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WO2018202199A1 - Procédés pour isoler des cellules sans utiliser de séquences de marqueurs transgéniques - Google Patents

Procédés pour isoler des cellules sans utiliser de séquences de marqueurs transgéniques Download PDF

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WO2018202199A1
WO2018202199A1 PCT/CN2018/085829 CN2018085829W WO2018202199A1 WO 2018202199 A1 WO2018202199 A1 WO 2018202199A1 CN 2018085829 W CN2018085829 W CN 2018085829W WO 2018202199 A1 WO2018202199 A1 WO 2018202199A1
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Prior art keywords
plant
tolerance
targeted
modification
resistance
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PCT/CN2018/085829
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Inventor
Caixia Gao
Rui Zhang
Jinxing LIU
Aaron HUMMEL
Zarir Vaghchhipawala
Mathias LABS
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Institute of Genetics and Developmental Biology of CAS
KWS SAAT SE and Co KGaA
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Institute of Genetics and Developmental Biology of CAS
KWS SAAT SE and Co KGaA
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Application filed by Institute of Genetics and Developmental Biology of CAS, KWS SAAT SE and Co KGaA filed Critical Institute of Genetics and Developmental Biology of CAS
Priority to AU2018263195A priority Critical patent/AU2018263195B2/en
Priority to US16/610,782 priority patent/US20210230616A1/en
Priority to JP2019560164A priority patent/JP2020518253A/ja
Priority to KR1020227033631A priority patent/KR20220137166A/ko
Priority to EA201992615A priority patent/EA201992615A1/ru
Priority to KR1020197036090A priority patent/KR20200004382A/ko
Priority to CA3062475A priority patent/CA3062475A1/fr
Priority to EP18794967.2A priority patent/EP3635118A4/fr
Priority to BR112019022737A priority patent/BR112019022737A2/pt
Publication of WO2018202199A1 publication Critical patent/WO2018202199A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety

Definitions

  • the present invention relates to methods for targeted editing in a plant, a plant cell or material, which is combined with the parallel introduction of a phenotypically selectable trait. Furthermore, methods are provided not comprising a step of introducing a transgenic selection marker sequence. The methods comprise introducing a targeted modification at a first genomic target site to obtain a selectable phenotype which does not rely on the provision of an exogenous polynucleotide template, nor does it rely on the introduction of a double-stand break at the target site.
  • the invention relates to the combination of specific method steps parallelizing transgenic marker-free selection and targeted editing at different genomic target site resulting in conferring a selectable or other phenotype enabling the isolation of plant material without a selection marker cassette to allow precision breeding comprising significantly reduced selection efforts for identifying a genotype of interest.
  • SSNs site-specific nucleases
  • Plant breeding and developments in agricultural technology such as agrochemicals has/have made remarkable progress in increasing crop yields for over a century.
  • plant breeders must constantly respond to many changes. Agricultural practices change, which creates the need for developing plants with genotypes carrying specific agronomic characteristics.
  • target environments and the organisms within them are constantly changing. For example, fungal and insect pests continually evolve and overcome resistance of a plant of interest. New land areas are regularly being used for farming, exposing plants to altered growing conditions.
  • consumer preferences and requirements change. Plant breeders therefore face the endless task of continually developing new crop varieties (Collard and Mackill, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2008 Feb 12; 363 (1491) : 557–572) .
  • MAS marker-assisted selection
  • DNA marker technology can dramatically enhance the efficiency of plant breeding by allowing selection on the basis of easy to assay markers, instead of determining phenotypical traits.
  • development of such markers with diagnostic or screening properties and the effectiveness of applying these markers is often a laborious and time consuming process as detailed above.
  • methods for detecting point mutations e.g. SNPs, only can identify a limited number of such point mutations and detect a limited repertoire (Slade et al., Nat. Biotech. 23, 75-81) .
  • selectable marker genes play an important role in plant for transgenic and transplastomic plant research or crop development. Selectable marker genes are often used in combination with reporter genes, which reporter genes do not provide a cell with a selective advantage, but which reporter genes can be used to monitor transgenic events, or to manually separate transgenic material from non-transformed material.
  • Transgenic selection marker genes can thus increase the efficiency of recovering plants regenerated from treated cells, but the introduction of transgenic sequencing into the plant genome is not always desirable. Furthermore, the elimination of transgenic marker genes after selection has been achieved is often very complicated.
  • Precision gene editing or genome engineering has evolved as one of the most important areas of genetic engineering allowing the targeted and site-directed manipulation of a genome of interest over the last years.
  • An indispensable prerequisite for site-directed genome engineering are programmable nucleases, which can be used to break a nucleic acid of interest at a defined position to induce either a double-strand break (DSB) or one or more single-strand breaks.
  • said nucleases can be chimeric or mutated variants, no longer comprising a nuclease function, but rather operating as recognition molecules in combination with another enzyme. Those nucleases or variants thereof are thus key to any gene editing or genome engineering approach.
  • nucleases especially tailored endonucleases comprising meganucleases, zinc finger nucleases, TALE nucleases, Argonaute nucleases, derived, for example, from Natronobacterium gregoryi, and CRISPR nucleases, comprising, for example, Cas, Cpf1, CasX or CasY nucleases as part of the Clustered Regularly lnterspaced Short Palindromic Repeats (CRISPR) system.
  • CRISPR Clustered Regularly lnterspaced Short Palindromic Repeats
  • CRISPRs Clustered Regularly lnterspaced Short Palindromic Repeats
  • the CRISPR system fulfils the role of an adaptive immune system to defend against viral attack.
  • short segments of viral DNA are integrated into the CRISPR locus.
  • RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. That RNA, which contains sequence complementary to the viral genome, mediates targeting of a CRISPR effector protein to a target sequence in the viral genome.
  • the CRISPR effector protein cleaves and thereby interferes with replication of the viral target.
  • the CRISPR system has successfully been adapted for gene editing or genome engineering also in eukaryotic cells. Editing in animal cells and therapeutic applications for human beings are presently of significant research emphasis. The targeted modification of complex animal and also plant genomes still represents a demanding task.
  • a CRISPR system in its natural environment describes a molecular complex comprising at least one small and individual non-coding RNA in combination with a Cas nuclease or another CRISPR nuclease like a Cpf1 nuclease (Zetsche et al., "Cpf1 Is a Single RNA-Guides Endonuclease of a Class 2 CRISPR-Cas System” , Cell, 163, pp. 1-13, October 2015) which can produce a specific DNA double-stranded break.
  • CRISPR systems are categorized into 2 classes comprising five types of CRISPR systems, the type II system, for instance, using Cas9 as effector and the type V system using Cpf1 as effector molecule (Makarova et al., Nature Rev. Microbial., 2015) .
  • a synthetic non-coding RNA and a CRISPR nuclease and/or optionally a modified CRISPR nuclease, modified to act as nickase or lacking any nuclease function can be used in combination with at least one synthetic or artificial guide RNA or gRNA combining the function of a crRNA and/or a tracrRNA (Makarova et al., 2015, supra) .
  • CRISPR-RNA CRISPR-RNA
  • crRNA CRISPR-RNA
  • the maturation of this guiding RNA which controls the specific activation of the CRISPR nuclease, varies significantly between the various CRISPR systems which have been characterized so far.
  • the invading DNA also known as a spacer, is integrated between two adjacent repeat regions at the proximal end of the CRISPR locus.
  • Type II CRISPR systems code for a Cas9 nuclease as key enzyme for the interference step, which systems contain both a crRNA and also a trans-activating RNA (tracrRNA) as the guide motif.
  • RNAse llI double-stranded RNA regions which are recognized by RNAse llI and can be cleaved in order to form mature crRNAs. These then in turn associate with the Cas molecule in order to direct the nuclease specifically to the target nucleic acid region.
  • Recombinant gRNA molecules can comprise both, the variable DNA recognition region and also the Cas interaction region, and can be specifically designed, independently of the specific target nucleic acid and the desired Cas nuclease.
  • PAMs protospacer adjacent motifs
  • the PAM sequence for the Cas9 from Streptococcus pyogenes has been described to be "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 PAM sequence for Cas9 from Staphylococcus aureus is "NNGRRT” or “NNGRR (N) " .
  • Further variant CRISPR/Cas9 systems are known.
  • a Neisseria meningitidis Cas9 cleaves at the PAM sequence NNNNGATT.
  • a Streptococcus thermophilus Cas9 cleaves at the PAM sequence NNAGAAW.
  • a further PAM motif NNNNRYAC has been described for a CRISPR system of Campylobacter (WO 2016/021973 A1) .
  • Cpf1 nucleases it has been described that the Cpf1-crRNA complex efficiently cleaves target DNA proceeded by a short T-rich PAM in contrast to the commonly G-rich PAMs recognized by Cas9 systems (Zetsche et al., supra) .
  • modified CRISPR polypeptides specific single-stranded breaks can be obtained.
  • the combined use of Cas nickases with various recombinant gRNAs can also induce highly specific DNA double-stranded breaks by means of double DNA nicking.
  • the specificity of the DNA binding and thus the DNA cleavage can be optimized.
  • Synthetic CRISPR systems consisting of two components, a guide RNA (gRNA) also called single guide RNA (sgRNA) and a non-specific CRISPR-associated endonuclease can be used to generate knock-out cells or animals by co-expressing a gRNA specific to the gene to be targeted and capable of association with the endonuclease Cas9.
  • gRNA guide RNA
  • sgRNA single guide RNA
  • non-specific CRISPR-associated endonuclease can be used to generate knock-out cells or animals by co-expressing a gRNA specific to the gene to be targeted and capable of association with the endonuclease Cas9.
  • the gRNA is an artificial molecule comprising one domain interacting with the Cas or any other CRISPR effector protein or a variant or catalytically active fragment thereof and another domain interacting with the target nucleic acid of interest and thus representing a synthetic fusion of crRNA and tracrRNA ( "single guide RNA” (sgRNA) or simply "gRNA” ; Jinek et al., 2012, supra) .
  • the genomic target can be any ⁇ 20 nucleotide DNA sequence, provided that the target is present immediately upstream of a PAM.
  • the PAM sequence is of outstanding importance for target binding and the exact sequence is dependent upon the species of Cas9 and, for example, reads 5'NGG 3'or 5'NAG 3' (Standard IUPAC nucleotide code) (Jinek et al., 2012, supra) for a Streptococcus pyogenes derived Cas9.
  • modified Cas nucleases targeted single strand breaks can be introduced into a target sequence of interest.
  • the combined use of such a Cas nickase with different recombinant gRNAs highly site specific DNA double strand breaks can be introduced using a double nicking system. Using one or more gRNAs can further increase the overall specificity and reduce off-target effects.
  • the Cas9 protein and the gRNA form a ribonucleoprotein complex through interactions between the gRNA "scaffold" domain and surface-exposed positively-charged grooves on Cas9.
  • the "spacer” sequence of the gRNA remains free to interact with target DNA.
  • the Cas9-gRNA complex will bind any genomic sequence with a PAM, but the extent to which the gRNA spacer matches the target DNA determines whether Cas9 will cut.
  • a "seed" sequence at the 3'end of the gRNA targeting sequence begins to anneal to the target DNA. If the seed and target DNA sequences match, the gRNA will continue to anneal to the target DNA in a 3'to 5'direction (relative to the polarity of the gRNA) .
  • the Type V system together with the Type II system belongs to the Class 2 CRISPR systems (Makarova and Koonin Methods. Mol. Biol., 2015, 1311: 47-753) .
  • the Cpf1 effector protein is a large protein (about 1,300 amino acids) that contains a RuvC like nuclease domain homologous to the corresponding domain of Cas9 along with a counterpart to the characteristic arginine-rich cluster of Cas9.
  • Cpf1 lacks the HNH nuclease domain that is present in all Cas9 proteins, and the RuvC-like domain is contiguous in the Cpf1 sequence, in contrast to Cas9 where it contains long inserts including the HNH domain (Chylinski, 2014; Makarova, 2015) .
  • Cpf1 effectors possess certain differences over Cas9 effectors, namely no requirement of additional trans-activating crRNAs (tracrRNA) for CRISPR array processing, efficient cleavage of target DNA by short T-rich PAMs (in contrast to Cas9, where the PAM is followed by a G-rich sequence) , and the introduction of staggered DNA double strand breaks by Cpf1.
  • the CRISPR systems per se lack the inherent capacity to create a point mutation at a desired position in a genome of interest in a target cell.
  • Genome engineering tools like CRISPR systems introducing a double-strand break (DSB) require a DSB repair mechanism. Said mechanisms have been divided into two major basic types, non-homologous end joining (NHEJ) and homologous recombination (HR) . Homology based repair mechanisms in general are usually called homology-directed repair (HOR) .
  • NHEJ non-homologous end joining
  • HR homologous recombination
  • HOR homology-directed repair
  • NHEJ is the dominant nuclear response in animals and plants which does not require homologous sequences, but is often error-prone and thus potentially mutagenic (Wyman C., Kanaar R. "DNA double-strand break repair: all's well that ends well” , Annu. Rev. Genet. 2006; 40, 363-83) .
  • Repair by HOR requires homology, but those HOR pathways that use an intact chromosome to repair the broken one, i.e., double-strand break repair and synthesis-dependent strand annealing, are highly accurate. In the classical DSB repair pathway, the 3'ends invade an intact homologous template then serve as a primer for DNA repair synthesis, ultimately leading to the formation of double Holliday junctions (dHJs) .
  • dHJs double Holliday junctions
  • dHJs are four-stranded branched structures that form when elongation of the invasive strand "captures" and synthesizes DNA from the second DSB end.
  • the individual HJs are resolved via cleavage in one of two ways. Synthesis-dependent strand annealing is conservative, and results exclusively in non-crossover events. This means that all newly synthesized sequences are present on the same molecule. Unlike the NHEJ repair pathway, following strand invasion and D loop formation in synthesis-dependent strand annealing, the newly synthesized portion of the invasive strand is displaced from the template and returned to the processed end of the non-invading strand at the other DSB end.
  • the 3'end of the non-invasive strand is elongated and ligated to fill the gap.
  • a central feature of this pathway is the presence of only one invasive end at a DSB that can be used for repair.
  • SSNs site-specific nucleases
  • GE genome engineering
  • a targeted modification is made at a first gene to confer a selectable or other phenotype on the cell and its progeny refraining from introducing a transgenic selectable marker sequence.
  • a targeted modification is made at a second gene of interest that may or usually may not confer a phenotype on the cell.
  • the cell and its progeny cells or plants can be isolated or regenerated from a background of untreated cells by applying a selection agent or other method that uses the phenotype conferred by the modification at the first gene to identify the cells that have undergone this first gene modification.
  • Cells or plants with the targeted modification at the second gene of interest, which second modification represents the actual aim to be achieved, are identified from this population to provide faster and thus cheaper selection without the need of a transgenic selectable marker sequence present, or to be introduced, in a genome of interest.
  • a method for isolating at least one modified plant cell or at least one modified plant tissue, organ, or whole plant comprising the at least one modified plant cell, without stably integrating a transgenic selectable marker sequence comprising: (a) introducing at least one first targeted base modification into a first plant genomic target site of at least one plant cell to be modified, wherein the at least one targeted base modification causes expression of at least one phenotypically selectable trait; (b) introducing at least one second targeted modification into a second plant genomic target site of the at least one plant cell to be modified, wherein the at least one second targeted modification is introduced using at least one of a site-specific effector to create the at least one second targeted modification at the second plant genomic target site, wherein the at least one second targeted modification is introduced simultaneously or subsequently to the introduction of the at least one first targeted base modification into the same at least one plant cell to be modified, or into at least one progeny cell, tissue, organ, or plant thereof
  • step (b) additionally comprises introducing a repair template to make a targeted sequence conversion or replacement at the at least second plant genomic target site.
  • the method according to the first aspect comprises a further step of (d) crossing at least one modified plant or plant material comprising the at least one first and the at least one second targeted modification with a further plant or plant material of interest to segregate the resulting progeny plants or plant material to achieve a genotype of interest, optionally wherein the genotype of interest does not comprise the at least one first targeted modification.
  • the at least one site-specific effector is temporarily or permanently linked to at least one base editing complex, wherein the base editing complex mediates the at least one first targeted base modification of step (a) .
  • the at least one site-specific effector is selected from at least one of a nuclease, comprising a CRISPR nuclease, including Cas or Cpf1 nucleases, a TALEN, a ZFN, a meganuclease, an Argonaute nuclease, a restriction endonuclease, including FokI or a variant thereof, a recombinase, or two site-specific nicking endonucleases, or a base editor, or any variant or catalytically active fragment of the aforementioned effectors.
  • a nuclease comprising a CRISPR nuclease, including Cas or Cpf1 nucleases, a TALEN, a ZFN, a meganuclease, an Argonaute nuclease, a restriction endonuclease, including FokI or a variant thereof, a recombinase, or two site
  • the at least one site-specific effector is a CRISPR-based nuclease, wherein the CRISPR-based nuclease comprises a site-specific DNA binding domain directing the at least one base editing complex, wherein the at least one CRISPR-based nuclease, or the nucleic acid sequence encoding the same, is selected from the group comprising (a) Cas9, including SpCas9, SaCas9, SaKKH-Cas9, VQR-Cas9, St1Cas9, (b) Cpf1, including AsCpf1, LbCpf1, FnCpf1, (c) CasX, or (d) CasY, or any variant or derivative of the aforementioned CRISPR-based nucleases, preferably wherein the at least one CRISPR-based nuclease comprises a mutation in comparison to the respective wild-type sequence so that the resulting CRISPR-based nuclease is converted to a
  • the at least one first targeted base modification according to the first aspect is made by at least one base editing complex comprising at least one base editor as component.
  • the base editing complex comprises at least one cytidine deaminase, or a catalytically active fragment thereof.
  • the at least one first targeted base modification is a conversion of any nucleotide C, A, T, or G, to any other nucleotide.
  • the base editing complex contains at least one of an APOBEC1 component, an UGI component, a XTEN component, or a PmCDA1 component.
  • the at least one base editing complex comprises more than one component, and the at least two components are physically linked.
  • the at least one base editing complex comprises more than one component, and the at least two components are provided as individual components.
  • the at least one component of the at least one base editing complex comprises at least one organelle localization signal to target the at least one base editing complex to a subcellular organelle.
  • the at least one organelle localization signal is a nuclear localization signal (NLS)
  • the at least one organelle localization signal is a chloroplast transit peptide.
  • the at least one organelle localization signal is a mitochondria transit peptide.
  • the first plant genomic target site of the at least one plant cell is a genomic target site encoding at least one phenotypically selectable trait, wherein the at least one phenotypically selectable trait is a resistance/tolerance trait or a growth advantage trait, and wherein the at least one first targeted base modification at the first plant genomic target site of the at least one plant cell confers resistance/tolerance or a growth advantage towards a compound or trigger to be added to the at least one modified plant cell, tissue or plant, or a progeny thereof.
