4):910-918), rabbit cells (Yang D et al. ., 2014, Journal of Molecular Cell Biology, 6(1):97-99), frog cells (Nakayama T et al., 2013, Genesis, 51(12):835-843), fish cells (Hwang WY et al., 2013, Nature Biotechnology, 31(3):227-229), plant cell (Shan Q et al., 2013, Nature Biotechnology, 31(8):686-688; Jiang W et al., 2013 , Nucleic Acids Research, 41(20):e188), drosophila cells (Yu Z et al., 2013, Genetics, 195(1):289- 291), nematode cells (Friedland AE et al., 2013, Nature Methods, 10(8):741-743), yeast cells (DiCarlo J, et al., 2013, Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Research 41(7):4336- 4343), but also bacterial cells (Jiang W et al., 2013, Nature Biotechnology, 31(3):233-239). On the other hand, this system has never been used to address the sites of meiotic recombination in any organism.

[0067] The inventors have shown that it is possible to modify the CRISPR-Cas9 system in order to induce targeted meiotic recombinations in a eukaryotic cell and, in particular, in yeast. They, in fact, showed that the combined expression of a Spo11-Cas9 fusion protein and one or more guide RNAs made it possible to address the action of the Spo11 transesterase, which is responsible for double-strand breaks during meiosis. The repair of these breaks using a chromatid from the homologous chromosome as a matrix induces the desired recombination phenomena.

[0068] Thus, the present invention relates to a method for inducing targeted meiotic recombinations in a eukaryotic cell comprising

[0069] - enter in said cell:

[0070] a) a fusion protein comprising a Cas9 domain and a Spo11 domain, or a nucleic acid encoding said fusion protein; It is

[0071] b) one or more guide RNAs or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising an RNA structure for binding to the Cas9 domain of the fusion protein and a sequence complementary to the chromosomal region addressed; It is

[0072] - induce said cell to enter meiotic prophase I.

[0073] As used herein, the term “eukaryotic cell” refers to a yeast, plant, fungal or animal cell, in particular, a mammalian cell such as a mouse cell or a rat cell, or a cell of insect. The eukaryotic cell is preferably non-human and/or non-embryonic.

[0074] According to a particular embodiment, the eukaryotic cell is a yeast cell, in particular, a yeast of industrial interest. Exemplary yeasts of interest include, but are not limited to, yeasts of the genus Saccharomyces sensu stricto, Schizosaccharomyces, Yarrowia, Hansenula, Kluyveromyces, Pichia or Candida, as well as hybrids obtained from a strain belonging to one of these genera.

[0075] Preferably, the yeast of interest belongs to the genus Saccharomyces. It may, notably, belong to a species selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces castelli, Saccharomyces eubayanus, Saccharomyces kluyveri, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces uvarum, Saccharomyces paradoxus and Saccharomyces pastorianus(also called of Saccharomyces carlsbergensis), or is a hybrid obtained from a strain that belongs to one of these species, such as, for example, a S. cerevisiae/S. paradoxus or a hybrid S. cerevisiae/S. uvarum.

[0076] According to another particular embodiment, the eukaryotic cell is a fungal cell, in particular, a fungal cell of industrial interest. Exemplary fungi include, but are not limited to, filamentous fungal cells. Filamentous fungi include fungi that belong to the Eumycota and Oomycota subdivisions. Filamentous fungal cells can be selected from the group consisting of Trichoderma, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora cells , Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium or Trametes.

[0077] According to yet another particular embodiment, the eukaryotic cell is a plant cell, in particular, a plant cell of agronomic interest. Exemplary plants include, but are not limited to, rice, wheat, soybeans, corn, tomatoes, Arabidopsis thaliana, barley, rapeseed, cotton, sugar cane and beets. According to a preferred embodiment, the eukaryotic cell is a rice cell.

[0078] Preferably, the eukaryotic cell is heterozygous for the gene(s) addressed by the guide RNA.

[0079] As used herein, the term “fusion protein” refers to a chimeric protein that comprises at least two domains derived from the combination of different proteins or protein fragments. The nucleic acid that encodes this protein is obtained by recombining the regions that encode proteins or protein fragments so that they are in phase and are transcribed into the same mRNA. The various domains of the fusion protein can be directly adjacent or can be separated by the binding of sequences (linkers) that introduce a certain structural flexibility into the construct.

[0080] The fusion protein used in the present invention comprises a Cas9 domain and a Spo11 domain.

[0081] The Cas9 domain is the fusion protein domain that has the ability to interact with guide RNAs and to direct the nuclease activity of the Spo11 domain towards a specific chromosomal region. The Cas9 domain may consist of a wild-type or modified Cas9 protein (also called Csn1 or Csx12), or a fragment of this protein capable of interacting with guide RNAs. The Cas9 protein can be notably modified in order to modulate its enzymatic activity. In this way, the nuclease activity of the Cas9 protein can be modified or inactivated. The Cas9 protein can also be truncated to remove protein domains not essential for the functions of the fusion protein, in particular, Cas9 protein domains that are not required for interaction with guide RNAs.

[0082] The Cas9 protein or fragment thereof as used in the present invention can be obtained from any known Cas9 protein (Makarova et al., 2008, Nat. Rev. Microbiol., 9, pages 466-477). Exemplary Cas9 proteins that may be used in the present invention include, but are not limited to, Cas9 proteins from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, llulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsonii, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina. Other Cas9 proteins that can be used in the present invention are also described in the article by Makarova et al. (Makarova et al., 2008, Nat. Rev. Microbiol., 9, pages 466-477). Preferably, the Cas9 domain comprises, or consists of, the Streptococcus pyogenes Cas9 protein (NCBI accession number: WP_010922251.1, SEQ ID NO: 8) or a fragment thereof capable of interacting with guide RNAs.

[0083] According to a particular embodiment, the Cas9 domain consists of an integral Cas9 protein, preferably the Cas9 protein from Streptococcus pyogenes.

[0084] In general, Cas9 proteins comprise two nuclease domains: a domain related to a RuvC domain and a domain related to an HNH domain. These two domains cooperate to create DNA double-strand breaks (Jinek et al., Science, 337: 816-821). Each of these nuclease domains can be inactivated by deletion, insertion or replacement according to techniques well known to a person skilled in the art such as targeted mutagenesis, PCR mutagenesis or total gene synthesis. Thus, the RuvC domain can be inactivated, for example, by the D10A substitution and the HNH domain can be inactivated, for example, by the H840A substitution (Jinek et al., Science, 337: 816-821), in which the positions indicated are those of SEQ ID NO: 8.

[0085] In the peptide sequences described in this document, amino acids are represented by their one-letter code according to the following nomenclature: C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine; G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M: methionine; N: asparagine; P: proline; Q: glutamine; A: arginine; S: serine; T: threonine; V: valine; W: tryptophan and Y: tyrosine.

[0086] According to one embodiment, the Cas9 domain is deficient in at least one nuclease activity. This domain can be obtained by inactivating at least one nuclease domain of the Cas9 protein as described above.

[0087] According to a particular embodiment, the Cas9 domain comprises, or consists of, a Cas9 protein or a Cas9 protein fragment, which lacks nuclease activity (also called Cas9* or dCas9). This catalytically inactive form can be obtained by inactivating the two nuclease domains of the Cas9 protein as mentioned above, for example, by introducing the two point mutations that replace the aspartate at position 10 and the histidine at position 840 with alanines.

[0088] According to a preferred embodiment, the Cas9 domain comprises, or consists of, a Cas9 protein, preferably the Cas9 protein from Streptococcus pyogenes (spCas9), which lacks nuclease activity (spCas9*).

[0089] According to a particular embodiment, the Cas9 domain comprises, or consists of, the sequence presented in SEQ ID NO: 8, in which the aspartate at position 10 and histidine at position 840 have been replaced by alanines.

[0090] Spo11 is a protein related to the catalytic A subunit of a type II topoisomerase present in archaea (Bergerat et al., Nature, vol. 386, page 414 7). It catalyzes the DNA double-strand breaks that initiate meiotic recombinations. It is a highly conserved protein for which homologues exist in all eukaryotes. Spo11 is active as a dimer made up of two subunits, each of which shares a strand of DNA. Although essential, Spo11 does not act alone to generate double-strand breaks during meiosis. In the yeast S. cerevisiae, for example, it cooperates with proteins Rec102, Rec103/Sk18, Rec104, Rec114, Mer1, Mer2/Rec107, Mei4, Mre2/Nam8, Mre11, Rad50, Xrs2/Nbs1, Hop1, Red1, Mek1, Set1 and Spp1 and with other partners described in the articles by Keeney et al. (2001 Curr. Top. Dev. Biol, 52, pages 1-53), Smith et al. (Curr. Opin. Genet. Dev, 1998, 8, pages 200-211) and Acquaviva et al. (2013 Science, 339, pages 215-8). However, it has recently been shown that targeting Spo11 to a particular site is sufficient to initiate the process of meiotic recombination (Pecina et al., 2002 Cell, 111, 173-184). It should be noted that several Spo11 protein homologs can coexist in the same cell, notably in plants. Preferably, the Spo11 protein is one of the Spo11 proteins of the eukaryotic cell of interest.

[0091] The Spo11 domain of the Cas9-Spo11 fusion protein is, in general, the domain responsible for double-strand breaks. This domain may consist of a Spo11 protein or a fragment thereof with the ability to induce DNA double-strand breaks.

[0092] The Spo11 protein or fragment thereof as used in the present invention can be obtained from any Spo11 protein such as the Spo11 protein of Saccharomyces cerevisiae (Gene ID: 856364, NCBI accession number: NP_011841 (SEQ ID NO: 9 ) Esposito and Esposito, Genetics, 1969, 61, pages 79-89), the AtSpo11-1 and AtSpo11-2 proteins from Arabidopsis thaliana (Grelon M. et al., 2001, Embo J., 20, pages 589-600) , the murine mSpo11 protein (Baudat F et al., Molecular Cell, 2000, 6, pages 989-998), the C. elegans Spo11 protein or the Drosophila meiW68 Spo11 protein (McKim et al., 1998, Genes Dev, 12(18), pages 2932-42). Of course, these examples are not limiting and any known Spo11 protein can be used in the method according to the invention.

