Recurrent selection to obtain optimal gene edit combinations

Abstract

The invention provides methods to select an optimal combination of multiple gene edits to confer a desired phenotype in a plant, through crossing populations of edited plants and selecting plants with the desired phenotype. In particular, the crossing is configured such that diversity of the edits within the population is promoted.

Description

[0001] BASF Agricultural Solutions US LLC 240242WO02

[0002] 1

[0003] RECURRENT SELECTION TO OBTAIN OPTIMAL GENE EDIT COMBINATIONS

[0004] Field of the Invention

[0005] This invention generally relates to the field of genetic improvement of plants and provides methods for the selection of optimal combinations of gene edits in multiplex gene editing approaches. of the Invention

[0006] Many traits are polygenic (quantitative) in character, e.g., yield being a classic example, with the final phenotype dependent on the activity of multiple genes across multiple genomic locations and / or multiple pathways. Because of this, it may be desirable to simultaneously edit multiple steps of a single pathway, and / or individual steps from multiple pathways to obtain the desired result (e.g. improved yield). Due to differing efficiencies in the nicking / cutting and repair processes, differences in chromatin structure and availability, location in the genome etc., genome editing rarely yields edits that are entirely uniform and / or homozygous in the primary transformants, and this situation is even less likely when applied across a diverse set of target genes. Therefore, the challenge in the simultaneous editing of multiple gene targets is to identify and stack the edited alleles of interest in a homozygous state in a single plant. The more genes that are tackled, the more problematic this process is, due to the need to follow and select for multiple (unique) alleles across multiple generations and multiple lines. This becomes highly restrictive in terms of both time and required resources as the number of genes increases, due to the need to design and implement assays for each gene target edit, at each stage of the stacking process and potential limitations in the number of seeds produced by an individual edited plant at each generation. In practice, these restrictions severely limit the application of multiplex gene editing in crops.

[0007] There are several scientific publications on multiplex gene editing in crops, such as Wolter et al (2019) BMC Plant Biology 19:176 and Zhou et al (2019) Plant Cell Reports 38:475, who describe multiplex gene editing to efficiently and quickly introduce modification of multiple targets.

[0008] Lorenzo et al (2023) Plant Cell 35:218 describe a multiplex genome editing strategy to improve complex quantitative traits, called “BREEDIT”. They describe generation of multiplex edits in maize, genotyping TO lines, and crossing lines with the highest number of edits for phenotyping. A drawback of this technology is that the final number of plants to be processed increases dramatically as the number of genes in the study grows. WO2024 / 218295 describes the use of “BREEDIT” in combination with haploid breeding.

[0009] Stuttmann et al (2021) Plant J 106:8 describe a method for efficient multiplex editing and identification of plants with all intended edits by phenotypic screening prior to genotype analysis. WC2018140899 describes methods to create a plurality of targeted modifications in the plant genome and selection based on phenotype without the step of separately identifying and selecting for the occurrence of individual mutations. Stuttmann et al and WC2018140899 fail to describe an approach to maximize number of possible combinations of GE alleles in individual plants or lines, which limits the possibility to identify the optimal combinations of edits. They also remain silent on selection and identification of the most beneficial GE allele combinations for a particular phenotypic trait. BASF Agricultural Solutions US LLC 240242WO02

[0010] 2

[0011] EP3521436 describes a method for multiplex editing, crossing and selecting progeny with a desired trait. EP3521436 however has not optimized the crossing steps of the edited plants to maximize the number of possible combinations of edits in a single plant; nor does EP3521436 enrich for desirable edits in a single plant.

[0012] There remains a need to efficiently select the optimal combination of gene edits for crop improvement.

[0013] In a first aspect, a method is provided to select an optimal combination of multiple gene edits to confer a desired phenotype in a plant, said method comprising a) providing a population of cells; b) providing said cells with editing machinery comprising a library of editing cassettes to create multiple gene edits; c) generating a population of plants from said cells, said population comprising edited plants, wherein said edited plants comprise one or more of said multiple edits; d) crossing edited plants of said population for at least one generation to generate a plurality of combinations of said multiple gene edits .wherein the crossing is configured to promote diversity of edits within the population; and e) selecting plants with the desired phenotype. In a further aspect, the edited plants of said population are intercrossed to promote diversity of edits within the population, and wherein a male sterility system is used to promote intercrossing. In yet another aspect, in only seeds from a male sterile plant are retained in at least one of the generations step d), whereas in yet another aspect, in step d) said plants are intercrossed for at least two generations, or at least three generations, or at least four generations, or at least five generations, or at least six generations, and wherein after at least one of the crossing steps plants with the desired phenotype are selected and retained.

[0014] In another embodiment, the method according to the invention is provided wherein said editing machinery is designed to be stably integrated into the plant genome; the editing machinery or a component thereof is linked to a selectable or screenable marker gene and to a male sterility gene; after step b) or after step c) cells or plants comprising the editing machinery in the plant genome are selected based on expression of the selectable or screenable marker gene; in a first crossing, said edited plants are cross-pollinated with pollen from plants not comprising a male sterility gene and not comprising the editing machinery; male fertile plants not comprising the selectable marker and the male sterility gene are eliminated by application of a selective agent after seeds are produced and before seeds are harvested; and only seeds from male sterile plants comprising the selectable marker and the male sterility gene are retained; in a next crossing, plants derived from said seeds are inter-crossed; male fertile plants not comprising the selectable marker and the male sterility gene are eliminated with a selective agent after seeds are produced and before seeds are harvested; seeds obtained from plants comprising the selectable marker and the male sterility gene are harvested; and only seeds from male sterile plants comprising the selectable marker and the male sterility gene are retained; and, optionally, said next crossing step is repeated for one or more generations, and wherein after at least one of the crossing steps plants with the desired phenotype are selected and retained.

[0015] In yet another embodiment, the method according to the invention is provided wherein the editing machinery consists of two components, such as an RNA-guided nuclease and an RNA, wherein the two components of the editing machinery are delivered separately, wherein only one of the two components of the editing machinery is linked to the male sterility system, and wherein plants comprising the two components of the editing machinery are selected after one or more initial crossings, BASF Agricultural Solutions US LLC 240242WO02

[0016] 3 and wherein after one or more subsequent crossings, plants are selected for the absence of the component of the editing machinery which is not linked to the transgenic dominant male sterility gene, and for the presence of the component of the editing machinery which is linked to the transgenic dominant male sterility gene.

[0017] In a further aspect, the library of editing cassettes contains an editing cassette for editing an endogenous male fertility gene, optionally a single dominant male fertility gene, and in a first crossing, said edited plants of said population are crossed; in a next crossing, progeny plants obtained from said first crossing are inter-crossed; male sterile plants comprising edits in both alleles of said single dominant male fertility gene are selected, and seeds from male sterile plants are retained; and optionally, said next crossing step is repeated for one or more generations, wherein after at least one of the crossing steps plants with the desired phenotype are selected and retained. In another aspect, said editing machinery is designed to be stably integrated into the plant genome, and wherein the editing machinery is linked to a selectable marker gene and, optionally, to a late pollen ablation gene, and plants comprising the editing machinery in the plant genome are selected after one or more initial crossings, and after one or more subsequent crossings, plants are selected for the absence of the editing machinery. In yet another aspect, said editing machinery is designed to be not stably integrated into the plant genome and, optionally, the editing machinery is delivered as ribonucleoprotein (RNP).

