DE102015006335A1 - Methods and constructs for targeted nucleic acid editing in plants - Google Patents

Abstract

The present invention relates to methods and constructs for the targeted modification of a nucleic acid target region in a plant target structure. Specifically, the present invention relates to methods and constructs for direct recovery of a plant or plant material comprising a previously deliberately engineered into a meristematic cell editing of a nucleic acid, but not for this purpose to produce the targeted alteration of the nucleic acid target region previously introduced recombinant constructs.

Description

Technical area

More particularly, the present invention relates to methods for producing a plant, plant material or plant cell comprising providing and introducing at least one gRNA and one Cas nuclease and / or effector domain or at least one recombinant construct comprising a gRNA and a cas Nuclease and / or an effector domain, or the sequences coding therefor, as well as at least one regulatory sequence and / or a localization sequence, into a plant target structure comprising at least one meristematic cell, thereby directly obtaining a plant, a plant material or a plant cell may be, wherein the at least one recombinant construct is preferably not integrated chromosomally or extrachromosomally. In addition, suitable recombinant constructs and vectors as well as methods for introducing these constructs and vectors into a plant targeting target of interest are disclosed. Finally, there is disclosed the use of a recombinant construct for targeting a nucleic acid target region in a plant cell, as well as plants, plant material or a plant cell obtainable or obtained by the methods of the invention.

Background of the invention

Genome editing is a molecular biology method by which targeted modifications such as insertions, deletions or point mutations or combinations thereof can be introduced into the genome of a living organism. For this purpose, targeted molecular instruments are needed which, on the one hand, have nuclease activity, but can also be conducted with sufficient specificity to the target sequence to be modified, in order to be able to plan and carry out targeted and site-directed mutagenesis. In plant biotechnology, targeted genome editing has become an alternative over the past few years to classical breeding and transgenic approaches. The tools available to date, such as zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), have only been used to a limited extent in the field of plant biotechnology, due in part to the low efficiency, but also to the difficult and complex design of the Constructs lies.

Another molecular tool that has been used more and more in recent years for precise and site-directed gene homeostasis are Cas nuclease-based systems. These nucleases are part of the system now referred to in the literature as "CRISPR / Cas" (Clustered regularly interspaced short palindromic repeat / CRISPR-associated genes). This system was originally identified in 1987 when analyzing the iap gene of E. coli, identifying naturally occurring repeat sequences in the bacterial genome. Later, these palindromic DNA repeats of 20 to 50 nucleotides were found to follow a pattern. This is where the acronym CRISPR was coined ( Jansen, R. et al., "Identification of genes that are associated with DNA repeats in prokaryotes", Mol. Microbiol., 2002, 43 (6), 1565-1575 ), with research still focused on bacteria. Finally, it has been reported that the CRISPR locus is a type of bacterial immune system and can confer acquired immunity to phages ( Barrangou et al. "CRISPR provides acquired resistance against viruses in procaryotes" Science 2007, 315: 1709.1712 ) by first inserting the invading phage DNA into a CRISPR locus as a protospacer, then transcribing the locus, and finally activating the CRISPR-mediated silencing mechanism.

The functional characterization also led to the gradual use of the system as a universal tool for the homologation of higher organisms. In the meantime, a variety of CRISPR / Cas systems, including a Type II system, have been described (see, for example, US Pat Van der Oost et al. "Unraveling the structural and mechanistic basis of CRISPR-Cas systems" Nature 2014, 482: 331-338 ), although the analyzes are far from complete.

The discovery and utilization of the bacterial Type-II CRISPR System provides another "genome editing" system with great potential.

The CRISPR / Cas system makes it possible to generate a targeted DNA double-strand break through a small, customizable non-coding RNA (guide RNA or gRNA) in combination with a Cas nuclease. The immune response mediated by CRISPR / Cas in natural systems requires CRISPR RNA (crRNA), whereby the maturation of this lead RNA, which controls the targeted activation of Cas nuclease, varies significantly between the different currently characterized CRISPR systems. First, will the invading DNA, also called "spacer", is integrated between two contiguous repeat regions at the proximal end of the CRISPR locus. Type II CRISPR systems encode a key enzyme for the interference step, a Cas9 nuclease, which requires both a crRNA and a trans-activating RNA (tracrRNA) as a leitmotif. These hybridize and form double-stranded (ds) RNA regions, which can be recognized and cleaved by RNAseIII to form mature crRNAs. These in turn associate with the Cas molecule to target the nuclease to the target nucleic acid region. Recombinant gRNA molecules can comprise both the variable DNA recognition and the Cas interaction region and thus be designed specifically depending on the specific target nucleic acid and the desired Cas nuclease. As a further security mechanism, in the nucleic acid target region, PAMs (protospacer adjacent motifs), DNA sequences, which follow DNA directly recognized by the Cas9 / RNA complex, must be present. For example, the PAM sequence for the Cas9 from Streptococcus pyogenes is approximately "NGG" or "NAG" (standard IUPAC nucleotide code) ( Jinek et al., "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity", Science 2012, 337: 816-821 ). The Staphylococcus aureus Cas9 PAM sequence is "NNGRRT" or "NNGRR (N)". Other variant CRISPR / Cas9 systems are known. Thus, a Neisseria meningitidis Cas9 cuts in the PAM sequence NNNNGATT. A Streptococcus thermophilus Cas9 cuts NNAGAAW in the PAM sequence. By using modified Cas polypeptides also targeted single-strand breaks can be achieved. The combined use of Cas nickases with different recombinant gRNAs can also induce highly targeted DNA double strand breaks by double DNA nicking. In addition, the use of two gRNAs can be used to optimize the specificity of DNA binding and thereby DNA cleavage.

The natural repair mechanism of the plant cell repair DNA double strand breakage by either non-homologous end-joining (NHEJ) or homology-directed repair (HDR) or homologous recombination (HR). In addition, so-called Alternative End-joining Pathways (AEJ) have been described in plants ( Charbonnel C, Allain E, Gallego ME, White Cl (2011) Kinetic analysis of DNA double-strand break repair pathways in Arabidopsis. DNA Repair (Amst) 10: 611-619 ).

Unlike the genetic modification of bacterial genomes, modification of complex eukaryotic genomes poses a greater challenge, because of the complexity of these genomes, molecular tools need to be provided which allow for targeted genome modification without undesired off-target effects, i.e., inefficiencies. H. cause undesired mutations or modifications within the genome or non-genomic DNA of the target cell.

Out US Pat. No. 8,697,359 B1 It is known that using the CRISPR / Cas technology, eukaryotic genomes, especially mammalian genomes and these, preferably for therapeutic purposes, can be altered. Here, the expression of a target gene is programmably suppressed by targeted introduction of a Cas9 endonuclease and a guide RNA (gRNA). This gRNA is an essential element of any Cas9 CRISPR system because it directs the actual Cas nuclease targeted to the (genomic) target DNA. For this purpose, a tracr (trans-activating CRISPR RNA) sequence and a tracr mate sequence is disclosed for Cas9-CRISPR systems, which may be encompassed by the gRNA, wherein the tracr sequences hybridize so as to be recognized. However, the use of CRISPR technology for the modification of complex plant genomes as well as the necessary molecular tools are not disclosed.

WO 2015/026885 A1 however, specifically dedicated to the application of CRISPR / Cas technology in plants. However, only one approach and suitable molecular tools are disclosed which need compellingly the subculture, selection and regeneration of plant calli after the introduction of the CRISPR / Cas tools and thus not the direct recovery of a plant or plant material containing the desired DNA. Change allows which / which contains the desired DNA change.

An overview of the current status of the development of the use of CRISPR / Cas technology for the genome editing of plant genomes can be found in Bortesi & Fischer ("The CR / SPR / Cas9 system for genome editing and beyond", Biotechnology Advances, 33, pages 41-52, 20.12.2014) , Among other things, the problem of providing specific gRNAs for targeting in corn is reported here. In addition, the problem of high off-target mutation rates is discussed, which is not only observed in the use of CRISPR / Cas in mammalian cells, but also in plant cells. Here, the design of the individual CRISPR / Cas tools is crucial in order to detect to achieve a targeted targeted change without off-target effects on the respective target cell / target organism. Furthermore, Bortesi & Fischer recognizes that the CRISPR / Cas system is also used for the epigenetic modification of DNA Since the CRISPR / Cas system can also be used to cleave methylated DNA, it notes that no applications in plants are known.

Further describes Guilinger et al. Fok nucleases , which are used like nickases, and thus produce a higher specificity ( Guilinger et al .; Fusion of catalytically inactive Cas9 to Fokl nuclease improves the specificity of genome modification, doi: 10.1038 / nbt.2909 ). Guilinger et al. however, only present data for human cells, not for plant cells.

Mali et al 2013 refers to the use of the CRISPR / Cas system in human cells, wherein here nuclease-null variants of Cas9 or aptamer-coupled gRNAs are used, which may be fused to transcriptional activator domains ( Mali, P., et al. (2013). "CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering ). Mali et al. however, does not mention how and to what extent a corresponding system could be used in plant cells.

In summary, many of the plant-based approaches used today are only temporary and / or spatially limited (transient) in that mutations in already differentiated cells, e.g. In leaves, but these mutations are not transmitted through the germ line. Furthermore, there are approaches that require a stable integration of the coding sequences, including the required regulatory sequences such as promoters and terminators into the genome, over which then a stable mutation can be generated, which is inherited from generation to generation. In this case, however, the CRISPR / Cas tools are still contained in the genome of the plant and thereby also potential plant products such as fruits and seeds, which is undesirable with regard to the risk assessment of the corresponding products.

Therefore, the CRISPR / Cas approach in plant biotechnology is still characterized by low efficiency, but also a difficult and expensive design of the constructs, as well as the frequent occurrence of off-target effects. In addition, many of the current approaches are based on either integrating the CRISPR / Cas tools stably into the genome of a plant cell or introducing them into cells of a differentiated tissue, such as leaves. Consequently, in the stable approach, on the one hand, the individual tools, such as the Cas9, and not only the targeted DNA changes they cause, are inherited to the offspring. When transformed into differentiated cells and tissues, the mutation introduced by the CRISPR / Cas tools will only be effective in the respective cells, but can not be inherited via the germline.