  • the at least one phenotypically selectable trait of interest is or is encoded by at least one endogenous gene, or the at least one phenotypic trait of interest is or is encoded by at least one transgene, wherein the at least one endogenous gene or the at least one transgene encode (s) at least one phenotypic trait selected from the group consisting of resistance/tolerance to a phytotoxin, preferably a herbicide, inhibiting, damaging or killing cells lacking the at least one modification at the at least one phenotypic trait of interest, or wherein the at least one phenotypic trait is selected from the group consisting of boosters of cell division, growth rate, embryogenesis, or another phenotypically selectable property that provides an advantage to a modified cell, tissue, organ, or plant compared to an unmodified cell, tissue, organ, or plant.
  • the at least one first plant genomic target site is at least one endogenous gene or a transgene encoding at least one phenotypically selectable trait selected from the group consisting of herbicide resistance/tolerance, wherein the herbicide resistance/tolerance is selected from the group consisting of resistance/tolerance to EPSPS-inhibitors, including glyphosate, resistance/tolerance to glutamine synthesis inhibitors, including glufosinate, resistance/tolerance to ALS-or AHAS-inhibitors, including imidazoline or sulfonylurea, resistance/tolerance to ACCase inhibitors, including aryloxyphenoxypropionate (FOP) , resistance/tolerance to carotenoid biosynthesis inhibitors, including inhibitors of carotenoid biosynthesis at the phytoene desaturase step, inhibitors of 4-hydroxyphenyl-pyruvate-dioxygenase (HPPD) , or inhibitors of other carotenoid biosynthesis targets
  • the at least one phenotypically selectable trait is a phytotoxic resistance/tolerance trait, preferably a herbicide resistance/tolerance trait, and wherein the at least one first targeted base modification at the first plant genomic target site of the at least one plant cell to be modified confers resistance/tolerance for a phytotoxic compound, preferably a herbicide, said compound being an exogenous compound to be added to the at least one modified plant cell, tissue, organ, or whole plant, or a progeny thereof.
  • the first plant genomic target site of the at least one plant cell is ALS. In another embodiment, the first plant genomic target site of the at least one plant cell is PPO. In yet another embodiment, the first plant genomic target site of the at least one plant cell is EPSPS, ALS, or PPO, and wherein the EPSPS, ALS or PPO comprises at least one nucleic acid conversion resulting in at least one corresponding amino acid conversion, wherein the at least one nucleic acid conversion is made by at least one base editor.
  • the methods of the present invention comprises introduction of a targeted modification into the first plant genomic target site of the at least one plant cell, wherein the first plant genomic target site is ALS, and wherein the targeted modification occurs at the sequence encoding A122 in comparison to an ALS reference sequence according to SEQ ID NO: 25, or at the sequence encoding P197 in comparison to an ALS reference sequence according to SEQ ID NO: 25, or at the sequence encoding A205 in comparison to an ALS reference sequence according to SEQ ID NO: 25, or at the sequence encoding D376 in comparison to an ALS reference sequence according to SEQ ID NO: 25, or at the sequence encoding R377 in comparison to an ALS reference sequence according to SEQ ID NO: 25.
  • a targeted modification occurs at the sequence encoding W574 in comparison to an ALS reference sequence according to SEQ ID NO: 25.
  • a targeted modification occurs at the sequence encoding S653 in comparison to an ALS reference sequence according to SEQ ID NO: 25.
  • a targeted modification occurs at the sequence encoding G654 in comparison to an ALS reference sequence according to SEQ ID NO: 25.
  • the first plant genomic target site of the at least one plant cell is PPO
  • a targeted modification occurs at the sequence encoding C215 in comparison to an PPO reference sequence according to SEQ ID NO: 26.
  • a targeted modification occurs at the sequence encoding A220 in comparison to an PPO reference sequence according to SEQ ID NO: 26.
  • a targeted modification occurs at the sequence encoding G221 in comparison to an PPO reference sequence according to SEQ ID NO: 26.
  • a targeted modification occurs at the sequence encoding N425 in comparison to an PPO reference sequence according to SEQ ID NO: 26, or at the sequence encoding Y426, or at the sequence encoding I475, in comparison to an PPO reference sequence according to SEQ ID NO: 26.
  • the first plant genomic target site of the at least one plant cell is EPSPS, and targeted modifications occur at the sequence encoding G101 and at G144, at the sequence encoding G101 and at A192, or at the sequence encoding T102 and at P106, all sequences in comparison to an EPSPS reference sequence according to SEQ ID NO: 27.
  • the at least one phenotypically selectable trait is a visible phenotype that is useful in identifying or isolating at least one modified plant cell, tissue, organ or whole plant.
  • the at least one phenotypically selectable trait can be a glossy phenotype, a golden phenotype, a growth advantage phenotype, or a pigmentation phenotype, or any other visually screenable phenotype.
  • a method for isolating at least one modified plant cell or at least one modified plant tissue, organ, or whole plant comprising the at least one modified plant cell, without stably integrating a transgenic selectable marker sequence comprising: (a) introducing at least one first targeted codon deletion modification into a first plant genomic target site of at least one plant cell to be modified using at least one first site-specific effector, comprising a nuclease, a recombinase, or a DNA modification reagent, wherein the at least one targeted codon deletion modification causes expression of at least one phenotypically selectable trait; (b) introducing at least one second targeted modification into a second plant genomic target site of the at least one plant cell to be modified, wherein the at least one second targeted modification is introduced using at least one second site-specific effector to create the at least one second targeted modification at the second plant genomic target site, wherein the at least one second targeted modification is introduced simultaneously or subsequently to the introduction of
  • a method for isolating at least one modified plant cell or at least one modified tissue, organ, or whole plant comprising the at least one modified plant cell, without stably integrating a transgenic selectable marker sequence
  • the method comprising: (a) introducing at least one first targeted frameshift or deletion modification into a first plant genomic target site of at least one plant cell to be modified using at least one first site-specific effector, wherein the at least one targeted frameshift or deletion modification causes expression of at least one phenotypically selectable trait; (b) introducing at least one second targeted modification into a second plant genomic target site of the at least one plant cell to be modified, wherein the at least one second targeted modification is introduced using at least one second site-specific effector, comprising a nuclease, a recombinase, or a DNA modification reagent, to create the at least one second targeted modification at the second plant genomic target site, wherein the at least one second targeted modification is introduced simultaneously or subsequently to the introduction
  • step (b) additionally comprises introducing a repair template to make a targeted sequence conversion or replacement at the at least one first and/or second plant genomic target site.
  • the at least one site-specific effector is selected from at least one of a CRISPR nuclease, including Cas or Cpf1 nucleases, a TALEN, a ZFN, a meganuclease, an Argonaute nuclease, a restriction endonuclease, including FokI or a variant thereof, a recombinase, or two site-specific nicking endonucleases, or any variant or catalytically active fragment of the aforementioned effectors.
  • a CRISPR nuclease including Cas or Cpf1 nucleases
  • TALEN TALEN
  • ZFN a meganuclease
  • an Argonaute nuclease including FokI or a variant thereof, a recombinase, or two site-specific nicking endonucleases, or any variant or catalytically active fragment of the aforementioned effectors.
  • the at least one site-specific effector is a CRISPR-based nuclease, wherein the CRISPR-based nuclease comprises a site- specific DNA binding domain, wherein the at least one CRISPR-based nuclease, or the nucleic acid sequence encoding the same, is selected from the group comprising (a) Cas9, including SpCas9, SaCas9, SaKKH-Cas9, VQR-Cas9, St1Cas9, (b) Cpf1, including AsCpf1, LbCpf1, FnCpf1, (c) CasX, or (d) CasY, or any variant or derivative of the aforementioned CRISPR-based nucleases, optionally wherein the at least one CRISPR-based nuclease comprises a mutation in comparison to the respective wild-type sequence so that the resulting CRISPR-based nuclease is converted to a single-strand
  • the at least site-specific effector, or at least one component of a complex comprising the at least one site-specific effector comprises at least one organelle localization signal to target the at least one base editing complex to a subcellular organelle, wherein the at least one organelle localization signal can be selected from a nuclear localization signal (NLS) , a chloroplast transit peptide, or a mitochondria transit peptide.
  • NLS nuclear localization signal
  • chloroplast transit peptide a chloroplast transit peptide
  • mitochondria transit peptide a mitochondria transit peptide
  • the first plant genomic target site of the at least one plant cell is a genomic target site encoding at least one phenotypically selectable trait, wherein the at least one phenotypically selectable trait is a resistance/tolerance trait or a growth advantage trait, and wherein the at least one first targeted base modification at the first plant genomic target site of the at least one plant cell confers resistance/tolerance or a growth advantage towards a compound or trigger to be added to the at least one modified plant cell, tissue or plant, or a progeny thereof.
  • the at least one phenotypically selectable trait of interest is or is encoded by at least one endogenous gene, or the at least one phenotypic trait of interest is or is encoded by at least one transgene, wherein the at least one endogenous gene or the at least one transgene encode (s) at least one phenotypic trait selected from the group consisting of resistance/tolerance to a phytotoxin, preferably a herbicide, inhibiting, damaging or killing cells lacking the at least one modification at the at least one phenotypic trait of interest, or wherein the at least one phenotypic trait is selected from the group consisting of boosters of cell division, growth rate, embryogenesis, or another phenotypically selectable property that provides an advantage to a modified cell, tissue, organ, or plant compared to an unmodified cell, tissue, organ, or plant.
  • the at least one first plant genomic target site is at least one endogenous gene or a transgene encoding at least one phenotypically selectable trait selected from the group consisting of herbicide resistance/tolerance, wherein the herbicide resistance/tolerance is selected from the group consisting of resistance/tolerance to EPSPS-inhibitors, including glyphosate, resistance/tolerance to glutamine synthesis inhibitors, including glufosinate, resistance/tolerance to ALS-or AHAS-inhibitors, including imidazoline or sulfonylurea, resistance/tolerance to ACCase inhibitors, including aryloxyphenoxypropionate (FOP) , resistance/tolerance to carotenoid biosynthesis inhibitors, including inhibitors of carotenoid biosynthesis at the phytoene desaturase step, inhibitors of 4-hydroxyphenyl-pyruvate-dioxygenase (HPPD) , or inhibitors of other organophosphate
  • HPPD 4-hydroxy
  • the at least one phenotypically selectable trait is a phytotoxic resistance/tolerance trait, preferably a herbicide resistance/tolerance trait, and the at least one first targeted codon deletion or frameshift or deletion modification at the first plant genomic target site of the at least one plant cell to be modified confers resistance/tolerance for a phytotoxic compound, preferably a herbicide, said compound being an exogenous compound to be added to the at least one modified plant cell, tissue, organ, or whole plant, or a progeny thereof.
  • the first plant genomic target site of the at least one plant cell is a homolog of the PPX2L gene product from Amaranthus tuberculatus for the purpose of selection.
  • the at least one first targeted base modification, targeted codon deletion, or targeted frameshift or deletion modification occurs at the position comparable to the G210 residue of the PPX2L gene product from Amaranthus tuberculatus according to SEQ ID NO: 28.
  • the at least one phenotypically selectable trait is a visible phenotype that is useful in identifying or isolating at least one modified plant cell, tissue, organ, or whole plant.
  • the at least one phenotypically selectable trait according to the various aspects of the present invention can be a glossy phenotype, a golden phenotype, a growth advantage phenotype or a pigmentation phenotype, or any other visually screenable phenotype.
  • the at least one plant cell to be modified is preferably being derived from a plant selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea spp., including Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticum durum, Secale cereale, Triticale, Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta spp., including Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Nicotian
  • Figure 1 illustrates how the methods according to the present invention can be implemented for isolating cells of interest during selection, for example, for plant breeding and targeted selection strategies.
  • Fig. 1 A shows the treatment of cells with a base editor (BE) , or a BE complex, and editing reagents, i.e. a site-specific effector comprising a site-specific nuclease (SSN) at two different genomic locations in parallel. Arrows indicate the target site, where the base editor (complex) and the site-specific effector will introduce two targeted site-specific modifications.
  • Fig. 1 B shows the result of the preceding step illustrated in Fig.
  • the BE complex
  • the site-specific effector introduces a targeted edit in a trait gene, highlighted in black. Therefore, the two distinct modifications within two different genomic target sites allow the isolation of plant cells or plants from treated cells. Plants can then be screened for an edit at a gene of interest, which is usually different from the modified phenotype used for screening purposes.
  • Fig. 1 C shows the result after segregating plants to achieve a desired genotype.
  • This desired genotype of interest comprises the targeted modification (black) introduced via a site-specific effector, but does no longer comprise the modified phenotype modification, the latter having been introduced for selection purposes, yet not as a genomic trait comprised by the genome of the resulting plant cell, tissue, organ or whole plant in this example.
  • FIG. 1 illustrates enhanced screening efficiency by co-editing TaALS S1 site.
  • Figure 3 illustrates generation of herbicide resistant wheat by editing TaALS-P173.
  • Figure 4 illustrates generation of herbicide resistant corn by editing ZmALS-P165.
  • Figure 5 illustrates the sequence structure and herbicide resistant sites to be edited in corn.
  • Figure 6 illustrates efficient editing of ZmALS-P197 and ZmALS-G654.
  • Figure 7 illustrates the efficiency of converting ZmALS-P197 and ZmALS-G654 to desired herbicide resistance-conferring residues.
  • SEQ ID NO: 1 is a nucleotide sequence of an APOBEC1 (rat cytidine deaminase) -XTEN linker (see, for example, Schellenberger et al., "A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner" , Nature Biotechnol. 27, 1186-1190 (2009) ) -nCas9 (D10A) -UGI (uracil DNA glycosylase inhibitor) -NLS encoding construct, which was not codon optimized.
  • the sequence includes a 3′stop codon TAA.
  • SEQ ID NO: 2 is a nucleotide sequence of an APOBEC1 -XTEN linker -nCas9 (D10A) -UGI -NLS encoding construct, which was codon optimized for use in cereal plants.
  • the sequence includes a 3′stop codon TAG.
  • SEQ ID NO: 3 represents an exemplary protospacer sequence for Zm_ALS1&2_P197S/L/F for base editing for a B73 reference genotype. The position is based on the coordinates of the residue in the Arabidopsis ALS homolog. The sequence applies for a SpCas9-derived (Streptococcus pyogenes Cas9-derived) based editor.
  • SEQ ID NO: 4 represents an exemplary protospacer sequence for Zm_ALS1&2_P197S/L/F for base editing for a B73 reference genotype. The position is based on the coordinates of the residue in the Arabidopsis ALS homolog. The sequence applies for a SaKKH-BE3-derived based editor (Staphylococcus aureus Cas9 (SaCas9) -derived mutant of SaCas9 with a relaxed PAM specificity) .
  • SEQ ID NO: 5 represents an exemplary protospacer sequence for Zm_ALS1&2_P197S/L/F for base editing for a B73 reference genotype. The position is based on the coordinates of the residue in the Arabidopsis ALS homolog. The sequence applies for a VQR-BE3-derived based editor (Staphylococcus aureus Cas9 (SaCas9) -derived mutant of SaCas9 with a different PAM specificity) .
  • SEQ ID NO: 6 represents an exemplary protospacer sequence for Zm_ALS1&2_S653N for base editing for a B73 reference genotype. The position is based on the coordinates of the residue in the Arabidopsis ALS homolog. The sequence applies for a SpCas9-derived based editor.
  • SEQ ID NO: 7 represents an exemplary protospacer sequence for Zm_PPO_A220_&_G221 for base editing for a B73 reference genotype. The position is based on the coordinates of the residue in the Arabidopsis PPO homolog. The sequence applies for a SpCas9-derived based editor.
  • SEQ ID NO: 8 represents an exemplary protospacer sequence for Zm_PPO_A220_&_G221 for base editing for a B73 reference genotype. The position is based on the coordinates of the residue in the Arabidopsis PPO homolog. The sequence applies for a SaKKH-BE3-derived based editor.
  • SEQ ID NO: 9 represents an exemplary protospacer sequence for Zm_PPO_A220_&_G221 for base editing for a B73 reference genotype. The position is based on the coordinates of the residue in the Arabidopsis PPO homolog. The sequence applies for a VQR-BE3-derived based editor.
  • SEQ ID NO: 10 represents an exemplary protospacer sequence for Zm_PPO_C215 for base editing for base editing for a B73 reference genotype. The position is based on the coordinates of the residue in the Arabidopsis PPO homolog. The sequence applies for a SpCas9-derived based editor.
  • SEQ ID NO: 11 represents an exemplary protospacer sequence for Zm_PPO_C215 for base editing for base editing for a B73 reference genotype. The position is based on the coordinates of the residue in the Arabidopsis PPO homolog. The sequence applies for a SaKKH-BE3-derived based editor.
  • SEQ ID NO: 12 represents an exemplary protospacer sequence for Zm_PPO_C215 for base editing for base editing for a B73 reference genotype. The position is based on the coordinates of the residue in the Arabidopsis PPO homolog. The sequence applies for a SaKKH-BE3-derived based editor.
  • SEQ ID NO: 13 represents an exemplary protospacer sequence for Zm_PPO_C215 for base editing for base editing for a B73 reference genotype. The position is based on the coordinates of the residue in the Arabidopsis PPO homolog. The sequence applies for a VQR-BE3-derived based editor.
  • SEQ ID NO: 14 is a nucleotide sequence of an APOBEC1 -XTEN linker -CasX1 -UGI -NLS encoding construct, which was codon optimized. The sequence includes a 3′stop codon TAG.
  • SEQ ID NO: 15 is a nucleotide sequence of an APOBEC1 -XTEN linker -AsCpf1 (R1226A) (Acidaminococcus sp. Cpf1 with R1226A mutation) -UGI -NLS encoding construct, which was codon optimized.
  • the sequence includes a 3′stop codon TAG.
  • SEQ ID NO: 16 is a nucleotide sequence of a construct encoding NLS -dCas9 -NLS -Linker -PmCDA1 (activation-induced cytidine deaminase (AID) ortholog PmCDA1 from sea lamprey, see Nishida et al. (Science 2016, vol. 353, issue 6305, aaf8729) ) -UGI.
  • the sequence includes a 3′stop codon TAG.
  • SEQ ID NO: 17 is a nucleotide sequence encoding an exemplary Cas9 nickase n (i) Cas9 (D10A) .
  • SEQ ID NO: 18 is a nucleotide sequence encoding an exemplary CasX.
  • SEQ ID NO: 19 is a nucleotide sequence encoding an exemplary AsCpf1 (R1226A) .
  • SEQ ID NO: 20 is a nucleotide sequence encoding an exemplary APOBEC1.
  • SEQ ID NO: 21 is a nucleotide sequence encoding an exemplary UGI.