[0018] In another aspect, the method according to the invention is provided in which activation of said editing machinery is inducible. In a further aspect, the method of the invention further comprises the step of genotyping the selected plants with the desired phenotype for the target site edits.

[0019] Also provided is a method to select an optimal combination of multiple edits to confer a desired phenotype in a plant, said method comprising a) providing a seed mixture comprising a plurality of combinations of multiple gene edits obtained from intercrossing of edited plants in a population for at least one generation, using a male sterility system, wherein said edited plants in said population are generated from cells to which editing machinery is provided, said editing machinery comprising a library of editing cassettes to create multiple edits, wherein said edited plants comprise one or more of said multiple edits; b) selecting a plant with the desired phenotype; and, optionally c) genotyping said selected plant with the desired phenotype for the target site edits.

[0020] In another aspect, the methods according to the invention are used to produce a plant with an optimal combination of multiple gene edits to confer a desired phenotype.

[0021] In a further aspect, a plant is provided, or cell, tissue, organ, material, or seed of said plant, obtained by or obtainable by methods according to the invention.

[0022] Another embodiment provides the methods according to the invention in which the plant is a Brassica plant, and the desired phenotype is podshatter reduction. BASF Agricultural Solutions US LLC 240242WO02

[0023] 4

[0024] Brief description of the drawings

[0025] Figure 1 : Recurrent selection for selecting optimal gene edit combinations using transgenic male sterility. Figure 1a construct to be transformed in the plant cells comprising Editing machinery for multiple target site edits (E); expression cassette for a dominant male sterility system (MS) and a selectable marker (S). This editing cassette is transformed into plant cells and a population of plants is generated from said cells. Figure 1b represents the population of plants that are generated from the step as shown in Figure 1 a. The three blocks in the cell refer to the transgene comprising Editing machinery, expression cassette for a dominant male sterility system, and selectable marker as shown in Figure 1 a. The population of plants comprises different combinations of target site edits (E1-E6). Plants with various different edits and the transgene are male sterile and can only be used as female plants in a crossing. The plants with the different edits and the transgene are intercrossed with plants not comprising the transgene and the edits (empty circle in Figure 1 b). After pollination, the plants not comprising the transgene are eliminated using treatment with a selective agent (H) and seeds are harvested from plants comprising the different edits and the transgene. Figure 1c represents the population of plants resulting from the crossing of the step of figure 1 b. 50% of the plants in the population will contain the transgene, are male sterile, and can only be used as female parent; 50% of the plants in the population will not contain the transgene and can act both as male and female parent. Said population will be crossed. Plants comprising the transgene will be cross-pollinated with other plants, which thus may comprise different edits which will increase edit diversity, whereas, for (mainly) selfpollinating species, plants not comprising the transgene will mainly be self-pollinated. To maximize the population variability with regard to the edits, the self-pollinated plants not comprising the transgene are eliminated after pollination using treatment with a selective agent (H) and seeds are harvested from the cross-pollinated plants comprising the different edits and the transgene. Figure 1d represents the population of plants resulting from the crossing of the step of figure 1c. Again, 50% of the plants in the population will contain the transgene, are male sterile, and can only be used as female parent; 50% of the plants in the population will not contain the transgene and can act both as male and female parent. Said population will be intercrossed as described above for the step of figure 1c. The crossing and selection step as described in Figure 1d can be repeated several times leading to additional edits or additional combinations of edits in one plant.

[0026] Figure 2: Recurrent selection for selecting optimal gene edit combinations using editing of an endogenous single dominant male fertility gene in stable transformation. Figure 2a Construct to be transformed into the plant cells comprising Editing machinery for multiple target site edits including a single dominant male fertility gene (E(ms)); expression cassette for a post- meiotic pollen ablation gene (PAG) and a selectable marker (S). This editing cassette is transformed into plant cells and a population of plants is generated from said cells. Figure 2b represents the population of plants that are generated from the step as shown in Figure 2a. The population of plants comprises different combinations of target site edits (E1-E6). Said population will comprise plants that are heterozygous for the edit in the male fertility gene (ms), and thus still contain one functional copy of the male fertility gene and are male fertile. Plants with various different edits and the transgene however will not be able to produce viable transgene-containing pollen (P) due to the presence of the post-meiotic pollen ablation gene, and thus can only be pollinated with pollen comprising various different edits (x) but not containing the transgene. In case the editing machinery edits both alleles of the male fertility gene, some plants in the population may already be homozygous for the edit in the male fertility gene (ms / ms; not shown in Figure 2b) and will not be male fertile. These plants will be pollinated by pollen from plants which are heterozygous for the edit in the male fertility gene (ms). In case the editing machinery is present, but no edits are made in the male fertility gene, pollen of these plants, which still may contain target BASF Agricultural Solutions US LLC 240242WO02

[0027] 5 site edits, will be used for cross pollinating other plants in the population. Seeds are harvested from the plants comprising the different edits and the transgene. Figure 2c represents the population of plants resulting from the crossing of the step of figure 2b. 50% of the plants in the population will contain the transgene. These plants are eliminated before pollination. Of the remaining 50% of the plants not containing the transgene 25% will have the edit in the male fertility gene in homozygous form and thus are male sterile; 50% will have the edit in the male fertility gene in heterozygous form and are male fertile, and 25% will not have the edit in the male fertility gene and will be male fertile. Intercrossing in the population will result in cross-pollination of the plants homozygous for the edit in the male fertility gene, and for (mainly) self-pollinating species, mainly self-pollination of the plants heterozygous for, or not comprising the edit in the male sterility gene. To maximize the population variability with regard to the edits, the self-pollinated plants that are heterozygous for, or which do not contain the edit in the male sterility gene are eliminated after pollination. Seeds are harvested from the cross-pollinated plants comprising the different edits in the multiple targets. Figure 2d represents the population of plants resulting from the crossing of the step of figure 2c. None of the plants will comprise the transgene, and the population will be a mixture of plants that are both homozygous and heterozygous for the edit in the male fertility gene. Said population will be intercrossed as described above for the step of figure 2c. The crossing and selection step as described in Figure 2d can be repeated several times.