There is thus a continuing need for the establishment of transient and also optionally inducible methods and constructs based u. a. on gRNAs and Cas nucleases or gRNAs and other effector domains, for effecting a desired alteration of a target sequence in a plant target cell, whereby only the alteration of the target sequence, but not the constructs, are transmitted to the progeny. In addition, there is a great need to provide a CRISPR / Cas based method that provides the ability to directly make a germline change in a plant cell or plant tissue so that the mutation is inheritable and on the plant derived from the altered plant cell or plant Tissue results, directly seed can be harvested, which contains the targeted genommodification, without having to go through expensive and costly intermediate steps. Finally, there is a need to selectively extend the RNA-directed DNA modification system provided by the CRISPR / Cas tools, by targeting not only genomic target structures but any nucleic acid targeting not only the genome of a cell but also the cell Cytosol or in plastids, can be modified in a controlled manner.

Summary of the invention

It is therefore an object of the present invention to provide methods and molecular tools which allow the transient transformation of plant tissue or cells, especially meristematic cells, and thereby the controlled alteration of any target nucleic acid region in any plant cellular compartment allow.

The aim was to combine the above advantages in a system that is broadly applicable in plant biotechnology.

This object is achieved by the methods and constructs provided herein according to the subject-matter of the appended claims and further as set forth in detail herein in the specification, figures and attached sequence listing. The object is specifically achieved by providing a method comprising the transformation or transfection of at least one meristematic cell. In addition, the object is achieved by the provision of recombinant constructs comprising specifically modified CRISPR / Cas tools and / or other effector domains. Finally, the object is achieved by the provision of suitable regulatory sequences and localization sequences which allow the recombinant construct of interest to be directed into an arbitrarily controllable compartment of a plant target of interest.

In this way, at least one targeted change in any nucleic acid target region in any compartment of a plant cell, in particular a meristematic plant cell can be achieved. Since the thus altered at least one meristematic cell can directly and directly pass on the targeted change in the nucleic acid target region to its offspring through the subsequent cell division and differentiation and / or if it has the potential for a whole altered plant organism to mature from the meristematic cell, Thus, the provision of a plant or plant material or a plant cell can be achieved without complex further cultivation or crossing and selection steps (especially complex backcrossing regimes) must take. Rather, a plant, plant material or a plant cell can be obtained directly and directly from the at least one so-modified meristematic target cell. This makes it possible to produce gRNA (s) and / or Cas nucleases) and / or other effector domains only transiently in the meristems or to provide or activate, wherein these recombinant macromolecules preferably then, d. H. after gRNA (s) and / or Cas nuclease (s) and / or further effector domain (s) have fulfilled the desired effect, since no integration thereof into the genome or endogenous extrachromosomal DNA takes place, due to regulatory aspects and the risk assessment of the plant product may be beneficial. The Cas nucleases used herein may also contain one or more mutations in the catalytic domain responsible for DNA (single strand) cleavage. This results in a broad spectrum of application of the Cas nuclease and, for instance in the case of Cas-based nickases, a higher specificity of binding since two CRISPR / Cas constructs are used to attach both single strands of DNA double strand at the desired site columns. Cas-null variants are also proposed herein, as well as their combined use with other effector domains to optimize targeted nucleic acid editing.

In addition, it has been found that by exploiting the mechanism of action of the CRISPR / Cas tools also other effector domains such as DNA or RNA or histone-modifying or DNA or RNA or histone-binding polypeptides or nucleic acids, includes, for example, all types of monomeric, dimeric or multimeric nucleases, including nickases, activators and repressors of transcription, phosphatases, glycosylases, or enzymes capable of causing epigenetic changes, such as methylases, acetylases, methyltransferases or histone deacetylases, aptamers comprising single-stranded ones DNA or RNA sequences, as well as peptides, fluorescent proteins, bioluminescence, marker nucleic acid or marker amino acid sequences, and the like, and combinations thereof can be employed according to the methods provided herein, thereby changing the spectrum of known targeted genome editing to a general one Nucleic acid editing that is not is restricted to genomic DNA per se.

The present disclosure thus offers the opportunity to extend the CRISPR / Cas mechanism to include not only nucleolytic cleavage of DNA, but also any modification of genomic DNA, e.g. As the epigenetic change, as well as RNA in plant cells (such as mRNA) is made possible.

In addition, by employing additional regulatory sequences including promoters, terminators, transcription factor binding sites or introns, and / or localization sequences comprising nuclear, mitochondrial and plastid localization sequences, the present disclosure provides the ability to purposely modify any target nucleic acid region of a plant targeting structure which, for example, also allows mitochondrial and plastid DNA to act as the target of editing. In addition, the possibility of targeted modification of RNA, z. MRNA, again exploiting gRNA-directed sequence recognition underlying the CRISPR / Cas system and "reprogramming" it in accordance with the present disclosure to extend the scope of CRISPR / Cas technology.

Also provided herein are methods and constructs wherein gRNA and / or Cas nuclease are already provided linked to another effector domain on a recombinant construct.

Furthermore, a method is provided in which the at least one gRNA and the at least one Cas nuclease and / or the at least one further effector domain are provided separately on different recombinant constructs. According to this method, the gRNA component can be provided as DNA or RNA, the Cas nuclease or variant thereof can be provided as DNA or RNA or as polypeptide sequence and the effector domain can be provided as DNA or RNA or as polypeptide sequence.

In addition, it was an object, by establishing plant-specific constructs and methods, to provide the possibility of providing the constructs in a targeted manner, such as inducible or tissue or organelle-specific, and to minimize undesired off-target effects. Finally, it was an object to design procedures and constructs that not only allow the possibility of efficient gene knock-ins, but also, for example, the very possibility of specific gene knock-outs, of insertions of genetic fragments, of specific epigenetic modifications, of introduction Of point mutations, acetylations, methylations, phosphorylation, glycosylations, markers by resistance markers or fluorescent proteins, activation or repression of transcription, targeted cleavage of double-stranded or single-stranded nucleic acids, the binding of nucleic acids and the like offer, so that a broad field of application in plant breeding is opened. From a breeding as well as a regulatory point of view, it is desirable to enable stable inheritance of a feature caused by the modification for at least one generation in the simultaneous absence of the necessary constructs of the CRISPR / Cas system in the resulting plant or the resulting plant cells.

Concrete aspects and embodiments of the present invention will become apparent from the following detailed description and examples, the drawings, the sequence listing, and more particularly the appended claims.

Brief description of the drawings

1A -F show maize embryos of different sizes. In the embryos analyzed here, the meristem is clearly recognizable as a disk-shaped structure in the center of the embryo. Depending on the size and stage of development of the embryos, the meristem is differently developed and recognizable to different degrees. The meristem is additionally marked by an asterisk (*).

2A and B show a direct comparison of the meristems of a 0.5 mm and a 1 mm maize embryo. In both cases, the meristem can be recognized as a disc-shaped structure in the center of the embryo, but in the 1 mm embryo, the meristem is already very much surrounded by leaf tissue. This complicates the accessibility of the meristem so that smaller embryos with a more exposed meristem are preferable.

3A -D reproduce prepared meristems in maize seedlings. Since the meristem in seedlings is completely surrounded by leaves, it must be dissected free to be accessible for bombardment, microinjection, etc. For this purpose, the outer tissue structures are completely removed so that the meristem (arrows) is exposed.

4A -C show prepared meristems in older corn plants. Since the meristem in older plants, as well as in seedlings, is completely surrounded by leaves, it must be prepared to be accessible for bombardment, microinjection, etc. For this purpose, the outer leaves are completely removed so that the meristem (arrows) is exposed.

5 shows the biolistic test bombardment of maize embryo meristems. On the left is a schematic image of an embryo with the disc-shaped meristem structure (highlighted by a double ring and an arrow). Right shows the fluorescence of RFP after test firing. For the test bombardment a gene coding for an RFP (red fluorescent protein) was used. It is a clear expression of the protein in the meristematic regions (double ring) detectable.

6 illustrates the preparation of "Tassel" meristems on adult corn plants. Through a window-like opening, the meristems (arrow) are uncovered on one side. The recombinant constructs can then z. B. be introduced via bombardment or microinjection. It is advantageous that the plant is not severely damaged and the meristems are not completely exposed (about 1-2 days later, the "Tassel Meristem" is no longer visible through the opening, as it is pushed further upwards) and thereby an oxidation and further damage is reduced.

7 shows the biolistic test bombardment of an uncovered maize "Tassel" -Meristems. On the left is a schematic picture of the meristem tissue of the maize flag ("Tassel"). Right shows the fluorescence of RFP after test firing. For the test bombardment, a gene coding for an RFP (red fluorescent protein) was used. Clear expression of the protein in the meristematic tissues is detectable.

Detailed description

definitions

The term plant or plant cell as used herein refers to plant organisms, plant organs, differentiated and undifferentiated plant tissues, plant cells, seeds and their progeny or progeny. Plant cells include, for. Cells of seeds, mature and immature embryos, meristematic tissues, seedlings, callus tissues, leaves, flowers, roots, shoots, gametophytes, sporophytes, pollen and microspores, protoplasts, macroalgae and microalgae.

Plant material, as used herein, refers to any material that can be recovered from a plant at any stage of development. The term thus includes plant cells, tissues and organs as well as trained plant structures as well as subcellular components such as nucleic acids, polypeptides, and all chemical plant substances that are present within a plant cell and / or can be produced by this.

The term chromosomally or extrachromosomally integrated, as used herein, refers to the transient introduction and / or design of the one or more recombinant constructs according to the present invention, and thus to the subsequent fate of the one or more recombinant constructs / Constructs in a plant target structure, eg. A cell, wherein both the one or more recombinant construct (s) and conditions for delivery thereof are such that no integration of the at least one recombinant construct into the endogenous nucleic acid material of a plant target structure comprising the genome or extrachromosomal Nucleic acids of the plant target structure, eg. As a cell takes place, so that the at least one recombinant construct is not chromosomally or extrachromosomally integrated into the endogenous DNA / RNA of the target cell and thus not passed on to the offspring of the cell. The one or more recombinant construct (s) or its transcription or translation products are thus only transient, d. H. transient, constitutive or inducible, active in the target cell, but are not inheritable to the progeny of the target cell, i. H. they are also not active in the offspring of a target cell.

The term progeny or descendant, as used herein, in the context of a recombinant microorganism, plant or cell according to the present disclosure, refers to the progeny of such organism or cell produced by natural reproductive asexual cell division and differentiation processes from the parent organism or the Origin cell emerge. It is known to those skilled in the art that in the course of cell division and differentiation naturally mutations can be introduced into the genome of an organism, whereby the descendant or offspring genomically differs from the parent organism, but still the same (sub-) Species can be assigned. Also, such naturally-altered progeny which, in addition to the deliberately introduced alteration, carry further alterations in other DNA regions are therefore included within the term progeny or descendant according to the present disclosure.