  • SEQ ID NO: 22 is a nucleotide sequence encoding an exemplary PmCDA1.
  • SEQ ID NO: 23 represents an exemplary protospacer sequence for Zm_PPO_N425_&Y426 for base editing for base editing for a B73 reference genotype. The position is based on the coordinates of the residue in the Arabidopsis PPO homolog. The sequence applies for a VQR-BE3-derived based editor.
  • SEQ ID NO: 24 is a sequence of Acidaminococcus sp BV3L6 Cpf1 (AsCpf1) , UniProtKB/Swiss-Prot identifier: U2UMQ6.1.
  • SEQ ID NO: 25 is a sequence of acetolactate synthase (ALS) (chloroplastic) from Arabidopsis thaliana, GenBank: AAW70386.
  • ALS acetolactate synthase
  • SEQ ID NO: 26 is a sequence of Arabidopsis thaliana protoporphyrinogen oxidase (PPO) .
  • SEQ ID NO: 27 is a sequence of Arabidopsis thaliana 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) , mature protein after chloroplast transit peptide removal; NCBI accession AAY25438.
  • EPSPS Arabidopsis thaliana 5-enolpyruvylshikimate-3-phosphate synthase
  • SEQ ID NO: 28 is a sequence of Amaranthus tuberculatus mitochondrial protoporphyrinogen oxidase (PPX2L) , cf. NCBI accession DQ386114.
  • Ranges may be expressed herein as from “about” or “approximately” or “substantially” one particular value and/or to “about” or “approximately” or “substantially” another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value and/or to the other particular value. Further, the term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about” can mean within an acceptable standard deviation, per the practice in the art.
  • “about” can mean a range of up to ⁇ 20%, preferably up 5 to ⁇ 10%, more preferably up to ⁇ 5%, and more preferably still up to ⁇ 1%of a given value.
  • the term can mean within an order of magnitude, preferably within 2-fold, of a value.
  • catalytically active fragment as used herein referring to amino acid sequences denotes the core sequence derived from a given template amino acid sequence, or a nucleic acid sequence encoding the same, comprising all or part of the active site of the template sequence with the proviso that the resulting catalytically active fragment still possesses the activity characterizing the template sequence, for which the active site of the native enzyme or a variant thereof is responsible. Said modifications are suitable to generate less bulky amino acid sequences still having the same activity as a template sequence making the catalytically active fragment a more versatile or more stable tool being sterically less demanding.
  • “Complementary” or “complementarity” as used herein describes the relationship between two DNA, two RNA, or, regarding hybrid sequences according to the present invention, between an RNA and a DNA nucleic acid region. Defined by the nucleobases of the DNA or RNA, two nucleic acid regions can hybridize to each other in accordance with the lock-and-key model. To this end the principles of Watson-Crick base pairing have the basis adenine and thymine/uracil as well as guanine and cytosine, respectively, as complementary bases apply.
  • non-Watson-Crick pairing like reverse-Watson-Crick, Hoogsteen, reverse-Hoogsteen and Wobble pairing are comprised by the term "complementary" as used herein as long as the respective base pairs can build hydrogen bonding to each other, i.e., two different nucleic acid strands can hybridize to each other based on said complementarity.
  • construct refers to a construct comprising, inter alia, plasmids or plasmid vectors, cosmids, artificial yeast chromosomes or bacterial artificial chromosomes (YACs and BACs) , phagemides, bacterial phage based vectors, an expression cassette, isolated single-stranded or double-stranded nucleic acid sequences, comprising DNA and RNA sequences, or amino acid sequences, viral vectors, including modified viruses, and a combination or a mixture thereof, for introduction or transformation, transfection or transduction into a target cell or plant, plant cell, tissue, organ or material according to the present disclosure.
  • a recombinant construct according to the present invention can comprise an effector domain, either in the form of a nucleic acid or an amino acid sequence, wherein an effector domain represents a molecule, which can exert an effect in a target cell and includes a transgene, an single-stranded or double-stranded RNA molecule, including a guideRNA, a miRNA, a single or duplexed CRISPR tracr/crRNA, or an siRNA, or an amino acid sequences, including, inter alia, an enzyme or a catalytically active fragment thereof, a binding protein, an antibody, a transcription factor, a nuclease, preferably a site specific nuclease, and the like.
  • the recombinant construct can comprise regulatory sequences and/or localization sequences.
  • the recombinant construct can be integrated into a vector, including a plasmid vector, and/or it can be present isolated from a vector structure, for example, in the form of a polypeptide sequence or as a non-vector connected single-stranded or double-stranded nucleic acid.
  • the genetic construct can either persist extrachromosomally, i.e. non integrated into the genome of the target cell, for example in the form of a double-stranded or single-stranded DNA, a double-stranded or single-stranded RNA or as an amino acid sequence.
  • the genetic construct, or parts thereof, according to the present disclosure can be stably integrated into the genome of a target cell, including the nuclear genome or further genetic elements of a target cell, including the genome of plastids like mitochondria or chloroplasts.
  • plasmid vector refers to a genetic construct originally obtained from a plasmid.
  • delivery construct refers to any biological or chemical means used as a cargo for transporting a nucleic acid, including a hybrid nucleic acid comprising RNA and DNA, and/or an amino acid sequence of interest into a target cell, preferably a eukaryotic cell.
  • delivery construct or vector as used herein thus refers to a means of transport to deliver a genetic or a recombinant construct according to the present disclosure into a target cell, tissue, organ or an organism.
  • a vector can thus comprise nucleic acid sequences, optionally comprising sequences like regulatory sequences or localization sequences for delivery, either directly or indirectly, into a target cell of interest or into a plant target structure in the desired cellular compartment of a plant.
  • a vector can also be used to introduce an amino acid sequence or a ribonucleo-molecular complex into a target cell or target structure.
  • a vector as used herein can be a plasmid vector.
  • a direct introduction of a construct or sequence or complex of interest is conducted.
  • the term direct introduction implies that the desired target cell or target structure containing a DNA target sequence to be modified according to the present disclosure is directly transformed or transduced or transfected into the specific target cell of interest, where the material delivered with the delivery vector will exert its effect.
  • the term indirect introduction implies that the introduction is achieved into a structure, for example, cells of leaves or cells of organs or tissues, which do not themselves represent the actual target cell or structure of interest to be transformed, but those structures serve as basis for the systemic spread and transfer of the vector, preferably comprising a genetic construct according to the present disclosure to the actual target structure, for example, a meristematic cell or tissue, or a stem cell or tissue.
  • vector is used in the context of transfecting amino acid sequences and/or nucleic sequences, including hybrid nucleic acid sequences, into a target cell the term vector implies suitable agents for peptide or protein transfection, like for example ionic lipid mixtures, cell penetrating peptides (CPPs) , or particle bombardment.
  • vector cannot only imply plasmid vectors but also suitable carrier materials which can serve as basis for the introduction of nucleic acid and/or amino acid sequence delivery into a target cell of interest, for example by means of particle bombardment.
  • Said carrier material comprises, inter alia, gold or tungsten particles.
  • vector also implies the use of viral vectors for the introduction of at least one genetic construct according to the present disclosure like, for example, modified viruses for example derived from the following virus strains: adenoviral or adeno-associated viral (AAV) vectors, lentiviral vectors, herpes simplex virus (HSV-1) , vaccinia virus, Sendai virus, Sindbis virus, Semliki forest alphaviruses, Epstein-Barr-Virus (EBV) , Maize Streak Virus (MSV) , Barley Stripe Mosaic Virus (BSMV) , Brome Mosaic virus (BMV, accession numbers: RNA 1: X58456; RNA2: X58457; RNA3: X58458) , Maize stripe virus (MSpV) , Maize rayado fino virus (MYDV) , Maize yellow dwarf virus (MYDV) , Maize dwarf mosaic virus (MDMV) , positive strand RNA viruses
  • vector also implies suitable chemical transport agents for introducing linear nucleic acid sequences (single-or double-stranded) , or amino sequences, or a combination thereof into a target cell combined with a physical introduction method, including polymeric or lipid-based delivery constructs.
  • Suitable delivery constructs or vectors thus comprise biological means for delivering nucleotide sequences into a target cell, including viral vectors, Agrobacterium spp., or chemical delivery constructs, including nanoparticles, e.g., mesoporous silica nanoparticles (MSNPs) , cationic polymers, including PEI (polyethylenimine) polymer based approaches or polymers like DEAE-dextran, or non-covalent surface attachment of PEI to generate cationic surfaces, lipid or polymeric vesicles, or combinations thereof.
  • Lipid or polymeric vesicles may be selected, for example, from lipids, liposomes, lipid encapsulation systems, nanoparticles, small nucleic acid-lipid particle formulations, polymers, and polymersomes.
  • prokaryotic or a eukaryotic cell preferably an animal cell and more preferably a plant or plant cell or plant material according to the present disclosure relates to the descendants of such a cell or material which result from natural reproductive propagation including sexual and asexual propagation. It is well known to the person having skill in the art that said propagation can lead to the introduction of mutations into the genome of an organism resulting from natural phenomena which results in a descendant or progeny, which is genomically different to the parental organism or cell, however, still belongs to the same genus/species and possesses mostly the same characteristics as the parental recombinant host cell.
  • derivatives or descendants or progeny resulting from natural phenomena during reproduction or regeneration are thus comprised by the term of the present disclosure.
  • derivative can imply, in the context of a substance or molecule rather than referring to a cell or organism, directly or by means of modification indirectly obtained from another. This might imply a nucleic acid sequence derived from a cell or a plant metabolite obtained from a cell or material.
  • derived derived from or amino acid
  • a biological sequence nucleic acid or amino acid
  • a molecule or a complex imply that the respective sequence is based on a reference sequence, for example from the sequence listing, or a database accession number, or the respective scaffold structure, i.e., originating from said sequence, whereas the reference sequence can comprise more sequences, e.g., the whole genome or a full polyprotein encoding sequence, of a virus, whereas the sequence "derived from” the native sequence may only comprise one isolated fragment thereof, or a coherent fragment thereof.
  • a cDNA molecule or a RNA can be said to be "derived from” a DNA sequence serving as molecular template.
  • the skilled person can thus easily define a sequence "derived from” a reference sequence, which will, by sequence alignment on DNA or amino acid level, have a high identity to the respective reference sequence and which will have coherent stretches of DNA/amino acids in common with the respective reference sequence (>75%query identity for a given length of the molecule aligned provided that the derived sequence is the query and the reference sequence represents the subject during a sequence alignment) .
  • the skilled person can thus clone the respective sequences based on the disclosure provided herein by means of polymerase chain reactions and the like into a suitable vector system of interest, or use a sequence as vector scaffold.
  • derived from is thus no arbitrary sequence, but a sequence corresponding to a reference sequence it is derived from, whereas certain differences, e.g., certain mutations naturally occurring during replication of a recombinant construct within a host cell, cannot be excluded and are thus comprised by the term "derived from” .
  • sequence stretches from a parent sequence can be concatenated in a sequence derived from the parent. The different stretches will have high or even 100%homology to the parent sequence.
  • a sequence of the artificial molecular complexes according to the present invention when provided or partially provided as nucleic acid sequence will then be transcribed and optionally translated in vivo and will possibly be further digested and/or processed within a host cell (cleavage of signal peptides, endogenous biotinylation etc. ) so that the term "derived from” indicates a correlation to the sequence originally used according to the disclosure of the present invention.
  • fusion can refer to a protein and/or nucleic acid comprising one or more non-native sequences (e.g., moieties) .
  • a fusion can be at the N-terminal or C-terminal end of the modified protein, or both, or within the molecule as separate domain.
  • the fusion molecule can be attached at the 5’or 3’end, or at any suitable position in between.
  • a fusion can be a transcriptional and/or translational fusion.
  • a fusion can comprise one or more of the same non-native sequences.
  • a fusion can comprise one or more of different non-native sequences.
  • a fusion can be a chimera.
  • a fusion can comprise a nucleic acid affinity tag.
  • a fusion can comprise a barcode.
  • a fusion can comprise a peptide affinity tag.
  • a fusion can provide for subcellular localization of the site-specific effector or base editor (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an 15 endoplasmic reticulum (ER) retention signal, and the like) .
  • a fusion can provide a non-native sequence (e.g., affinity tag) that can be used to track or purify.
  • a fusion can be a small molecule such as biotin or a dye such as alexa fluor dyes, Cyanine3 dye, Cyanine5 dye.
  • a fusion can comprise a detectable label, including a moiety that can provide a detectable signal.
  • Suitable detectable labels and/or moieties that can provide a detectable signal can include, but are not limited to, an enzyme, a radioisotope, a member of a specific binding pair; a fluorophore; a fluorescent reporter or fluorescent protein; a quantum dot; and the like.
  • a fusion can comprise a member of a FRET pair, or a fluorophore/quantum dot donor/acceptor pair.
  • a fusion can comprise an enzyme.
  • Suitable enzymes can include, but are not limited to, horse radish peroxidase, luciferase, beta-25 galactosidase, and the like.
  • a fusion can comprise a fluorescent protein.
  • Suitable fluorescent proteins can include, but are not limited to, a green fluorescent protein (GFP) , (e.g., a GFP from Aequoria victoria, fluorescent proteins from Anguilla japonica, or a mutant or derivative thereof) , a red fluorescent protein, a yellow fluorescent protein, a yellow-green fluorescent protein (e.g., mNeonGreen derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum) any of a variety of fluorescent and colored proteins.
  • GFP green fluorescent protein
  • red fluorescent protein e.g., a GFP from Aequoria victoria, fluorescent proteins from Anguilla japonica, or a mutant or derivative thereof
  • red fluorescent protein
  • a fusion can comprise a nanoparticle.
  • Suitable nanoparticles can include fluorescent or luminescent nanoparticles, and magnetic nanoparticles, or nanodiamonds, optionally linked to a nanoparticle Any optical or magnetic property or characteristic of the nanoparticle (s) can be detected.
  • a fusion can comprise a helicase, a nuclease (e.g., Fokl) , an endonuclease, an exonuclease (e.g., a 5'exonuclease and/or 3'exonuclease) , a ligase, a nickase, a nuclease-helicase (e.g., Cas3) , a DNA methyltransferase (e.g., Dam) , or DNA demethylase, a histone methyltransferase, a histone demethylase, an acetylase (including for example and not limitation, a histone acetylase) , a deacetylase (including for example and not limitation, a histone deacetylase) , a phosphatase, a kinase, a transcription (co-) activator, a transcription (co-) factor, an
  • genetic (ally) manipulated is used in a broad sense herein and means any modification of a nucleic acid sequence or an amino acid sequence, a target cell, tissue, organ or organism, which is accomplished by human intervention, either directly or indirectly, to influence the endogenous genetic material or the transciptome or the proteinome of a target cell, tissue, organ or organism to modify it in a purposive way so that it differs from its state as found without human intervention.
  • the human intervention can either take place in vitro or in vivo/in planta, or also both. Further modifications can be included, for example, one or more point mutation (s) , e.g.
  • nucleic acid molecule or an amino acid molecule or a host cell or an organism including a plant or a plant material thereof which is/are similar to a comparable sequence, organism or material as occurring in nature, but which have been constructed by at least one step of purposive manipulation.
  • a “targeted genetic manipulation” or “targeted (base) modification” as used herein is thus the result of a “genetic manipulation” , which is effected in a targeted way, i.e. at a specific position in a target cell and under the specific suitable circumstances to achieve a desired effect in at least one cell, preferably a plant cell, to be manipulated, wherein the term implies that the sequence to be targeted and the corresponding modification are based on preceding sequence considerations so that the resulting modification can be planned in advance, e.g., based on available sequence information of a target site in the genome of a cell and/or based on the information of the target specificity (recognition or binding properties of a nucleic acid or an amino acid sequence, complementary base pairing and the like) of a molecular tool of interest.
  • the term “genome” refers to the entire complement of genetic material (genes and non-coding sequences) that is present in each cell of an organism, or virus or organelle; and/or a complete set of chromosomes inherited as a (haploid) unit from one parent.
  • the term “particle bombardment” as used herein, also named “biolistic transfection or “microparticle-mediated gene transfer” refers to a physical delivery method for transferring a coated microparticle or nanoparticle comprising a nucleic acid or a genetic construct of interest into a target cell or tissue. The micro or nanoparticle functions as projectile and is fired on the target structure of interest under high pressure using a suitable device, often called gene-gun.
  • the transformation via particle bombardment uses a microprojectile of metal covered with the gene of interest, which is then shot onto the target cells using an equipment known as "gene gun” (Sandford et al. 1987) at high velocity fast enough ( ⁇ 1500 km/h) to penetrate the cell wall of a target tissue, but not harsh enough to cause cell death.
  • gene gun Systemandford et al. 1987
  • the precipitated nucleic acid or the genetic construct on the at least one microprojectile is released into the cell after bombardment, and integrated into the genome.
  • the acceleration of microprojectiles is accomplished by a high voltage electrical discharge or compressed gas (helium) .
  • metal particles used it is mandatory that they are non-toxic, non-reactive, and that they have a lower diameter than the target cell.
  • the most commonly used are gold or tungsten.
  • gene editing and “genome engineering” are used interchangeably herein and refer to strategies and techniques for the targeted, specific modification of any genetic information or genome of a living organism.
  • the terms comprise gene editing, but also the editing of regions other than gene encoding regions of a genome. It further comprises the editing or engineering of the nuclear (if present) as well as other genetic information of a cell.
  • the terms “genome editing” and “genome engineering” also comprise an epigenetic editing or engineering, i.e., the targeted modification of, e.g., methylation, histone modification or of non-coding RNAs possibly causing heritable changes in gene expression.
  • germplasm is a term used to describe the genetic resources, or more precisely the DNA of an organism and collections of that material. In breeding technology, the term germplasm is used to indicate the collection of genetic material from which a new plant or plant variety can be created.
  • guide RNA refers to a synthetic fusion of a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) , or the term refers to a single RNA molecule consisting only of a crRNA and/or a tracrRNA, or the term refers to a gRNA individually comprising a crRNA or a tracrRNA moiety.
  • crRNA CRISPR RNA
  • tracrRNA trans-activating crRNA
  • tracr and the crRNA moiety thus do not necessarily have to be present on one covalently attached RNA molecule, yet they can also be comprised by two individual RNA molecules, which can associate or can be associated by non-covalent or covalent interaction to provide a gRNA according to the present disclosure.
  • gDNA or "sgDNA” or “guide DNA” are used interchangeably herein and either refer to a nucleic acid molecule interacting with an Argonaute nuclease.
  • guiding nucleic acids or “guide nucleic acids” due to their capacity to interacting with a site-specific nuclease and to assist in targeting said site-specific nuclease to a genomic target site.