[0028] Figure 3: Recurrent selection for selecting optimal gene edit combinations by editing an endogenous single dominant male fertility gene with Ribonucleoprotein (RNP) delivery. Figure 3a RNP to be delivered to the plant cells comprising Editing machinery for multiple target site edits including a single dominant male fertility gene (E(ms)). The RNP is delivered to plant cells and a population of plants is generated from said cells. Figure 3b represents the population of plants that are generated from the step as shown in Figure 3a. The population of plants comprises different combinations of target site edits (E1-E6). Said plants are heterozygous for the edit in the male fertility gene (ms), and thus still contain one functional copy of the male fertility gene and are male fertile. Plants are intercrossed in the population. Seeds in said first intercrossing step will mainly be the result of self-pollination. Seeds are harvested from the plants comprising the different edits. Figure 3c represents the population of plants resulting from the crossing of the step of figure 3b. 25% of the plants will have the edit in the male fertility gene in homozygous form and thus are male sterile; 50% will have the edit in the male fertility gene in heterozygous form and are male fertile, and 25% will not have the edit in the male sterility gene and will be male fertile. Intercrossing in the population will result in cross-pollination of the plants homozygous for the edit in the male fertility gene, and in the case of (mainly) self-pollinating species, will result mainly in self-pollination of the plants that are heterozygous for, or do not contain the edit in the male sterility gene. To maximize the population variability with regard to the edits, the self-pollinated plants that are heterozygous for, or do not contain the edit in the male sterility gene are eliminated after pollination. Seeds are harvested from the cross-pollinated plants comprising the various different edits. Figure 3d represents the population of plants resulting from the crossing of the step of figure 3c. The population will be a mixture of plants homozygous and heterozygous for the edit in the male fertility gene. Said population will be intercrossed as described above for the step of figure 3c. The crossing and selection step as described in Figure 3d can be repeated several times.

[0029] Detailed Description

[0030] Disclosed herein are methods to generate a population of genome edited plants containing edits in multiple targets / genes generated following multiplexed editing, and then to use recurrent mass selection, combined with phenotyping and BASF Agricultural Solutions US LLC 240242WO02

[0031] 6 selection / cu 11 i n g to allow the population to enrich itself for the beneficial allele-combinations. Specifically then, a diverse set of genome edited lines or events is intermingled and allowed to intermate over several generations which, when combined with selection on trait performance, will be leading to the accumulation of allele combinations and fixation of those edits that lead to trait improvement.

[0032] The methods of the invention have the clear advantage that there is no need in advance, to have knowledge on the particular combination of genes to be edited, to achieve an improved phenotype.

[0033] In a first aspect, a method is provided to select an optimal combination of multiple gene edits to confer a desired phenotype in a plant, said method comprising a) providing a population of cells; b) providing said cells with editing machinery comprising a library of editing cassettes to create multiple gene edits; c) generating a population of plants from said cells, said population comprising edited plants, wherein said edited plants comprise one or more of said multiple edits; d) crossing edited plants of said population for at least one generation to generate a plurality of combinations of said multiple gene edits, wherein the crossing is configured to promote diversity of edits within the population; and e) selecting plants with the desired phenotype.

[0034] Suitable to the invention is a method to select a plant with an optimal combination of multiple gene edits to confer a desired phenotype, said method comprising a) providing a population of cells; b) providing said cells with editing machinery comprising a library of editing cassettes to create multiple gene edits; c) generating a population of plants from said cells, said population comprising edited plants, wherein said edited plants comprise one or more of said multiple edits, wherein the crossing is configured to promote diversity of edits within the population; d) crossing edited plants of said population for at least one generation to generate a plurality of combinations of said multiple gene edits in individual lines; and e) selecting plants with the desired phenotype.

[0035] Suitable to the invention is a method to produce a plant comprising an optimal combination of multiple gene edits for a desired phenotype, said method comprising a) providing a population of cells; b) providing said cells with editing machinery comprising a library of editing cassettes to create multiple gene edits; c) generating a population of plants from said cells, said population comprising edited plants, wherein said edited plants comprise one or more of said multiple edits; d) crossing edited plants of said population for at least one generation to generate a plurality of combinations of said multiple gene edits, wherein the crossing is configured to promote diversity of edits within the population; and e) selecting plants with the desired phenotype.

[0036] In a further aspect, the method of the invention further comprises the step of genotyping the selected plants with the desired phenotype for the target site edits, said the editing machinery may be designed to be stably integrated into the plant genome, and the method of the invention may comprises in step c) selection of said edited plants are for the presence of the editing machinery in the plant genome.

[0037] Creation of gene edits

[0038] Gene edits can be generated using different techniques known in the art. The editing can be based on the use of site-specific nucleases, such as RNA-guided nucleases as CRISPR / Cas, including Cas9, Cas12a / Cpf1 , homing endonucleases, transcription activator-like effector nucleases (TALENs), or zinc-finger nucleases (ZFNs). BASF Agricultural Solutions US LLC 240242WO02

[0039] 7

[0040] CRISPR technology for gene editing, and in particular Cas9, is disclosed in, for example, WO20131746772, WO14093595, WO14065596 and by Jinek et al Science 2012; 337(6096): 816-821. Cas12a (formerly called Cpf1) endonuclease is disclosed US2016 / 0208243, WO2016205711 and by Zetsche et al Ce / / 2015; 163(3): 759-771.

[0041] Other CRISPR nucleases in addition to Cas9 and Cas12a have been developed over the last years. Examples include Cas3 (Brouns et al Science 2008; 321 (5891): 960-964), Cas10 (Marraffini et al Science 2008; 322(5909): 1843-1845), Csf1 (Koonin et al Genome Biology and Evolution. 2017; 9(10): 2812-2825, C2cl (Yanhg et al, Cell 2016; 167(7): 1814-1828), C2c2 (Abudayyeh et al., Nature 2017; 550(7675): 280-284) Cas13 (Abudayyeh et al., supra), and Cas14 (Harrington et al Science 2018; 362(6416): 839-842). An overview of the mechanism and applications of different CRISPR / Cas systems is provided in Hillary and Ceasar (2023) Mol Biotechnol 65(3): 311-325.

[0042] It is well known in the art that CRISPR / Cas proteins interact with a guide RNA (gRNA) to direct the proteins to the target site and to cleave the DNA at the target site. The target site comprises the sequence recognized by the gRNA, and a protospacer adjacent motif (PAM) which is a short DNA sequence usually 2-6 base pairs in length that is flanking the DNA region targeted for cleavage by the CRISPR system. The PAM sequence is specific for a given Cas endonuclease. A gRNA may comprise two separate RNA molecules, i.e. a pre-crisprRNA (pre-crRNA) and a trans-activating crispr RNA (tracrRNA), which are partially complementary and therefore form a RNA duplex via base pairing. Said RNA duplex can be cleaved by RNase III, a RNA-specific ribonuclease, resulting in a crRNA / tracrRNA hybrid. Preferably, the functional guide RNA according to the present invention is a single guide RNA (sgRNA) molecule comprising both a tracrRNA component and a crRNA component joined by tetraloop. The skilled person in the relevant technical field is aware of the fact that a naturally occurring CRISPR nuclease and the cognate guiding RNA are mutually compatible. Further, the skilled person knows that a different CRISPR / Cas effector is guided by a different type of guiding RNA.