The term vector or vector system as used herein refers to a means of transport to a recombinant construct comprising nucleic acids or also polypeptides and optionally further sequences such as regulatory sequences or localization sequences, directly or indirectly into a desired target cell or plant target structure in the desired To be able to bring in cellular compartment. The direct introduction takes place directly into a plant target cell or plant target structure, which contains nucleic acids which are to be selectively modified according to the present disclosure. Indirect incorporation involves incorporation into a structure, e.g. B. cells of leaves or other plant organs and tissues, While not directly encompassing the plant target cell of interest, this does not allow the systemic spread and propagation of the vector comprising a recombinant construct according to the present disclosure into the plant target structure, e.g. As meristematic tissue or cells or stem cells is guaranteed. The term vector or vector system as used herein, in the context of transfection of amino acid sequences, includes suitable agents for peptide or protein transfection, such as e.g. As ionic lipid mixtures or agents that are suitable for the transfection of a nucleic acid, such as. B. carrier materials through which nucleic acid and amino acid sequences can be introduced by means of particle bombardment in a cell, such as. As gold and tungsten particles. In addition, this term, especially when using the methods and constructs disclosed herein, also encompasses viral vectors, ie modified viruses, such as those derived from one of the following viruses: Maize Streak Virus (MSV), Barley Stripe Mosaic Virus (BSMV), Maize yellow dwarf virus (MYDV), Maize dwarf mosaic virus (MDMV) and bacterial vectors such as Agrobacterium spp., Such as Example Agrobacterium tumefaciens. Finally, the term also includes suitable means of delivery for introducing linear nucleic acids (single or double stranded) into a target cell. Those of skill in the art, knowing the constructs disclosed herein, are aware of any further sequences which a vector must have in order to be functional in a desired target cell. The customary preparation, processing and application of such vectors is also known to the person skilled in the art.

The term vector system, as used herein, refers to a system consisting of or including at least one or more vector (s). Thus, a vector system may comprise a vector which contains / encodes two different recombinant constructs, including nucleic acid and / or amino acid sequences. A vector system may also contain multiple vectors which in turn contain / encode at least one nucleic acid or amino acid sequence according to the present disclosure.

The term nucleic acid or nucleic acid sequence as used herein refers to natural as well as synthetic deoxyribonucleic acids (DNA) and ribonucleic acids (RNA), which may also contain synthetic nucleotide analogs. The nucleic acids used according to the present invention for the synthesis of a desired product, such as protein or RNA, or for the targeted control thereof, e.g. A Cas nuclease or a gRNA, may optionally be "adapted for use in a plant targeting". In one embodiment, the aforementioned sequences may be codon optimized, i. H. the codon usage of a gene or RNA is specifically adapted to that of the target cell / organism. It is known to those skilled in the art that a desired target gene encoding a protein of interest can be modified without changing the translated protein sequence to account for specific species-dependent codon usage. Thus, the nucleic acids of the present invention are specific to the codon usage of Hordeum vulgare, Sorghum bicolor, Secale cereale, Triticale, Saccharum officinarium, Zea mays, Setaria italic, Oryza sativa, Oryza minuta, Oryza australiensis, Oryza alta, Triticum aestivum , Triticum durum, Hordeum bulbosum, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Malus domestica, Beta vulgaris, Helianthus annuus, Daucus glochidiatus, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Erythranthe guttata, Genlisea aurea, Nicotiana sylvestris, Nicotiana tabacum, Nicotiana tomentosiformis, Solanum lycopersicum, Solanum tuberosum, Coffea canephora, Vitis vinifera, Cucumis sativus, Morus notabilis, Arabidopsis thaliana, Arabidopsis lyrata, Arabidopsis arenosa, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardamine flexuosa, Lepidium virginicum, Capsella bursa-pastoris, Olmarabidopsis pumila, Arabis hirsuta, Brassica napus, Brassica oleracea, Bra ssica rapa, Brassica juncacea, Brassica nigra, Raphanus sativus, Eruca vesicaria sativa, Citrus sinensis, Jatropha curcas, Glycine max, Gossypium ssp. or adapted to Populus trichocarpa. Further, according to the present disclosure, the sequence of the gRNA or the gRNA coding sequence must be adapted to the nucleic acid target region within a plant target structure. In a further embodiment, the gRNA or the sequence coding for the gRNA can additionally be adapted in the region which is responsible for the interaction or coupling with a Cas nuclease and / or an effector domain.

The term sequences as used herein refers to nucleic acid sequences as well as amino acid sequences, wherein the particular sequence may contain as well as natural nucleotides and amino acids also synthetic analogs or synthetic linkages as building blocks.

The terms polypeptide, polypeptide sequence, protein sequence and amino acid sequence are used interchangeably herein.

The term regulatory sequence as used herein refers to a nucleic acid or protein sequence which can direct transcription and / or translation of a disclosed nucleic acid sequence into cis or trans.

The term construct or recombinant construct (used interchangeably herein) as used herein refers to a plasmid vector comprising, among other plasmids, cosmids, yeast or bacterial artificial chromosomes (YACs and BACs), an expression cassette, single stranded or linear nucleic acid sequences or amino acid sequences, which can be introduced into a target cell according to the present disclosure. A recombinant construct according to the invention may contain CRISPR / Cas tools or parts thereof comprising at least one gRNA or at least one Cas nuclease variant and / or at least one further effector domain either in the form of a nucleic acid or an amino acid sequence. Furthermore, the recombinant construct may comprise regulatory sequences and / or localization sequences. The recombinant construct may be integrated into a plasmid vector and / or isolated from a plasmid vector, e.g. In the form of a polypeptide sequence or a non-plasmid vector linked single- or double-stranded nucleic acid. Upon introduction, the construct is preferably extrachromosomal and not integrated into the genome, and usually in the form of a double-stranded or single-stranded DNA, a double-stranded or single-stranded RNA or a polypeptide. Recombinant as used herein means a sequence of nucleic acids or amino acids, such as does not occur naturally, in particular in their entirety.

Whenever the present disclosure refers to sequence homologies or sequence identities of nucleic acid or protein sequences in terms of percentages, these references refer to values as determined using EMBOSS Water Pairwise Sequence Alignments (Nucleotides) ( http://www.ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html ) for Nucleic Acid Sequences or EMBOSS Water Pairwise Sequence Alignments (Protein) ( http://www.ebi.ac.uk/Tools/psa/emboss_water/ ) can be calculated for amino acid sequences. At Local Molecular Aligning tools provided by the European Molecular Biology Laboratory (EMBL), the European Bioinformatics Institute (EBI) uses a modified Smith-Waterman algorithm (see http://www.ebi.ac.uk/Tools/psa/ and Smith, TF & Waterman, MS "Identification of Common Molecular Subsequences" Journal of Molecular Biology, 1981 147 (1): 195-197 ). Furthermore, in carrying out the respective pairwise alignment of two sequences using the modified Smith-Waterman algorithm, reference is hereby made to the default parameters currently specified by the EMBL-EBI. These are (i) for amino acid sequences: matrix = BLOSUM62, gap open penalty = 10 and gap extend penalty = 0.5 and (ii) for nucleic acid sequences: matrix = DNA full, gap open penalty = 10 and gap extend penalty = 0.5.

For the purposes of the present invention, "homologous sequences" or "homologues" or comparable terms are understood as meaning nucleic acid sequences which have the same phylogenetic origin. Preferably, proteins encoded by these nucleic acid sequences perform the same function. Homologous nucleic acid sequences have at least 70%, preferably at least 75%, at least 80%, at least 85% or at least 90%, more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

Nucleic acid target region, as used herein, refers to any genomic and extrachromosomal DNA or RNA, in particular mRNA, a target organism or a target cell, the modification of which is desired and which can be effected by the methods and constructs disclosed herein, and is deliberately not gene Regions, d. H. Regions that carry the information for the transcription of mRNA limited.

Complementary or complementarity, as used herein, describes the relationship between two DNA or RNA nucleic acid regions which, by virtue of their nucleobases, fit together according to the key-and-lock principle and hydrogen-bond with each other. In this case, according to the Watson-Crick base pairing, the bases adenine and thymine / uracil or guanine and cytosine are considered to be complementary. Other non-Watson-Crick pairings, such as Reverse-Watson-Crick, Hoogsteen, Reverse-Hoogsteen, and Wobble-pairing, are also complementary to the term provided that the corresponding base pairs form hydrogen bonds with each other. H. two different nucleic acid strands can hybridize to each other due to their complementarity.

By "hybridizing" or "hybridization" is meant a process in which a single-stranded nucleic acid molecule attaches to a largely complementary strand of nucleic acid, ie, base pairings are received. Standard methods for hybridization are, for example, in Sambrook et al. 2001 described. Preferably, it is understood that at least 60%, more preferably at least 65%, 70%, 75%, 80% or 85%, more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98% or 99% of the bases of the nucleic acid sequence base pair with the largely complementary nucleic acid strand. The possibility of such attachment depends on the stringency of the hybridization conditions. The term "stringency" refers to the hybridization conditions. High stringency is given when base pairing is difficult, low stringency when base pairing is facilitated. The stringency of the hybridization conditions depends, for example, on the salt concentration or ionic strength and the temperature. In general, the stringency can be increased by increasing the temperature and / or lowering the salt content. By "stringent hybridization conditions" are meant those conditions in which a hybridization occurs predominantly only between homologous nucleic acid sequences. The term "hybridization conditions" does not only refer to the conditions prevailing in the actual attachment of the nucleic acids, but also to the conditions prevailing during the subsequent washing steps. Stringent hybridization conditions are, for example, conditions under which predominantly only those nucleic acids which hybridize to at least 70%, preferably at least 75%, at least 80%, at least 85% or at least 90%, particularly preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. Stringent hybridization conditions are, for example: hybridization in 4 x SSC at 65 ° C followed by multiple washes in 0.1 x SSC at 65 ° C for a total of about 1 hour. The term "stringent hybridization conditions" as used herein can also mean hybridization at 68 ° C in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours and then washing twice at 2x SSC and 0.1% SDS at 68 ° C. Preferably, hybridization takes place under stringent conditions.