  • mutation and “modification” are used interchangeably to refer to a deletion, insertion, addition, substitution, edit, strand break, and/or introduction of an adduct in the context of nucleic acid manipulation in vivo or in vitro.
  • a deletion is defined as a change in a nucleic acid sequence in which one or more nucleotides is absent.
  • An insertion or addition is that change in a nucleic acid sequence which has resulted in the addition of one or more nucleotides.
  • substitution results from the replacement of one or more nucleotides by a molecule which is a different molecule from the replaced one or more nucleotides.
  • a nucleic acid may be replaced by a different nucleic acid as exemplified by replacement of a thymine by a cytosine, adenine, guanine, or uridine.
  • Pyrimidine to pyrimidine e.g., C to Tor T to C nucleotide substitutions
  • purine to purine e.g., G to A or A to G nucleotide substitutions
  • transitions whereas pyrimidine to purine or purine to pyrimidine (e.g., G to T or G to C or A to T or A to C) are termed transversions.
  • a nucleic acid may be replaced by a modified nucleic acid as exemplified by replacement of a thymine by thymine glycol. Mutations may result in a mismatch.
  • mismatch refers to a non-covalent interaction between two nucleic acids, each nucleic acid residing on a different nucleotide sequence or nucleic acid molecule, which does not follow the base-pairing rules. For example, for the partially complementary sequences 5'-AGT-3'and 5'-AAT-3', a G-A mismatch (a transition) is present.
  • nucleotide and nucleic acid with reference to a sequence or a molecule are used interchangeably herein and refer to a single or double-stranded DNA or RNA of natural or synthetic origin.
  • nucleotide sequence is thus used for any DNA or RNA sequence independent of its length, so that the term comprises any nucleotide sequence comprising at least one nucleotide, but also any kind of larger oligonucleotide or polynucleotide.
  • the term (s) thus refer to natural and/or synthetic deoxyribonucleic acids (DNA) and/or ribonucleic acid (RNA) sequences, which can optionally comprise synthetic nucleic acid analoga.
  • a nucleic acid according to the present disclosure can optionally be codon optimized.
  • Codon optimization implies that the codon usage of a DNA or RNA is adapted to that of a cell or organism of interest to improve the transcription rate of said recombinant nucleic acid in the cell or organism of interest.
  • the skilled person is well aware of the fact that a target nucleic acid can be modified at one position due to the codon degeneracy, whereas this modification will still lead to the same amino acid sequence at that position after translation, which is achieved by codon optimization to take into consideration the species-specific codon usage of a target cell or organism.
  • Nucleic acid sequences according to the present application can carry specific codon optimization for the following non limiting list of organisms: Hordeum vulgare, Sorghum bicolor, Secale cereale, Saccharum officinarium, Zea mays, Setaria italic, Oryza sativa, Oryza minuta, Oryza australiensis, Oryza a/ta, Triticum aestivum, Triticum durum, Triticale, Hordeum bulbosum, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Ma/us 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,
  • nucleotide can thus generally refer to a base-sugar-phosphate combination.
  • a nucleotide can comprise a synthetic nucleotide.
  • a nucleotide can comprise a synthetic nucleotide analog.
  • Nucleotides can be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) ) .
  • nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP) , uridine triphosphate (UTP) , cytosine triphosphate (CTP) , guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof.
  • Such derivatives can include, for example and not limitation, [ ⁇ S] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them.
  • nucleotide as used herein can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
  • ddNTPs dideoxyribonucleoside triphosphates
  • Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.
  • a nucleotide may be unlabeled or detectably labeled by well-known techniques. Labeling can also be carried out with quantum dots.
  • Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.
  • Fluorescent labels of nucleotides may include but are not limited to fluorescein, 5-carboxyfluorescein (FAM) , 2'7'-5 dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE) , rhodamine, 6-carboxyrhodamine (R6G) , N, N, N', N'-tetramethyl-6-carboxyrhodamine (TAMRA) , 6-carboxy-X-rhodamine (ROX) , 4- (4'dimethylaminophenylazo) benzoic acid (DABCYL) , Cascade Blue, Oregon Green, Texas Red, Cyanine and 5- (2'-aminoethyl) aminonaphthalene-l-sulfonic acid (EDANS) .
  • FAM 5-carboxyfluorescein
  • JE 2'7'-5 dimethoxy-4'5-dichloro-6-carboxyfluoresc
  • non-native or “non-naturally occurring” or “artificial” can refer to a nucleic acid or polypeptide sequence, or any other biomolecule like biotin or fluorescein that is not found in a native nucleic acid or protein.
  • Non-native can refer to affinity tags.
  • Non-native can refer to fusions.
  • Non-native can refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and/or deletions.
  • a non-native sequence may exhibit and/or encode for an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitinating activity, etc.
  • a non-native nucleic acid or polypeptide sequence may be linked to a naturally-occurring nucleic acid or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid and/or polypeptide sequence encoding a chimeric nucleic acid and/or polypeptide.
  • a non-native sequence can refer to a 3'hybridizing extension sequence.
  • phytotoxic or "phytotoxicity” as used herein in the context of plant cells, tissues, organs or plants, refers to a cytotoxic effect or cytotoxicity in general for a plant, or any plant cell.
  • the term thus implies a toxic effect by a compound or trigger on a plant inhibiting, damaging or even killing a plant cell, tissue, organ or whole plant.
  • damage may be caused by a wide variety of compounds, including herbicides, pesticides, trace metals, toxic effectors induced by a pathogen, salinity phytotoxins or allelochemicals.
  • the term also refers to plant phytohormones, for example, but not restricted to hormones for the regulation of plant immune responses, like ethylene, jasmonic acid, and salicylic acid, or plant hormones, such as auxins, abscisic acid (ABA) , cytokinins, gibberellins, and brassinosteroids, that regulate plant development and growth.
  • hormones for the regulation of plant immune responses like ethylene, jasmonic acid, and salicylic acid
  • plant hormones such as auxins, abscisic acid (ABA) , cytokinins, gibberellins, and brassinosteroids, that regulate plant development and growth.
  • plant refers to a whole plant organism, a plant organ, differentiated and undifferentiated plant tissues, plant cells, seeds, and derivatives and progeny thereof.
  • Plant cells include without limitation, for example, cells from seeds, from mature and immature embryos, meristematic tissues, seedlings, callus tissues in different differentiation states, leaves, flowers, roots, shoots, gametophytes, sporophytes, pollen and microspores, protoplasts, macroalgae and microalgae.
  • the different plant cells can either be haploid, diploid or multiploid.
  • plant organ refers to plant tissue or a group of tissues that constitute a morphologically and functionally distinct part of a plant.
  • a "plant material” as used herein refers to any material which can be obtained from a plant during any developmental stage.
  • the plant material can be obtained either in planta or from an in vitro culture of the plant or a plant tissue or organ thereof.
  • the term thus comprises plant cells, tissues and organs as well as developed plant structures as well as sub-cellular components like nucleic acids, polypeptides and all chemical plant substances or metabolites which can be found within a plant cell or compartment and/or which can be produced by the plant, or which can be obtained from an extract of any plant cell, tissue or a plant in any developmental stage.
  • the term also comprises a derivative of the plant material, e.g., a protoplast, derived from at least one plant cell comprised by the plant material.
  • the term therefore also comprises meristematic cells or a meristematic tissue of a plant.
  • a "plasmid” refers to a circular autonomously replicating extrachromosomal element in the form of a double-stranded nucleic acid sequence.
  • these plasmids are routinely subjected to targeted modifications by inserting, for example, genes encoding a resistance against an antibiotic or an herbicide, a gene encoding a target nucleic acid sequence, a localization sequence, a regulatory sequence, a tag sequence, a marker gene, including an antibiotic marker or a fluorescent marker, and the like.
  • the structural components of the original plasmid like the origin of replication, are maintained.
  • the localization sequence can comprise a nuclear localization sequence, a plastid localization sequence, preferably a mitochondrion localization sequence or a chloroplast localization sequence.
  • Said localization sequences are available to the skilled person in the field of plant biotechnology.
  • a variety of plasmid vectors for use in different target cells of interest is commercially available and the modification thereof is known to the skilled person in the respective field.
  • PCR Polymerase chain reaction
  • PCR is a technique for synthesizing a specific DNA segment. PCR comprises a series of repetitive denaturation, annealing, and extension cycles. Typically, a double-stranded DNA is heat denatured, and two primers complementary to the 3′boundaries of the target segment are annealed to the DNA at low temperature, and then extended at an intermediate temperature. One set of these three consecutive steps is referred to as a "cycle" .
  • Progeny comprises any subsequent generation of a plant, plant cell or plant tissue.
  • regulatory sequence refers to a nucleic acid or an amino acid sequence, which can direct the transcription and/or translation and/or modification of a nucleic acid sequence of interest.
  • protein protein
  • amino acid amino acid sequence
  • polypeptide amino acid sequence
  • amino acid molecule comprises any natural or chemically synthesized protein, peptide, polypeptide and enzyme or a modified protein, peptide, polypeptide and enzyme, wherein the term “modified” comprises any chemical or enzymatic modification of the protein, peptide, polypeptide and enzyme, including truncations of a wild-type sequence to a shorter, yet still active portion.
  • selectable phenotypes or “phenotypically selectable” or “phenotypically screenable” defines alterations in the cell or organism’s performance or visual characteristics with respect to growth, metabolism, sensitivity to a phytotoxic (e.g., herbicide) or other compound, or consumption of nutrients.
  • a “selectable phenotype” also includes the visible or invisible appearance as observed by eye or using special equipment.
  • a phenotypically selectable trait is thus encoded by at least one genomic region and results in a phenotype which can be screened visually microscopically, or by any means of molecular or analytical biology.
  • nucleic acid or amino acid sequences Whenever the present disclosure relates to the percentage of the homology or identity of nucleic acid or amino acid sequences these values define those as obtained by using the EMBOSS Water Pairwise Sequence Alignments (nucleotide) programme (www. ebi. ac. uk/Tools/psa/emboss_water/nucleotide. html) nucleic acids or the EMBOSS Water Pairwise Sequence Alignments (protein) programme (www. ebi. ac. uk/Tools/psa/emboss_water/) for amino acid sequences.
  • strand break when made in reference to a double-stranded nucleic acid sequence, e.g., a genomic sequence as DNA target sequence, includes a single-strand break and/or a double-strand break.
  • a single-strand break (a nick) refers to an interruption in one of the two strands of the double-stranded nucleic acid sequence. This is in contrast to a double-strand break which refers to an interruption in both strands of the double-stranded nucleic acid sequence.
  • Strand breaks according to the present disclosure may be introduced into a double-stranded nucleic acid sequence by enzymatic incision at a nucleic acid base position of interest using a suitable endonuclease, including a CRISPR endonuclease or a variant thereof, where the variant can be a mutated or truncated version of the wild-type protein or endonuclease, which still can exert the enzymatic function of the wild-type protein.
  • a suitable endonuclease including a CRISPR endonuclease or a variant thereof, where the variant can be a mutated or truncated version of the wild-type protein or endonuclease, which still can exert the enzymatic function of the wild-type protein.
  • target region refers to a target which can be any genomic or epigenomic region within any compartment of a target cell.
  • targeted or site-specific or site-directed refers to an action of molecular biology which uses information on the sequence of a genomic region of interest to be modified, and which further relies on information of the mechanism of action of molecular tools, e.g., nucleases, including CRISPR nucleases and variants thereof, TALENs, ZFNs, meganucleases or recombinases, DNA-modifying enzymes, including base modifying enzymes like cytidine deaminase enzymes, histone modifying enzymes and the like, DNA-binding proteins, cr/tracr RNAs, guide RNAs and the like, which allow the in silico prediction of at least one modification to be effected within a genomic target region of interest. Therefore, the relevant molecular tools can be designed and constructed ex vivo or in silico.
  • nucleases including CRISPR nucleases and variants thereof, TALENs, ZFNs, meganucleases or recombinases
  • transgene or “transgenic” as used herein refer to at least one nucleic acid sequence that is taken from the genome of one organism, or produced synthetically, and which is then introduced into host a cell or organism or tissue of interest and which is subsequently integrated into the host's genome by means of “stable” transformation or transfection approaches.
  • transient transformation or transfection or introduction refers to a way of introducing molecular tools including at least one nucleic acid (DNA, RNA, single-stranded or double-stranded or a mixture thereof) and/or at least one amino acid sequence, optionally comprising suitable chemical or biological agents, to achieve a transfer into at least one compartment of interest of a cell, including, but not restricted to, the cytoplasm, an organelle, including the nucleus, a mitochondrion, a vacuole, a chloroplast, or into a membrane, resulting in transcription and/or translation and/or association and/or activity of the at least one molecule introduced without achieving a stable integration or incorporation and thus inheritance of the respective at least one molecule introduced into the genome of a cell.
  • transient introduction refers to the transient introduction of at least one nucleic acid sequence according to the present disclosure, preferably incorporated into a delivery vector or into a recombinant construct, with or without the help of a delivery vector, into a target structure, for example, a plant cell, wherein the at least one nucleic acid sequence is introduced under suitable reaction conditions so that no integration of the at least one nucleic acid sequence into the endogenous nucleic acid material of a target structure, the genome as a whole, occurs, so that the at least one nucleic acid sequence will not be integrated into the endogenous DNA of the target cell.
  • the introduced genetic construct will not be inherited to a progeny of the target structure, for example a prokaryotic, an animal or a plant cell.
  • the at least one nucleic acid sequence or the products resulting from transcription or translation thereof are only present temporarily, i.e., in a transient way, in constitutive or inducible form, and thus can only be active in the target cell for exerting their effect for a limited time. Therefore, the at least one nucleic acid sequence introduced via transient introduction will not be heritable to the progeny of a cell. The effect which a nucleic acid sequence introduced in a transient way can, however, potentially be inherited to the progeny of the target cell.
  • a “variant” of any site-specific effector or base editor disclosed herein represents a molecule comprising at least one mutation, deletion or insertion in comparison to the respective wild-type enzyme to alter the activity of the wild-type enzyme as naturally occurring.
  • a “variant” can, as non-limiting example, be a catalytically inactive Cas9 (dCas9) , or a site-specific nuclease, which has been modified to function as nickase.
  • the present invention provides methods for targeted editing in a plant cell, tissue, organ or material, which methods specifically combined and use a parallel introduction strategy.
  • the methods provided herein thus rely on the parallel introduction of a phenotypically selectable trait at a first genomic target site, wherein this phenotypically selectable trait as such allows for an easy screening and does not comprise the introduction of a transgenic marker sequence or marker cassette.
  • the introduction of a targeted modification at a first genomic target site to obtain a selectable phenotype does not rely on the provision of an exogenous polynucleotide template, nor does it rely on the introduction of a double-stand (ds) break at the target site, which steps are usually needed for a variety of genome editing approaches using site-specific nucleases (SSNs) introducing a double-strand break at a genomic target site, which is often cured by providing a repair template for homologous repair (HR) as exogenous nucleic acid material.
  • SSNs site-specific nucleases
  • the methods of the present invention allow precision breeding strategies comprising significantly reduced selection efforts for identifying a genotype of interest, which in turn helps to reduce time and costs necessary to identify relevant modifications within a plant cell or germplasm of interest.
  • a method for isolating at least one modified plant cell or at least one modified plant tissue, organ, or whole plant comprising the at least one modified plant cell, without stably integrating a transgenic selectable marker sequence comprising: (a) introducing at least one first targeted base modification into a first plant genomic target site of at least one plant cell to be modified, wherein the at least one targeted base modification causes expression of at least one phenotypically selectable trait; (b) introducing at least one second targeted modification into a second plant genomic target site of the at least one plant cell to be modified, wherein the at least one second targeted modification is introduced using at least one of a site-specific effector to create the at least one second targeted modification at the second plant genomic target site, wherein the at least one second targeted modification is introduced simultaneously or subsequently to the introduction of the at least one first targeted base modification into the same at least one plant cell to be modified, or into at least one progeny cell, tissue, organ, or plant thereof comprising the at least one first targeted modification
  • a "phenotypically selectable trait” as used herein refers to a trait encoded by at least one gene causing a visible or otherwise selectable phenotype after expression of the relevant genomic trait. Selection for said trait can be accomplished visually, or by using a selection agent, compound or trigger to be applied to a plant cell, tissue, organ, material or whole plant.
  • the first and the second plant genomic target site can be the same, or different genomic loci.
  • the first and the second plant genomic target site reside within different genomic loci, which genomic loci can be located on the same, or on different chromosomes.
  • a parallel introduction strategy of a first and a second targeted modification is made, wherein this parallelization of the different targeted modifications introduced at a first and at a second plant genomic target site significantly improves the later screening steps.
  • the second modification will have no opportunity for selection because the phenotype it confers will not be expressed or relevant in the process of generating the plants.
  • the purpose underlying the methods of the present invention is to use the first modification causing a phenotypically selectable phenotype as a tool to enable selection.
  • the methods disclosed herein have the advantage of not incorporating a transgenic marker gene.
  • the methods according to the various aspects of the present invention rely on the simultaneous or subsequent introduction of the at least one first targeted base modification, codon deletion or frameshift or deletion modification into the same at least one plant cell to be modified also receiving the at least one second targeted modification into a second plant genomic target site of interest.
  • the modifications at the first and the second target site are thus preferably introduced at the same time into the same cell, i.e., in a simultaneous way, i.e., in parallel.
  • the subsequent introduction in this sense thus refers to the fact that the different tools introduced comprising at least one base editing complex and/or at least one site-specific effector might act shortly before each other.
  • the term subsequently in this context implies that the parallel and simultaneous introduction of the tools of interest within the same cell.
  • the methods according to the present invention thus make it possible for cells to select for cells that did, or did not receive the at least one first modification by selecting for the phenotypically selectable trait targeted with the first targeted modification by suitable reagents, or by visual screening. Therefore, this screening eliminates cells not comprising the at least one first modification, or the screening allows the visual inspection and separation of cells into modified cells having received, or not having received the first targeted modification. Of the cells having successfully received the first targeted modification, a reasonable number can be expected to also have the at least one second targeted modification as well due to the parallel introduction and delivery approach according to the present invention.
  • “Reasonable” in this context implies any improvement, i.e., a decrease, of the number of cells to be screened for the presence of the at least one second targeted modification by selecting for the at least one phenotypically selectable trait caused by the at least one first targeted base modification.
  • the actual frequency of the presence of the at least one second targeted modification is usually hard to predict as it will be variable depending on several factors. This makes screening for any modification introduced via genome engineering cumbersome to screen for using common molecular techniques, e.g., relying on PCR:
  • the frequency of cells having received both, the first and the second targeted modification can be in the range of between 2: 1 and 1,000: 1 plant cells or plants having the first modification compared to those having the first and second modifications.
  • the first modification can be removed by crossing the derived plant and genetically segregating it away from the second modification.