[0093] According to a preferred embodiment, the Spo11 domain comprises, or consists of, a Spo11 protein, preferably a Spo11 protein from Saccharomyces cerevisiae, such as, for example, the protein having the sequence SEQ ID NO: 9.

[0094] According to a particular embodiment, the Spo11 domain is nuclease deficient. In particular, the Spo11 domain may comprise, or consist of, the Spo11-Y135F mutant protein, a mutant protein lacking the ability to induce DNA double-strand breaks (Neale MJ, 2002, Molecular Cell, 9, 835-846). The position indicated is that of SEQ ID NO: 9.

[0095] The ability of the fusion protein according to the invention to induce DNA double-strand breaks may originate from the Cas9 domain or the Spo11 domain. Thus, the fusion protein comprises at least one domain, Cas9 or Spo11, which has nuclease activity, preferably the Spo11 domain.

[0096] According to a particular embodiment, several fusion proteins according to the invention comprising several Spo11 domains can be introduced into the same cell. In particular, when several Spo11 homologs exist in the eukaryotic cell of interest, the various fusion proteins may each comprise a different Spo11 homolog. By way of example, the two fusion proteins according to the invention which respectively comprise the Arabidopsis thaliana Spo11-1 and Spo11-2 domains can be introduced into the same cell, preferably the same Arabidopsis thaliana cell. Still by way of example, one or more fusion proteins according to the invention comprising the rice Spo11-1, Spo11-2, Spo11-3 and/or Spo11-4 domains can be introduced into the same cell, preferably, in the same rice cell. Numerous Spo11 homologs have been identified in several species, in particular, in plant species (Sprink T and Hartung F, Frontiers in Plant Science, 2014, Vol. 5, article 214, doi: 10.3389/fpls.2014.00214; Shingu Y et al. , BMC Mol Biol, 2012, doi: 10.1186/1471-2199-13-1). A person skilled in the art can readily identify Spo11 homologs in a given species, notably through well-known bioinformatics techniques.

[0097] The fusion protein according to the invention comprises a Spo11 domain and a Cas9 domain as defined above.

[0098] According to one embodiment, the Spo11 domain is at the N-terminal site and the Cas9 domain is at the C-terminal site of the fusion protein. According to another embodiment, the Spo11 domain is at the C-terminal site and the Cas9 domain is at the N-terminal site of the fusion protein.

[0099] The fusion protein may also comprise a nuclear localization signal (NLS) sequence. NLS sequences are well known to a person skilled in the art and generally comprise a short sequence of basic amino acids. By way of example, the NLS sequence may comprise the sequence PKKKRKV (SEQ ID NO: 3). The NLS sequence may be present at the N-terminal end, at the C-terminal end or in an internal region of the fusion protein, preferably at the N-terminal end of the fusion protein.

[00100] The fusion protein may also comprise an additional cell penetration domain, that is, a domain that facilitates entry of the fusion protein into the cell. This type of domain is well known to a person skilled in the art and may comprise, for example, a penetrating peptide sequence derived from the HIV-1 TAT protein such as GRKKRRQRRRPPQPKKKRKV (SEQ ID NO: 4), derived from the TLM sequence of the virus. of human hepatitis B as PLSSIFSRIGDPPKKKRKV (SEQ ID NO: 5) or a polyarginine peptide sequence. Such a cell-penetrating domain may be present at the N-terminal end or the C-terminal end or may be within the fusion protein, preferably at the N-terminal end.

[00101] The fusion protein may additionally comprise one or more binding sequences (linkers) between the Cas9 and Spo11 domains and, optionally, between these domains and other domains of the protein such as the nuclear localization signal sequence or the cell penetration. The length of these linkers is readily adjustable by a person skilled in the art. In general, such sequences comprise between 10 and 20 amino acids, preferably about 15 amino acids, and more preferably 12 amino acids. The linkers between the various domains can be of identical or different lengths.

[00102] According to a particular embodiment, the fusion protein comprises, or consists of, successively, the N-terminal end to the C-terminal end: a nuclear localization signal, a first linker (ligand1), a Cas9 domain , a second linker (ligand2) and a Spo11 domain.

[00103] According to another particular embodiment, the fusion protein comprises, or consists of, successively, from the N-terminal end to the C-terminal end: a nuclear localization signal, a first linker (linker1), a domain Spo11, a second ligand (ligand2) and a Cas9 domain.

[00104] The fusion protein may additionally comprise a tag that is a defined amino acid sequence. This tag can notably be used to detect fusion protein expression, to identify proteins that interact with the fusion protein, or to distinguish fusion protein binding sites in the genome. Detection of the tag attached to the fusion protein can be performed with an antibody specific to said tag or by means of any other technique well known to a person skilled in the art. The identification of proteins that interact with the fusion protein can be performed, for example, by coimmunoprecipitation techniques. Characterization of fusion protein binding sites in the genome can be performed, for example, by immunoprecipitation, chromatin immunoprecipitation coupled to real-time quantitative PCR (ChlP-qPCR), chromatin immunoprecipitation coupled to sequencing techniques (ChIP-Seq ), cartography using oligonucleotide mapping (oligo) or any other technique well known to a person skilled in the art.

[00105] This tag may be present at the N-terminal end of the fusion protein, at the C-terminal end of the fusion protein, or in a non-terminal position on the fusion protein. Preferably, the tag is present at the C-terminal end of the fusion protein. The fusion protein may comprise one or more tags, which may be identical or different.

[00106] Labels, as used in the present invention, can be selected from a variety of labels well known to a person skilled in the art. In particular, the tags used in the present invention can be peptide tags and/or protein tags. Preferably, the tags used in the present invention are peptide tags. Exemplary peptide tags that can be used in the present invention include, but are not limited to, tags consisting of repeats of at least six histidines (His), in particular, tags consisting of six or eight histidines, as well as Flag. , polyglutamate, hemagglutinin (HA), calmodulin, Strep, E-tag, myc, V5, Xpress, VSV, S-tag, Avi, SBP, Softag 1, Softag 2, Softag 3, isopetag, SpyTag and tetracysteine tags and combinations of the same. Exemplary protein tags that may be used in the present invention include, but are not limited to, glutathione S-transferase (GST) tags, Staphylococcus aureus protein A, Nus A, chitin binding protein (CBP), thioredoxin, maltose binding protein (MBP), biotin carboxyl carrier protein (BCCP), and immunoglobulin constant fragment (Fc), wherein the tags comprise a fluorescent protein such as green fluorescent protein (GFP), red fluorescent protein (RFP), protein cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), and combinations thereof.

[00107] According to a preferred embodiment, the fusion protein comprises a tag consisting of six histidines and/or one or more Flag motifs, preferably three Flag motifs. According to a particular embodiment, the fusion protein comprises a tag consisting of six histidines and three Flag motifs.

[00108] Alternatively, the Spo11 domain of the Cas9-Spo11 fusion protein can be replaced by one of the Spo11 partners capable of recruiting Spo11, i.e., a protein that forms a complex with Spo11 and thereby induces the formation of cleavages. double filament. This partner can be selected from the proteins mentioned in the articles by Keeney et al. (2001 Curr. Top. Dev. Biol, 52, pages 1-53), Smith et al. (Curr. Opin. Genet. Dev, 1998, 8, pages 200 211) and Acquaviva et al. (2013 Science, 339, pages 215-8), and more particularly, the group consisting of Rec102, Rec103/Sk18, Rec104, Rec114, Mer1, Mer2/Rec107, Mei4, Mre2/Nam8, Mre11, Rad50, Xrs2/Nbs1 , Hop1, Red1, Mek1, Set1 and Spp1. Preferably, the partner replacing the Spo11 domain is Mei4 or Spp1.

[00109] All modalities described for the Cas9-Spo11 fusion protein also apply to fusion proteins in which the Spo11 domain is replaced by one of its partners.

[00110] The fusion protein as described above can be introduced into the cell in protein form, notably in mature form or in precursor form, preferably in mature form, or in the form of a nucleic acid encoding said protein.

[00111] When the fusion protein is introduced into the cell in protein form, protecting groups can be added at the C- and/or N-terminal ends in order to improve the resistance of the fusion protein to peptidases. For example, the protecting group at the N-terminal end may be an acylation or acetylation and the protecting group at the C-terminal end may be an amidation or an esterification. The action of the protease can also be prevented by the use of amino acids that have the D configuration, cyclization of the protein by the formation of disulfide bonds, lactam rings or bonds between the N- and C-terminal ends. The fusion protein of the invention can also be prevented. understand pseudopeptide linkages that replace “conventional” peptide linkages (CONH) and that confer increased resistance to peptidases such as CHOH-CH2, NHCO, CH2-O, CH2CH2, CO-CH2, N-N, CH=CH, CH2NH, and CH2-S. The fusion protein may also comprise one or more amino acids that are rare amino acids, notably hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylsparagine, allo-isoleucine, N-methylisoleucine, N -methylvaline, pyroglutamine, aminobutyric acid; or synthetic amino acids, notably, ornithine, norleucine, norvaline, and cyclohexyl-alanine.

[00112] The fusion protein according to the invention can be obtained by conventional chemical synthesis (solid phase or homogeneous liquid phase) or by enzymatic synthesis (Kullmann W, Enzymatic peptide synthesis, 1987, CRC Press, Florida, USA). It can also be obtained by a method that consists of growing a host cell that expresses a nucleic acid that encodes the fusion protein and recovering said protein from those cells or from the culture medium.

[00113] As used in the present application, the term “guide RNA” or “gRNA” refers to an RNA molecule capable of interacting with the Cas9 domain of the fusion protein in order to guide it in the direction of a target chromosomal region.