Detailed description

One aspect of the present invention relates to a method for producing a plant, a plant material or a plant cell, comprising the following steps: (i) providing a plant target structure which comprises at least one meristematic cell, wherein the at least one meristematic cell comprises at least one nucleic acid Target region includes; (ii) providing at least one gRNA or providing one or more recombinant constructs, said recombinant construct (s) comprising at least one gRNA or a sequence encoding a gRNA, and optionally at least one regulatory sequence and / or a localization sequence and providing at least one Cas nuclease and / or an effector domain or providing one or more recombinant constructs / constructs, wherein the recombinant construct (s) comprises at least one Cas nuclease or one for a Cas nuclease Nuclease encoding sequence and / or at least one effector domain or an effector domain coding sequence, and optionally at least one regulatory sequence and / or a localization sequence, wherein the gRNA is capable of both a portion of the nucleic acid Hybridize target region as well as to interact with the Cas nuclease and / or the effector domain; wherein when the gRNA or the gRNA coding sequence and the Cas nuclease or the Cas nuclease coding sequence and / or the effector domain or the coding for an effector domain sequence by one or more recombinant (s) Kontrukt (e ), the gRNA or the gRNA coding sequence and the Cas nuclease or the Cas nuclease coding sequence and / or the effector domain or the coding for an effector domain sequence on or in the same, or on or can be located in different recombinant constructs; optionally: wherein the gRNA or the gRNA coding sequence and / or the Cas nuclease or the Cas nuclease coding sequence and / or the effector domain or an effector domain coding sequence for application in a plant cell out are adjusted; (iii) optionally: providing at least one vector for introducing the recombinant construct (s); (iv) optionally, providing at least one further recombinant construct comprising a recombinant nucleic acid portion for targeted homology-directed repair of the nucleic acid target region in the plant target structure or insertion into the nucleic acid target region in the plant target structure preferably comprising at least one regulatory sequence and optionally at least one further vector for introducing the at least one further recombinant construct; (v) introducing the gRNA, the Cas nuclease and / or the effector domain and / or the recombinant construct (s) into the plant target structure; (vi) cultivating the plant target structure under conditions which include activating the introduced gRNA, Cas nuclease and / or the effector domain and / or introduced recombinant construct (s) and thereby targeting the target nucleic acid region plant target structure to obtain a plant target structure comprising at least one meristematic cell comprising the targeted alteration of the target nucleic acid region; (vii) recovering a plant, plant material or plant cell from the targeted altered at least one meristematic cell, (viii) wherein the Plant, the plant material or the plant cell directly by cell division and differentiation and optionally cross-pollination or self-recovery of the targeted altered at least one meristematic cell, and wherein the recovered plant, the plant material or the plant cell comprises the targeted change of the target nucleic acid region wherein the or the recombinant construct (s) containing at least one gRNA or a sequence coding for a gRNA, and / or at least one Cas nuclease or a sequence coding for a Cas nuclease and / or at least one effector Domain or coding for an effector domain coding sequence include, preferably not chromosomally or extrachromosomally integrated / are.

Meristematic cells belong to a tissue type within a plant, which is referred to as meristem or also forming tissue. Like stem cells in animal organisms, plant-derived meristematic cells, as undifferentiated cells, have the ability to differentiate into any specialized cell type, depending on their environmental impact. Meristems are present in plant organisms not only during embryonic development but throughout the life cycle, so that targeted alteration of meristematic cells and tissues according to the present disclosure is not limited to plant embryos or seedlings, but also in larger seedlings and mature plants, for example Meristems, from which generative plant organs (eg corn in the flag or the piston) emerge, can be performed.

In one embodiment, the meristematic cell is a mature or immature plant cell of a plant embryo or a seedling or a plant comprising at least one meristematic cell or meristematic tissue.

In one embodiment, the meristematic cell is a cell of a monocotyledonous or a dicotyledonous plant.

In accordance with the present invention, therefore, there is provided a specific method which can selectively target, either directly or indirectly, the low population of meristematic cells in a plant at all stages of its development as a plant targeting structure. The at least one meristematic target cell can be controlled directly or indirectly. be, d. H. At least one recombinant construct according to the present disclosure can be introduced directly into the at least one meristematic target cell, or the at least one recombinant construct can be introduced by means of a suitable vector into any plant cell or tissue, wherein the at least one recombinant construct can subsequently be transported to the plant target structure. This is achieved, for example, by the systemic spread of at least one recombinant construct introduced by a vector into a plant cell or into a plant tissue.

A plant targeting structure as used herein comprises at least one plant-derived meristematic cell, which may be tissue, plant matter, whole plant, or isolated cell, wherein the plant-derived meristematic cell also contains at least one target nucleic acid region. The at least one nucleic acid target region contained in the plant target structure comprises DNA and RNA sequences and may be present chromosomally or extrachromosomally within the target structure.

In one aspect of the present invention, which relates to the introduction of targeted nucleic acid alteration into a non-chromosomal target structure, the term plant target structure as used herein includes at least one plant cell, which may be tissue, plant material, whole plant, or isolated cell, wherein the plant cell also contains at least one nucleic acid target region comprising DNA and RNA.

According to one aspect of the present invention, at least one target nucleic acid region in a plant meristematic cell as the target structure is altered by transiently introduced CRISPR / Cas tools and / or optionally other effector domains. Since the thus modified at least one meristematic cell can directly and directly pass on the targeted change in the nucleic acid target region to its offspring by the subsequent cell division and differentiation, the method according to the present invention requires no further crossing and selection steps to obtain a plant, plant material or to provide a plant cell with the desired targeted change. Rather, from the embryonic or secondary meristems, such. As pollen or ovaries, optionally be carried out by performing a self-fertilization or cross-pollination, plant organisms or plant targets, which carry the targeted introduced change.

In one embodiment, the method according to the present invention offers the further advantage that the CRISPR / Cas tools and / or optionally further effector domains are transiently introduced into the plant target structure, preferably a meristematic cell or a meristematic tissue, so that no Stable integration of the CRISPR / Cas tools such as Cas nuclease and gRNA and optionally regulatory sequences and possibly further effector domains into the endogenous chromosomal or endogenous extrachromosomal nucleic acids of the plant target structure takes place.

In accordance with the present disclosure, it has been found that by exploiting the mechanism of action of RNA-directed DNA modification of the CRISPR-Cas tools, other effector domains can also be employed, in accordance with the methods provided herein, thereby broadening the spectrum of targeted genome editing leaves. Either the Cas-nuclease variant or the gRNA or both thereof can be linked to an effector domain.

An effector domain as used herein includes DNA- or RNA-modifying or DNA- or RNA-binding polypeptides or nucleic acids comprising all types of monomeric, dimeric or multimeric nucleases, such as e.g. TALE nucleases, meganucleases, zinc finger nucleases, ribonuclease, deoxyribonucleases, exonucleases, endonucleases and type I, II, III or IV restriction endonucleases and the like, and comprising nickases, transcriptional activators and repressors, phosphatases, glycosylases, or enzymes, which may cause epigenetic changes, such as acetylases, methylases, methyltransferases, proteins capable of binding methylated DNA, or histone deacetylases, aptamers comprising single-stranded DNA or RNA sequences, and peptides, fluorescent proteins, marker nucleic acid or marker amino acid sequences, and the like , and combinations of them. With respect to enzymes or polypeptides in general, the term "effector domain" also includes a catalytic domain or core domain of the particular enzyme or polypeptide, e.g. B. a binding protein, wherein the catalytic domain or core domain is still able to fulfill the enzymatic or the binding function of the respective native enzyme or polypeptide. The design of such truncated domains and their adaptation to the desired function is known to those skilled in the art.

Methods and constructs are provided herein in which gRNA and / or Cas nuclease are already provided linked to another effector domain as or on a recombinant construct.

In a further embodiment, a method is provided in which the at least one gRNA and the at least one Cas nuclease and / or at least one further effector domain are provided separately on different recombinant constructs. According to this method, the gRNA component can be provided as DNA or RNA, the Cas nuclease or variant thereof can be provided as DNA or RNA or as polypeptide sequence, and the effector domain can be provided as DNA or RNA or as polypeptide sequence.

According to one embodiment of the present invention, which comprises the simultaneous introduction of at least one gRNA and at least one Cas nuclease variant together with at least one effector domain, the effector domain with the gRNA or the Cas nuclease variant can be replaced by a nucleic acid variant Amino acid linker linked to one another to ensure an ideal arrangement of the domains and thus their functionality by adequate flexibility of the domains to each other.

Activators and repressors which can be used according to the present invention preferably comprise SEQ ID NOs: 1-4 as well as sequences with at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%. , 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% Sequence homology to these sequences, which despite modification still perform the same function as the sequences with the corresponding SEQ ID NOs ,

Methods for growing and culturing the microorganisms and viruses which can be used as vectors according to the present disclosure are known to those skilled in the art.

In one embodiment, the recombinant construct according to the present invention is introduced into the plant target structure with the aid of at least one vector or a vector system.

In another embodiment, the recombinant construct according to the present invention is introduced directly into the target cell without an additional vector, preferably by mechanical methods, by transfection or by the use of endocytosis.

Also contemplated, according to one embodiment of the present invention, is the introduction of at least one recombinant construct into a plant targeting structure.

Vectors and vector systems of the present invention include those selected from the group consisting of SEQ ID NOs: 12-15 and 25-38, and sequences of at least 66%, 67%, 68%, 69%, 70%, 71%. , 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% Sequence homology to these sequences, which despite modification still perform the same function as the / the unmodified vector or vector system with the corresponding SEQ ID NO. The said vectors and vector systems may comprise, for example, codon-optimized or truncated recombinant constructs, or they may contain targeted point mutations in order to ensure their activity in different target cells. The sequence according to SEQ ID NO: 31 is a hybrid sequence in which the region between the BclI and the BssHII sites of the Fescue segment RNA3 of the bromine mosaic virus (see NCBI: DQ530425) is replaced by the corresponding section of the R_BMV_RNA3_S113'A / G ( Hema & Kao 2004, Journal of Virology ) Fragment was replaced. Further, according to the present disclosure, an Agrobacterium spp. as a vector and may be used alone or in combination with other delivery agents or vectors. According to one embodiment, the above-mentioned vectors and vector systems according to SEQ ID NOs: 12-15 and 25-38 or sequences with the above-mentioned sequence homology thereto are used as scaffold structure to recombinant constructs of the invention comprising at least one gRNA and at least one Cas nuclease and / or to introduce at least one effector domain into a plant target structure. The molecular biological methods and procedures required for this purpose are known to the person skilled in the art.

Recombinant constructs according to the present invention include those selected from the group consisting of SEQ ID NOs: 23 and 24 and sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to these sequences, which, despite modification, still perform the same function as the respective unmodified recombinant construct with the corresponding SEQ ID NO. Said recombinant constructs may comprise, for example, codon-optimized or truncated sequences, or they may contain targeted point mutations to ensure or modify their activity or binding properties in different target cells. In SEQ ID NO: 23, positions 16239-16258 correspond to the position of interest for the particular gRNA, which can and must be modified depending on the target nucleic acid sequence. In SEQ ID NO: 24, positions 16645-16664 correspond to the position of interest for the particular gRNA, which can and must be modified depending on the target nucleic acid sequence.