  • the methods disclosed herein can thus be used for enriching recovery of plants with the targeted modification at a second gene of interest by eliminating or removing the cells that did not receive the editing reagents or did not undergo the targeted modification as screened for the at least one first targeted modification of interest.
  • a targeted base modification refers to a to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template (cf. Komor et al., Nature, Vol. 533, 2016) .
  • the methods according to the first aspect of the present invention additionally comprise, within step (b) , introducing a repair template to make a targeted sequence conversion or replacement at the at least second plant genomic target site.
  • a repair template (RT) represents a single-stranded or double-stranded nucleic acid sequence, which can be provided during any genome editing causing a double-strand or single-strand DNA break to assist the targeted repair of said DNA break by providing a RT as template of known sequence assisting homology-directed repair.
  • the size of the at least one repair template nucleic acid sequence according to the present invention as part can vary. It can be in the range from about 20 bp to about 5,000 bp or even 8,000 bp depending on the DNA target sequence to be modified in a site-directed way.
  • the RT can be provided as individual physical entity, or as part of a complex according to the present invention. The use of a RT might be favorable for certain applications to avoid undesired insertions or deletions due to a cellular NHEJ repair mechanism
  • the methods provided herein comprise a further step of (d) crossing at least one modified plant or plant material comprising the at least one first and the at least one second targeted modification with a further plant or plant material of interest to segregate the resulting progeny plants or plant material to achieve a genotype of interest, optionally wherein the genotype of interest does not comprise the at least one first targeted modification.
  • the further plant or plant material of interest can be any plant material comprising genomic material of interest, wherein this material, comprising, for example, an elite event or any trait of interest, is intended, e.g., for subsequent rounds of breeding to create a genotype and thus a plant of interest.
  • the genotype of interest is thus the result of preceding breeding steps combining traits from different plants of interest.
  • the final genotype of interest does not comprise the at least one first targeted modification, i.e. the at least one phenotypically selectable trait.
  • the methods of the present invention are particularly suitable to remove the first targeted modification causing a phenotypically selectable trait by crossing the derived plant and genetically segregating it away from the second targeted modification (cf. Fig. 1 C) , if desired for certain applications.
  • the first targeted modification encoding a phenotypically selectable trait of interest can be kept in the genotype of interest in case that the phenotypically selectable trait as such has a value for the resulting genotype of interest and the corresponding plant or plant material.
  • the at least one site-specific effector is temporarily or permanently linked to at least one base editing complex
  • the base editing complex mediates the at least one first targeted base modification of step (a) .
  • the at least one site-specific effector can thus be non-covalently (temporarily) or covalently (permanently) be attached to at least one base editing complex. Any component of the at least one base editing complex can be temporarily or permanently linked to the at least one site-specific effector.
  • temporaryly and “permanently” are thus to be construed broadly and comprise both covalent and/or non-covalent bonds or attachments to achieve physical proximity of the at least one site-specific effector and the least one base editing complex.
  • the linkage of at least on component of the at least one base editing complex and the at least one site-specific effector, or also the any other component, for example a gRNA or a RT associated with the at least one site-specific effector, might be of interest in case the at least one first and the at least one second genomic target site are in close proximity within a genome of interest.
  • the at least one site-specific effector is selected from at least one of a nuclease, comprising a CRISPR nuclease, including Cas or Cpf1 nucleases, a TALEN, a ZFN, a meganuclease, an Argonaute nuclease, a restriction endonuclease, including FokI or a variant thereof, a recombinase, or two site-specific nicking endonucleases, or a base editor, or any variant or catalytically active fragment of the aforementioned effectors.
  • a nuclease comprising a CRISPR nuclease, including Cas or Cpf1 nucleases, a TALEN, a ZFN, a meganuclease, an Argonaute nuclease, a restriction endonuclease, including FokI or a variant thereof, a recombinase, or two site
  • a "site-specific effector” as used herein can thus be defined as any nuclease, nickase, recombinase, or base editor, having the capacity to introduce a single-or double-strand cleavage into a genomic target site, or having the capacity to introduce a targeted modification, including a point mutation, an insertion, or a deletion, into a genomic target site of interest.
  • the at least one "site-specific effector” can act on its own, or in combination with other molecules as part of a molecular complex.
  • the "site-specific effector” can be present as fusion molecule, or as individual molecules associating by or being associated by at least one of a covalent or non-covalent interaction so that the components of the site-specific effector complex are brought into close physical proximity.
  • a “base editor” as used herein refers to a protein or a fragment thereof having the same catalytical activity as the protein it is derived from, which protein or fragment thereof, alone or when provided as molecular complex, referred to as base editing complex herein, has the capacity to mediate a targeted base modification, i.e., the conversion of a base of interest resulting in a point mutation of interest which in turn can result in a targeted mutation, if the base conversion does not cause a silent mutation, but rather a conversion of an amino acid encoded by the codon comprising the position to be converted with the base editor.
  • the at least one base editor according to the present invention temporarily or permanently linked to at least one site-specific effector, or optionally to a component of at least one site-specific effector complex.
  • the linkage can be covalent and/or non-covalent.
  • Any base editor or site-specific effector, or a catalytically active fragment thereof, or any component of a base editor complex or of a site-specific effector complex as disclosed herein can be introduced into a cell as a nucleic acid fragment, the nucleic acid fragment representing or encoding a DNA, RNA or protein effector, or it can be introduced as DNA, RNA and/or protein, or any combination thereof.
  • a key toolset that eliminates the requirement for making selectable modifications with an endonuclease, a DSB, and a repair template is the use of base editors or targeted mutagenesis domains.
  • Multiple publications have shown targeted base conversion, primarily cytidine (C) to thymine (T) , using a CRISPR/Cas9 nickase or non-functional nuclease linked to a cytidine deaminase domain, Apolipoprotein B mRNA-editing catalytic polypeptide (APOBEC1) , e.g., APOBEC derived from rat.
  • C cytidine
  • T thymine
  • APOBEC1 Apolipoprotein B mRNA-editing catalytic polypeptide
  • cytosine (C) is catalysed by cytidine deaminases and results in uracil (U) , which has the base-pairing properties of thymine (T) .
  • U uracil
  • T thymine
  • Most known cytidine deaminases operate on RNA, and the few examples that are known to accept DNA require single-stranded (ss) DNA.
  • ss single-stranded
  • Studies on the dCas9-target DNA complex reveal that at least nine nucleotides (nt) of the displaced DNA strand are unpaired upon formation of the Cas9-guide RNA-DNA ‘R-loop’ complex (Jore et al., Nat. Struct. Mol. Biol., 18, 529-536 (2011) ) .
  • Any base editing complex according to the present invention can thus comprise at least one cytidine deaminase, or a catalytically active fragment thereof.
  • the at least one base editing complex can comprise the cytidine deaminase, or a domain thereof in the form of a catalytically active fragment, as base editor.
  • the at least one first targeted base modification is a conversion of any nucleotide C, A, T, or G, to any other nucleotide.
  • Any one of a C, A, T or G nucleotide can be exchanged in a site-directed way as mediated by a base editor, or a catalytically active fragment thereof, to another nucleotide.
  • the at least one base editing complex can thus comprise any base editor, or a base editor domain or catalytically active fragment thereof, which can convert a nucleotide of interest into any other nucleotide of interest in a targeted way.
  • the present invention provides methods combining the knowledge of the base editor tools as such and uses this technology in a combined method for achieving a phenotypically selectable phenotype of interest to avoid the need of a transgenic marker, as the base edit can artificially create an endogenous marker having a phenotypical output being selectable.
  • a base editor is combined with a modified site-specific effector that retains the ability to recognize and bind a genomic target region, optionally guided by a gRNA for CRISPR-based nucleases, to mediate the conversion of C to U, or G to A, to introduce a site directed mutagenesis.
  • targeted mutations can be effected which result in a phenotype of interest.
  • This approach allows marker-free selection and screening for a modification or a genotype of interest in a synergistic way, without the need to introduce a DSB or a RT for the at least one first modification according to the various aspects of the present invention, i.e., for a targeted base modification, a targeted codon deletion, or a targeted frameshift or deletion modification.
  • uracil DNA glycosylase (UGI) domain further increased the base-editing efficiency.
  • a nuclear localization signal (NLS) or any other organelle targeting signal, can be further required to ensure proper targeting of the complex.
  • the at least one site-specific effector is a CRISPR-based nuclease, wherein the CRISPR-based nuclease comprises a site-specific DNA binding domain directing the at least one base editing complex, wherein the at least one CRISPR-based nuclease, or the nucleic acid sequence encoding the same, is selected from the group comprising (a) Cas9, including SpCas9, SaCas9, SaKKH-Cas9, VQR-Cas9, St1Cas9, (b) Cpf1, including AsCpf1, LbCpf1, FnCpf1, (c) CasX, or (d) CasY, or any variant or derivative of the aforementioned CRISPR-based nucleases, preferably wherein the at least one CRISPR-based nuclease comprises a mutation in comparison to the respective wild-type sequence so that the resulting CRISPR-based nuclease
  • CRISPR-based nuclease is any nuclease which has been identified in a naturally occurring CRISPR system, which has subsequently been isolated from its natural context, and which preferably has been modified or combined into a recombinant construct of interest to be suitable as tool for targeted genome engineering.
  • Any CRISPR-based nuclease can be used and optionally reprogrammed or additionally mutated to be suitable for the various embodiments according to the present invention as long as the original wild-type CRISPR-based nuclease provides for DNA recognition, i.e., binding properties. Said DNA recognition can be PAM dependent.
  • CRISPR nucleases having optimized and engineered PAM recognition patterns can be used and created for a specific application.
  • Cpf1 variants can comprise at least one of a S542R, K548V, N552R, or K607R mutation, preferably mutation S542R/K607R or S542R/K548V/N552R in AsCpf1 from Acidaminococcus (cf. SEQ ID NO: 24) .
  • modified Cas variant e.g., Cas9 variants, can be used according to the methods of the present invention as part of a base editing complex, e.g.
  • CRISPR nucleases are envisaged, which might indeed not be any "nucleases” in the sense of double-strand cleaving enzymes, but which are nickases or nuclease-dead variants, which still have inherent DNA recognition and thus binding ability.
  • Exemplary Cas-or Cpf1-based constructs suitable for the purpose of the present invention are disclosed in SEQ ID NOs: 17 to 19.
  • An AsCpf1 wild-type sequence is disclosed in SEQ ID NO: 24.
  • Other suitable Cpf1-based effectors for use in the methods of the present invention are derived from Lachnospiraceae bacterium (LbCpf1, e.g., NCBI Reference Sequence: WP_051666128.1) , or from Francisella tularensis (FnCpf1, e.g., UniProtKB/Swiss-Prot: A0Q7Q2.1) .
  • Variants of Cpf1 are known (cf. Gao et al., BioRxiv, dx. doi. org/10.1101/091611) .
  • Variants of AsCpf1 with the mutations S542R/K607R and S542R/K548V/N552R that can cleave target sites with TYCV/CCCC and TATV PAMs, respectively, with enhanced activities in vitro and in vivo are thus envisaged as site-specific effectors according to the present invention.
  • Genome-wide assessment of off-target activity indicated that these variants retain a high level of DNA targeting specificity, which can be further improved by introducing mutations in non-PAM-interacting domains.
  • the at least one first targeted base modification is made by at least one base editing complex comprising at least one base editor as component.
  • the base editing complex according to the present invention comprises the base editor as well as further optional components.
  • the base editing complex contains an APOBEC1 component, preferably a rat APOBEC1.
  • the base editing complex can comprise any cytidine/cytosine deaminase enzyme as base editor, for example a human AID, e.g., UniProtKB/Swiss-Prot: Q9GZX7.1, a human APOBEC3G, e.g., GenBank: CAK54752.1, or a lamprey CDA1, e.g. GenBank: ABO15150.1, but any enzyme or catalytically active fragment thereof is envisaged within the scope of the present invention.
  • An exemplary APOBEC component suitable for use in the methods of the present invention is represented by SEQ ID NO: 20.
  • a modified base editor can be used according to the methods of the present invention, preferably a base editor having a narrow editing width of below 6 nt, below 5 nt, below 4 nt, below 3 nt, or event 2 nt or 1 nt.
  • the narrower the editing window the more precise an edit can be introduced at a genomic target site of interest.
  • the base editing complex contains an UGI (uracil DNA glycosylase inhibitor) component.
  • UGI uracil DNA glycosylase inhibitor
  • a UGI derived from Bacillus subtilis can be used, or any other domain inhibiting UDG activity to repress the activity of endogenous base-excision repair (BER) active in certain cells.
  • An exemplary UGI component suitable for use in the methods of the present invention is represented by SEQ ID NO: 21.
  • the base editing complex contains a XTEN component i.e., a specific linker to provide optimum deamination activity of the at least one base editor linked to the at least one site-specific effector.
  • a XTEN component i.e., a specific linker to provide optimum deamination activity of the at least one base editor linked to the at least one site-specific effector.
  • Other linkers having a length of at least 2 nucleotide (nt) between the base editor and the site-specific effector can be used, which do not influence the binding activity as conferred by the site-specific effector and/or the base editing activity of the base editor.
  • a suitable XTEN linker sequence is provided with SEQ ID NO: 1 (position 688 to 735) , SEQ ID NO: 2 (position 706 to 753) , SEQ ID NO: 14 (position 706 to 753) , or SEQ ID NO: 15 (position 706 to 753) .
  • further linkers known to the skilled person as well as literature on linker design. Both, rigid as well as flexible linkers can thus be used according to the various methods of the present invention.
  • Exemplary fusion constructs according to the present invention are provided with SEQ ID NOs: 1, 2, 14, 15, or 16.
  • the at least one base editing complex comprises more than one component, and wherein the at least two components are physically linked.
  • a physical linkage can comprise a covalent linkage, e.g., by fusing DNA fragments to each other to create a fusion protein after expression, or by chemically crosslinking different components of a complex according to the present disclosure to each other.
  • a physical linkage can additionally comprise a non-covalent interaction. Non-covalent interactions or attachments thus comprise electrostatic interactions, van der Waals forces, TT-effects and hydrophobic effects. Of special importance in the context of nucleic acid molecules are hydrogen bonds as electrostatic interaction.
  • a hydrogen bond is a specific type of dipole-dipole interaction that involves the interaction between a partially positive hydrogen atom and a highly electronegative, partially negative oxygen, nitrogen, sulfur, or fluorine atom not covalently bound to said hydrogen atom.
  • the base editing complex contains a PmCDA1 (activation-induced cytidine deaminase (AID) ortholog PmCDA1 from sea lamprey, see Nishida et al. (Science 2016, vol. 353, issue 6305, aaf8729) ) component as base editor.
  • PmCDA1 activation-induced cytidine deaminase (AID) ortholog PmCDA1 from sea lamprey, see Nishida et al. (Science 2016, vol. 353, issue 6305, aaf8729)
  • An exemplary PmCDA1 for use according to the methods of the present invention is provided with SEQ ID NO: 22.
  • CRISPR-based nucleases act via recognition of a protospacer-adjacent motif (PAM) present within a genomic target region of interest to be modified.
  • PAM protospacer-adjacent motif
  • wild-type CRISPR nucleases have intrinsic PAM specificities varying from nuclease to nuclease.
  • CRISPR-based nucleases are this envisaged, which have an altered PAM specificity and thus a modified targeting range, for example, SpCas9 mutants that accept NGA (VQR-Cas9) , NGAG (EQR-Cas9) , or NGCG (VRER-Cas9) PAM sequences, as well as an engineered SaCas9 variant containing three mutations (SaKKH-Cas9) that relax the variant’s PAM requirement to NNNRRT (Kleinstiver et al., Nat. Biotechnol. 33, 1293-1298 (2015) ) .
  • Exemplary PAM sequences according to the present invention suitable for different CRISPR-based nucleases are represented by SEQ ID NOs: 3 to 13 and 23.
  • the at least one base editing complex comprises more than one component, wherein the at least two components are provided as individual components. This approach can be suitable for certain transformation or transfection strategies.
  • At least one component of any complex according to the present invention can comprise a part or portion, which can specifically interact or associate with a cognate binding partner within a cell of interest so that a complex will form within the cell, or the complex can be formed ex vivo before transformation or transfection.
  • the binding pairs can associate via a docking domain or association domain, or the nucleic acid sequence encoding the same, which is selected from at least one of biotin, an aptamer, a DNA, RNA or protein dye, comprising fluorophores, comprising fluorescein, or a variant thereof, maleimides, or Tetraxolium (XTT) , a guide nucleic acid sequence specifically configured to interact with a at least one repair template nucleic acid sequence, a streptavidin, or a variant thereof, preferably a monomeric steptavidin, an avidin, or a variant thereof, an affinity tag, preferably a streptavidin-tag, an antibody, a single-chain variable fragment (scFv) , an antigen specific for a given antibody or scFv, a single-domain antibody (nanobody) , an anticalin, an Agrobacterium VirD2 protein or a domain thereof, a Picornavirus VPg, a topoisomerase or
  • the cognate binding partners have a high affinity constant or bonding affinity and thus a low dissociation constant (K d ) for each other under physiological conditions, i.e. a K d value in the low ⁇ M, or preferably nM range, and preferably below to assist in complex formation of the at least one base editing complex, or the at least one site-specific effector complex according to the present invention.
  • K d dissociation constant
  • At least one component of the at least one base editing complex, and/or at least one component of the at least one site-specific effector complex comprises at least one organelle localization signal to target the at least one base editing complex to a subcellular organelle.
  • the at least one organelle localization signal is a nuclear localization signal (NLS) .
  • the at least one organelle localization signal is a chloroplast transit peptide.
  • the at least one organelle localization signal is a mitochondria transit peptide.
  • One or more localization signal (s) can be present being associated with at least one component of the base editing, or the site-specific effector complex.
  • the first plant genomic target site of the at least one plant cell is a genomic target site encoding at least one phenotypically selectable trait, wherein the at least one phenotypically selectable trait is a resistance/tolerance trait or a growth advantage trait, and wherein the at least one first targeted base modification at the first plant genomic target site of the at least one plant cell confers resistance/tolerance or a growth advantage towards a compound or trigger to be added to the at least one modified plant cell, tissue or plant, or a progeny thereof.
  • a “growth advantage” as used herein refers to any physiologically or metabolically favourable property during all stages of plant development and reproduction, for example, favouring the resistance to biotic and abiotic stress, or influencing plant growth and development, e.g. under stress conditions like drought, salinity, and the like.