[00114] Each gRNA comprises two regions:

[00115] - a first region (commonly called the “SDS” region), at the 5' end of the gRNA, which is complementary to the target chromosomal region and which mimics the crRNA of the endogenous CRISPR system, and

[00116] - a second region (commonly called the “handling” region), at the 3' end of the gRNA, which mimics the base pair interactions between the trans-activating crRNA (tracrRNA) and the endogenous CRISPR system crRNA and has a double-stranded stem-loop structure that terminates in the 3' direction with an essentially single-stranded sequence. This second region is essential for the binding of the gRNA to the Cas9 domain of the fusion protein.

[00117] The first region of the gRNA varies according to the chromosomal sequence addressed. On the other hand, the handling regions of the various gRNAs used can be identical or different. According to a particular embodiment, the handling region comprises, or consists of, the 3' sequence of 82 nucleotides of sequences SEQ ID NO: 10 to 16 (sequence in lowercase letters in Figure 13).

[00118] The SDS region of the gRNA, which is complementary to the target chromosomal region, generally comprises between 10 and 25 nucleotides. Preferably, this region is 19, 20 or 21 nucleotides long, and particularly preferably 20 nucleotides.

[00119] The second region of the gRNA has a stem-loop (or hairpin) structure. Rod and loop lengths may vary. Preferably, the loop is 3 to 10 nucleotides long and the stem is 6 to 20 nucleotides long. The stem may optionally have incompatible regions (which form “bulges”) of 1 to 10 nucleotides. Preferably, the total length of this handling region is 50 to 100 nucleotides and, more particularly, preferably 82 nucleotides.

[00120] The total length of a gRNA is, in general, 50 to 140 nucleotides, preferably 80 to 125 nucleotides and, more particularly, preferably 90 to 110 nucleotides. According to a particular embodiment, a gRNA as used in the present invention has a length of 102 nucleotides.

[00121] The gRNA is, preferably, formed from a single RNA molecule that comprises the two domains. Alternatively, the gRNA can be formed from two distinct RNA molecules, wherein the first molecule comprises the SDS region and half of the stem of the second region, and wherein the second molecule comprises the second half of the stem of the gRNA. In this way, the pairing of the two RNA molecules by their complementary sequences in the stem forms a functional gRNA.

[00122] A person skilled in the art can, by use of well-known techniques, readily define the sequence and structure of gRNAs according to the chromosomal region to be addressed (see, for example, the article by Di Carlo et al., Nucleic Acids Research 2013, 1-8).

[00123] In the method according to the invention, one or more gRNAs can be used simultaneously. These different gRNAs can address identical or different, preferably different, chromosomal regions.

[00124] gRNAs can be introduced into the eukaryotic cell as mature gRNA molecules, as precursors, or as one or more nucleic acids that encode said gRNAs.

[00125] When gRNAs (gRNA) are introduced into the cell directly as RNA molecules (mature or precursor), these gRNAs may contain modified nucleotides or chemical modifications that allow them, for example, to increase their resistance to nucleases and, being thus, increase their useful life in the cell. They may notably include at least one non-natural or modified nucleotide such as, for example, wherein a nucleotide comprises a modified base, such as inosine, methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine, deoxyuridine, diamino-2,6- purine, bromo-5-deoxyuridine or any other modified base that allows hybridization. The gRNAs used according to the invention can also be modified at the internucleotide linkage such as, for example, phosphorothioates, H-phosphonates or alkyl phosphonates, or at the backbone such as, for example, alpha-oligonucleotides, 2'-O-alkyl -ribose or peptide nucleic acid (PNA) (Egholm et al., 1992 J. Am. Chem. Soc., 114,1895-1897).

[00126] gRNAs can be natural RNA, synthetic RNA or RNA produced by recombination techniques. These gRNAs can be prepared by any methods known to a person skilled in the art, such as, for example, chemical synthesis, amplification techniques or in vivo transcription.

[00127] According to one embodiment, the method comprises introducing, into the eukaryotic cell, the fusion protein and one or more gRNAs capable of directing the action of the fusion protein in the direction of a certain chromosomal region. The protein and gRNAs can be introduced into the cytoplasm or nucleus of the eukaryotic cell by any method known to a person skilled in the art, for example, by microinjection. The fusion protein can notably be introduced into the cell as one element of a protein-RNA complex comprising at least one gRNA.

[00128] According to another embodiment, the method comprises introducing, into the eukaryotic cell, the fusion protein and one or more nucleic acids that encode one or more gRNAs.

[00129] According to yet another embodiment, the method comprises introducing, into the eukaryotic cell, a nucleic acid that encodes the fusion protein and one or more gRNAs.

[00130] According to yet another embodiment, the method comprises introducing, into the eukaryotic cell, a nucleic acid that encodes the fusion protein and one or more nucleic acids that encode one or more gRNAs.

[00131] The fusion protein, or the nucleic acid encoding said fusion protein, and the gRNA(gRNAs), or the nucleic acid(nucleic acids) encoding said gRNA(gRNAs), can be introduced into the cell simultaneously or sequentially.

[00132] Alternatively, and more particularly in relation to plant cells, the nucleic acid encoding the fusion protein and the nucleic acid (nucleic acids) encoding the gRNA (gRNAs) can be introduced into a cell by crossing two cells into which the nucleic acid encoding the fusion protein and the nucleic acid (nucleic acids) encoding the gRNA (gRNAs) were respectively introduced.

[00133] Alternatively, and more particularly in relation to plant cells, the nucleic acid encoding the fusion protein and the nucleic acid (nucleic acids) encoding the gRNA (gRNAs) can be introduced into a cell by mitosis of a cell into which the nucleic acid encoding the fusion protein and the nucleic acid (nucleic acids) encoding the gRNA (gRNAs) have previously been introduced.

[00134] In embodiments in which the fusion protein and/or gRNA(gRNAs) are introduced into the eukaryotic cell as a nucleic acid encoding said protein and/or said gRNA(gRNAs), the expression of said acids nucleic acids enable the production of the fusion protein and/or gRNA(gRNAs) in the cell.

[00135] In the context of the invention, “nucleic acid” means any molecule based on DNA or RNA. These molecules may be synthetic or semi-synthetic, recombinant, optionally amplified or cloned into vectors, chemically modified, comprising non-natural bases or modified nucleotides comprising, for example, a modified bond, a modified pyrimidine or purine base or a modified sugar. Preferably, codon usage is optimized according to the nature of the eukaryotic cell.

[00136] The nucleic acids encoding the fusion protein and those encoding the gRNAs can be placed under the control of identical or different promoters, which can be constitutive or inducible, in particular, meiosis-specific promoters. According to a preferred embodiment, the nucleic acids are placed under the control of constitutive promoters such as the ADH1 promoter or the RNA polymerase III-dependent pRPR1 and SNR52 promoters, more preferably, the pRPR1 promoter.

[00137] The nature of the promoter may also depend on the nature of the eukaryotic cell. According to a particular embodiment, the eukaryotic cell is a plant cell, preferably a rice cell, and the nucleic acids are placed under the control of a promoter selected from the maize ubiquitin promoters (pZmUbi) and the polymerase III U3 and U6 promoters. According to a preferred embodiment, the nucleic acid encoding the fusion protein is placed under the control of the pZmUbi promoter and the nucleic acids encoding the gRNAs are placed under the control of the U3 or U6 promoter, preferably the U3 promoter.

[00138] The nucleic acids encoding the fusion protein and the gRNA(gRNAs) may be arranged in the same construct, in particular, in the same expression vector, or in distinct constructs. Alternatively, nucleic acids can be inserted into the eukaryotic cell genome in identical or identical regions. According to a preferred embodiment, the nucleic acids encoding the fusion protein and the gRNA (gRNAs) are arranged in the same expression vector.

[00139] Nucleic acids as described above can be introduced into the eukaryotic cell by any method known to a person skilled in the art, in particular, by microinjection, transfection, electroporation and biolistics.

[00140] Optionally, the expression or activity of the eukaryotic cell's endogenous Spo11 protein can be suppressed in order to better control the phenomena of meiotic recombination. This inactivation can be carried out by techniques well known to a person skilled in the art, notably by inactivating the gene encoding the endogenous Spo11 protein or by inhibiting its expression by means of interfering RNA.

[00141] After introducing, into the eukaryotic cell, the fusion protein and one or more gRNAs, or nucleic acids that encode it, the method according to the invention comprises inducing said cell to enter meiotic prophase I.

[00142] This induction can be done according to various methods well known to a person skilled in the art.

[00143] By way of example, when the eukaryotic cell is a mouse cell, the cells can be induced to enter meiotic prophase I by the addition of retinoic acid (Bowles J et al., 2006, Science, 312(5773), pages 596-600).

[00144] When the eukaryotic cell is a plant cell, the induction of meiosis is carried out according to a natural process. According to a particular embodiment, after transformation of a callus comprising one or more plant cells, a plant is regenerated and placed in conditions that promote the induction of a reproductive phase and, thereby, the meiotic process. These conditions are well known to a person skilled in the art.

[00145] When the eukaryotic cell is a yeast, this induction can be carried out by transferring the yeast to the sporulation medium, in particular, from the rich medium to the sporulation medium, wherein said sporulation medium preferably lacks from a fermentable carbon source or a nitrogen source, and by incubating the yeast in the sporulation medium for a period of time sufficient to induce Spo11-dependent double-strand breaks. The initiation of the meiotic cycle depends on several signals: the presence of the two type-corresponding alleles MATa and MATα, the absence of a nitrogen source and a fermentable carbon source.

[00146] As used in this document, the term “rich medium” refers to a culture medium that comprises a fermentable carbon source and a nitrogen source as well as all the nutritional elements necessary for yeast to multiply by mitotic division. Such a medium can be readily selected by a person skilled in the art and can, for example, be selected from the group consisting of YPD medium (1% yeast extract, 2% bactopeptone and 2% glucose), YPG medium (1% yeast extract, 2% bactopeptone and 3% glycerol) and synthetic complete medium (SC) (Treco and Lundblad, 2001, Curr. Protocol. Mol. Biol., Chapter 13, Unit 13.1).