[0043] CRISPR arrays can be designed to contain one or multiple guide RNA sequences corresponding to a desired target DNA sequence. Many computational tools have now been developed to design sgRNAs (see, for example, Li et al., Genomics, Proteomics & Bioinformatics, 2023, 21 (1): 108-126. The design of guide RNAs for use in plant genome editing is disclosed for example in US2015 / 0082478.

[0044] Editing machinery, as used herein, can thus comprise or consist of site-specific nucleases, such as RNA-guided nucleases as CRISPR / Cas, including Cas9, Cas12a / Cpf1 , homing endonucleases, transcription activator-like effector nucleases (TALENs), or zinc-finger nucleases (ZFNs).

[0045] Said multiple gene edits can be single or multiple gene edits in multiple genes in a single pathway, or single or multiple gene edits in multiple different genes in different pathways leading to a desired phenotype, and can include edits in different genes, different homeologous genes, different genes in a family, or different genes from different families. Multiple gene edits can be created by expressing multiple gRNAs or a library of gRNAs. Multiple gRNAs can be expressed from a single recombinant DNA construct, or from multiple recombinant DNA constructs. Multiple gRNAs can have a specificity for different target sequences. Each one of the multiple gRNAs can be under its own promoter. Each one of the multiple gRNAs can also be on a single transcript which can be processed through for example pre-crRNA processing, tRNA-based processing or ribozyme-based processing. A tool for multiplex genome engineering in plants is described, for example, in Vazquez-Vilar et al (2021) Front Plant Sci 12:689937.

[0046] Said multiple edits can be more than one edit, such 5 edits or more, 7 edits or more, 8 edits or more, 10 edits or more, 12 edits or more, 15 edits or more, 20 edits or more, 25 edits or more, or even more edits. BASF Agricultural Solutions US LLC 240242WO02

[0047] 8

[0048] The term “target sequence” as used herein, e.g. in the context of endonucleases, describes a nucleic acid sequence or part of a nucleic acid sequence, which can be subjected to one or more modification(s) via a method according to the present invention, and wherein said modification(s) may include one or more deletion(s) of one or more nucleotide(s) and / or one or more insertion(s) of one or more nucleotide(s) and / or one or more substitution(s) of one or more nucleotide(s). Said target sequence can be a genomic DNA sequence, such as a gene sequence, a coding sequence, or a gene regulatory sequence. The target sequence is preferably located in close proximity, and preferably directly adjacent, to a PAM sequence. Thus, it is preferred that up to 20 nucleotides, preferably up to 15 nucleotides, preferably up to 10 nucleotides, preferably up to 9 nucleotides, preferably up to 8 nucleotides, preferably up to 7 nucleotides, preferably up to 6 nucleotides, preferably up to 5 nucleotides, preferably up to 4 nucleotides, preferably up to 3 nucleotides, preferably up to 2 nucleotides, preferably up to 1 nucleotides, preferably 0 nucleotides (i.e. directly adjacent), between the target sequence and the PAM sequence.

[0049] A target site edit is an edit in the target sequence. Each of the multiple gene edits according to the invention each are thus at a target sequence.

[0050] Upon cleavage of the site-specific nucleases, the cut or nick in the DNA can be repaired via non-homologous end joining (NHEJ) or homologous recombination (HR).

[0051] Prime editing or base editing can also be used to generate edits (see, for example, Li et al (2023) J. Integr. Plant Biol. 65: 444-467).

[0052] In one aspect of the invention, the gene edits are knock-outs. In another aspect of the invention, the knock-outs are created through cleavage with site-specific nucleases, and NHEJ. Suitable DNA programmable nucleases for NHEJ are CRISPR / Cas9 and CRISPR / Cas12a.

[0053] A library of editing cassettes, as used herein, comprises at least two (2) editing cassettes. It may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, at least 10, at least 15, at leat 20, at least 30, at least 40, at least 50 or at least 100 editing cassettes. An editing cassette may comprise or consist of a site-specific nuclease, or comprise or consist of a RNA-guided nuclease and an RNA, such as a CRISPR / Cas enzyme and a gRNA. A library of editing cassettes may comprise or consist of a library of site-specific nucleases, or a single CRISPR / Cas enzyme with a library of gRNAs. Said library of gRNAs may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, at least 10, at least 15, at leat 20, at least 30, at least 40, at least 50 or at least 100 gRNAs. Said library of gRNAs may be expressed from a single recombinant DNA construct, or from multiple recombinant DNA constructs.

[0054] Providing said cells with editing machinery

[0055] The editing machinery can be provided to the cells (e. g. a plant cell, plant tissue or plant protoplast) in several ways. See, for example, Sandhya et al (2020), Journal of Genetic Engineering and Biotechnology 18(1): 25.

[0056] The editing machinery can be delivered to the plant cells on a T-DNA using Agrobacterium, such as Agrobacterium tumefaciens or Agrobacterium rhizogenes. The T-DNA can be stably integrated in the genome, or transiently be expressed in the case of agroinfiltration. The binary vector (T-DNA) containing the editing machinery is transformed into the Agrobacterium strain. Further, the Agrobacterium-mediated genetic transformation of editing machinery is delivered into the desired explant such as immature embryos or scutella, meristematic tissue, callus, leaf, and floral organs of plants. BASF Agricultural Solutions US LLC 240242WO02

[0057] 9

[0058] The T-DNA encodes the editing machinery, and contains expression cassettes for the site-specific nuclease, such as a RNA- guided nuclease, and the gRNA or gRNAs. The sequences encoding the RNA-guided nuclease and the sequences encoding the gRNAs may be on the same T-DNA or on a different T-DNA. The multiple gRNAs may be on the same T-DNA or on different T-DNAs.

[0059] The editing machinery can also be delivered by direct delivery, such as PEG-mediated delivery, particle bombardment or electroporation. Direct delivery can be performed into plant protoplasts. The editing machinery may be provided as plasmid DNA encoding the editing machinery. Alternatively, it may be provided as ribonucleoprotein (RNP) complex. In case an RNA- guided nuclease is used, the RNA-guided nuclease can be provided simultaneously with the gRNA, or in a separate step that precedes or follows the step of providing the gRNA. The gRNA may be provided as RNA or as DNA molecule encoding the gRNA. The RNA-guided nuclease may be provided as a ribonucleoprotein (RNP) complex, such as a RNP that includes the RNA-guided nuclease complexed with a polynucleotide including the gRNA.

[0060] In one embodiment, the editing machinery is designed to be stably integrate into the plant genome. Stable integration in the plant genome can be obtained throught delivery of T-DNA via Agrobacterium, or through delivery of plasmid DNA or of DNA fragments through direct delivery of DNA through, for example, PEG-mediated delivery, particle bombardment or electroporation.

[0061] When the editing machinery is a RNA-guided nuclease, the RNA-guided nuclease can be provided simultaneously with the gRNA, or in a separate step that precedes or follows the step of providing the gRNA.

[0062] Alternatively, plants can be provided with editing machinery through crossing. gRNAs can be provided to cells already containing CRISPR / Cas. Alternatively, gRNAs are provided to cells, plants are generated from said cells, and CRISPR / Cas is introduced through crossing plants comprising gRNAs and stably transformed plants comprising CRISPR / Cas.