In one aspect, the present invention relates to methods for producing a plant, a plant material or a plant cell, wherein the recombinant construct is selected from SEQ ID NOs: 23 and 24, as well as sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to these sequences.

In another aspect, the present invention relates to a plant, plant material or plant cell obtainable or obtained by a method comprising introducing a recombinant construct of SEQ ID NOs: 23 and 24, and sequences of at least 66%, 67%. , 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to these sequences into a plant target structure comprising at least one meristematic cell.

In yet another aspect, the present invention relates to the use of at least one recombinant construct of SEQ ID NOs: 23 and 24, and sequences of at least 66%, 67%, 68%, 69%, 70%, 71%, 72 %, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to these sequences, for the targeted alteration of at least one nucleic acid target region in a plant cell.

Suitable for use in the present invention are all Cas polypeptide sequences and Cas-encoding nucleic acid sequences which have been optimized especially for use in a plant cell or their coding constructs carry suitable regulatory sequences, the adequate transcription and / or translation in a plant cell in the appropriate cellular compartment comprising the nucleus, the cytosol, a mitochondrion, a plastid comprising a chloroplast, can effect. Furthermore, in one embodiment, the respective Cas nucleases must have their intrinsic nuclease function.

Alternatively, according to one aspect of the present disclosure, Cas nickases may also be used, i. H. Cas polypeptides that have been altered to cleave only one DNA strand and do not generate DNA double strand breakage like a native Cas nuclease. On the one hand, this offers the possibility of increased specificity, since two recombinant constructs comprising a Cas nickase must be used to achieve a double strand break. On the other hand, it is possible to introduce a deliberately offset double strand break instead of a "blunt" cut.

Finally, in accordance with one aspect of the present disclosure, Cas-zero nucleases, i. H. Variants which no longer have any nuclease activity are used. This opens up the possibility of the Cas nuclease together with another effector domain according to the present disclosure, z. A further DNA- or RNA-modifying or DNA- or RNA-binding polypeptide or nucleic acids, according to the present disclosure, thereby expanding the spectrum of introduction of targeted alterations in a plant target structure.

It is known in the art to introduce targeted mutations into the catalytic domain of a Cas nuclease of interest in order to "reprogram" them into a nickase or Cas null nuclease.

Exemplary Cas nucleases or Cas nuclease encoding sequences for use in the present disclosure are disclosed in SEQ ID NOs 16-22 and as UniProtKB / Swiss-Prot database access Q99ZW2.1 (SEQ ID NO: 39) and also include such sequences with at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% , 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 % Sequence homology to these sequences, which despite modification still perform the same function as the respective unmodified sequences with the corresponding SEQ ID NO or accessible under the said database depository number.

Yet another aspect of the present invention utilizes the RNA-directed DNA modification mechanism of action underlying the CRISPR / Cas system in that an effector domain instead of or together with the Cas nuclease is targeted to a desired position by a targeted gRNA a nucleic acid target region is directed in a plant target structure so that the effector domain can be targeted to effect the desired nucleic acid editing.

In one embodiment of this aspect, the target nucleic acid region is genomic DNA.

In a further embodiment of this aspect, the nucleic acid target region is a mitochondrial or plastid DNA, wherein the recombinant construct comprises a localization sequence which determines the location of the recombinant construct in the corresponding target compartment, e.g. A mitochondrion or a chloroplast.

In one embodiment, the Cas nuclease or the Cas nuclease coding sequence and / or the effector domain or the effector domain coding sequence additionally comprises a sequence selected from a nuclear localization sequence, a plastid localization sequence, preferably a mitochondrial localization sequence and a chloroplast localization sequence.

In a further embodiment, the gRNA or the sequence encoding the gRNA additionally comprises a sequence selected from a nuclear localization sequence, a plastid localization sequence, preferably a mitochondrial localization sequence and a chloroplast localization sequence.

In yet another embodiment of this aspect, the nucleic acid target region is a ribonucleic acid (RNA) in any plant compartment, e.g. B. the cytosol. In accordance with this embodiment, a targeted modified gRNA capable of interacting with an RNA targeting structure may be provided. The gRNA can also be another effector domain, z. An aptamer.

It is known to those skilled in the art that the design of the corresponding at least one gRNA, which is used together with at least one Cas nuclease and / or with at least one effector domain, of the specificity and especially the binding and recognition properties of each used Cas nuclease and / or the effector domain and the nucleic acid target region, which is to be targeted to change dependent.

Wild type Cas nucleases, especially Cas9 type, produce a blunt double strand break in the target DNA sequence, i. H. without single-stranded DNA overhang. As a result, the cell's own DNA repair mechanism of the target cell is activated by the so-called non-homologous repair (non-homologous end joining or NHEJ) induced. However, this mechanism is susceptible to error, so it can lead to the establishment of unwanted mutations at the sites of reunification of the individual DNA strands. By means of NHEJ, single or multiple gene knock-outs can be mediated, whereby the DNA strands are brought together again after targeted DNA breakage by cell-specific mechanisms. Another repair mechanism is homology-directed repair (HDR) or homologous recombination (HR)). This mechanism also serves to reunite DNA strands in DNA lesions. However, unlike NHEJ, HDR requires the presence of a template, i. H. a homologous DNA piece that mediates the alloying of the individual DNA strands. This allows, for example, insertions, gene exchange or targeted assembly of the individual strands without the addition of undesirable, non-controllable mutations to be achieved. Both repair mechanisms, NHEJ and HDR / HR, are naturally occurring DNA repair mechanisms that occur as natural repair mechanisms in every cell disclosed herein.

Critical to the site-directed introduction of alteration of a nucleic acid target region, according to the type II CRISPR / Cas system mechanism set forth above, is the targeted selection and targeted design of the gRNA sequences to target the cleavage of off-target regions other than the target region avoid. The identification of suitable PAM motifs depending on the CRISPR / Cas tools used and optionally further effector domains and the use of this information for the design of suitable recombinant constructs is known to the person skilled in the art.

Therefore, according to one embodiment of the present disclosure, the genome or extrachromosomal nucleic acid target region of a cell is first screened for suitable PAM sequences to specifically design a suitable gRNA.

The term guide RNA or gRNA as used herein refers to a nucleic acid molecule which may consist of natural or synthetic RNA and / or of natural or synthetic DNA and which has the function of being able to form a complex with a Cas nuclease, whereby the Cas nuclease is only able to recognize a nucleic acid target region. In addition, in addition to the Cas nuclease interaction domain, a gRNA has the function of a recognition domain for targeted hybridization to a complementary target nucleic acid molecule of interest. Thus, gRNAs can form an intrinsic hairpin region by complementary base pairing, mimicking the natural tracrRNA / crRNA hairpin structure (see Jinek et al., 2012 above) and also include a suitable recognition domain depending on the desired target sequence.

According to the present disclosure, gRNAs can be used which have been specially adapted for use in a plant cell.

In accordance with the present invention, it has also been found that any desired gRNA, as described herein, can additionally be conjugated to at least one effector domain, such as e.g. As an aptamer, coupled or can be introduced together with the effector domain, so as to expand the functionality of the gRNA. The recombinant construct comprising a gRNA and at least one effector domain can be introduced as a DNA or RNA construct into the plant target structure using a suitable recombinant construct and / or vector. In addition, the effector domain can not only consist of a nucleic acid, but also be present as a polypeptide or a sequence coding therefor.

In one embodiment, the gRNA is coupled to the Cas nuclease and / or the effector domain, e.g. For example, the DNA or RNA modifying or the DNA or RNA binding polypeptide or nucleic acid is introduced into the plant target structure.

In a further embodiment, the gRNA is used as a separate recombinant construct independent of the Cas nuclease and / or the effector domain, e.g. As the DNA- or RNA-modifying or DNA- or RNA-binding polypeptide or nucleic acid introduced into the plant target structure.

The gRNA can be introduced into the plant target structure as a DNA or RNA molecule. Thus, according to one embodiment, the gRNAs may be directly in the form of a synthetic nucleic acid, e.g. B. as RNA, optionally also in complex with a Cas nuclease, or in another embodiment in the form of an activatable and transcribable recombinant DNA construct are introduced into the target cell. Further, according to the present disclosure, a single gRNA may be employed or dual or multiple gRNAs in one or more recombinant construct (s) may be simultaneously introduced into a cell, the gRNAs having the same or individual regulatory sequence (s). feature. Since the interaction domain of a classical CRISPR / Cas gRNA will always interact with the same Cas nuclease, the use of individual gRNAs, which carry a different recognition sequence as a further component, in one approach, a multiplexing, ie the targeted change of multiple target regions in one approach be achieved. It may always be necessary for a PAM sequence to be located adjacent to or within the target region. The design of a suitable gRNA can be determined by those of skill in the art having regard to the Cas nuclease used, the nucleic acid target region, the type of nucleic acid alteration desired, selected from mutation, insertion or deletion, as well as the desired target cell in silico. However, the efficacy of these gRNAs in vivo as well as possible off-target effects must be evaluated separately for each gRNA. In addition, for non-established systems, as in the transient transformation of plant meristem cells, suitable vectors and methods for introducing at least one gRNA together with at least one suitable Cas nuclease and / or at least one effector domain, eg. A DNA / RNA modifying or a DNA / RNA binding polypeptide or nucleic acid, to ensure the concerted activity of both molecules in the target cell.

In one embodiment of the present invention, the methods of the invention, and thereby the plants, plant materials or cells produced thereby, are based on the naturally occurring DNA repair mechanism of the target cell.

In another embodiment of the present disclosure, repair of a single-stranded or double-stranded DNA break previously mediated by a Cas nuclease and / or another effector domain is cured by one or more HDR template (s) does not occur naturally but was introduced into the target cell (s).