[00147] As used herein, the term “sporulation medium” refers to any medium that induces yeast cells to enter meiotic prophase without vegetative growth, in particular, a culture medium that does not comprise a carbon source fermentable or a nitrogen source, but comprising a carbon source that can be metabolized by respiration, such as acetate. Such a medium can be readily selected by a person skilled in the art and can, for example, be selected from the group consisting of 1% KAc medium (Wu and Lichten, 1994, Science, 263, pages 515-518), medium SPM (Kassir and Simchen, 1991, Meth. Enzymol., 194, 94-110) and the sporulation media described in Sherman's article (Sherman, Meth. Enzymol., 1991, 194, 3-21).

[00148] According to a preferred embodiment, before being incubated in the sporulation medium, the cells are cultivated for a few division cycles in a pre-sporulation medium so as to obtain effective and synchronous sporulation. The pre-sporulation medium can be readily selected by one skilled in the art. For example, this medium may be SPS medium (Wu and Lichten, 1994, Science, 263, pages 515-518).

[00149] The choice of media (rich medium, pre-sporulation medium, sporulation medium) depends on the physiological and genetic characteristics of the yeast strain, notably if that strain is auxotrophic for one or more compounds.

[00150] Once the cell is involved in meiotic prophase I, the meiotic process can continue until the four daughter cells that have the required recombinations are produced.

[00151] Alternatively, when the eukaryotic cell is a yeast and, in particular, a yeast of the genus Saccharomyces, the cells can be returned to growth conditions in order to restart a mitotic process. This phenomenon, called “resumption of growth” or “RTG”, was previously described in patent application WO 2014/083142 and occurs when cells that have entered meiosis in response to a nutritional deficiency are placed in the presence of a carbon and energy source. nitrogen after the formation of Spo11-dependent double-strand breaks but before the first meiotic division (Honigberg and Esposito, Proc. Nat. Acad. Sci USA, 1994, 91, 6559-6563). Under these conditions, they stop progressing through the stages of meiotic differentiation to resume a mitotic mode of growth while inducing the desired recombinations during the repair of double-strand breaks caused by Spo11 (Sherman and Roman, Genetics, 1963, 48, 255 -261; Esposito and Esposito, Proc. Nat. Sci, 1974, 71, pages 3172-3176;

[00152] The method may further comprise obtaining a cell or cells that have the desired recombination(s).

[00153] The method according to the invention can be used in all applications in which it is desired to improve and control the phenomena of meiotic recombination. In particular, the invention enables the association, preferably, of genetic traits of interest. This preferential association makes it possible, on the one hand, to reduce the time needed to select them and, on the other hand, to generate possible, but unlikely, natural combinations. Finally, depending on the modality selected, the organisms obtained by this method can be considered as non-genetically modified organisms (non-GMO).

[00154] According to another aspect, the present invention relates to a method for generating variants of a eukaryotic organism, with the exception of humans, preferably, a yeast or a plant, more preferably, a yeast, notably, a strain of yeast of industrial interest, which comprises

[00155] - introduce into a cell of said organism:

[00156] a) a fusion protein comprising a Cas9 domain and a Spo11 domain, or a nucleic acid encoding said fusion protein; It is

[00157] b) one or more guide RNAs or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising an RNA structure for binding to the Cas9 domain and a sequence complementary to an addressed chromosomal region ; It is

[00158] - inducing said cell to enter meiotic prophase I;

[00159] - obtain a cell or cells that have the desired recombination (recombinations) in the addressed chromosomal region (addressed chromosomal regions); It is

[00160] - generating a variant of the organism of said recombinant cell.

[00161] In this method, the term “variant” should be understood broadly to refer to an organism that has at least one difference in phenotype or genotype from the parent organisms.

[00162] Recombinant cells can be obtained by allowing meiosis to continue until spores are obtained, or, in the case of yeast, by returning the cells to growth conditions after inducing double-strand breaks in order to summarize a mitotic process.

[00163] When the eukaryotic cell is a plant cell, a plant variant can be generated by fusion of plant gametes, wherein at least one of the gametes is a recombinant cell by the method according to the invention.

[00164] The present invention also relates to a method for identifying or locating genetic information encoding a characteristic of interest in the genome of a eukaryotic cell, preferably a yeast, which comprises:

[00165] - introduce into the eukaryotic cell:

[00166] a) a fusion protein comprising a Cas9 domain and a Spo11 domain, or a nucleic acid encoding said fusion protein; It is

[00167] b) one or more guide RNAs or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising an RNA structure for binding to the Cas9 domain and a sequence complementary to an addressed chromosomal region ; It is

[00168] - inducing said cell to enter meiotic prophase I;

[00169] - obtain a cell or cells that have the desired recombination (recombinations) in the addressed chromosomal region (addressed chromosomal regions); It is

[00170] - analyze the genotypes and phenotypes of recombinant cells in order to identify or locate the genetic information that encodes the characteristics of interest.

[00171] Preferably, the trait of interest is a quantitative tract of interest (QTL).

[00172] According to another aspect, the present invention relates to a fusion protein comprising a Cas9 domain and a Spo11 domain as described above.

[00173] The present invention also relates to a nucleic acid encoding said fusion protein according to the invention.

[00174] The nucleic acid according to the invention can be in the form of double-stranded or single-stranded DNA and/or RNA. According to a preferred embodiment, the nucleic acid is an isolated DNA molecule, synthesized by recombinant techniques well known to a person skilled in the art. The nucleic acid according to the invention can be deduced from the sequence of the fusion protein according to the invention and the use of codons can be appropriate according to the host cell in which the nucleic acid is to be transcribed.

[00175] The present invention further relates to an expression cassette comprising a nucleic acid according to the invention operably linked to the sequences necessary for its expression. Notably, the nucleic acid can be under the control of a promoter that allows its expression in a host cell. In general, an expression cassette comprises, or consists of, a promoter for initiating transcription, a nucleic acid according to the invention, and a transcription terminator.

[00176] The term “expression cassette” refers to a nucleic acid construct that comprises a coding region and a regulatory region, operably linked. The expression “operationally linked” indicates that the elements are combined so that expression of the coding sequence is under the control of the transcriptional promoter. Typically, the promoter sequence is placed upstream of the gene of interest, at a distance from it compatible with controlling its expression. Spacer sequences may be present between the regulatory elements and the gene, as they do not impede expression. The expression cassette may also comprise at least one activation sequence (“enhancer”) operably linked to the promoter.

[00177] A wide variety of promoters that can be used for the expression of genes of interest in host cells or organisms are available to a person skilled in the art. They include constitutive promoters as well as inducible promoters that are activated or suppressed by exogenous chemical or physical stimuli.

[00178] Preferably, the nucleic acid according to the invention is placed under the control of a constitutive promoter or a meiosis-specific promoter.

[00179] Exemplary meiosis-specific promoters that can be used in the context of the present invention include, but are not limited to, endogenous Spo11 promoters, promoters of Spo11 partners to form double-strand breaks, the Rec8 promoter (Murakami & Nicolas, 2009, Mol . Cell. Biol, 29, 3500-16), or the Spo13 promoter (Malkova et al., 1996, Genetics, 143, 741-754).

[00180] Other inducible promoters can also be used such as the estradiol promoter (Carlile & Amon, 2008 Cell, 133, 280-91), the methionine promoter (Care et al., 1999, Molecular Microb 34, 792-798) , promoters induced by heat shock, metals, steroids, antibiotics and alcohol.

[00181] The constitutive promoters that can be used in the context of the present invention are, by way of non-limiting example: the cytomegalovirus (CMV) immediate early gene promoter, the simian virus (SV40) promoter, the major late gene promoter of adenovirus, the Rous sarcoma virus (RSV) promoter, the mouse mammary tumor virus (MMTV) promoter, the phosphoglycerate kinase (PGK) promoter, the elongation factor ED1-alpha promoter, ubiquitin promoters, promoters actin promoters, tubulin promoters, immunoglobulin promoters, alcohol dehydrogenase 1 (ADH1) promoter, RNA polymerase III-dependent promoters such as U6, U3, H1, 7SL, pRPR1 (“Ribonuclease P RNA 1”), SNR52 promoters (“ small nuclear RNA”), or the pZmUbi promoter.

[00182] The transcription terminator can be readily selected by a person skilled in the art. Preferably, this terminator is RPR1t, the 3' flanking sequence of the Saccharomyces cerevisiae SUP4 gene or the nopaline synthase (tNOS) terminator.

[00183] The present invention further relates to an expression vector comprising a nucleic acid or an expression cassette according to the invention. Such an expression vector can be used to transform a host cell and to express the nucleic acid according to the invention in said cell. Vectors can be constructed by conventional molecular biology techniques well known to a person skilled in the art.

[00184] Advantageously, the expression vector comprises regulatory elements for expressing the nucleic acid according to the invention. These elements may comprise, for example, transcription promoters, transcription activators, terminator sequences, initiation codons and termination codons. Methods for selecting such elements as a function of the host cell in which expression is desired are well known to a person skilled in the art.

[00185] In a particular embodiment, the expression vector comprises a nucleic acid encoding the fusion protein according to the invention, placed under the control of a constitutive promoter, preferably the ADH1 promoter (pADH1). It may also comprise a terminator sequence such as the ADH1 terminator (tADH1).

[00186] The expression vector may comprise one or more bacterial or eukaryotic origins of replication. The expression vector may, in particular, include a bacterial origin of replication functional in E. coli as the ColE1 origin of replication. Alternatively, the vector may comprise a eukaryotic origin of replication, preferably functional in S. cerevisiae.