  • a “compound” or “trigger” according to the present invention can thus be a herbicide, for example being selected from cell metabolism inhibitors, for example: EPSPS inhibition (glycines, e.g., glyphosate) ; ALS/AHAS (branched amino acid production) inhibition (for example, imidazolines, sulfonylurea) ; lipid synthesis inhibition/ACCases (aryloxyphenoxypropionate (FOPs) , cyclohexanedione (DIMs) , phenylpyrazolin (DENs) ; inhibitors of glutamine synthetase (glufosinate/phosphinotricin) , growth/cell division inhibitors, for example, disruptors of plant cell growth (phenoxycarboxylic acids, e.g., 2, 4-D) , synthetic auxins (benzoic acid e.g., dicamba) , auxin transport inhibition (phtalamates) ; and interference with light processes
  • a “compound” or “trigger” can be a plant growth factor or any other substance, endogenously produced by a plant, or exogenously applied, which influences plant metabolism.
  • the compound or trigger can be exogenously applied to allow selection for a trait of interest, the phenotypically selectable trait encoded by the at least one plant cell, tissue, organ, material or whole plant, an modified in a targeted way according to the various methods of all aspects of the present invention.
  • the at least one phenotypically selectable trait of interest is or is encoded by at least one endogenous gene, or wherein the at least one phenotypic trait of interest is or is encoded by at least one transgene, wherein the at least one endogenous gene or the at least one transgene encode (s) at least one phenotypic trait selected from the group consisting of resistance/tolerance to a phytotoxin, preferably a herbicide, inhibiting, damaging or killing cells lacking the at least one modification at the at least one phenotypic trait of interest, or wherein the at least one phenotypic trait is selected from the group consisting of boosters of cell division, growth rate, embryogenesis, or another phenotypically selectable property that provides an advantage to a modified cell, tissue, organ, or plant compared to an unmodified cell, tissue, organ, or plant.
  • the at least one first plant genomic target site is at least one endogenous gene or a transgene encoding at least one phenotypically selectable trait selected from the group consisting of herbicide resistance/tolerance, wherein the herbicide resistance/tolerance is selected from the group consisting of resistance/tolerance to EPSPS-inhibitors, including glyphosate, resistance/tolerance to glutamine synthesis inhibitors, including glufosinate, resistance/tolerance to ALS-or AHAS-inhibitors, including imidazoline or sulfonylurea, resistance/tolerance to ACCase inhibitors, including aryloxyphenoxypropionate (FOP) , resistance/tolerance to carotenoid biosynthesis inhibitors, including inhibitors of carotenoid biosynthesis at the phytoene desaturase step, inhibitors of 4-hydroxyphenyl-pyruvate-dioxygenase (HPPD) , or
  • the at least one endogenous gene or the at least one transgene encode (s) at least one phenotypic trait selected from the group consisting of resistance/tolerance to biotic stress, including pathogen resistance/tolerance, wherein the pathogen is selected from a virus, a bacterial, fungal, or an animal pathogen, resistance/tolerance to abiotic stress, including chilling resistance/tolerance, drought stress resistance/tolerance, osmotic resistance/tolerance, heat stress resistance/tolerance, cold stress resistance/tolerance, oxidative stress resistance/tolerance, heavy metal stress resistance/tolerance, salt stress or waterlogging resistance/tolerance, lodging resistance/tolerance, shattering resistance/tolerance, or wherein the at least one phenotypic trait of interest is selected from the group consisting of the modification of a further agronomic trait of interest, including yield increase, flowering time modification, seed color modification, endosperm composition modification, nutritional
  • the at least one phenotypically selectable trait is a phytotoxic resistance/tolerance trait, preferably a herbicide resistance/tolerance trait, and wherein the at least one first targeted base modification at the first plant genomic target site of the at least one plant cell to be modified confers resistance/tolerance for a phytotoxic compound, preferably a herbicide, said compound being an exogenous compound to be added to the at least one modified plant cell, tissue, organ, or whole plant, or a progeny thereof.
  • Any further phenotypically selectable trait encoded by the genome of a plant cell of interest can be made the target of the at least one first targeted modification according to the various aspects of the present invention provided that at least one gene is known encoding a phenotypically selectable trait of interest, and provided that a corresponding and complementary compound or trigger is available or can be designed to screen for a targeted modification.
  • a suitable read-out and determination strategy based on the observation of visually screenable traits has to be at hand.
  • the first plant genomic target site of the at least one plant cell is a gene conferring resistance or tolerance to a herbicide or a phytotoxic compound, wherein the first plant genomic target site comprises at least one nucleic acid conversion resulting in at least one corresponding amino acid conversion, wherein the at least one nucleic acid conversion is made by at least one base editor.
  • the first plant genomic target site of the at least one plant cell is ALS.
  • Any ALS sequence is suitable for the purpose of the present invention.
  • An exemplary ALS sequence is represented by SEQ ID NO: 25.
  • the first plant genomic target site of the at least one plant cell is PPO.
  • Any PPO sequence is suitable for the purpose of the present invention.
  • An exemplary PPO sequence is represented by SEQ ID NO: 26.
  • the first plant genomic target site of the at least one plant cell is EPSPS.
  • Any EPSPS sequence is suitable for the purpose of the present invention.
  • An exemplary EPSPS sequence is represented by SEQ ID NO: 27.
  • the first plant genomic target site of the at least one plant cell is EPSPS, ALS, or PPO, or any allelic or plant variant thereof, and wherein the EPSPS, ALS or PPO comprises at least one nucleic acid conversion resulting in at least one corresponding amino acid conversion, wherein the at least one nucleic acid conversion is made by at least one base editor.
  • EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
  • ALS acetolactate synthase
  • Another target is the acetolactate synthase (ALS) gene, for which a variety of single amino acid mutations have been linked to tolerance to one or more herbicides from the classes triazolopyrimidines, sulfonylureas, pyrimidinylthiobenzonates, imidazolinones, and sulfonylaminocarbonyltriazolinone.
  • Suitable residue substitutions for the purpose of the present invention include A122, P197, A205, D376, W574, and S653) .
  • PPO protoporphyrinogen oxidase
  • the technology presented in the present application allows for the precise amino acid modification and deletion as well as the introduction of stop codons to alter or interrupt the sequence of gene that gives rise to a selectable phenotype.
  • stop codons Of 61 codons that encode for amino acids, five amino acids can be converted to a stop codon by at least one cytosine/cytidine to thymine/thymidine conversion on either strand.
  • CRISPR nuclease by itself.
  • CRISPR nucleases that were shown to provide single or multiple base pair deletions include Cas9, Cpf1, CasX, and CasY. Although these are the most convenient options at this point, future development of site-directed nucleases will easily be adaptable to the procedures described in this document.
  • the first plant genomic target site of the at least one plant cell is ALS
  • a targeted modification occurs at the sequence encoding A122 in comparison to an ALS reference sequence according to SEQ ID NO: 25, or a targeted modification occurs at the sequence encoding P197 in comparison to an ALS reference sequence according to SEQ ID NO: 25, or a targeted modification occurs at the sequence encoding A205 in comparison to an ALS reference sequence according to SEQ ID NO: 25, or a targeted modification occurs at the sequence encoding D376 in comparison to an ALS reference sequence according to SEQ ID NO: 25, or a targeted modification occurs at the sequence encoding R377 in comparison to an ALS reference sequence according to SEQ ID NO: 25, or a targeted modification occurs at the sequence encoding W574 in comparison to an ALS reference sequence according to SEQ ID NO: 25, or a targeted modification occurs at the sequence encoding S653 in comparison to an ALS reference sequence according to SEQ ID NO: 25, or a targeted modification occurs at the sequence encoding
  • the first plant genomic target site of the at least one plant cell is PPO, and a targeted modification occurs at the sequence encoding C215, A220, G221, N425, or Y426 in comparison to an PPO reference sequence according to SEQ ID NO: 26, or any combination of the aforementioned mutations.
  • the first plant genomic target site of the at least one plant cell is PPX2L gene product from Amaranthus tuberculatus for the purpose of selection.
  • the first targeted modification comprising a targeted base modification, a targeted codon deletion, or a targeted frameshift or deletion modification, occurs at the position comparable to the G210 residue of the PPX2L gene product from Amaranthus tuberculatus according to SEQ ID NO: 28.
  • the first plant genomic target site of the at least one plant cell is EPSPS, and at least one targeted modification occurs at any one of targeted modifications occurs at the sequence encoding G101, T102, P106, G144, or A192 in comparison to an EPSPS reference sequence according to SEQ ID NO: 27, or any combination of the aforementioned mutations.
  • targeted modifications occur at the sequence encoding G101 and at G144 in comparison to an EPSPS reference sequence according to SEQ ID NO: 27, or targeted modifications occur at the sequence encoding G101 and at A192 in comparison to an EPSPS reference sequence according to SEQ ID NO: 27, or targeted modifications occur at the sequence encoding T102 and at P106 in comparison to an EPSPS reference sequence according to SEQ ID NO: 27.
  • the at least one phenotypically selectable trait is a visible phenotype that is useful in identifying or isolating at least one modified plant cell, tissue, organ or whole plant.
  • a "visible" phenotype is any phenotype which can be detected by means of observation with the eyes, either macroscopically or microscopically, so that no screening by means of molecular biology becomes necessary.
  • the at least one phenotypically selectable trait is a glossy phenotype, a golden phenotype, a pigmentation phenotype, or a growth advantage phenotype.
  • a glossy phenotype is known to the skilled person. Said visible phenotypes will vary depending on the plant or plant cell of interest due to its genetic background.
  • a method for isolating at least one modified plant cell or at least one modified plant tissue, organ, or whole plant comprising the at least one modified plant cell, without stably integrating a transgenic selectable marker sequence comprising: (a) introducing at least one first targeted codon deletion modification into a first plant genomic target site of at least one plant cell to be modified using at least one first site-specific effector, comprising a nuclease, a recombinase, or a DNA modification reagent, wherein the at least one targeted codon deletion modification causes expression of at least one phenotypically selectable trait; (b) introducing at least one second targeted modification into a second plant genomic target site of the at least one plant cell to be modified, wherein the at least one second targeted modification is introduced using at least one second site-specific effector to create the at least one second targeted modification at the second plant genomic target site, wherein the at least one second targeted modification is introduced simultaneously or subsequently to the introduction of
  • a method for isolating at least one modified plant cell or at least one modified tissue, organ, or whole plant comprising the at least one modified plant cell, without stably integrating a transgenic selectable marker sequence
  • the method comprising: (a) introducing at least one first targeted frameshift or deletion modification into a first plant genomic target site of at least one plant cell to be modified using at least one first site-specific effector, wherein the at least one targeted frameshift or deletion modification causes expression of at least one phenotypically selectable trait; (b) introducing at least one second targeted modification into a second plant genomic target site of the at least one plant cell to be modified, wherein the at least one second targeted modification is introduced using at least one second site-specific effector, comprising a nuclease, a recombinase, or a DNA modification reagent, to create the at least one second targeted modification at the second plant genomic target site, wherein the at least one second targeted modification is introduced simultaneously or subsequently to the introduction
  • the methods according to the present invention provide a new way of combining two different molecular complexes, one complex being configured to introduce at least one first targeted modification resulting in a selectable phenotype without inserting a transgenic marker, and the other complex configured to introducing at least one second targeted modification, wherein the first modification serves for screening purposes, whilst the second modification represents a genomic edit to be introduced. Therefore, the methods of the present invention synergistically combine genome editing strategies at different genomic target sites to achieve different targeted modifications ultimately resulting in an efficient breeding process to achieve a plant having a genotype of interest.
  • step b. of the methods of the present invention additionally comprises introducing a repair template (RT) to make a targeted sequence conversion or replacement at the at least one first and/or second plant genomic target site.
  • RT repair template
  • This RT adds another level of precision to the genome editing approach, as the provision of a suitable RT, provided separately, or as part of at least one complex according to the present invention, as the break resulting from a nuclease or nickase can be repaired in a predetermined way by providing a RT of interest to assist homology-directed repair instead of relying on an error prone endogenous NHEJ pathway as repair mechanism.
  • a CRISPR-based nuclease is used as site-specific effector interacting with a gRNA, wherein the gRNA can be covalently linked to a RT, or wherein the CRISPR-based nuclease and/or the gRNA interact non-covalently with the RT.
  • the RT is provided separately, including addition on a construct encoding a RT of interest, and the RT will associate with a site-specific effector complex by means of complementary base pairing mediated by homology arms within the RT annealing to at least one genomic target site of interest.
  • a fusion protein or a non-covalently associated active Cpf1 and an inactive dCas9 as interaction domain can be provided as site-specific effector.
  • the gRNA for Cas9 can target the repair template or an extension thereof, forming a Cpf1-dCas9-RT complex.
  • the crRNA (Cpf1) targets the genomic locus defined for the double strand cut to initiate HDR.
  • a highly active zinc finger protein, a megaTAL or an inactive meganuclease can be used.
  • a plant cell, tissue, organ, material or whole plant, or a progeny thereof, obtainable by any one of the methods disclosed herein is provided.
  • the methods provided herein are specifically designed to assist in the provision of new plants having agronomically favorable traits, but do not comprise a transgenic marker sequence, the methods disclosed herein are suitable for creating a variety of different plant genotypes in a fast and reliable way.
  • the at least one plant cell to be modified is preferably being derived from a plant selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea spp., including Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticum durum, Secale cereale, Triticale, Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta spp., including Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Nicotiana bent
  • the present invention provides a method of generating a genetically modified plant by genome editing, the method comprising the steps of:
  • first genome editing system can target and modify a plant selectable marker gene
  • second genome modification system can target and modify a gene of interest in the plant
  • step d) selecting plants in which the selectable marker gene has been modified from the plants regenerated in step d) ;
  • step f) identifying a plant whose target gene is modified from the plants selected in step e) .
  • the cells or tissues of the plant include any cells or tissues that can be regenerated into intact plants, such as protoplasts, callus, explants, immature embryos, and the like.
  • genetical modification includes altering the sequence of a gene and/or altering the expression of a gene.
  • the term "gene of interest” means any nucleotide sequence to be modified in a plant, including both structural and non-structural genes.
  • the gene of interest is associated with a trait of the plant, preferably a agronomic trait.
  • selectable marker gene means a plant endogenous gene that, after suitably modified, confers the plant a selectable trait that can be selected. Preferably, when suitably modified, the selectable marker gene does not substantially altering other traits of the plant.
  • the selectable marker gene may be a plant endogenous herbicide resistance gene, which confer herbicide resistance to the plant when suitably modified.
  • the plant endogenous herbicide resistance genes include but are not limited to PsbA, ALS, EPSPS, ACCase, PPO, and HPPD, PDS, GS, DOXPS, and P450.
  • the ALS mutation sites capable of conferring herbicide resistance include, but are not limited to, A122, P197, A205, and S653 (the amino acid numbering refers to the amino acid sequence of the ALS in Arabidopsis thaliana) .
  • the EPSPS mutation sites capable of conferring herbicide resistance include, but are not limited to, T102, P106 (amino acid numbering refers to the EPSPS amino acid sequence in Arabidopsis thaliana) .
  • ACCase mutation sites capable of conferring herbicide resistance include, but are not limited to, I1781, W2027, I2041, D2078, and G2096 (amino acid numbering refers to the amino acid sequence of the chloroplast ACCase in Alopecurus myosuroides) .
  • HPPD mutation sites capable of conferring herbicide resistance include, but are not limited to, P277, L365, G417, and G419 (amino acid numbering refers to the amino acid sequence of the HPPD enzyme in rice) .
  • the ALS mutation site capable of conferring herbicide resistance in wheat includes TaALS P173. In some embodiments, the ALS mutation site capable of conferring herbicide resistance in corn includes ZmALS P165. In some embodiments, the ALS mutation site capable of conferring herbicide resistance in rice includes OsALS P171.
  • the selectable marker gene may be a gene that, when modified appropriately, causes the plant to produce visually-observable trait changes, such as genes controlling ligule, leaf color, leaf wax, including but not limited to LIG, PDS, zb7, and GL2.
  • transgenic methods require the application of certain selective pressures during plant regeneration (eg, screening using different antibiotics depending on the transgene vector used) to increase the efficiency.
  • certain selective pressures e.g, screening using different antibiotics depending on the transgene vector used
  • this will lead to the integration of foreign genes, in particular antibiotic resistance genes, in the plant genome, resulting in potential safety issues.
  • the genome editing system can achieve the target gene modification without integration into the plant genome.
  • the regeneration of step d) is preferably carried out without selective pressure. This avoids the integration of foreign genes and results in genetically modified (genomically edited) transgenic plants. However, regeneration of plants without selective pressure will greatly reduce screening efficiency.
  • This problem is solved in the present invention by co-transforming a genome editing system that targets the gene of interest and a genome editing system that targets the endogenous selectable marker gene.
  • a genome editing system that targets the gene of interest and a genome editing system that targets the endogenous selectable marker gene are co-transformed into a plant (such as a plant cell or tissue) , then editing of the gene of interest and endogenous selectable marker genes will tend to occur together. Therefore, a plant selected based on an endogenous selectable marker gene will have a high probability that its gene of interest will also be modified.
  • the first screen for the editing of endogenous selectable marker genes will greatly improve the screening efficiency of editing of the gene of interest. And, because only endogenous selectable marker genes are used, transgene concerns are avoided.
  • the endogenous selectable marker gene preferably does not affect the trait of interest after being modified, for example, does not reduce yield and the like. More preferably, the modification of the endogenous selectable marker gene confers the plant additional traits of interest, such as herbicide resistance. That is, it is preferred that the traits available for selection of plants in the present invention are also agronomically useful traits such as herbicide resistance.
  • the method of performing the selection in step e) depends on the nature of the selectable marker gene. For example, if the selectable marker gene is modified to confer herbicide resistance to the plant, the regenerated plant can be placed at a suitable concentration at which the plant having the wild-type selectable marker gene cannot survive or grow poorly. Then, plants that survive or grow well at this concentration of herbicide are selected.
  • the identification in step f) can be performed by, for example, PCR/RE, or sequencing methods.
  • PCR/RE PCR/RE
  • sequencing methods The person skilled in the art is well acquainted with how to identify whether a gene has been mutated or not.
  • Suitable methods for transforming a plant (cell or tissue) of the present invention include, but are not limited to, particle bombardment, PEG-mediated protoplast transformation, and Agrobacterium-mediated transformation.
  • genome editing systems suitable for use with the present invention include, but are not limited to, precise base editor (PBE) systems, CRISPR-Cas9 systems, CRISPR-Cpfl systems, CRISPRi systems, zinc finger nuclease systems, and TALEN systems.
  • PBE precise base editor
  • CRISPR-Cas9 systems CRISPR-Cpfl systems
  • CRISPRi systems zinc finger nuclease systems
  • TALEN systems TALEN systems.
  • CRISPR systems are produced by bacteria during evolution to protect against foreign gene invasion. It has been modified and widely used in genome editing of eukaryotes.
  • CRISPR-Cas9 system refers to a Cas9 nuclease-based genome CRISPR editing system.