[0063] Said population of cells may comprise plant cells, plant tissue or plant protoplasts. Preferably, said population of cells is homogeneous, originates from the same variety, preferably from the same plant so that all cells have the same genotype. Optionally, the cells may be inbred lines with a high level of homozygosity.

[0064] Plant Regeneration

[0065] Plants can be generated from the cells to which the editing machinery was provided using regeneration methods known in the art. For example, callus can be produced from the plant cell, and plantlets and plants produced from such callus. Alternatively, whole seedlings or plants are grown directly from the plant cell without a callus stage.

[0066] Cells or plants generated from said cells may also be selected. Selection may be on selective media if the editing machinery was transformed together with a selectable marker. Selection may also be performed through molecular analysis, e.g. using molecular analysis to detect the presence or absence of the editing machinery or of the edit. In one embodiment, a population of plants is generated from the cells to which the editing machinery was provided. Said population of plants comprises multiple plants, regenerated from the cells to which the editing machinery was provided. Said multiple plants can be at least 2 plants, at least 3 plants, at least 5 plants, at least 10 plants, at least 20 plants, at least 30 plants, at least 50 plants, at least BASF Agricultural Solutions US LLC 240242WO02

[0067] 10

[0068] 80 plants, or at least 100 plants. Said multiple plants may comprise different edits. Said multiple plants may comprise different gRNAs.

[0069] The term “plant cell” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, and single cells thereof. Further, the plant cell may be selected from the group consisting of protoplasts, suspension cultures, callus tissue, scutella tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, preferably wherein the plant cell is from a plant that can be obtained, analyzed, treated in line with the disclosure provided herein.

[0070] In a particularly preferred embodiment of the method according to the present invention, the cell is a plant cell, preferably a plant cell belonging to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp . , Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica juncea, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, or Ziziphus spp. BASF Agricultural Solutions US LLC 240242WO02

[0071] 11

[0072] Preferred plants are Abelmoschus spp. , Allium spp., Apium graveolens, Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica juncea, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Capsicum spp., Citrullus lanatus, Cucumis spp., Cynara spp., Daucus carota, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hordeum spp. (e.g. Hordeum vulgare), Lactuca sativa, Medicago sativa, Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Pennisetum sp., Saccharum spp., Secale cereale, Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Zea mays.

[0073] Crossing and selection

[0074] The edited plants of the population as described herein are crossed for at least one generation to generate a plurality of combinations of edits. Crossing is configured to promote diversity of edits within the population. Diversity of edits within the population means to the variation among individuals in their edits, meaning that the individuals in the population differ with regard to the edits they contain. To configure crossings to promote diversity of edits within the population, preferably, different individuals of the population of plants are intercrossed or different individuals undergo inter-sib mating. The plants can also be crossed by hand crossing. Preferably, the different individuals of the population that are intercrossed comprise different edits. The resulting offspring of the intercrossing has a combination of edits, and different individuals of the offspring have different combinations of edits. The plants can be crossed for at least one generation, or for two generations, or for three generations, or for four generations, or for five generations, or for six generations, or for seven generations, or for eight generations, or for nine generations, or for ten generations, or for at least ten generations. The plants in the population can be plants that are cross-pollinating, or plants that are mainly cross-pollinating. The plants in the population are fertile or can segregate for male fertility and sterility. Certain plants in the population can also be emasculated before anther dehiscence to promote cross pollination.

[0075] Generation time can be shortened using speed breeding techniques, for example by manipulated controlled-environment growth conditions in order to reduce time to floral initiation and hasten embryo development and seed maturity, such as, for example, day / night temperature, available light spectrum and intensity, and photoperiod duration.

[0076] Phenotyping

[0077] The phenotype of a plant refers to its observable physical and biochemical characteristics. A desired phenotype, as used herein, is a phenotype of interest. The desired phenotype can be any phenotype of interest, including but not limited to an agronomic phenotype of interest. Examples of phenotypes for which the method according to the invention can be used, including desired phenotypes, are yield, seed size, seed number, seed quality, fatty acids, oil content, oil composition, seed shattering, yield potential, plant height, leaf area, days to maturity, root depth, root architecture, abiotic stress tolerance, such as drought tolerance, frost tolerance, heat tolerance, salt tolerance; nutrient use efficiency, flowering time, water use efficiency, grain quality, stem strength, chlorophyll content, biotic stress tolerance such as virus resistance, fungal resistance, disease resistance, insect tolerance and nematode resistance, biomass, plant architecture, establishment, nitrogen use efficiency. BASF Agricultural Solutions US LLC 240242WO02

[0078] 12

[0079] Methods to determine the phenotype of a plant may comprise both traditional phenotyping techniques and advanced molecular biology or biochemistry tools. Phenotyping may include morphological measurements, such as, for example, plant height, leaf size, and flower characteristics, pod strength, as well as physiological evaluations, such as, for example, photosynthetic efficiency and water use efficiency. Methods for phenotyping may incorporate the use of high-throughput phenotyping technologies, such as imaging systems and sensors that capture detailed data on plant growth and development over time. This technology allows for the non-destructive monitoring of plant traits, facilitating the collection of large datasets that are essential for robust phenotype characterization.

[0080] Pod strength and podshatter resistance can be determined in many ways. The level of resistance to pod shattering is positively correlated with and can, for example, be measured by determining the force needed to break pods in the 'tensile separation test’ (Davies and Bruce, 1997, J Mat Sci 32: 5895-5899; Morgan et al., 1998, Fields Crop Research 58, 153- 165), the number of intact pods remaining after e.g. 20 sec (‘IP20’; Morgan et al., 1998, supra), 9.7 or 17 sec (Bruce et al., 2002, Biosystems Eng 81 (2): 179-184) in a 'random impact test’, the pod sample half-life (hereinafter also referred to as ‘LD50’) in a random impact test, i.e. the treatment time needed to cause the opening of 50% of the pods in tested pod samples, and the 'field score for shattering’ (Morgan et al., 1998, supra). Random impact tests (RITs) and algorithms to define the pod sample half-lives in such RITs have been described in Bruce et al., 2002 (supra) and Morgan et al., 1998 (supra). Both publications are hereby incorporated by reference. Briefly, a sample of intact mature pods is placed in a closed drum together with steel balls and the drum is then vigorously agitated for increasing periods of times (e.g. 10 s, 20 s, 40 s, 80 s). After each period, the drum is opened and the number of broken and damaged pods is counted. The most accurate estimation of the level of shattering resistance for each line is calculated by fitting a linear x linear curve to all the available data and estimating the time taken for half of the pods within a sample to be broken (“pod sample half-life” or “LD50”). It is important however that pods open mainly along the dehiscence zone, and are not simply pulverized, as may occur with indehiscent pods.