[00187] The vector may additionally comprise elements that allow its selection in a bacterial or eukaryotic host cell, such as, for example, an antibiotic resistance gene or a selection gene that ensures the complementation of the respective deleted gene in the host cell genome. . Such elements are well known to a person skilled in the art and are extensively described in the literature.

[00188] In a particular embodiment, the expression vector comprises one or more antibiotic resistance genes, preferably a gene for resistance to ampicillin, kanamycin, hygromycin, geneticin and/or nourseothricin.

[00189] The expression vector may also comprise one or more sequences that allow targeted insertion of the vector, expression cassette or nucleic acid into the genome of a host cell. Preferably, the insertion is performed in a gene whose inactivation allows the selection of host cells that have integrated the vector, cassette or nucleic acid, such as the TRP1 locus.

[00190] The vector can be circular or linear, single or double stranded. It is advantageously selected from plasmids, phages, phagemids, viruses, cosmids and artificial chromosomes. Preferably, the vector is a plasmid.

[00191] The present invention relates, in particular, to a vector, preferably a plasmid, which comprises a bacterial origin of replication, preferably the ColE1 origin, a nucleic acid as defined above under the control of a promoter, preferably, a constitutive promoter such as the ADH1 promoter, a terminator, preferably the ADH1 terminator, one or more selection markers, preferably resistance markers such as the gene for kanamycin or ampicillin resistance, and one or more more sequences that allow targeted insertion of the vector, the expression cassette or the nucleic acid into the host cell genome, preferably into the TRP1 locus of the yeast genome.

[00192] In a particular embodiment, the nucleic acid according to the invention carried out by the vector encodes a fusion protein comprising one or more tags, preferably comprising a tag consisting of six histidines and/or one or more motifs Flag, preferably three Flag motifs. Preferably, the tag or tags are C-terminated.

[00193] According to a particular embodiment, the expression vector is the P1 plasmid that has the nucleotide sequence SEQ ID NO: 1 or the P2 plasmid that has the nucleotide sequence SEQ ID NO: 2.

[00194] The present invention also relates to the use of a nucleic acid, an expression cassette or an expression vector according to the invention to transform or transfect a cell. The host cell may be transformed/transfected in a stable or transient manner and the nucleic acid, cassette or vector may be contained in the cell as an episome or may be integrated into the host cell genome.

[00195] The present invention relates to a host cell comprising a fusion protein, a nucleic acid, an expression cassette or an expression vector according to the invention.

[00196] Preferably, the cell is a eukaryotic cell, in particular, a yeast, plant, fungal or animal cell. Particularly preferably, the host cell is a yeast cell. In a particular embodiment, the host cell is non-human and/or non-embryonic.

[00197] According to a particular embodiment, the eukaryotic cell is a yeast cell, in particular, a yeast of industrial interest. Exemplary yeasts of interest include, but are not limited to, yeasts of the genus Saccharomyces sensu stricto, Schizosaccharomyces, Yarrowia, Hansenula, Kluyveromyces, Pichia or Candida, as well as hybrids obtained from a strain belonging to one of these genera.

[00198] Preferably, the yeast of interest belongs to the genus Saccharomyces, preferably a yeast selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces castelli, Saccharomyces eubayanus, Saccharomyces kluyveri, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces uvarum, Saccharomyces paradoxus, Saccharomyces pastorianus (also called Saccharomyces carlsbergensis), and hybrids obtained from at least one strain belonging to one of these species, more preferably, said eukaryotic host cell is Saccharomyces cerevisiae.

[00199] According to another particular embodiment, the eukaryotic cell is a fungal cell, in particular, a fungal cell of industrial interest. Exemplary fungi include, but are not limited to, filamentous fungal cells. Filamentous fungi include fungi that belong to the Eumycota and Oomycota subdivisions. Filamentous fungal cells can be selected from the group consisting of Trichoderma, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora cells , Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium or Trametes.

[00200] In another preferred embodiment, the cell is a plant cell, preferably a plant cell selected from the group consisting of rice, wheat, soybeans, corn, tomatoes, Arabidopsis thaliana, barley, rapeseed, cotton , sugar cane and beet, more preferably, said eukaryotic host cell is a rice cell.

[00201] The present invention also relates to the use of the fusion protein, nucleic acid, expression cassette or expression vector according to the invention to (i) induce targeted meiotic recombinations in a eukaryotic cell, (ii ) generate variants of a eukaryotic organism, and/or (iii) identify or locate the genetic information that encodes a characteristic of interest in a eukaryotic cell genome.

[00202] The present invention further relates to a kit comprising a fusion protein, a nucleic acid, an expression cassette or an expression vector according to the invention, or a host cell transformed or transfected with a nucleic acid, an expression cassette or an expression vector according to the invention. It also relates to the use of said kit to implement a method according to the invention, in particular, to (i) induce the addressed meiotic recombinations in a eukaryotic cell, (ii) generate variants of a eukaryotic organism, and/or (iii) identify or locate the genetic information that encodes a characteristic of interest in a eukaryotic cell genome.

[00203] The methods according to the invention can be in vitro, in vivo or ex vivo methods.

[00204] The following examples are presented for illustrative and non-limiting purposes.

EXAMPLES 1. DESIGN, SYNTHESIS AND CLONING OF A NUCLEOTIDE SEQUENCE THAT CODES THE SPCAS9 PROTEIN AND ITS NUCLEASE DEFICIENT FORM SPCAS9*.

[00205] The SpCas9 gene that encodes the Cas9 protein comes from the bacterial strain Streptococcus pyogenes. The catalytically inactive form of SpCas9 (SpCas9*) is distinguished from SpCas9 by two-point mutations: the aspartate at position 10 and histidine at position 840 have both been replaced by alanines (Asp1^Ala1 and His 4 ^Ala 4 ).

[00206] Because of variations in the frequency of use of genetic codons between Streptococcus pyogenes and Saccharomyces cerevisiae, the SpCas9 and SpCas9* gene sequences were adapted in order to optimize their expression in yeast (yeast_optim_SpCas9 and yeast_optim_SpCas9*). The amino acid sequences of the two proteins were not modified.

2. MANIPULATION OF THE SEQUENCES YEASTOPTIM SPCAS9 AND YEASTOPTIM SPCAS9* IN ORDER TO FUSE THE PROTEINS SPCAS9 AND SPCAS9* WITH MEIOTIC TRANSESTERASE SPO11.

[00207] The manipulation of the yeast_optim_SpCas9 and yeast_optim_SpCas9* sequences made it possible to fuse the SpCas9 and SpCas9* proteins with a nuclear localization signal (NLS) associated with an N-terminal ligand (ligand 1) and a second C-terminal ligand (ligand 2 ) (which will separate the SpCas9 and SpCas9* proteins from the Spo11 protein in the final construct). The nucleotide sequences obtained in this way and encoding the protein sequences NLS-ligandel—SpCas9-ligand2 and NLS—ligantel-SpCas9*-ligand2 were then cloned into an integrative plasmid, which contains the complete form of the Spo11 protein from Saccharomyces cerevisiae marked with a sequence that encodes the C-terminal double 6xHis-3*Flag motif and whose expression is controlled by the constitutive pADH1 promoter. The resulting plasmid constructs, P1 and P2, thus contained the N-terminal in-phase fusion of NLS-ligandel-SpCas9-ligand2 and NLS-ligandel-SpCas9*-ligand2 to the Spo11 protein. Consequently, P1 and P2 respectively allowed the constitutive expression in yeast of the fusion proteins NLS-SpCas9-Spo11-6xHis-3xFlag (SEQ ID NO: 6) and NLS-SpCas9*-Spo11-6xHis-3xFlag (SEQ ID NO: 7) (Figure 1).

3. MANIPULATION OF MULTIPLE AND SINGLE GUIDE RNA EXPRESSION VECTORS.

[00208] Starting with a 2 micron (2μ) plasmid (Farzadfard F et al., 2013, ACS Synth. Biol., 2, pages 604 613; DiCarlo JE et al., 2013, Nucleic Acids Res., 41(7 ), pages 4336-4343) which contains the handling region (82 nucleotides) of a guide RNA (gRNA), placed under the control of a constitutive RNA polymerase III-dependent promoter such as pRPR1 or SNR52, the expression vector for a A single 102-nucleotide gRNA was constructed by cloning the 20-nucleotide specificity determination sequence (SDS region) of the gRNA into a restriction site located immediately 5' of the sequence encoding the linearized vector handling region, by the assembly method. Gibson (Figure 2A).

[00209] This expression vector contained a sequence comprising several unique restriction sites (multiple cloning site (MCS)) downstream of the terminator (RPR1t or SUP4 3' flanking sequence). Also, in order to obtain a system that allows multiplexed addressing of meiotic recombination sites, several gRNA expression cassettes were inserted into the expression vector in its MCS. The gRNA expression cassettes consist of a constitutive RNA polymerase III-dependent promoter (pRPR1 or SNR52), the specific gRNA and a terminator (RPR1t or the 3' flanking sequence of SUP4). These gRNA expression cassettes were first cloned into unique gRNA expression vectors (see above), then were amplified by PCR before being successively cloned into the multiple cloning site (MCS) of the expression vector for a single gRNA by techniques conventional insertion/ligation method (Figure 2B). This strategy ends in the concatenation of several gRNA cassettes into a single expression vector.

4. COEXPRESSION OF SPCAS9-SPO11 AND SPCAS9*-SPO11 FUSION PROTEINS WITH GRNAS IN YEAST.

[00210] In order to introduce the NLS-SpCas9-Spo11 or NLS—SpCas9*—Spo11 fusions into the chromosomal TRP1 locus, Saccharomyces cerevisiae yeast strains were transformed by heat shock with the linearized P1 or P2 vectors. These fusion proteins, which carry a C-terminal 3*Flag tag, were placed under the control of the constitutive ADH1 promoter. After transformation, the cells were plated in Petri dishes containing selective medium (adapted for the selection markers carried by the P1 and P2 plasmids) in order to select the transformants that integrated the fusion into their genome.