  • Cas9 nuclease and “Cas9” are used interchangeably herein and refer to an RNA Guided nuclease that include a Cas9 protein or fragment thereof (eg, a protein comprising the active DNA cleavage domain of Cas9 and/or the gRNA binding domain of Cas9) .
  • Cas9 is a component of the prokaryotic immune system of CRISPR/Cas that can target and cleave DNA target sequences to form DNA double-strand breaks (DSBs) under the guidance of guide RNA.
  • DSBs DNA double-strand breaks
  • CRISPR-Cas9 systems suitable for use in the present invention include, but are not limited to, those described in Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31, 686-688 (2013) .
  • guide RNA and “gRNA” can be used interchangeably herein, which typically are composed of crRNA and tracrRNA molecules forming complexes through partial complement, wherein crRNA comprises a sequence that is sufficiently complementary to a target sequence for hybridization and directs the CRISPR complex (Cas9+crRNA+tracrRNA) to specifically bind to the target sequence.
  • CRISPR complex Cas9+crRNA+tracrRNA
  • sgRNA single guide RNA
  • the CRISPR-Cas9 system of the present invention may include one of the following:
  • an expression construct comprising a nucleotide sequence encoding a Cas9 protein, and a guide RNA;
  • a Cas9 protein a Cas9 protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA
  • an expression construct comprising a nucleotide sequence encoding a Cas9 protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
  • v) an expression construct comprising a nucleotide sequence encoding a Cas9 protein and a nucleotide sequence encoding a guide RNA.
  • the CRISPR-Cpf1 system is a CRISPR genome editing system based on the Cpf1 nuclease.
  • the difference between Cpf1 and Cas9 is that the molecular weight of the Cpf1protein is small, and only crRNA is required as the guide RNA, and the PAM sequence is also different.
  • the CRISPR-Cpf1 system suitable for use in the present invention includes, but is not limited to, the system described in Tang et al., 2017.
  • the CRISPR-Cpfl system of the present invention may include one of the following:
  • an expression construct comprising a nucleotide sequence encoding a Cpf1 protein, and a guide RNA;
  • a Cpf1 protein a Cpf1 protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA
  • an expression construct comprising a nucleotide sequence encoding a Cpf1 protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
  • v an expression construct comprising a nucleotide sequence encoding a Cpf1 protein and a nucleotide sequence encoding a guide RNA.
  • CRISPR interference is a gene silencing system derived from the CRISPR-Cas9 system that uses a nuclease-inactivated Cas9 protein. Although this system does not change the sequence of the target gene, it is also defined herein as a genome editing system. CRISPRi systems suitable for use with the present invention include, but are not limited to, the system described in Seth and Harish, 2016.
  • the CRISPRi system of the present invention may include one of the following:
  • nuclease-inactivated Cas9 protein i) a nuclease-inactivated Cas9 protein, and a guide RNA
  • an expression construct comprising a nucleotide sequence encoding a nuclease-inactivated Cas9 protein, and a guide RNA;
  • nuclease-inactivated Cas9 protein a nuclease-inactivated Cas9 protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
  • an expression construct comprising a nucleotide sequence encoding a nuclease-inactivated Cas9 protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
  • v) an expression construct comprising a nucleotide sequence encoding a nuclease-inactivated Cas9 protein and a nucleotide sequence encoding a guide RNA.
  • the precise base editor system is a system that has recently been developed based on CRISPR-Cas9, which enables accurate single-base editing of a genome using a nuclease-inactivated fusion protein of Cas9 protein and cytidine deaminase.
  • Nuclease-inactivated Cas9 (due to mutations in the HNH subdomain and/or RuvC subdomain of the DNA cleavage domain) retains gRNA-directed DNA-binding ability, and the cytidine deaminase can catalyze deamination of cytidine (C) on DNA to form uracil (U) .
  • the nuclease-inactivated Cas9 is fused with a cytidine deaminase.
  • the fusion protein can target the target sequence in the plant genome. Due to the absence of the Cas9 nuclease activity, the DNA double strand is not cleaved.
  • the deaminase domain in the fusion protein converts the cytidine of the single-stranded DNA produced in the formation of the Cas9-gRNA-DNA complex to U, and the substitution of C to T is achieved by base mismatch repair.
  • the precise base editor system suitable for use in the present invention includes, but is not limited to, the system described in Zong et al., 2017.
  • the precise base editor system of the present invention may include one of the following:
  • a fusion protein of nuclease-inactivated Cas9 and cytidine deaminase, and guide RNA i) a fusion protein of nuclease-inactivated Cas9 and cytidine deaminase, and guide RNA;
  • an expression construct comprising the nucleotide sequence encoding a fusion protein of a nuclease-inactivated Cas9 protein and a cytidine deaminase, and a guide RNA;
  • a fusion protein of nuclease-inactivated Cas9 protein and cytidine deaminase a fusion protein of nuclease-inactivated Cas9 protein and cytidine deaminase, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
  • an expression construct comprising a nucleotide sequence encoding a fusion protein of a nuclease-inactivated Cas9 protein and a cytidine deaminase, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
  • v) an expression construct comprising a nucleotide sequence encoding a fusion protein of a nuclease-inactivated Cas9 protein and a cytidine deaminase and a nucleotide sequence encoding a guide RNA.
  • the nuclease-inactivated Cas9 protein comprises amino acid substitutions D10A and/or H840A relative to wild-type Cas9 (S. pyogenes SpCas9) .
  • the cytidine deaminase include, but are not limited to, APOBEC1 deaminase, activation-induced cytidine deaminase (AID) , APOBEC3G, or CDA1 (PmCDA1) .
  • ZFN Zac finger nuclease
  • ZFN Zac finger nuclease
  • the zinc finger DNA binding domain of a single ZFN typically contains 3-6 individual zinc finger repeats, each zinc finger repeat recognizing, for example, 3 bp.
  • ZFN systems suitable for use in the present invention can be obtained, for example, from Shukla et al., 2009 and Townsend et al., 2009.
  • Transactivator-like effector nucleases are restriction enzymes that can be engineered to cleave specific DNA sequences, usually prepared by fusion of the DNA binding domain of the transcriptional activator-like effector (TALE) and a DNA cleavage domain. TALE can be engineered to bind almost any desired DNA sequences.
  • TALE transcriptional activator-like effector
  • the TALEN system suitable for use in the present invention can be obtained, for example, from Li et al., 2012.
  • Those skilled in the art can appropriately determine the combination of the first genome editing system and the second genome editing system in the method of the present invention according to the respective characteristics of different genome editing systems and the specific type of genome editing desired to be implemented, for example, selecting a suitable combination to avoid interference with each other, for example, interference between different systems that can share a same gRNA.
  • the CRISPR-Cas9 system is generally not used to target the gene of interest because the two systems can share a same gRNA and thus Cas9 for knockout of the gene of interest may also knock out the endogenous selectable marker gene, vice versa.
  • both the first and second genome editing systems are precise base editor systems.
  • the components of the first and second genome editing systems may be expressed by the same expression construct or by different expression constructs, which can be conveniently selected by those skilled in the art.
  • guide RNAs for a gene of interest and a selectable marker gene can be transcribed with the same expression construct.
  • the components of the first and second genome editing systems are expressed by the same expression construct.
  • the first and second genome editing systems are both precise base editor systems, and wherein fusion protein of nuclease-inactivated Cas9 protein and cytidine deaminase and guide RNAs for gene of interest and the selectable marker gene are expressed by a same expression construct.
  • the plant is monocotyledonous or dicotyledonous.
  • the plant is selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea spp., including Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticum durum, Secale cereale, Triticale, Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta spp., including Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Nico
  • the plant is a crop plant.
  • the method further comprises obtaining progeny of the genetically modified transgene-free plant.
  • the present invention also provides a genetically modified plant or a progeny thereof or a part thereof, wherein the plant is obtained by the above method of the present invention.
  • the present invention also provides a plant breeding method comprising crossing a first genetically modified plant obtained by the above method of the present invention with a second plant not containing the genetic modification, thereby introducing said genetic modification into the second plant.
  • the screening efficiency of genetically modified transgene-free plants can be greatly improved.
  • the screening efficiency of transgene-free mutants can be improved by about 10-100 times for a gene of interest having a mutation rate of less than 1%.
  • a variety of suitable delivery techniques for introducing genetic material into a plant cell are known to the skilled person., e.g. by choosing direct delivery techniques ranging from polyethylene glycol (PEG) treatment of protoplasts (Potrykus et al. 1985) , procedures like electroporation (D′Halluin et al., 1992) , microinjection (Neuhaus et al., 1987) , silicon carbide fiber whisker technology (Kaeppler et al., 1992) , viral vector mediated approaches (Gelvin, Nature Biotechnology 23, "Viral-mediated plant transformation gets a boost” , 684-685 (2005) ) and particle bombardment (see e.g. Sood et al., 2011, Biologia Plantarum, 55, 1-15) .
  • PEG polyethylene glycol
  • Physical means finding application in plant biology are particle bombardment, also named biolistic transfection or microparticle-mediated gene transfer, which refers to a physical delivery method for transferring a coated microparticle or nanoparticle comprising a nucleic acid or a genetic construct of interest into a target cell or tissue.
  • Physical introduction means are suitable to introduce nucleic acids, i.e., RNA and/or DNA, and proteins.
  • specific transformation or transfection methods exist for specifically introducing a nucleic acid or an amino acid construct of interest into a plant cell, including electroporation, microinjection, nanoparticles, and cell-penetrating peptides (CPPs) .
  • CPPs cell-penetrating peptides
  • chemical-based transfection methods exist to introduce genetic constructs and/or nucleic acids and/or proteins, comprising inter alia transfection with calcium phosphate, transfection using liposomes, . e.g., cationic liposomes, or transfection with cationic polymers, including DEAD-dextran or polyethylenimine, or combinations thereof.
  • Said delivery methods and delivery vehicles or cargos thus inherently differ from delivery tools as used for other eukaryotic cells, including animal and mammalian cells and every delivery method has to be specifically fine-tuned and optimized so that a construct of interest for mediating genome editing can be introduced into a specific compartment of a target cell of interest in a fully functional and active way.
  • the above delivery techniques can be used to insert the at least one molecular complex according to the present invention, i.e., a base editor complex and/or a site-specific effector complex, or at least one subcomponent thereof, i.e., at least one SSN, at least one gRNA, at least one RT, or at least one base editor, or the sequences encoding the aforementioned subcomponents, according to the present invention into a target cell, in vivo or in vitro.
  • a base editor complex and/or a site-specific effector complex i.e., at least one SSN, at least one gRNA, at least one RT, or at least one base editor, or the sequences encoding the aforementioned subcomponents, according to the present invention into a target cell, in vivo or in vitro.
  • Physical and chemical delivery methods are particularly preferred according to the present invention, as said methods allow the co-delivery and thus the parallel introduction of various tools of interest into at least one plant cell.
  • the crRNA portion of the gRNA comprises a stem loop or an optimized stem loop structure or an optimized secondary structure.
  • the mature crRNA comprises a stem loop or an optimized stem loop structure in the direct repeat sequence, wherein the stem loop or optimized stem loop structure is important for cleavage activity.
  • the mature crRNA preferably comprises a single stem loop.
  • the direct repeat sequence preferably comprises a single stem loop.
  • the cleavage activity of the effector protein complex is modified by introducing mutations that affect the stem loop RNA duplex structure.
  • mutations which maintain the RNA duplex of the stem loop may be introduced, whereby the cleavage activity of the effector protein complex is maintained.
  • mutations which disrupt the RNA duplex structure of the stem loop may be introduced, whereby the cleavage activity of the effector protein complex is completely abolished.
  • the methods according to the various aspects of the present invention are not restricted to a first and/or second targeted modification being a modification within a coding region encoding an amino acid.
  • the modification of a regulatory sequence is envisaged as well. Any modification having an epigenetic effect can also be addressed by the methods of the present invention.
  • the at least one genomic target sequence to be modified can be a regulatory sequence such as a promoter wherein the editing of the promoter comprises replacing the promoter, or promoter fragment with a different promoter (also referred to as replacement promoter) or promoter fragment (also referred to as replacement promoter fragment) , wherein the promoter replacement results in any one of the following or any one combination of the following: an increased promoter activity, an increased promoter tissue specificity, a decreased promoter activity, a decreased promoter tissue specificity, a new promoter activity, an inducible promoter activity, an extended window of gene expression, a modification of the timing or developmental progress of gene expression in the same cell layer or other cell layer , for example, extending the timing of gene expression in the tapetum of anthers, a mutation of DNA binding elements and/or a deletion or addition of DNA binding elements.
  • a regulatory sequence such as a promoter wherein the editing of the promoter comprises replacing the promoter, or promoter fragment with a different promoter (also referred to as replacement promoter
  • the promoter (or promoter fragment) to be modified can be a promoter (or promoter fragment) that is endogenous, artificial, pre-existing, or transgenic to the cell that is being edited.
  • the replacement promoter or fragment thereof can be a promoter or fragment thereof that is endogenous, artificial, pre-existing, or transgenic to the cell that is being edited.
  • the at least one genomic target sequence can be a promoter wherein the editing of the promoter comprises replacing a native EPSPS1 promoter from with a plant ubiquitin promoter.
  • the at least one genomic target sequence to be modified can be a promoter wherein the promoter to be edited is selected from the group comprising a Zea mays-PEPC1 promoter (Kausch et al., Plant Molecular Biology, 45: 1-15, 2001) , a Zea mays ubiquitin promoter (UBI1ZM PRO, Christensen et al., plant Molecular Biology 18: 675-689, 1992) , a rice actin promoter (McElroy et al., The Plant Cell, Vol 2, 163-171, February 1990) , a Zea mays-GOS2 promoter (U.S. Pat. No. 6,504,083) , or a Zea mays oleosin promoter (U.S. Pat. No. 8,466,341) .
  • the at least one site-specific effector complex can be used in combination with a co-delivered RT to allow for the insertion of a promoter or promoter element into a genomic nucleotide sequence of interest without incorporating a selectable transgene marker, wherein the promoter insertion (or promoter element insertion) results in any one of the following or any one combination of the following: an increased promoter activity. i.e., increased promoter strength, increased promoter tissue specificity, a decreased promoter activity, a decreased promoter tissue specificity, a new promoter activity, an inducible promoter activity, an extended window of gene expression, a modification of the timing or developmental progress of gene expression a mutation of DNA binding elements and/or an addition of DNA binding elements.
  • an increased promoter activity i.e., increased promoter strength, increased promoter tissue specificity, a decreased promoter activity, a decreased promoter tissue specificity, a new promoter activity, an inducible promoter activity, an extended window of gene expression, a modification of the timing
  • Promoter elements to be inserted can be, but are not limited to, promoter core elements, such as, but not limited to, a CAAT box, a CCAAT box, a Pribnow box, a and/or TATA box, translational regulation sequences and/or a repressor system for inducible expression, such as TET operator repressor/operator/inducer elements, or sulphonylurea repressor/operator/inducer elements.
  • promoter core elements such as, but not limited to, a CAAT box, a CCAAT box, a Pribnow box, a and/or TATA box, translational regulation sequences and/or a repressor system for inducible expression, such as TET operator repressor/operator/inducer elements, or sulphonylurea repressor/operator/inducer elements.
  • the dehydration-responsive element was first identified as a cis-acting promoter element in the promoter of the drought-responsive gene rd29A, which contains a 9 bp conserved core sequence, TACCGACAT (Yamaguchi-Shinozaki, K., and Shinozaki, K. (1994) Plant Cell 6, 251-264) . Insertion of DRE into an endogenous promoter may confer a drought inducible expression of the downstream gene.
  • Another example is ABA-responsive elements (ABREs) which contains a (C/T) ACGTGGC consensus sequence found to be present in numerous ABA and/or stress-regulated genes (Busk P. K., Pages M. (1998) Plant Mol. Biol. 37: 425-435) .
  • the promoter, or promoter element, to be inserted can be a promoter, or promoter element, that is endogenous, artificial, pre-existing, or transgenic to the cell that is being edited.
  • the at least one site-specific effector complex can be used to insert an enhancer element, such as but not limited to a Cauliflower Mosaic Virus 35 S enhancer, in front of an endogenous FMT1 promoter to enhance expression of the FTM1.
  • an enhancer element such as but not limited to a Cauliflower Mosaic Virus 35 S enhancer
  • the at least one site-specific effector complex can be used to insert a component of the TET operator repressor/operator/inducer system, or a component of the sulphonylurea repressor/operator/inducer system into plant genomes to generate or control inducible expression systems without incorporating a selectable transgene marker.
  • the at least one site-specific effector complex can be used to allow for the deletion of a promoter or promoter element, wherein the promoter deletion (or promoter element deletion) results in any one of the following or any one combination of the following: a permanently inactivated gene locus, an increased promoter activity (increased promoter strength) , an increased promoter tissue specificity, a decreased promoter activity, a decreased promoter tissue specificity, a new promoter activity, an inducible promoter activity, an extended window of gene expression, a modification of the timing or developmental progress of gene expression, a mutation of DNA binding elements and/or an addition of DNA binding elements.
  • Promoter elements to be deleted can be, but are not limited to, promoter core elements, promoter enhancer elements or 35S enhancer elements.
  • the promoter or promoter fragment to be deleted can be endogenous, artificial, pre-existing, or transgenic to the cell that is being edited.
  • the at least one genomic target site of interest to be modified can be a terminator wherein the editing of the terminator comprises replacing the terminator, also referred to as a "terminator swap" or “terminator replacement” , or terminator fragment with a different terminator, also referred to as replacement terminator, or terminator fragment, also referred to as replacement terminator fragment, wherein the terminator replacement results in any one of the following or any one combination of the following: an increased terminator activity, an increased terminator tissue specificity, a decreased terminator activity, a decreased terminator tissue specificity, a mutation of DNA binding elements and/or a deletion or addition of DNA binding elements.
  • the terminator or fragment thereof to be modified can be a terminator that is endogenous, artificial, pre-existing, or transgenic to the cell that is being edited.
  • the replacement terminator can be a terminator or fragment thereof that is endogenous, artificial, pre-existing, or transgenic to the cell that is being edited.
  • the at least one genomic target site of interest to be modified can be a terminator wherein the terminator to be edited is selected from the group comprising terminators from maize Argos 8 or SRTF18 genes, or other terminators, such as potato PinII terminator, sorghum actin terminator (WO 2013/184537 A1) , rice T28 terminator (WO 2013/012729 A2) , AT-T9 TERM (WO 2013/012729 A2) or GZ-W64A TERM (U.S. Pat. No. 7,053,282) .
  • terminators from maize Argos 8 or SRTF18 genes or other terminators, such as potato PinII terminator, sorghum actin terminator (WO 2013/184537 A1) , rice T28 terminator (WO 2013/012729 A2) , AT-T9 TERM (WO 2013/012729 A2) or GZ-W64A TERM (U.S. Pat. No. 7,053,282) .