[0081] Selecting plants with the desired phenotype can be based on eliminating plants with an undesired phenotype (e.g. culling), or selecting the 5%, or 10%, or 15%, or 20%, or 25%, or 35% , or 40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 95%, or 98%, or 99% best performing plants for the desired phenotype.

[0082] Plants with the desired phenotype can be selected in every generation. Alternatively, plants with the desired phenotype can be selected after the second generation and every subsequent generation, or after the third generation and any subsequent generation. site edits

[0083] In one aspect, the selected plants with the desired phenotype are genotyped for the target site edits. Genoypting the target site edits will reveal the combination of target site edits underlying the desired phenotype. Thus, by genotyping the target site edits, it will be clear which combination of target site edits confers the optimal combination of target site edits to confer the desired phenotype.

[0084] Genotyping of the target site edits can be performed using methods known in the art. For example, DNA extracted from the plants can be amplified in a polymerase chain reaction (PCR) to selectively target regions of interest that are target for BASF Agricultural Solutions US LLC 240242WO02

[0085] 13 editing. This amplification can be performed using specific primers designed to flank the edit site, ensuring that only the relevant genomic regions are amplified, and next-generation sequencing (NGS) to determine the precise sequence of the edited regions.

[0086] The skilled person will understand that the methods according to the invention are optimal in a multiplex editing strategy where the plants in the population used in recurrent selection contain a large variation with regard to the combination of edits present in the different individual plants. Preferably, the design of the editing machinery is not directed to a high efficiency of gene editing in order to create a maximum number of target site edits in one generation in a single plant; rather the methods will rely on the fact that not all target site edits are generated in the same plants in one single generation or in one single step. This results in multiple plants in a population, wherein individual plants comprise different combinations of target site edits.

[0087] Editing efficiency of the different target sites can be tested in advance. For example, if the CRISPR / Cas system is used, gRNA efficiency can be tested before setting up the multiplex editing experiment. In case of high editing efficiency, the methods according to the invention preferably remove the editing machinery at an early step in the process, for example by eliminating plants comprising the editing cassettes, or by transient expression of the editing cassettes. In case of lower editing efficiency, the editing machinery may remain active in the initial steps in the process to increase the number and variation of target site edits.

[0088] In a further aspect, said editing machinery is designed to be stably integrated into the plant genome. The editing machinery may be linked to a selectable or screenable marker gene, and after step b) or after step c) cells or plants comprising the editing machinery in the plant genome can be selected based on expression of the selectable or screenable marker gene. The editing machinery may also be linked to a selectable or screenable marker gene and to a male sterility gene. The editing machinery may be linked to a selectable marker gene and to a male sterility gene, and, in a first crossing, said edited plants are crossed to plants not comprising male sterility and not comprising the editing machinery to produce F1 plants, and wherein after each crossing step, the plants not comprising the selectable marker and male sterility gene are eliminated with a selective agent. In a further aspect, in a first crossing, said edited plants are cross-pollinated with pollen from plants not comprising a male sterility gene and not comprising the editing machinery; male fertile plants not comprising the selectable marker and the male sterility gene are eliminated by application of a selective agent after seeds are produced and before seeds are harvested; and only seeds from male sterile plants comprising the selectable marker and the male sterility gene are retained; in a next crossing, plants derived from said seeds are inter-crossed; male fertile plants not comprising the selectable marker and the male sterility gene are eliminated with a selective agent after seeds are produced and before seeds are harvested; seeds obtained from plants comprising the selectable marker and the male sterility gene are harvested; and only seeds from male sterile plants comprising the selectable marker and the male sterility gene are retained; and optionally, said next crossing step is repeated for one or more generations. In another aspect, after each crossing step plants with the desired phenotype are selected and retained.

[0089] It will be clear that every time the next crossing step is repeated, plants derived from seeds from the latest previous crossing step are used for inter-crossing, so that subsequent generations are obtained.

[0090] In a further aspect, the library of editing cassettes contains an editing cassette for editing an endogenous male fertility gene, optionally a single dominant male fertility gene, and in a first crossing, said edited plants of said population are crossed; in a BASF Agricultural Solutions US LLC 240242WO02

[0091] 14 next crossing, progeny plants obtained from said first crossing are inter-crossed; male sterile plants comprising edits in both alleles of said single dominant male fertility gene are selected, and seeds from male sterile plants are retained; and optionally, said next crossing step is repeated for one or more generations, wherein after at least one of the crossing steps plants with the desired phenotype are selected and retained. In another aspect, said editing machinery is designed to be stably integrated into the plant genome, and wherein the editing machinery is linked to a selectable marker gene and, optionally, to a late pollen ablation gene, and plants comprising the editing machinery in the plant genome are selected after one or more initial crossings, and after one or more subsequent crossings, plants are selected for the absence of the editing machinery. In yet another aspect, said editing machinery is designed to be not stably integrated into the plant genome and, optionally, the editing machinery is delivered as ribonucleoprotein (RNP). In another aspect, in step d) only seeds from a male sterile plant are retained.

[0092] In another aspect, the method according to the invention is provided wherein in step d) said plants are crossed for at least two generations, or at least three generations, or at least four generations, or at least five generations, or at least six generations, and wherein after each crossing step plants with the desired phenotype are selected and retained.

[0093] Selectable marker genes are useful for selection of cells comprising the editing machinery from cells not comprising the editing machinery (i.e. to select transformants or transformed cells with the editing machinery). Selectable marker genes are introduced into plant genome to express a protein generally with an enzymatic activity, which allows distinguishing transformed from non-transformed cells. Genes conferring resistance to selective agents such as antibiotics or herbicides have widely been employed to select transformants. These selectable marker genes enable the transformed cells to survive on medium containing the selective agent, while non-transformed cells and tissues die. Genes encoding resistance to specific antibiotics or herbicides have proved particularly effective for selection and provide a means of rapidly identifying transformed cells, tissues and regenerated shoots that have integrated foreign DNA and that express the selectable gene product and by inference, the gene(s) of interest. Selectable marker genes can be divided into several categories depending on whether they confer positive or negative selection and whether selection is conditional or non-conditional in the presence of external substrates. Positive selectable marker genes are defined as those that allow the growth of transformed tissues, whereas negative selectable marker genes result in the death of the transformed tissue. See also Sundar and Sakthivel (2008) J Plant Physiol 165: 1698-1716.

[0094] Examples of selectable markers are genes conferring resistance to antibiotics, such genes conferring kanamycin resistance such as the nptll gene, or the Atwbd O gene, or the aaC3 gene; genes conferring neomycin resistance such as the nptll gene or the aac3 gene; the genes conferring resistance to paramomycin, G418 such as the nptl gene; genes conferring resistance to Aminoglycosides such as the aaC3 gene; genes conferring resistance to spectinomycin such as the aadA gene; genes conferring resistance to streptomycin such as the SPT gene; genes conferring resistance to Hybromycin such as the hph gene. Other examples of selectable marker genes are genes conferring resistance to herbicides, such as genes conferring resistance to phosphinothricin (PPT), such as the pat gene or the bar gene; genes conferring resistance to glyphosate, including the epsps, aroA, cp4epsps or gox gene; genes conferring resistance to methionine sulfoximine, such as the pat gene; genes conferring resistance to sulfonylureas such as the csr1-1 gene; genes conferring resistance to Imidazolinones, such as csr1 -2 genes. Promoters for expression of such selectable marker genes are known in the art and include promoters conferring constitutive expression and promoters conferring tissue specific expression. In one embodiment, the selectable marker is the double mutated 5-enol-pyruvylshikimate-3-phosphate synthase under control of a BASF Agricultural Solutions US LLC 240242WO02

[0095] 15

[0096] Histone promoter or a 35S promoter or a Ubiquitin promoter. In another embodiment, the selectable marker is the bar gene under control of a Histone promoter or a 35S promoter or a Ubiquitin promoter.