[00211] The expression vector for the gRNA (gRNAs) was then introduced into diploid yeast strains expressing the NLS-SpCas9-Spo11 or NLS-SpCas9*-Spo11 fusion proteins by heat shock transformation. The cells were then plated in medium selective for the selection markers for the gRNA expression plasmids. The gRNA expression plasmids comprised a 2 micron (2μ) origin of replication that allowed them to be maintained at a high copy number in each yeast cell (50-100 copies/cell).

[00212] The formation of double-stranded meiotic breaks generated in a single or multiplexed manner by the SpCas9-Spo11 or SpCas9*-Spo11 fusion proteins at the genomic sites addressed by multiple and single gRNAs is then detected by DNA Southern Blot analysis genomics extracted from diploid cells grown in sporulation medium.

5. COMPLEMENTATION OF SPORE DERIVED FROM THE SPORULATION OF STRAINS INACTIVATED BY THE SPO11 GENE BY THE EXPRESSION OF SPCAS9*—SPO11 FUSION PROTEINS

[00213] The inventors analyzed the viability of spores derived from meiosis of the following diploid strains of Saccharomyces cerevisiae:

[00214] - a SPO11/SPO11 strain (ORD7339) comprising two copies of the wild-type allele of the SPO11 gene,

[00215] - a spo11/spo11 strain (AND2822) comprising two copies of the mutated allele of the SPO11 gene. This mutated allele corresponds to the wild-type allele in which a genetic marker that completely inactivates the gene has been inserted,

[00216] - a spo11/spo11 strain dCAS9-SPO11/0 (AND2820) comprising two copies of the mutated allele of the SPO11 gene and one copy of the gene encoding the SpCas9*-Spo11 fusion protein integrated into one of the two chromosome copies IV at the TRP1 locus, and

[00217] - a spo11/spo11 dCAS9-SPO11/dCAS9-SPO11 (AND2823) strain comprising two copies of the mutated allele of the SPO11 gene and two copies of the gene encoding the SpCas9*-Spo11 fusion protein integrated into both copies of chromosome IV at the TRP1 locus.

[00218] The results presented in Figure 5 show that expression of the SpCas9*-Spo11 fusion protein complements the inviability of spores derived from sporulating strains inactivated by the SPO11 gene.

6. ADDRESSING DOUBLE FILAMENT MEIOTIC BREAKS BY THE SP CAS9*—SPO11 FUSION PROTEIN AND A YCR048W REGION-SPECIFIC GUIDE RNA

[00219] The SpCas9*-Spo11 (dCAS9-SPO11) expression cassette was integrated into the chromosomal TRP1 locus (chromosome IV). The UAS1-YCR048W guide RNA (sgRNA) (SEQ ID NO: 10) was expressed by the multicopy replication (non-integrative) plasmid as described in Figure 2A. The fusion gene carrying the dCAS9-SPO11 construct was expressed under the control of the constitutive ADH1 promoter. The guide RNA was expressed under the control of the constitutive RPR1 promoter. Yeast cells were transformed by conventional electroporation method. Integration of the cassette carrying the dCAS9-SPO11 construct was confirmed by Southern blot.

[00220] The following strains were used:

[00221] - SPO11/SPO11 (ORD7304),

[00222] - SPO11/SPO11 that expresses the UAS1-YCR48W guide RNA (sgRNA) (ANT2524),

[00223] - spo11/spo11 dCAS9-SPO11/0 which expresses guide RNA handling, that is, a guide RNA without the SDS region that is specific for the chromosomal address (ANT2527),

[00224] - spo11/spo11 dCAS9-SPO11/0 that expresses the UAS1-YCR48W guide RNA (sgRNA) (ANT2528), and

[00225] - spo11/spo11 dCAS9-SPO11/dCAS9—SPO11 expressing the UAS1-YCR48W guide RNA (sgRNA) (ANT2529).

[00226] Cells were collected after transfer to sporulation medium (1% KAc) and were taken at the indicated times (hours). The strains are homozygous for deletion of the SAE2 gene that inhibits the repair of DNA double-strand breaks (DSBs). The accumulation of DSBs was detected by Southern blot after digestion of genomic DNA by AseI restriction enzyme. The DNA was probed with an internal fragment for the YCR048W locus. Bands were quantified using ImageJ software.

[00227] The results presented in Figure 6 show that expression of the construct SpCas9*-Spo11 (dCAS9-SPO11) induces meiotic DNA double-strand breaks (DSBs) at the natural cleavage sites of the Spo11 protein (YCR043C-YCR048W region of the chromosome III (DSBs I to VI symbolized by black squares in Figure 6) and at the UAS1 site

(DSB VII, black triangle) addressed by the UAS1-YCR048W guide RNA and located in the coding region of the YCR048W gene. 7. ADDRESSING DOUBLE FILAMENT MEIOTIC BREAKS BY THE SP CAS9*-SPO11 FUSION PROTEIN AND A YCR048W REGION-SPECIFIC GUIDE RNA

[00228] The SpCas9*-Spo11 (dCAS9-SPO11) expression cassette was integrated into the chromosomal TRP1 locus (chromosome IV). The guide RNA (sgRNA) UAS1-YCR048W or UAS2-YCR048W (SEQ ID NO: 11) was expressed by the multicopy replication (non-integrative) plasmid as described in Figure 2A. The fusion gene carrying the dCAS9-SPO11 construct was expressed under the control of the constitutive ADH1 promoter. The guide RNA was expressed under the control of the constitutive RPR1 promoter. Yeast cells were transformed by conventional electroporation method. Integration of the cassette carrying the dCAS9-SPO11 construct was confirmed by Southern blot.

[00229] The following strains were used:

[00230] - SPO11/SPO11 (ORD7304),

[00231] - SPO11/SPO11 that expresses the UAS1-YCR048W guide RNA (sgRNA) (ANT2524),

[00232] - SPO11/SPO11 dCAS9—SPO11/0 which expresses guide RNA handling, that is, a guide RNA without the SDS region that is specific for the chromosomal address (ANT2518),

[00233] - SPO11/SPO11 dCAS9—SPO11/0 expressing the UAS1-YCR048W guide RNA (sgRNA) (ANT2519),

[00234] - SPO11/SPO11 dCAS9-SPO11/dCAS9-SPO11 expressing the UAS1-YCR48W guide RNA (sgRNA) (ANT2522),

[00235] - SPO11/SPO11 dCAS9-SPO11/0 expressing the UAS2-YCR048W guide RNA (sgRNA) (ANT2520),

[00236] - SPO11/SPO11 dCAS9-SPO11/dCAS9-SPO11 that expresses the UAS2-YCR048W guide RNA (sgRNA) (ANT2523), and

[00237] - spoll/spoll dCAS9-SPO11/0 that expresses the UAS1-YCR048W guide RNA (sgRNA) (ANT2528).

[00238] Cells were collected after transfer to sporulation medium (1% KAc) and were taken at the indicated times (hours). The strains are homozygous for deletion of the SAE2 gene that inhibits the repair of DNA double-strand breaks (DSBs). The accumulation of DSBs was detected by Southern blot after digestion of genomic DNA by AseI and SacI restriction enzymes. The DNA was probed with an internal fragment for the YCR048W locus. Bands were quantified using ImageJ software.

[00239] The results presented in Figure 7 indicate that expression of the dCAS9-SPO11 construct induces meiotic DNA double-strand breaks (DSBs) at the natural cleavage sites of the Spoil protein (YCR047C-YCR048W region of chromosome III (DSBs V and VI symbolized by squares) and at the UAS1-YCR048W site (DSB VII symbolized by a triangle) addressed by the UAS1-YCR048W guide RNA in the 5' coding region of the YCR048W gene and at the UAS2-YCR048W site (DSB VIII symbolized by a circle) addressed by the UAS2-YCR048W guide RNA in the 3' coding region of the YCR048W gene. These results show that targeting is effective in strains carrying the wild-type SPO11 gene or the mutated SPO11 gene.

8. ADDRESSING DOUBLE FILAMENT MEIOTIC BREAKS BY THE SP CAS9*—SPO11 FUSION PROTEIN AND A SPECIFIC GUIDE RNA FOR THE GAL2 REGION

[00240] The SpCas9*-Spo11 (dCAS9-SPOll) expression cassette was integrated into the chromosomal TRPl locus (chromosome IV). the UAS D/E-GAL2 guide RNA (sgRNA) (SEQ ID NO: 12) was expressed by the multicopy replication (non-integrative) plasmid as described in Figure 2A. The fusion gene carrying the dCAS9-SPOll construct was expressed under the control of the constitutive ADH1 promoter. The guide RNA was expressed under the control of the constitutive RPR1 promoter. Yeast cells were transformed by conventional electroporation method. The integration of the cassette carrying the dCAS9-SPOll construct was confirmed by Southern blot.

[00241] The following strains were used:

[00242] - SPO11/SPO11 (ORD7304),

[00243] - spoll/spoll GAL4/GAL4 dCAS9—SPO11/0 which expresses guide RNA handling, that is, a guide RNA without the SDS region that is specific for the chromosomal address (ANT2527),

[00244] - spoll/spoll gal4/gal4 dCAS9-SPO11/0 expressing guide RNA handling (ANT2536) (both alleles of the GAL4 gene are mutated and thus inactive),

[00245] - spoll/spoll GAL4/GAL4 dCAS9—SPOll/0 that expresses the UAS guide RNA D/E-GAL2 (ANT2530), and

[00246] - spoll/spoll gal4/gal4 dCAS9-SPOll/0 that expresses the UAS guide RNA D/E-GAL2 (ANT2533).

[00247] Cells were collected after transfer to sporulation medium (1% KAc) and were taken at the indicated times (hours). The strains are homozygous for deletion of the SAE2 gene that inhibits the repair of DNA double-strand breaks (DSBs). The accumulation of DSBs was detected by Southern blot after digestion of genomic DNA by the XbaI restriction enzyme. The DNA was probed with the terminal portion of the GAL2 gene. Bands were quantified using ImageJ software.