  • the at least one site-specific effector complex according to the present invention can be used in combination with a co-delivered RT sequence to allow for the insertion of a terminator or terminator element into a genomic nucleotide sequence of interest, wherein the terminator (element) insertion results in any one of the following or any one combination of the following: an increased terminator activity, i.e., increased terminator strength, an increased terminator tissue specificity, a decreased terminator activity, a decreased terminator tissue specificity, a mutation of DNA binding elements and/or an addition of DNA binding elements.
  • an increased terminator activity i.e., increased terminator strength, an increased terminator tissue specificity, a decreased terminator activity, a decreased terminator tissue specificity, a mutation of DNA binding elements and/or an addition of DNA binding elements.
  • the terminator or element or fragment thereof to be inserted can be a terminator (or terminator element) that is endogenous, artificial, pre-existing, or transgenic to the cell that is being edited.
  • the at least one site-specific effector complex can be used to allow for the deletion of a terminator or terminator element, wherein the terminator deletion (or terminator element deletion) results in any one of the following or any one combination of the following: an increased terminator activity (increased terminator strength) , an increased terminator tissue specificity, a decreased terminator activity, a decreased terminator tissue specificity, a mutation of DNA binding elements and/or an addition of DNA binding elements.
  • the terminator or terminator fragment to be deleted can be endogenous, artificial, pre-existing, or transgenic to the cell that is being edited.
  • the at least one site-specific effector complex of the present invention can be used to modify or replace a regulatory sequence in the genome of a cell without incorporating a selectable transgene marker.
  • a regulatory sequence is a segment of a nucleic acid molecule which is capable of increasing or decreasing the expression of specific genes within an organism and/or is capable of altering tissue specific expression of genes within an organism.
  • regulatory sequences include, but are not limited to, 3′UTR (untranslated region) region, 5′UTR region, transcription activators, transcriptional enhancers transcriptions repressors, translational repressors, splicing factors, miRNAs, siRNA, artificial miRNAs, promoter elements, CAMV 35 S enhancer, MMV enhancer elements, SECIS elements, polyadenylation signals, and polyubiquitination sites.
  • the editing in the form of at least one targeted modification of the present invention, or the replacement of a regulatory element results in altered protein translation, RNA cleavage, RNA splicing, transcriptional termination or post translational modification.
  • regulatory elements can be identified within a promoter and these regulatory elements can be edited or modified do to optimize these regulatory elements for up or down regulation of the promoter.
  • the at least one genomic target site of interest to be modified is a polyubiquitination site, wherein the modification of the polyubiquitination sites results in a modified rate of protein degradation.
  • the ubiquitin tag condemns proteins to be degraded by proteasomes or autophagy. Proteasome inhibitors are known to cause a protein overproduction. Modifications made to a DNA sequence encoding a protein of interest can result in at least one amino acid modification of the protein of interest, wherein said modification allows for the polyubiquitination of the protein (a post translational modification) resulting in a modification of the protein degradation.
  • the at least one genomic target site of interest to be modified is a polyubiquitination site on a maize EPSPS gene, wherein the polyubiquitination site modified resulting in an increased protein content due to a slower rate of EPSPS protein degradation.
  • the at least one genomic target site of interest to be modified is a an intron site, wherein the modification consist of inserting an intron enhancing motif into the intron which results in modulation of the transcriptional activity of the gene comprising said intron.
  • a plasmid encoding APOBEC–XTEN–Cas9 (nickase) –UGI (SEQ ID NO: 1 and SEQ ID NO: 2) was constructed by standard methods and the base editor and sgRNA were transiently expressed in cells derived from Zea mays tissues. Together with the complex, gRNAs designed for examples 2 to 6 were tested. Furthermore, specific PAM motifs (see SEQ ID NOs: 3 to 13 and 23) were defined in relation to a target site of interest.
  • the SaKKH-BE3 and VQR-BE3 proteins ( Komor A. et al., Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusion, Nat. Biotech. (2017) ) was be codon-optimized for expression in corn, synthesized, and cloned into a plasmid together with the appropriate sgRNAs for expression in the same corn cell systems.
  • Total genomic DNA was extracted from the cell populations 12-96 hours after treatment with the base editor expressing plasmids and subjected to targeted deep sequencing to analyze the frequency and pattern of base conversions at the targets.
  • Example 2 Transformation of base editing components and selection against sulfonylureas or imidazolinones
  • the base editors described in Example 1 and using several specifically designed gRNAs targeted to the corn ALS1, ALS2 genes that were validated by NGS in Example 1 were transformed into tissues from Zea mays and regenerated on selection media either containing a sulfonylurea (for P197 or S653 substitutions) or an imidazolinone (for S653 substitutions) .
  • a herbicide resistant plant will have undergone a base conversion due to the action of the base editor, resulting in a substitution of the proline in position 197 or the serine in position 653, depending on which base editor was delivered.
  • the ALS genes in herbicide resistant plants was selected using the complementary herbicide and it was analyzed using molecular techniques.
  • Example 3 Co-selection for herbicide resistance due to action of a base editor to enrich the frequency of a non-selectable modification at an unlinked locus
  • Example 2 To demonstrate that the transgene-free selection for isolating plants with gene editing events provides a suitable and straightforward tool during genome engineering, the methodology described in Example 2 was combined with the co-delivery of a site-specific nuclease to simultaneously generate base-conversions of a herbicide gene and targeted modifications of a gene of interest in the same cell in parallel.
  • a nuclease On the same plasmid, or a second plasmid, a nuclease is encoded together with a sgRNA and optionally a repair template to make a targeted modification in the same cells undergoing a base conversion due to the action of the base editor.
  • plants can be regenerated under herbicide selection as described in Example 2, and then screened by molecular and other appropriate techniques for targeted modifications at the gene of interest, whereas the herbicide selection allows a significant decrease in the number of cells to be screened for the at least one second modification, i.e., the at least one targeted modification at the second genomic locus representing the gene of interest to be modified.
  • Example 4 Design of a functional CRISPR/Cpf1 base editor and definition of the base editing window
  • CRISPR/Cas9 a second CRISPR protein, Cpf1
  • CRISPR/Cpf1 also forms an R loop like structure when binding its DNA target, leaving the non-target strand available in single-strand form for base conversions.
  • the base conversion window can be defined by targeted NGS on GC-rich sequences of the corn genome, after delivery of Cpf1 based editors targeted to those sequences in cell populations as described in Example 1. For other target plants, the strategy can be adapted accordingly.
  • Example 5 Use of a single-nucleotide deletion in a PPO gene to produce a selectable modification without a repair template or homologous recombination
  • a single amino acid deletion of glycine at position 210 of the PPO gene in Amaranthus tuberculatus has rendered this weed resistant to PPO-inhibiting herbicides (Patzoldt, W.L. et al. (2006) . "A codon deletion confers resistance to herbicides inhibiting protoporphyrinogen oxidase” PNAS 103 (33) : 12329-12334) . This isoform is also called PPX2L.
  • the equivalent amino acid in Nicotiana tabacum is a glycine in position 178 of the PPO2 gene. In Zea mays, the equivalent amino acid is an alanine, but the surrounding residues are highly conserved and likely still constitute a functional active site that would become resistant due to deletion of the alanine.
  • a site-directed nuclease such as Cas9 or Cpf1 can be used with appropriate crRNA or sgRNA to make a double-strand cut near the codon for this amino acid.
  • a site-directed nuclease such as Cas9 or Cpf1
  • crRNA or sgRNA can be used with appropriate crRNA or sgRNA to make a double-strand cut near the codon for this amino acid.
  • Three-base deletions that preserve an active PPO enzyme while inhibiting herbicide binding will result in herbicide resistant plants.
  • this selectable modification can be made without the use of a repair template or homologous recombination thus providing a transgenic marker free strategy.
  • Targets for selection based on herbicide-resistance also include other amino acid deletions, introduction of early stop codons, or amino acid changes in the PPO, ALS, and EPSPS genes as described earlier.
  • gRNA protospacer sequences suitable for base editing in the PPO gene are provided (see SEQ ID NOs: 7 to 13) .
  • sequence for a CasX-linked base editing complex (SEQ ID NO: 14)
  • sequence for a AsCpf1-linked base editing complex (SEQ ID NO: 15)
  • sequence for incorporation of the cytidine deaminase PmCDA1 into a Cas9-linked base editing complex (SEQ ID NO: 16) .
  • any order and combination of the following components can be used: niCas9 (D10A; SEQ ID NO: 17) , CasX (SEQ ID NO: 18) , niAsCpf1 (R1226A; SEQ ID NO: 19) , APOBEC1 (SEQ ID NO: 20) , UGI (SEQ ID NO: 21) , PmCDA1 (SEQ ID NO: 22) , as well as linkers, including XTEN linkers, and nuclear localization signals or other organelle targeting signals depending on the genomic site of interest, or any combination of the aforementioned components.
  • sgRNA Target sequence sgRNA-OsALS-S1 CAGGTCCCCCGCCGCATGAT CGG sgRNA-OsALS-S2 CCT ACCCGGGCGGCGCGTCCATG
  • the pH-nCas9-PBE-OsALS-S1/S2 binary vector was transformed into Agrobacterium strain AGL1 by electroporation.
  • Agrobacterium-mediated transformation, tissue culture and regeneration in rice cultivar Zhonghua 11 were performed according to Shan et al. (Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31, 686-688 (2013) ) .
  • Hygromycin selection 50 ⁇ g/ml was used during tissue culture. (This experiment is a proof of concept, so the plants were selected with hygromycin first, then by nicosulfuron. The objective was to first obtain transgenic plants and then screen for herbicide resistance) .
  • TaALS S1 site was used as a herbicide selection site. If the site is mutated, plants will gain herbicide (such as nicosulfuron) resistance, while only mutation in TaALS S2 site will not confer resistance (Tranel and Wright, 2002) .
  • the sgRNA target sequence used in the experiment is shown in Table 2.
  • sgRNA Target sequence sgRNA-TaALS-S1 CAGGTCCCCCGCCGCATGAT CGG sgRNA-TaALS-S2 CCT ACCCTGGCGGCGCGTCCATG sgRNA-TaACCase TTCAGCTACTAAGACAGCGC AGG
  • Plasmid DNA (mixture of equal proportions of pnCas9-PBE and pTaU6 vector series) was used to bombard young embryos of Konun 199 as previously described for transformation (Zhang, K., Liu, J., Zhang, Y., Yang, Z. &Gao, C. Biolistic genetic transformation of a wide range of Chinese elite wheat (Triticum aestivum L. ) varieties. J. Genet. Genomics. 42, 39-42 (2015) . After bombardment, embryos were processed according to the literature and no selective agent was used during tissue culture.
  • PCR/RE For wheat plants obtained by solely targeting the S2 site of the TaALS gene of B genome, every 3-4 plants were pooled as one sample to detect mutations by PCR/RE. 258 samples were detected by PCR/RE (approximately 1000 individual plants) and no mutation was detected.
  • every 3-4 plants were pooled as one sample and subjected to Sanger sequencing. 64 samples (about 256 individual plants) were sequenced, and no mutation was detected.
  • Wheat plants (approximately 800 plants) obtained by targeting the TaALS gene S1 and S2 sites in parallel were first grown on a selection medium containing 0.13 PPM Nicosulfuron (on which wild-type plants were unable to survive) . 30 days later, twelve seedlings survived, and 9 of them had base mutations at the TaALS-S2 site. The efficiency of selecting the ALS-S2 site mutant plants using nicosulfuron selection medium was 75% (9/12) . The mutation types of five mutants are shown in Figure 2B.
  • the experimental results show that for the target gene whose mutation rate is very low (eg, the target gene has a mutation rate of 0.5%) , the method of the invention can increase the probability of obtaining target mutation by 10-100 times.
  • the sgRNA site corresponding to TaALS-P173 was used to establish the herbicide selection system during wheat transformation.
  • PnCas9-PBE and TaALS-P173-sgRNA constructs were delivered into 640 immature embryo cells of the bread wheat variety Kenong 199 by particle bombardment.
  • PCR-RE assay PCR restriction enzyme digestion assay
  • the same seedlings were transfer to the media containing 0.27 ppm nicosulfuron ( Figure 3) .
  • Ten (1.56%) out of fourteen (2.1%) mutant seedlings which are identified using PCR-RE assay showed resistance after 3 weeks growth on the herbicide containing media, and three sensitive mutants did not contain any amino acid substitution (Table 3) .
  • SM Silent mutation
  • S Sensitive
  • R Resistant
  • Homo homozygous
  • Hetero heterozygous
  • TaALS-P173 substitution can be recognized from herbicide containing media.
  • the inventors then tested whether this site can also be used to select for other genome edited events. So three other sites (TaALS-A98, TaALS-A181, as well as TaACCase-A2004) were combined with TaALS-P173 separately.
  • the regenerated seedlings co-bombarded with TaALS-P173 site targeting systems were place on media containing nicosulfuron and the survived seedlings were submitted for genotyping.
  • Targeted mutants were detected at all three sites (Table 4) at selection efficiencies up to 78%. In sites TaALS-A181 and TaACCase-A2004, the selection efficiencies were relative low ( ⁇ 25%) , which was possibly caused by the low conversion ability of deaminase APOBEC1 at GC context.
  • APOBEC1 was replaced by another deaminase-PmCDA1, which has different sequence preference compared with APOBEC1.
  • Newly generated base editor pPmCDA1-PBE, TaACCase-A2004-sgRNA and TaALS-P173-sgRNA constructs were delivered into 640 immature embryo cells by particle bombardment. Out of 2 survived seedlings, both (100%) contained mutant alleles at target site TaACCase-A2004 (Table 4) .
  • Example 10 Development of base co-editing system in corn based on ZmALS-P165
  • acetolactate synthase site corresponding TaALS-P173 was targeted to test the herbicide resistance. It has been reported single edited allele on ZmALS2 could confer plants herbicide resistance (Svitashev et al, 2016) . So the binary vector targeting ZmALS-P165 was transformed to immature embryos (ZmALS-P165 site is conserved in both ZmALS1 and ZmALS2) . Three independent mutants were obtained from the regenerated plants and their genotypes are same. Two ZmALS1 alleles and one ZmALS2 allele containing C to T substitutions resulted in the single amino acid residue change: proline to leucine at position 165. One mutant plant with heterozygous P165L substitution on ZmALS2 showed resistance to Mesosulfuron-methyl, a sulfonylurea class of herbicides ( Figure 4) .
  • Example 11 Development of base co-editing system in rice based on OsALS-P171
  • acetolactate synthase site corresponding TaALS-P173 was targeted to test the herbicide resistance. It has been reported single edited allele could confer plants herbicide resistance (Kawai, K., Kaku, K., Izawa, N., Shimizu, M., Kobayashi, H., &Shimizu, T. (2008) . Herbicide sensitivities of mutated enzymes expressed from artificially generated genes of acetolactate synthase. Journal of pesticide science, 33 (2) , 128-137. ) . So the binary vector targeting OsALS-P171 was transformed to immature embryos. Mutants were obtained from the regenerated plants.
  • OsALS-P171 site could work well as a selectable marker
  • other three sites -OsAccase W2125, OsBDAH2 Stop and OsSbe2 Stop were combined with this selectable site separately. Both biolistic and Agrobacterium-mediated delivery were used for transformation. Surviving seedling showed target site mutation.
  • Example 12 Development of base co-editing system in corn based on ZmALS-P197 or ZmALS-G654
  • Target amino acids of Zea mays were chosen for conversion to amino acids that have been seen in weeds resistant to the herbicide groups like imidazolinones and sulfonylureas.
  • the green arrows in Fig. 5 are guide sequences to the coding or non-coding strand for obtaining desired conversion. Note: coordinates of the amino acid residues numbered in this Example are standardized to the archetypal ALS gene from Arabidopsis thaliana. Positions of these residues in the corn and wheat peptide sequences will be somewhat different.
  • Pol III promoter for sgRNA -Guides were made to modify ALS1 and ALS2 genes at the P197 locus (Fig. 6, left graph , top) and G654 locus (Fig. 6, Right graph, top) .
  • the base-editor system is a single vector system, in this case with a pUbi1 driven base editor and a ZmU3-driven guide RNA.
  • the results shown above is a %C to T conversion frequency calculated for every C in the guide RNA and minus the background from the negative control for both ALS1 and ALS2.
  • the frequency shown here does not specify whether one or both C’s at P197 codon changed in the same cell.
  • the changes were also evident but to a lesser extent.
  • the herbicide-sensitive residue is converted to herbicide-resistant up to 6%frequency of treated cells (Fig 7)
  • FIG. 6 Another way of analyzing the data shown in Fig. 6 is by counting the number of reads which show the desired amino acid codon conversion. The final %data is normalized to the protoplast transformation efficiency.
  • Top panel Shows the %of reads where the proline197 has been converted to a Leucine or a Serine at both ALS1 and ALS2 loci. The data is from experiment where the Pol III promoter was used.
  • Middle Panel Shows the %of reads where the proline197 has been converted to a Leucine or a Serine at both ALS1 and ALS2 loci.
  • the data is from experiment where the Pol II promoter and Ribozyme delivery strategy for sgRNA was used.
  • Bottom Panel Shows the %of reads where the Glycine654 has been converted to an Aspartic Acid at both ALS1 and ALS2 loci.
  • the data is from experiment where the Pol III promoter and Ribozyme delivery strategy for sgRNA was used.

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Abstract

L'invention concerne des procédés d'édition ciblée dans une plante, une cellule ou un matériau végétal, qui sont combinés à l'introduction parallèle d'une caractéristique phénotypiquement sélectionnable. Les procédés ne comprennent pas une étape d'introduction d'une séquence de marqueurs de sélection transgénique.
PCT/CN2018/085829 2017-05-05 2018-05-07 Procédés pour isoler des cellules sans utiliser de séquences de marqueurs transgéniques Ceased WO2018202199A1 (fr)

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AU2018263195A AU2018263195B2 (en) 2017-05-05 2018-05-07 Methods for isolating cells without the use of transgenic marker sequences
US16/610,782 US20210230616A1 (en) 2017-05-05 2018-05-07 Methods for isolating cells without the use of transgenic marker sequences
JP2019560164A JP2020518253A (ja) 2017-05-05 2018-05-07 トランスジェニックマーカー配列を使用せず細胞を単離する方法
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EA201992615A EA201992615A1 (ru) 2017-09-01 2018-05-07 Способы выделения клеток без использования трансгенных маркерных последовательностей
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CA3062475A CA3062475A1 (fr) 2017-05-05 2018-05-07 Procedes pour isoler des cellules sans utiliser de sequences de marqueurs transgeniques
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WO2023245113A1 (fr) * 2022-06-16 2023-12-21 Intellia Therapeutics, Inc. Procédés et compositions pour modifier génétiquement une cellule
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