[0097] As an alternative to selectable markers, screenable markers, also called visual markers, scorable markers or reporter genes can be used, useful for selection of cells comprising the editing machinery from cells not comprising the editing machinery (i.e. to select transformants or transformed cells with the editing machinery). Screenable marker genes are introduced into plant genome to express a protein conferring a visual phenotype, which allows distinguishing transformed from nontransformed cells. Genes conferring a visual phenotype include egfp, GFP, BFP, luciferase, GUS, dsRED. Transformed cells expressing a screenable marker can be selected visually, or using cell or seed sorting technologies.

[0098] Male sterility

[0099] The use of male sterility in the methods according to the invention has the advantage that intercrossing between plants in the population is favoured over selfing, especially for self-pollinating plants or mainly self-pollinating plants. Ensuring intercrossing enhances the genetic diversity with regard to the edits in the offspring.

[0100] Male sterility refers to the inability of an organism to produce functional male gametes, rendering it incapable of fertilization. In plants, male sterility can be induced through various means, including EMS mutations, gamma radiation, genetic transformation and cytoplasmic male sterility (CMS). Genetic male sterility is often controlled by specific genes that ensure that only the desired female parent contributes to the next generation. CMS, on the other hand, involves the interaction between nuclear genes and cytoplasmic factors, leading to the production of sterile pollen.

[0101] Dominant and recessive male sterility systems known in the art are suitable for the recurrent selection methods of the current invention. Nearly 200 male-sterility genes have been identified and characterized in plants (see, for example, Wan et al., (2021) Crop J 9: 1219 or Farinati et al (2023) Front Plant Sci. 4: 1223861).

[0102] Examples of nuclear male sterility genes suitable to the invention are ZmbHLH51 , ZmMYB84, OsTDR, CsMYB80, GmAMSI , ZmMs23, TaMsl , OsLTPg29, ZmLTPg11, ZmLTPx2, ZmMs2, ZmlPE1 / ZmMs20, osgptl , ZmMs33 / ZmGPAT6, zma- miR319, zma-miR159 ZmMs7, ZmLCB30, ZmGAMYB (as described in Wan et al., (2021) Crop J 9: 1219.

[0103] Examples of dominant male sterility systems are the wheat Ms2, Ms3 and Ms4 male sterility genes (Yang et al (2021) Int J Mol Sci 22:8541); the rice dominant male sterility from “Jiabuyu” as described by Pang et al (2017) Euphytica 213: 268; Examples of recessive male sterility genes are the wheat ms1 and ms5 male sterility genes (Yang et al (2021) Int J Mol Sci 22:8541); maize ZmMs45, ZmMs7, ZmMs26 or ZmMs30, or rice OsNP1 or OsCYPO703A3. Recessive male sterility genes can be dominant male fertility genes, or can be single dominant male fertility genes.

[0104] Male sterility can also be transgenic, such as germline-specific expression of lethality genes, such as expression of the barnase gene under control of a tapetum-specific promoter.

[0105] Male sterility can also be cytoplasmic, and can be restored using a fertility restorer. BASF Agricultural Solutions US LLC 240242WO02

[0106] 16

[0107] In one aspect of the invention, the edits are generated in a plant comprising cytoplasmic male sterility, and intercrossing is obtained using a nuclear fertility restorer.

[0108] In one aspect, intercrossing can be promoted by linking the editing machinery to a male sterility system, such as a dominant male sterility system, preferably a transgenic dominant male sterility, such as for example transgenic barnase under control of a tapetum-specific promoter. It is important to realize that plants comprising the editing cassette are male sterile and can only be used as female plant in a crossing. Therefore, in one specific aspect, the plants comprising the editing machinery linked to a selectable marker and a male sterility gene are crossed in a first crossing with plants not comprising the editing machinery (and the male sterility gene associated therewith). See Figure 1 b for an illustrative example. Plants comprising the editing machinery can only be cross-pollinated with pollen from the plants not comprising the editing machinery. Plants not comprising the editing machinery can self-pollinate and produce seeds obtained from selfing and not having the edits. To avoid that progeny obtained from selfing of the plants not comprising the editing machinery, and not comprising the edits, are part of the population for the next crossing step, the plants are treated with the selective agent after pollination, resulting in elimination of plants not comprising the editing machinery (and the male sterility gene and selectable marker associated therewith). The plants that are resistant to the selective agent which are heterozygous for the editing machinery will produce seeds comprising edits but not the editing machinery (and not the male sterility gene), and seeds comprising edits but still comprising the editing machinery (and the male sterility gene). See Figure 1c for an illustrative example.

[0109] In a subsequent round of crossing the population of plants derived from said seeds, i.e. comprising a mixture of plants with edits and without editing machinery, and plants with edits and with editing machinery, are inter-crossed. See Figure 1c for an illustrative example. The plants with edits and comprising the editing machinery will serve as female plants, as they contain the male sterility linked to the editing machinery. The plants not comprising the editing machinery will serve as male plants and pollen donors. The plants not comprising the editing machinery will also produce seeds from selfing. To eliminate the use of selfed seeds in the crossing scheme, the plants not comprising the editing machinery are eliminated by treatment with a selective agent after pollination. See Figure 1d for an illustrative example. The retained plants will have seeds comprising edits but not the editing machinery, and seeds comprising edits and comprising the editing machinery. Subsequent rounds of crossings within the population of progeny derived from said seeds can be performed essentially as described above.

[0110] Optionally, the editing machinery consists of two components, such as an RNA-guided nuclease and an RNA, such as a gRNA. The two components, such as the RNA-guided nuclease and the RNA, can be delivered separately, such as for example on two different T-DNAs. Optionally, one of the two components of the editing machinery, such as either the RNA- guided nuclease, or the RNA, can be linked to the male sterility system.

[0111] Separate delivery means that the delivery is configured such that the two components can be integrated at different positions in the genome. Separate delivery means that the two components are not on the same DNA fragment to be integrated in the genome. For example, they can be on two different vectors, such as two different T-DNA vectors. Separate delivery can be performed in one transformation experiment, or in two different transformation experiments.