In the context of the present disclosure, therefore, in one embodiment, an HDR template is disclosed, which can optionally be introduced into a target cell together with or with a time offset to the CRISPR / Cas constructs and / or a further at least one effector domain in order to target an HDR template. To induce mechanism and thereby insert targeted nucleic acid sequences as an insertion at the site of the double strand break. As a result, both targeted knock-ins and the targeted repair of the DNA lesion can be brought about to prevent an undesired mutation at the site of DNA breakage. A knock-in may include the targeted insertion of at least one nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 500 nucleotides or at least 1000 nucleotides into the target nucleic acid in the plant cell. A knock-in can also mean the targeted exchange of at least one nucleotide for another nucleotide. Further, knock-in may also be achieved by two, three, four or more exchanges or a combination of insertion and replacement. Insertions mean the targeted insertion of at least one nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 500 nucleotides, at least 1000 nucleotides, at least 2000 nucleotides, at least 5000 nucleotides, at least 10000 nucleotides or at least 15000 nucleotides into the target nucleic acid in the plant cell. A nucleic acid sequence as an insertion may be a sequence of a transcription factor binding site, a regulatory sequence, a polypeptide coding sequence, an intron sequence, a sequence encoding a noncoding RNA (eg, IncRNA), an expression cassette, or an RNAi construct. Furthermore, a knock-in can also be brought about by the deletion of sequence segments which disrupt the functionality of a gene (for example, the deletion of transposon insertions). The HDR template may be introduced into the plant target of interest as a single-stranded or double-stranded nucleic acid.

In one embodiment, a target nucleic acid in a plant cell is deliberately switched off (knock-out), ie the transcription and possibly translation of the nucleic acid is prevented. This can be done by the targeted mutation or deletion of a regulatory sequence such as promoter or terminator sequence of a target nucleic acid or by the targeted mutation or deletion of the target nucleic acid itself or parts thereof. Furthermore, a knock-out can also be achieved by targeted mutation or deletion, which change the reading frame of a target nucleic acid, or by targeted mutation or deletion of potential splice signals. In one embodiment, this knock-out occurs without the use of an HDR template, in another embodiment, an HDR template is introduced into the target cell in addition to the CRISPR / Cas constructs and / or a further effector domain. A targeted mutation is, for example, an exchange of at least one nucleotide for another nucleotide, preferably with the consequence that the codon concerned then codes for another amino acid. A targeted nucleic acid in a plant cell which has been deliberately switched off has at least one targeted mutation or deletion, but may also have two, three, four or more targeted mutations and / or deletions.

In yet another embodiment, in addition to the introduction of a targeted alteration, a target nucleic acid sequence of the natural NHEJ mechanism of a plant cell can be deliberately suppressed by addition of a corresponding inhibitor, thereby repressing the NHEJ mechanism of the cell. In one embodiment of the present invention in which the plant target structure is an isolated meristematic cell of a seedling / plant or a plant embryo or an unearthed meristemastic cell of a plant at a later stage of development, the plant target structures comprising the meristematic cells are treated before, during and after the Introducing the at least one recombinant construct according to the present disclosure such that oxidation of the isolated structures is prevented. In one embodiment, this involves the addition of an antioxidant.

Table 1 below lists suitable media for cultivating different plant target structures comprising the most matic cells. Other suitable reaction conditions, such as buffers, additives, temperature and pH conditions, and optionally other necessary additives, may be readily determined by one skilled in the art, having regard to a method and constructs disclosed herein, in accordance with any aspect of the present disclosure. Table 1: Media compositions for cultivating different plant target structures with meristematic cells (MS Medium = Murashige Skoog Medium, MS Salts = Murashige Skoog Salts (Toshio Murashige, Folke Skoog: A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures) In: Physiologia Plantarum, Jg. 15 (1962), Vol. 3, pp. 473-497, ISSN 0031-9317, doi: 10.1111 / j.1399-3054.1962.tb08052.x). embryo embryo Embryo bombardment Embryo bombardment Agro-embryo Agro-embryo Plants meristem exposure MM1 (MM = Maturation Medium) Rewinding Medium 1 MM2 maturation medium 2 MM1OSM (Osmotic) MM1MOD (MOD = modified MM1Tim (Tim = Timentin) MM1ACE Meristem peeling antioxidant MS salts MS medium MS salts MS salts MS salts MS salts MS salts 30.8 g / l 3.4 g / l 30.8 g / l 30.8 g / l 30.8 g / l 30.8 g / l 95 mg / L sucrose sucrose sucrose sucrose sucrose sucrose cysteine 36.4 g / l sorbitol 95 mg / l L-cysteine 150 mg / l timetin 19.62 g / l ACE 100 mg / L ascorbic acid 36.4 g / l mannitol 4.25 mg / l silver nitrate 95 mg L-cysteine 4.25 mg / l L-silver nitrate

The transient introduction of the constructs disclosed herein into meristematic cells or tissue has the advantage of developing reproductive tissue from which the targeted alteration can then be stably relayed to the next generation, the next generation being free of the previously introduced constructs. The methods and constructs disclosed herein further allow to harvest directly on the plant thus modified seed, which carries the stable DNA change, without going as an intermediate step on a cell culture in the form of callus production and regeneration, which also on the selection necessary for this - And Regenrationsschritte and the necessary media and additives can be bypassed.

The methods disclosed herein are capable of producing targeted DNA alterations in both monocot and dicotyledonous plants. Examples of monocots are grasses and cereals such as Hordeum vulgare, Sorghum bicolor, Secale cereale, Triticale, Saccharum officinarium, Zea mays, Setaria italic, Oryza sativa, Oryza minuta, Oryza australiensis, Oryza alta, Triticum aestivum, Traticum durum, Hordeum bulbosum, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii. Examples of dicotyledons are Malus domestica, Beta vulgaris, Helianthus annuus, Daucus glochidiatus, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Erythranthe guttata, Genlisea aurea, Nicotiana sylvestris, Nicotiana tabacum, Nicotiana tomentosiformis, Solanum lycopersicum, Solanum tuberosum, Coffea canephora, Vitis vinifera, Cucumis sativus, Morus notabilis, Arabidopsis thaliana, Arabidopsis lyrata, Arabidopsis arenosa, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardamine flexuosa, Lepidium virginicum, Capselia bursa pastoris, Olmarabidopsis pumila, Arabis hirsuta, Brassica napus, Brassica oleracea, Brassica rapa, Brassica juncacea, Brassica nigra, Raphanus sativus, Eruca vesicaria sativa, Citrus sinensis, Jatropha curcas, Glycine max, Gossypium ssp. or Populus trichocarpa.

In one embodiment, the nucleic acid sequence used to target a nucleic acid target region comprises at least one or more regulatory sequences.

In one embodiment, the nucleic acid sequence which is used as a regulatory sequence for targeting a nucleic acid target region comprises at least one or more promoter (s), optionally a plant- and tissue-specific, a phenotypic, a constitutive or inducible promoter, which is suitable for induction is the transcription in a desired target cell. A promoter is a nucleic acid region involved in the recognition and binding of RNA polymerases as well as other proteins to direct transcription. Suitable promoters for either the gRNA or the Cas nuclease are well known to those skilled in the art. Induction of an inducible promoter may be induced by stimuli such as temperature, chemicals, pH, light, endogenous plant signals, e.g. B. distributed after wounding of the plant, and the like. Exemplary promoters are selected from the group consisting of SEQ ID Nos: 5-11, and also include such sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74 %, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% Sequence homology to these sequences, which despite modification still perform the same function as the respective unmodified sequence with the corresponding SEQ ID NO. Further advantageous promoters are selected from the group consisting of promoters of the Wall-Associated Kinases (WAKs) 1 and 2 (see, for example, US Pat. Wagner TA, Kohorn BD. Wall-Associated Kinases are Expressed throughout Plant Development and Are Required for Cell Expansion. The Plant Cell. 2001; 13 (2): 303-318 as well as NCBI entry: NCBI Reference Sequence: NC_003070.9), a promoter of the SCARECROW1 (scr1) gene (see eg. Tissue Specificity and Evolution of Meristematic WOX3 Function; Rena Shimizu, Jiabing Ji, Eric Kelsey, Kazuhiro Ohtsu, Patrick S. Schnable and Michael J Scanlon Plant Physiology February 2009 vol. 149 no. 2 841-850 and z. NCBI entry: NCBI Reference Sequence: NC_003070.9), a promoter of the FAF2 and FAF4 genes (see eg. The FANTASTIC FOUR Proteins influence shoot meristem size in Arabidopsis thaliana Vanessa Wahl, Luise H Brand, Va-Long Guo, Markus Schmid Wahl et al. BMC Plant Biology 2010, 10: 285 http://www.biomedcentral.com/1471-2229/10/285 as well as NCBI entry: NCBI Reference Sequence: NC_003070.9), a promoter of the OSH1 gene (see eg. Sato et al. (1996) Proc. Natl. Acad. Sci. USA, 93: 8117-8122 as well as Genbank entry: GenBank: CP002688.1 or GenBank: AP008209.2) or a promoter of a metalloprotein gene, e.g. From rice (eg GenBank: BAD87835.1). The promoters of the present invention may be naturally occurring, synthetic or chimeric promoters, or a combination thereof. A preferred promoter according to the present disclosure is a promoter active in a plant meristem cell or a promoter active in plant cell plastids. In one embodiment, the nucleic acid sequence which is used for the targeted modification of a target nucleic acid region also comprises at least one terminator as a regulatory sequence.

In one embodiment, the nucleic acid or amino acid sequence used to target a nucleic acid target region comprising a gRNA and a Cas nuclease, or a sequence coding therefor, comprises at least one or more nuclear localization sequence (s) (KLS) effect the nuclear localization of the nucleic acids and polypeptides used to target a nucleic acid target region.

In one embodiment, the recombinant construct comprises a nucleic acid or amino acid sequence which is used for the targeted alteration of a nucleic acid target region, a gRNA and a Cas nuclease, or a sequence coding for same, one or more plastid localization sequence (s) (PLS), for example, a mitochondrial or chloroplast localization sequence (s) (MLS or CLS) which effects the localization of the nucleic acids and polypeptides used to target a nucleic acid target region in the corresponding plant plastids.

In one embodiment, the nucleic acid sequence encoding a Cas nuclease disclosed herein or a Cas nuclease disclosed herein further comprises a tag sequence. A tag sequence is a nucleic acid or protein portion which may be preceded and / or sequenced by the Cas nuclease or gRNA, or the nucleic acid sequence encoding the Cas nuclease or gRNA, and / or located within the sequence, optionally including their localization, to allow visualization within a target cell. Especially preferred are tag sequences selected from the following list: polyhistidine (His) tag, glutathione S-transferase (GST) tag, thioredoxin tag, FLAG tag, a tag with fluorescence properties selected from a green fluorescent protein (GFP) tag, DsRed tag, mCherry tag and the like, streptavidin or strep tag, maltose binding protein (MBP) tag, chloroplast transit peptide, mitochondrial transit peptide, snap tag, and / or secretion tag.