[00248] The results presented in Figure 8 show that expression of the dCAS9-SPO11 construct induces meiotic DNA double-strand breaks (DSBs) at the UAS D/E site of the GAL2 gene promoter addressed by the UAS D/E- guide RNA. GAL2.

9. ADDRESSING DOUBLE FILAMENT MEIOTIC BREAKS BY THE SPCAS9*—SPO11 FUSION PROTEIN AND A GUIDE RNA SPECIFIC FOR THE SWC3 REGION

[00249] The SpCAS9*-SPO11 (dCas9-SPO11) expression cassette was integrated into the chromosomal TRP1 locus (chromosome IV). The guide RNA (sgRNASWC3) SWC3 (SEQ ID NO: 13) was expressed by the multicopy replication (non-integrative) plasmid as described above (Figure 2A). The fusion gene carrying the SpCAS9*-SPO11 construct was expressed under the control of the constitutive ADH1 promoter. The guide RNA was expressed under the control of the constitutive RPR1 promoter. Yeast cells were transformed by conventional electroporation method. Integration of the cassette carrying the SpCAS9*-SPO11 construct was confirmed by Southern blot.

[00250] The following strains were used:

[00251] -SPO11/SPO11 (ORD7304),

[00252] -spo11/spo11 SpCAS9*-SPO11/0 which expresses guide RNA handling, that is, a guide RNA without the SDS region that is specific for the chromosomal address (ANT2527),

[00253] -spo11/spo11 SpCAS9*-SPO11/0 expressing the guide RNA (sgRNASWC3) SWC3 (ANT2564).

[00254] -Cells were collected after transfer to sporulation medium (1% KAc) and were taken at the indicated times (hours). The strains are homozygous for deletion of the SAE2 gene that inhibits the repair of DNA double-strand breaks (DSBs). The accumulation of DSBs was detected by Southern blot after digestion of genomic DNA by the restriction enzymes PacI and AvrII. The DNA was probed with an internal fragment for the SPO7 locus. Bands were quantified using ImageJ software.

[00255] -The results presented in Figure 9 show that expression of the SpCAS9*-Spo11 construct induces meiotic DNA double-strand breaks (DSBs) at the natural cleavage sites of the Spoil protein (SIN8-SWC3 region) of chromosome I (DSBs I, II and III symbolized by black squares in Figure 9) and at the address site (DSBs IV symbolized by a circle) by the SWC3 guide RNA (sgRNASWC3) and located in the coding region of the SWC3 gene.

10. ADDRESSING MEIOTIC DNA DOUBLE-STREAD RUPTURES BY THE SP PROTEIN CAS9*—SPO11 AND VARIOUS MULTIPLEXED GUIDE RNAS SPECIFIC FOR THE GAL2 REGION

[00256] The SpCAS9*-Spo11 (dCas9-SPO11) expression cassette was integrated into the chromosomal TRP1 locus (chromosome IV). The UAS-A guide RNA (UAS-A sgRNA) (SEQ ID NO: 14), the UAS-B guide RNA (UAS-B sgRNA) (SEQ ID NO: 15) and the UAS-D/guide RNA E (UAS-D/E sgRNA) (SEQ ID NO: 12) were expressed individually and in multiplex (Multi-gRNAs) by the multicopy (non-integrative) replication plasmid as described above (Figure 2A). The fusion gene carrying the SpCAS9*-SPO11 construct was expressed under the control of the constitutive ADH1 promoter. Guide RNAs were expressed under the control of the constitutive RPR1 promoter. Yeast cells were transformed by conventional electroporation method. Integration of the cassette carrying the SpCAS9*-SPO11 construct was confirmed by Southern blot.

[00257] The following strains were used:

[00258] - spoll/spoll GAL4/GAL4 SpCAS9*-SPO11/0which expresses guide RNA handling, that is, a guide RNA without the SDS region that is specific for the chromosomal address (ANT2527),

[00259] - spoll/spoll GAL4/GAL4 SpCAS9*-SPO11/0 that expresses the UAS-A guide RNA (UAS-A sgRNA) (ANT2532),

[00260] - spoll/spoll gal4/gal4 SpCAS9*-SPOll/0 that expresses the UAS-A guide RNA (UAS-A sgRNA) (ANT2534),

[00261] - spoll/spoll GAL4/GAL4 SpCAS9*—SPOll/0 that expresses the UAS-D/E guide RNA (sgRNAUAS-D/E) (ANT2530),

[00262] - spoll/spoll gal4/gal4 SpCAS9*-SPOll/0 that expresses the UAS-D/E guide RNA (sgRNAUAS-D/E) (ANT2533),

[00263] - spoll/spoll GAL4/GAL4 SpCAS9*—SPOll/0 that expresses in multiplex the UAS-A guide RNA (sgRNAUAS-A), the UAS-B guide RNA (sgRNAUAS-B) and the guide RNA UAS-D/E (sgRNAUAS-D/E) (Multi-gRNAs) (ANT2551),

[00264] - spoll/spoll gal4/gal4 SpCAS9*-SPOll/0 that expresses in multiplex the UAS-A guide RNA (sgRNAUAS-A), the UAS-B guide RNA (sgRNA UAS-B) and the RNA- UAS-D/E guide (UAS—D/E sgRNA) (Multi-gRNAs) (ANT2552).

[00265] Cells were collected after transfer to sporulation medium (1% KAc) and were taken at the indicated times (hours). The strains are homozygous for deletion of the SAE2 gene that inhibits the repair of DNA double-strand breaks (DSBs). The accumulation of DSBs was detected by Southern blot after digestion of genomic DNA by the XbaI restriction enzyme. The DNA was probed with the terminal portion of the GAL2 gene. Bands were quantified using ImageJ software.

[00266] The results presented in Figure 10 indicate that expression of the construct SpCAS9*-Spo11 (dCas9-SPO11) induces meiotic DNA double-strand breaks (DSBs) at the address sites in the GAL2 gene promoter on chromosome XII by the ) guide RNA(s). In particular, coexpression of Sp Cas9*-Spo11 with the individual sgRNAs sgRNA UAS- A and sgRNAUAS—D/E leads to the generation of DSBs, respectively, at the UAS-A and UAS—D/E sites. Interestingly, addressing Sp Cas9*-Spo11 by coexpression of sgRNA UAS-A , sgRNAUAS-B , and sgRNAUAS—D/E leads to the formation of multiplex DSBs at the various address sites.

11. STIMULATION OF MEIOTIC RECOMBINATION BY THE PROTEIN SPCAS9*—SPO11 AND SEVERAL GUIDE RNAS MULTIPLEXED IN THE GAL2 ADDRESS REGION

[00267] In order to detect crossovers induced by the expression of CRISPR/SpCas9*-Spo11, the NatMX and HphMX cassettes (which respectively confer resistance to nourseothricin and hygromycin) were trans inserted upstream and downstream of the GAL2 gene in diploid cells (see Figure 11A).

[00268] The SpCAS9*-Spo11 (dCAS9-SPO11) expression cassette was integrated into the chromosomal TRP1 locus (chromosome IV). The UAS—D/E guide RNA (UAS-D/E sgRNA) (SEQ ID NO: 12) was expressed by the multicopy (non-integrative) replication plasmid as described above (Figure 2A). Multiplex expression of UAS-A guide RNA (UAS-A sgRNA) (SEQ ID NO: 14), UAS-B guide RNA (UAS-B sgRNA) (SEQ ID NO: 15) and UAS guide RNA - D/E (UAS—D/E sgRNA) (SEQ ID NO: 12) was performed from the same guide RNA expression plasmid described above (Multi-gRNAs). The fusion gene carrying the SpCAS9*-SPO11 construct was expressed under the control of the constitutive ADH1 promoter. The guide RNA was expressed under the control of the constitutive RPR1 promoter. Yeast cells were transformed by conventional electroporation method. The integration of the cassette carrying the SpCAS9*-SPO11 construct was confirmed by Southern blot.

[00269] The following strains were used:

[00270] - SPO11/SPO11 pEMP46::NatMX/0 tGAL2::KanMX/0 (ANT2527),

[00271] - spo11/spo11 pEMP46::NatMX/0 tGAL2::KanMX/0 SpCAS9*-SPO11/0 which expresses the handling guide RNA, that is, a guide RNA without the SDS region that is specific for the chromosomal address (ANT2539),

[00272] - spo11/spo11 pEMP46::NatMX/0 tGAL2::KanMX/0 SpCAS9*-SPO11/0 expressing the UAS-D/E guide RNA (sgRNAUAS-D/E) (ANT2540),

[00273] - spo11/spo11 pEMP46::NatMX/0 tGAL2::KanMX/0 SpCAS9*-SPO11/0 that expresses in multiplex the UAS-A guide RNA (UAS-A sgRNA), the UAS-B guide RNA (sgRNA UAS-B ) and the guide RNA UAS-D/E (sgRNA UAS—D/E) (Multi-gRNAs) (ANT2557).

[00274] After sporulation, the tetrads composed of 4 spores were dissected and the spores genotyped after germination for segregation of nourseothricin and hygromycin. The number of tetrads showing a parental ditype (PD) was compared with those showing a tetratype (T) and a non-parental ditype (NPD). The genetic distance in centiMorgans was determined according to the formula cM = 100(T+6NPD)/2(PD+T+NPD). The increase in the number of tetratypes in cells expressing SpCAS9*-SPO11 (strains ANT2540 and ANT2557) was statistically tested with Fisher's test by calculating the p-value in relation to cells coexpressing SpCAS9*-SPO11 and handling guide RNA (strain ANT2539).

[00275] The results presented in Figure 11B show that expression of the SpCas9*-SPO11 (dCAS9-SPO11) construct stimulates meiotic recombination in the GAL2 address region.