In an embodiment according to the present invention, the method for producing a plant, a plant material or a plant cell further comprises a screening step. In this step, by methods for analyzing the nucleic acid sequence of a target region, e.g. By polymerase chain reaction or by probes, whether the incorporation, activation, and subsequent reaction of the at least one recombinant construct according to the present disclosure results in the desired targeted alteration of a target nucleic acid region. Methods for performing this screening are known to those skilled in the art for any plant targeting site as well as nucleic acid targeting region.

In accordance with the present disclosure, the vectors and / or recombinant constructs may be used to target a nucleic acid target region in a plant cell by mechanical methods including particle bombardment, microinjection and electroporation, or by induced endocytosis, suitable vectors, direct transfection, and the like be introduced. In one embodiment of the present disclosure, the vectors and / or recombinant constructs are introduced by particle bombardment into the target cell or plant target structure. Here, the vectors are first z. B. on gold or tungsten particles precipitated and the target cell / plant target structure is then bombarded with the thus obtained optionally further processed particles by a suitable apparatus. In another embodiment of the present disclosure, the vectors and / or recombinant constructs are introduced directly or indirectly by microinjection into the target cell or plant target structure. In another embodiment of the present disclosure, the vectors and / or recombinant constructs are obtained by spraying followed by uptake, e.g. As in the course of a viral infection, or infiltration into the target cell or plant target structure introduced.

In one embodiment, the vectors and / or recombinant constructs are introduced by particle bombardment into a meristematic cell. This method is suitable both for introducing recombinant constructs comprising double-stranded plasmid DNA, linear single-stranded or double-stranded DNA, single-stranded or double-stranded RNA, and polypeptides and combinations thereof in all types of plant meristems at different stages of development of a plant. As carrier material for the recombinant constructs, inter alia gold and tungsten can be used.

In a further embodiment according to the present disclosure, the introduction of the vectors according to the invention and / or of the recombinant constructs is carried out by microinjection directly into the target cell or the desired compartment of a target cell.

According to this further embodiment, the vectors and / or recombinant constructs are introduced by microinjection into a meristematic cell. This type of insertion is suitable for all types of meristems (see Example 2 below). In addition, the introduction is suitable according to this embodiment for introducing recombinant constructs comprising double-stranded plasmid DNA, linear single-stranded or double-stranded DNA, single-stranded or double-stranded RNA, and polypeptides, and combinations thereof.

In yet another embodiment according to the present disclosure, the introduction of the vectors of the invention is accomplished by electroporation by high voltage pulses.

In yet another embodiment according to the present disclosure, the introduction of the vectors of the invention is by endocytosis, i. H. a cell-specific mechanism by which non-cellular material can be absorbed into the cell.

In one embodiment, the vector is a viral vector comprising the at least one recombinant construct. The introduction by a viral vector offers the possibility of spreading the vector and comprising at least one recombinant construct. Suitable viral vectors which may be employed or modified for use in accordance with the present disclosure are selected from the group comprising, but not limited to, SEQ ID NOs: 12-15 and 25-38 and also include such sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83% , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to these sequences. It is known to those skilled in the art that the sequence must be adapted to a naturally occurring virus to be used as a viral vector, according to the desired recombinant construct to be introduced and the plant targeting structure of interest.

In another embodiment, the recombinant constructs are amplified by Agrobacterium spp. -mediated transformation, in particular Agrobacterium tumefaciens or Agrobacterium rhizogenes-mediated transformation, introduced into a plant target structure. This mode of introduction is well known to those skilled in the art for different target plants as well as different plant target structures thereof.

The person skilled in the art knows that the choice of the method of introduction u. a. may depend on the particular plant target cell as well as the construct to be introduced, which may require different modes of delivery depending on the target cell.

In a further embodiment according to the present disclosure, the introduction of the vectors according to the invention and / or recombinant constructs is carried out by a combination of the above-mentioned introduction methods. Thus, for example, a viral vector containing the at least one recombinant construct of interest can be introduced into the plant target structure by Agrobacterium tumefaciens as another vector, or by particle bombardment or microinjection, for example.

In one aspect of the present invention, specific methods for introducing vectors and / or recombinant constructs according to the present invention into meristematic plant cells and tissues are disclosed. Their accessibility is crucial for the transformation or transfection of meristematic cells and tissues, whereby the accessibility of the different types of plant meristems in the different stages of development of a plant differ considerably.

In one embodiment of the present invention, therefore, there is disclosed a method for providing a plant target structure comprising at least one meristematic cell, wherein at least one recombinant construct according to the present disclosure can be introduced into the plant target structure. The method according to this embodiment comprises, as a crucial step, making available a meristematic structure of interest comprising not yet differentiated meristematic cells. When the plant target structure is meristems in the embryo, it is essential to select a plant embryo of the correct size and the deeper, ie internal, meristematic cells as the target of transformation or transfection with at least one recombinant construct according to the present invention to drive since the meristematic cells located in the outer layers may have already reached a certain degree of differentiation and thus are no longer suitable according to the present invention. Preferably, the plant embryos are to be sized to be more exposed to a meristem. For maize embryos, this means that embryos of less than 1.5 mm maximum diameter, preferably less than 1 mm maximum diameter, more preferably less than 0.7 mm maximum diameter and most preferably less than 0.5 maximum diameter in can advantageously be used according to the invention. A meristematic cell in the sense of the present invention is thus a meristematic cell whose degree of differentiation still allows for the cells, after targeted alteration of a nucleic acid region of interest, to have all desired types of plant cells, in particular those types from which directly or indirectly A fertile plant can be regenerated, can arise.

In a further embodiment, there is disclosed a method for providing a plant target structure comprising at least one meristematic cell into which at least one recombinant construct according to the present disclosure can be introduced, wherein the meristematic cell is a cell of a seedling or an elder Plant acts.

According to this embodiment, the meristem must be completely or almost completely dissected free. It should also be noted that the lower lying, d. H. in the interior, meristematic cells are targeted as a target of transformation or transfection with at least one recombinant construct according to the present invention, since the located in the outer layers meristematic cells may have reached a certain degree of differentiation and thus no longer according to the present invention are suitable. According to this embodiment, the exposed meristems are subject to oxidation. In order to prevent damage to the meristematic cells, therefore, suitable antioxidant protective measures are preferably used, such. Example, the use of an anti-oxidant or other protective measures to ensure further development of the plant target structure comprising at least one meristematic cell.

Sequence Listing - free text

The following information represents the German translation of the information contained in the Sequence Listing as free text (numerical index <223>), in each case listed for the corresponding sequence index.

Examples

The present invention will be further illustrated by the following non-limiting examples.

Example 1: Preparation of CRISPR / Cas constructs

The construction of the constructs is based on the publication of Mali et al. 2003 , The promoters were targeted to be replaced with specific plant promoters and the gRNA adapted to the respective target genes.

Constructs for monolotyledonous plants:

The promoters include the maize Ubi intron promoter (SEQ ID NO: 7), the maize U3 promoter (SEQ ID NO: 10), the wheat U6 promoter (SEQ ID NO: 8), the rice U3 promoter (SEQ ID NO: 9) and the Brachipodium EF1 promoter (SEQ ID NO: 40). An exemplary construct used has SEQ ID NO: 23 (Vektor1_TaU6 standard).

Constructs for dicotyledonous plants:

Here, the promoters used were a Parsley Ubi4 (SEQ ID NO: 5) and an Arabidopsis U6 promoter (SEQ ID NO: 6). An exemplary construct used has SEQ ID NO: 24 (vector 1_ZmU3 standard).

The different gRNAs were specifically prepared for the respective target genes and cloned into the corresponding position in the vectors given above. The position of the gRNA sequence corresponds to the nucleotides identified as "n" in SEQ ID NOs: 23 and 24.

Example 2: Introduction of CRISPR / Cas Constructs

The constructs described above in Example 1 were introduced into the meristems by different methods. The basis for this is the accessibility of the meristems, which was guaranteed by different methods depending on the material used (see example 4).

The following methods were used:

- Particle bombardment:

Particle bombardment can be used on all meristems used. It is bombarded with plasmid DNA, linear dsDNA, RNA and protein. As carrier material gold and tungsten can be used. Bursts of embryo meristems ( ) and "Tassel" Meristemen ( With the help of the red fluorescent protein it could be proven that it is possible to introduce DNA by particle bombardment into these cells.

- microinjection:

The microinjection can take place in all meristems, preferably using a micromanipulator microscope. However, due to the size of certain meristem structures such as prepared "tassel" and "ear" meristems, microinjection may also be performed under microscopic control. The injection can take place by different methods and as with the particle bombardment (Example 1) indicated with different molecules. On the one hand dsplasmid DNA, linear dsDNA, RNA and protein in liquid solution are injected into the meristematic cells through a micro or nano-cannula, on the other hand dsplasmid-DNA, linear dsDNA, RNA and protein on micro- or nano-needles applied and transferred by puncturing the meristematic cells with the needles.

Example 3: Transient meristematic transformation of maize seedlings and / or embryos / treatment according to the invention of the meristematic tissues

The accessibility of the meristems in each stage differs considerably. So in the embryo ( 1 and 2 ) the meristems are relatively easily accessible, provided one uses embryos of the correct size. It is important to transform the lower-lying cells of the meristems, since the upper cells have already undergone a certain differentiation and are no longer suitable.

Meristems in seedlings and older plants must be prepared completely freely, since they are already surrounded by so many layers of tissue that they are not accessible for bombardment or microinjection. 3 and 4 show the prepared meristems that can be used for the transformation. Again, as with the embryo meristems, the upper cells have already undergone some differentiation and are no longer appropriate. So the cells have to be transformed further inside in the meristems. Since the isolated meristems are exposed and thus subject to high oxidation and consequent death, they were treated with an anti-oxidant to allow further development of the seedling to the plant.

In order to make the "Tassel" Meristems accessible, we have developed a method to harm the plant and the meristems as little as possible. For this purpose, a kind of window is cut through the leaves at the height of the "Tassel" meristems ( 6 ). This ensures that the leaves do not die off and the plant develops normally and that the meristem is protected from the remaining leaves.

• – Die Transfektion findet dann mittels der oben angegebenen Methoden (s. Beispiel 2) statt. Die Embryonen keimen aus und Pflanzen werden kultiviert bis zur Selbstung und Ernte. Vergleichbar bei den Keimlingen und den adulten Pflanzen, nur ohne Keimung.

In addition, the meristem pushes very quickly (within a few hours / days) further up, so that it is then fully protected again. This reduces the likelihood of oxidation and thus a death of the meristem.

• The transfection then takes place by means of the abovementioned methods (see Example 2). The embryos sprout and plants are cultivated until they become self-fertile and harvest. Comparable to seedlings and adult plants, only without germination.

Example 4: Detection of the targeted genetic modification

The proof is possible through different procedures and at different times:
The presence of the desired targeted alteration of a nucleic acid target region can be analyzed in early stages of seedling, the developing plants and the pollen so that one already receives indications of the mutations that have occurred. However, a clear result can only be obtained by analyzing the offspring of selfing, as this provides evidence of an inherited mutation.

Enrichment PCR:

This method is used when the targeted mutation destroys a restriction enzyme site. Digested isolated genomic or extrachromosomal DNA is then digested with the enzyme which cuts at this site to cut wild-type DNA. This is followed by a PCR with primers genomic upstream and downstream of the Restiktionsenzymschnittstelle. In the ideal case, you only get one product if a mutation has taken place and the DNA has not been cut at the site. Since digestion of the genomic or extrachromosomal DNA is usually not 100%, the resulting PCR amplificate is again digested with the enzyme to ensure that a mutation has occurred and the restriction enzyme site has mutated. The undigested fragments are subsequently cloned and sequenced to perform a precise analysis of the mutation. If the nucleic acid target region is an RNA, this can first be rewritten into DNA by methods known to the person skilled in the art, before an enrichment PCR is carried out.

- Sequencing:

If an Enrichment PCR is not possible, the specific region is sequenced via a Next Generation Sequencing (NGS) approach and the resulting sequences are examined for their mutations.

- Sequencing of whole genomes (WGS) for the identification of off-target effects:

In order to rule out that there have been undesired mutations, a WGS of the candidates with the desired mutations is performed.

Additional analyzes are the absence detection of the constructs and viruses used by means of specific PCR and qPCR systems.

Example 5: Viral Vectors

Viruses have the advantage that they can be introduced as finished virus particles, but also as DNA or RNA in a plant target structure. In addition, they offer the possibility of spreading in the cells, if this function was not destroyed by the modification of their RNA / DNA sequence. This has the advantage that on the one hand, one does not have to directly infect the meristem, or it may be sufficient to infect only a few cells and, nevertheless, it spreads to several cell or tissue types. In addition, DNA viruses offer the advantage of providing a large amount of DNA templates for HR.

This is followed by a sequence protocol according to WIPO St. 25. This can be downloaded from the official publication platform of the DPMA.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 8697359 B1 [0009]
• WO 2015/026885 A1 [0010]

Cited non-patent literature

• Jansen, R. et al., "Identification of genes that are associated with DNA repeats in prokaryotes", Mol. Microbiol., 2002, 43 (6), 1565-1575 [0003]
• Barrangou et al. "CRISPR provides acquired resistance against viruses in procaryotes" Science 2007, 315: 1709.1712 [0003]
• Van der Oost et al. "Unraveling the structural and mechanistic basis of CRISPR-Cas systems" Nature 2014, 482: 331-338 [0004]
• Jinek et al., "A programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity", Science 2012, 337: 816-821 [0006]
• Charbonnel C, Allain E, Gallego ME, White Cl (2011) Kinetic analysis of DNA double-strand break repair pathways in Arabidopsis. DNA Repair (Amst) 10: 611-619 [0007]
• Bortesi & Fischer ("The CR / SPR / Cas9 system for genome editing and beyond", Biotechnology Advances, 33, pp. 41-52, 20.12.2014) [0011]
• Guilinger et al. Fokl nucleases [0012]
• Guilinger et al .; Fusion of catalytically inactive Cas9 to Fokl nuclease improves the specificity of genome modification, doi: 10.1038 / nbt.2909 [0012]
• Guilinger et al. [0012]
• Mali et al 2013 [0013]
• Mali, P., et al. (2013). CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering [0013]
• Mali et al. [0013]
• http://www.ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html [0046]
• http://www.ebi.ac.uk/Tools/psa/emboss_water/ [0046]
• http://www.ebi.ac.uk/Tools/psa/ [0046]
• Smith, TF & Waterman, MS "Identification of Common Molecular Subsequences" Journal of Molecular Biology, 1981 147 (1): 195-197 [0046]
• Sambrook et al. 2001 [0050]
• Hema & Kao 2004, Journal of Virology [0070]
• Jinek et al., 2012 [0090]
• Wagner TA, Kohorn BD. Wall-Associated Kinases are Expressed throughout Plant Development and Are Required for Cell Expansion. The Plant Cell. 2001; 13 (2): 303-318 [0105]
• Tissue Specificity and Evolution of Meristematic WOX3 Function; Rena Shimizu, Jiabing Ji, Eric Kelsey, Kazuhiro Ohtsu, Patrick S. Schnable and Michael J Scanlon Plant Physiology February 2009 vol. 149 no. 2 841-850 [0105]
• The FANTASTIC FOUR Proteins influence shoot meristem size in Arabidopsis thaliana Vanessa Wahl, Luise H Brand, Va-Long Guo, Markus Schmid Wahl et al. BMC Plant Biology 2010, 10: 285 http://www.biomedcentral.com/1471-2229/10/285 [0105]
• Sato et al. (1996) Proc. Natl. Acad. Sci. USA, 93: 8117-8122 [0105]
• Mali et al. 2003 [0126]

Claims (15)

A process for producing a plant, a plant material or a plant cell, comprising the following steps: (i) providing a plant target structure, which comprises at least one meristematic cell, wherein the at least one meristematic cell comprises at least one target nucleic acid region; (ii) providing at least one gRNA or providing one or more recombinant constructs / constructs, wherein the recombinant construct (s) (a) at least one gRNA or a sequence coding for a gRNA, and (b) optionally comprising at least one regulatory sequence and / or a localization sequence, and providing at least one Cas nuclease and / or effector domain or providing one or more recombinant constructs / constructs, wherein the recombinant construct (s) (a) at least one Cas nuclease or a sequence coding for a Cas nuclease and / or at least one effector domain or an effector domain coding sequence, and (b) optionally comprising at least one regulatory sequence and / or a localization sequence, wherein the gRNA is capable of both hybridizing to a portion of the nucleic acid target region and interacting with the Cas nuclease and / or the effector domain; when the gRNA or the gRNA coding sequence and the Cas nuclease or the Cas nuclease coding sequence and / or the effector domain or the coding for an effector domain sequence are provided by one or more recombinant constructs / constructs, the gRNA or the gRNA coding sequence and the Cas nuclease or the Cas nuclease coding sequence and / or the effector domain or the effector domain coding sequence on or in the same, or on or in different recombinant constructs are localized; (iii) introducing the gRNA, the Cas nuclease and / or the effector domain or the recombinant construct (s) into the plant target structure; (iv) cultivating the plant target structure under conditions involving activation of the introduced gRNA, Cas nuclease and / or the effector domain or introduced recombinant construct (s) and thereby targeting the target nucleic acid target region to obtain a plant target structure comprising at least one meristematic cell comprising targeted alteration of the target nucleic acid region; (v) obtaining a plant, a plant material or a plant cell from the specifically altered at least one meristematic cell, wherein the plant, the plant material or the plant cell is obtained directly by cell division and differentiation and optionally cross-fertilization or selfing from the specifically modified at least one meristematic cell, and wherein the recovered plant, the plant material or the plant cell comprises the targeted alteration of the nucleic acid target region. The method of claim 1, wherein in the plant, plant material or plant cell of step (v), the recombinant construct (s) which (a) at least one gRNA or a sequence coding for a gRNA, or (b) at least one Cas nuclease or a sequence coding for a Cas nuclease and / or at least one effector domain or an effector domain coding sequence include, are not chromosomally or extrachromosomally integrated / are. A method according to claim 1 or 2, wherein in step (ii) the gRNA or the gRNA coding sequence and / or the Cas nuclease or the Cas nuclease coding sequence and / or the effector domain or an effector domain coding sequence adapted for use in a plant cell. A method according to any one of claims 1 to 3, wherein between step (ii) and (iii) there is provided at least one vector for introducing the recombinant construct (s). The method of any one of the preceding claims, wherein between steps (ii) and (iii), providing at least one further recombinant construct comprising a recombinant nucleic acid portion for targeted homology-directed repair of the target nucleic acid region in the plant Target structure or insertion into the nucleic acid target region in the plant target structure and optionally at least one further vector for introducing the at least one further recombinant construct takes place. A method according to any one of the preceding claims wherein the meristematic cell is a mature or immature plant cell of a plant embryo or a seedling or a plant comprising at least one meristematic cell or meristematic tissue. A method according to any one of claims 1 or 2, wherein the meristematic cell is a cell of a monocotyledonous or a dicotyledonous plant. A method according to any one of the preceding claims, wherein said at least one vector is selected from the group consisting of Agrobacterium spp., A virus selected from the group consisting of SEQ ID NOs: 12-15 and 25-38, and sequences of at least 66%. , 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to those sequences, or to an agent suitable for transfecting a peptide or polypeptide sequence or nucleic acid sequences, or a combination thereof. Method according to one of the preceding claims, wherein the gRNA is introduced directly as natural or synthetic nucleic acid and / or the Cas nuclease directly as a polypeptide and / or the effector domain directly as a nucleic acid or polypeptide in the plant target structure. Method according to one of the preceding claims, wherein the gRNA or the gRNA coding sequence or the Cas nuclease or the Cas nuclease coding sequence or the effector domain or the effector domain coding sequence additionally comprises a localization sequence selected from a Nuclear localization sequence, a plastid localization sequence, preferably a mitochondrial localization sequence and a chloroplast localization sequence. Method according to one of the preceding claims, wherein additionally an inhibitor of endogenous non-homologous end-joining (NHEJ) repair mechanism is introduced into the plant target structure. Method according to one of the preceding claims, wherein the recombinant construct is selected from SEQ ID NOs: 23 and 24, as well as sequences with at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to these sequences. Plant, plant material or plant cell, obtainable or obtained by a method according to any one of the preceding claims. A recombinant construct comprising a nucleic acid selected from SEQ ID NOs: 23 and 24, and sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76 %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the aforementioned sequences. Use of at least one recombinant construct according to claim 14 or a vector comprising a nucleic acid selected from the group consisting of SEQ ID NOS: 12-15 and 25-38, as well as sequences with at least 66%, 67%, 68%, 69%, 70 %, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to these sequences for targeted alteration of at least one nucleic acid target region in a plant target structure comprising at least one meristematic cell, wherein the at least one meristematic cell comprises at least one nucleic acid target region.

Images