12. ADDRESSING MEIOTIC DNA DOUBLE-STREAD RUPTURES BY THE SP CAS9*-SPO11 FUSION PROTEIN AND A GUIDE RNA SPECIFIC FOR THE SEQUENCE CODING THE PUT4 GENE

[00276] The SpCAS9*-Spo11 (dCAS9-SPO11) expression cassette was integrated into the chromosomal TRP1 locus (chromosome IV). The PUT4 guide RNA (sgRNAPUT4) (SEQ ID NO: 16) was expressed by the multicopy replication (non-integrative) plasmid as described above (Figure 2A). The fusion gene carrying the SpCAS9*-SPOll construct was expressed under the control of the constitutive ADH1 promoter. The guide RNA was expressed under the control of the constitutive RPR1 promoter. Yeast cells were transformed by conventional electroporation method. The integration of the cassette carrying the SpCAS9*-SPOll construct was confirmed by Southern blot.

[00277] The following strains were used:

[00278] - SPO11/SPO11 (ORD7304),

[00279] - spoll/spoll SpCAS9*-SPO11/0 which expresses guide RNA handling, that is, a guide RNA without the SDS region that is specific for the chromosomal address (ANT2527),

[00280] - spoll/spoll SpCAS9*-SPO11/0 that expresses the PUT4 guide RNA (sgRNAPUT4) (ANT2547).

[00281] Cells were collected after transfer to sporulation medium (1% KAc) and were taken at the indicated times (hours). The strains are homozygous for deletion of the SAE2 gene that inhibits the repair of DNA double-strand breaks (DSBs). The accumulation of DSBs was detected by Southern blot after digestion of genomic DNA by the restriction enzymes BamHI and XhoI. The DNA was probed with an internal fragment for the CIN1 locus. Bands were quantified using ImageJ software.

[00282] The results presented in Figure 12 show that expression of the construct SpCAS9*-SPO11 (dCas9-SPO11) induces meiotic DNA double-strand breaks (DSBs) at the natural cleavage sites of the Spo11 protein (PYK2 - PUT4 region) of the chromosome .

13. CONCLUSION

[00283] The results presented in Figures 5 to 12 show that:

[00284] - expression of the SpCas9*- Spoil fusion protein (dCAS9-SPO11) complements the inviability of spores derived from sporulation of strains inactivated by the SPO11 gene,

[00285] - expression of the SpCas9*- Spoil fusion protein (dCAS9-SPO11) induces the formation of double-stranded meiotic breaks at natural DSB sites,

[00286] - co-expression of the SpCas9*- Spoil fusion protein (dCAS9-SPO11) and a gRNA induces the formation of double-stranded meiotic breaks at natural DSB sites and at the address site,

[00287] - co-expression of the SpCas9*- Spoil fusion protein (dCAS9-SPO11) and multiplexed gRNA induces the formation of meiotic double-strand breaks (DSBs) at the various address sites, and

[00288] - addressing is effective in strains that have wild-type SPO11 and the SPO11tic mutated gene background.

14. INDUCTION OF DOUBLE FILAMENT MEIOTIC RUPTURES BY SPCAS9*—SPO11 FUSION PROTEIN IN RICE a) PREPARATION OF DCAS9- SPO11 TRANSFORMATION VECTOR (SEE FIGURE 14)

[00289] Cas9 is a protein of prokaryotic origin, the codons used are optimized for the plant species in which the protein must be expressed. The codons of the Streptococcus pyogenes Cas9 protein are thus optimized for its expression in rice (see Miao et al., Cell Research, 2013, pages 1-4). Additionally, the Cas9 protein is inactivated by mutation of two catalytic sites, RuvC and HNH (Asp"^Ala" and His 4^Ala 4). The catalytically inactive form of SpCas9 is called SpCas9* or dCas9.

[00290] First, the dCas9 stop codon is removed and a linker is added in phase at the C-terminal end of dCas9. The ligand may be a sequence already known in the literature for use in the plant species in question or an optimized sequence. The ligand CCGGAATTTATGGCCATGGAGGCCCCGGGGATCCGT (SEQ ID NO: 17) used in yeast is also compatible with use in rice.

[00291] A nuclear localization signal (NLS) is also added at the N-terminal end of dCas9. Optionally, a linker can be added between the NLS and dCas9, such as, for example, the sequence GGTATTCATGGAGTTCCTGCTGCG (SEQ ID NO: 18).

[00292] The SPO11 sequence is then added in-phase at the C-terminal end of the NLS-dCas9-Linker construct. You can use a complementary DNA sequence (cDNA), genomic DNA (gDNA), or a complementary DNA sequence with the addition of several introns. It is possible to use SPO11-1 and/or SPO11-2 from rice.

[00293] The nopaline synthase (tNOS) terminator, adapted for rice, is added in phase to the NLS-dCas9-Ligante-SPO11 construct.

[00294] The maize ubiquitin promoter pZmUbi1 (Christensen AH et al., 1992, Plant Mol Biol, 18(4), pages 675-689 or Christensen AH and Quail PH, 1995, Transgenic Res, 5(3), pages 213-218), is a promoter that allows for ubiquitous and strong expression in rice.

[00295] For stable transformation of rice cells, transfection is performed with a binary vector, for example, the pCAMBIA5300 binary vector that carries a hygromycin resistance gene interrupted by an intron of the catalase gene. This resistance gene makes it possible to effectively select, in a selective environment, individuals who have integrated dCas9-SPO11 into their genome. This vector also contains a kanamycin resistance gene, which facilitates cloning and manipulation in bacterial hosts.

B) PREPARATION OF THE CONSTRUCTION THAT CARRIES GUIDE RNA

[00296] Regarding the “handling” region of the guide RNA (gRNA), the “native” sequence of the bacteria S. pyogenes is used. The SDS region that determines gRNA specificity is selected as a function of the zone of interest to be addressed using software freely available on the Internet (e.g., CRISPR PLANT).

[00297] The guide RNA is placed under the control of the rice polymerase III U3 promoter (see Miao et al. Cell Research, 2013, pages 1-4). Alternatively, it is placed under the control of the U6 promoter.

[00298] - Single binary vector

[00299] The construct comprising the guide RNA placed under the control of the U3 promoter is integrated into the vector comprising dCAS9-SPO11.

[00300] - Separate binary vectors

[00301] In order to address multiple regions, the guide RNAs are carried by a separate vector, a binary vector carrying geneticin resistance (pCAMBIA2300), which makes it possible to apply a double selection for the presence of the dCas9 T-DNA -SPO11 and gRNA T-DNA.

[00302] Several transformation strategies are possible:

[00303] Cotransformation: Starting with two binary vectors, one that carries dCas9-SPO11 and the other the gRNA (gRNAs). They are introduced into the same bacterial strain or into two different bacterial strains which are then mixed before co-culture with the plant cells.

[00304] Sequential transformations: Stable transformants carrying the dCas9-SPO11 construct are produced and their seeds used to produce calli which are then used for transformation with the gRNA construct by Agrobacterium or by bombardment.

[00305] Independent transformations: Plants carrying dCAS9-SPO11 without a guide and plants carrying gRNAs alone are generated. The stable transformants are then crossed in order to produce multiple combinations.

[00306] Rice transformation is performed from mature seed embryo callus according to the protocol detailed in Sallaud C et al., 2003, Theor Appl Genet, 106(8), pages 1396-1408.

[00307] Use of dCas9-SPO11 technology to induce targeted recombination in wild-type SPO11

[00308] The dCas9-Spo11 fusion protein is produced with the native Spo11 protein (SPO11-1 or SPO11-2), as gDNA or cDNA.

[00309] - by direct transformation of callus derived from F1 seeds: calli are induced from mature F1 seed embryos obtained by castration and manual fertilization between two parental lines of agronomic interest. Calli are transformed simultaneously with T-DNA or T-DNAs carrying dCas9-SPO11 and gRNA(gRNAs) (cotransformation). Transformants without gRNA or with a gRNA that addresses another region are used as controls to test the effectiveness of the system. Recombination analysis is performed on F2 plants.

[00310] - by separate transformation of calli from each relative: one lineage is transformed stably and homozygously with the dCas9-SPO11 construct and crossed with the other lineage that carries the gRNA(gRNAs) with heterozygous insertion. F1 plants carrying both constructs or just the dCas9-SPO11 construct are selected. Recombinations at the addressed locus (loci) are quantified in F2 populations derived from the two plant types.

[00311] Use of dCAS9-SPO11 technology to induce targeted recombination in mutant spo11

[00312] One of the parental lines (seeds carried by a SPO11/spo11 heterozygote) is transformed with the dCas9-SPO11 construct and plant homozygotes for the transgene and the spo11 mutation are obtained in T1 generation. The second parental line (seeds carried by a SPO11/spo11 heterozygote) is transformed with the construct carrying the gRNA (gRNAs). Plants heterozygous for the gRNA construct and the spo11 mutation are obtained in T1 generation. The two types of plants are crossed: 4 genotypes of F1 seeds are obtained, all carrying the dCas9-SPO11 construct, but carrying or not the gRNA and producing or not endogenous SPO11. Recombination analysis is performed on the F2 populations.

Claims (5)

1. Fusion protein characterized by the fact that it comprises a Cas9 protein and a Spo11 protein. 2. Fusion protein according to claim 1, characterized by the fact that the Cas9 protein is a nuclease-deficient Cas9 protein. 3. Host cell characterized by the fact that it comprises a fusion protein as defined in claim 1 or 2, wherein said host cell is a yeast cell. 4. Host cell according to claim 3, characterized in that said host cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces castelli, Saccharomyces eubayanus, Saccharomyces kluyveri, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces uvarum, Saccharomyces paradoxus, Saccharomyces pastorianus(also called Saccharomyces carlsbergensis), and hybrids obtained from at least one strain belonging to one of these species. 5. Host cell according to claim 4, characterized by the fact that said host cell is Saccharomyces cerevisiae.