[0112] In an alternative aspect, intercrossing can be promoted by including an editing cassette for editing an endogenous male fertility gene, optionally a single dominant male fertility gene, such as the Ms1 gene of wheat. Said editing cassette for said endogenous male fertility gene results in an edit in said male fertility gene which results in loss of function of said endogenous male fertility gene, such as said endogenous single dominant male fertility gene. BASF Agricultural Solutions US LLC 240242WO02

[0113] 17

[0114] In one aspect, plants comprising the editing machinery are sorted from plants not comprising the editing machinery; here, plants comprising edits, but not the editing machinery are selected. For an illustrative example, see Figure 2c. Preferably, plants comprising the edit in the single dominant male fertility gene, but not the editing machinery, are selected. Said plants can be selected, for example, using molecular screening methods. Alternatively, a screenable marker can be linked to the editing machinery, such as a seed colour marker of hypocotyl colour marker, and plants not comprising the editing machinery can be selected based on phenotypic screening. Alternatively, a selectable marker, preferably a non-lethal selectable marker is linked to the editing machinery and plants can be selected or sorted, for example using leaf dip tests for herbicides or antibiotics. It is understood that, once the editing machinery is provided to the cell, the editing machinery can expressed transiently to introduce edits. Resulting plants therefore may contain edits but not the editing machinery in the genome. Selected plants comprising the edit in the single dominant male fertility gene but not the editing machinery may have the edit in the single dominant male fertility gene in a heterozygous form. Said plants having the edit in the single dominant male fertility gene in a heterozygous form will still be male fertile and can be used in a intercrossing step. The resulting plants will have seeds not comprising the edits in the single dominant male fertility gene (25%) which are fertile; comprising the edits in the single dominant male fertility gene in heterozygous form (50%) which are fertile, and comprising the edits in the single dominant male fertility gene in homozygous form (25%) which are male sterile. See Figures 2c and 2d for an illustrative example. The edits in other genes than the single dominant male fertility gene will also segregate in the population.

[0115] Said seeds will be used in a subsequent round of intercrossing in a population of which theoretically 75% of the plants is male fertile and 25% of the plants is male sterileln subsequent generations, theoretically 50% of the plants will be male fertile, and 50% of the plants will be male sterile.

[0116] In a particular embodiment, after one or more of the crossing steps, in particular after the second and subsequent crossing steps, only seeds from male sterile plants are selected and retained. In case a single dominant male fertility gene is edited, plants comprising the edits in the single dominant male fertility gene in homozygous form, or comprising the edits in both alleles of the single dominant male fertility gene, are selected and retained. See Figure 2d for an illustrative example. Such selection can be performed using methods known in the art and can comprise, for example, molecular screening methods. Such selection can be performed through identification of plants not comprising the single dominant male fertility gene in homozygous form, and eliminating said plants. Alternatively, such selection can be performed through identification of plants comprising the single dominant male fertility gene in homozygous form, and retaining seeds from said plants. Said seeds will be used in a subsequent round of intercrossing in a population of which 50% of the plants is male fertile (heterozygous for the edit in the male fertility gene) and 50% of the plants is male sterile (homozygous for the edit in the male fertility gene). Optionally, seeds from male sterile plants of naturally self-pollinating plants are selected through (1) bagging of one spike or flower or flowering organ of the plants in the population before flowering; (2) selecting plants which do not produce seeds from the bagged spike or flower or flowering organ; and (3) harvesting seeds from the non-bagged spike or flower or flowering organ of said selected plant.

[0117] Alternatively, said editing editing cassette for editing said endogenous male fertility gene contains a screenable or selectable marker to be inserted into said endogenous male fertility gene. Plants comprising the edit in said endogenous male fertility gene can be selected based on expression of said selectable or screenable marker.

[0118] Optionally, in the method according to the invention in which an editing cassette for editing an endogenous single dominant male fertility gene is used, the editing machinery can be linked to a pollen lethality gene, such as a post meiotic pollen ablation gene, to ensure that any pollen containing the editing machinery are eliminated. See Figure 2b for an illustrative BASF Agricultural Solutions US LLC 240242WO02

[0119] 18 example. Post meiosis pollen promoters can be used as described by Chent et al (2023) Plant Mol Biol Rep 41 :630 or Wang et al (2020) J Integr Plant Biol 62: 1246. This ensures inheritance of the editing machinery through the female germline only. When the editing machinery is present as a single copy single locus in a hemizygous state, 50% of the pollen containing the transgene will be eliminated leaving 50% fertile pollen on the plant. Said plants can be used as random pollinator in the above-described crossing scheme to introduce new edits via the male germline but will not transfer the transgene and all cross-pollinated progeny will be non-transgenic and may contain new edits.

[0120] In another aspect of the invention, the editing machinery is not stably integrated into the genome. Edits can be made, for example, using delivery of ribonucleoprotein complexes (RNPs). RNPs are well known in the art for the use of CRISPR / Cas in which the RNA-guided nuclease is provided as protein together with gRNAs. Upon transient expression of the editing machinery, edits are made in the cells, and edited cells will be used to generate a population of plants for crossing and selection. Preferably, the edited cells contain an edit in an endogenous male fertility gene, such as in an endogenous single dominant male fertility gene. Selected plants comprising the edit in the single dominant male fertility gene but not the editing machinery may have the edit in the single dominant male fertility gene in a heterozygous form. Said plants having the edit in the single dominant male fertility gene in a heterozygous form will still be male fertile and can be used in a intercrossing step. Theoretically, the resulting plants have seeds not comprising the edits in the single dominant male fertility gene (25%) which are fertile; comprising the edits in the single dominant male fertility gene in heterozygous form (50%) which are fertile, and comprising the edits in the single dominant male fertility gene in homozygous form (25%) which are male sterile. See Figures 3c and 3d for an illustrative example. The edits in other genes than the single dominant male fertility gene also segregate in the population.

[0121] Said seeds will be used in a subsequent round of intercrossing in a population of which theoretically 75% of the plants is male fertile and 25% of the plants is male sterile. In subsequent generations, theoretically 50% of the plants will be male fertile and 50% male sterile.

[0122] In a particular embodiment, after each crossing step, only seeds from male sterile plants comprising the edits in the single dominant male fertility gene in homozygous form, or comprising the edits in both alleles of the single dominant male fertility gene, are selected and retained. See Figure 3d for an illustrative example. Such selection can be performed using methods known in the art and can comprise, for example, molecular screening methods. Such selection can be performed through identification of plants not comprising the edit in the single dominant male fertility gene in homozygous form, or not comprising an edit in both allelels of the single dominant male fertility gene, and eliminating said plants. Alternatively, such selection can be performed through identification of plants comprising the edit in the single dominant male fertility gene in homozygous form, or comprising an edit in both alleles of the single dominant male fertility gene, and retaining seeds from said plants. Plants comprising the edit in the single dominant male fertility gene in homozygous form, or comprising an edit in both alleles of the single dominant male fertility gene, can be identified by (1) bagging of one spike or flower or flowering organ of the plants in the population before flowering; (2) selecting plants which do not produce seeds from the bagged spike or flower or flowering organ; and (3) harvesting seeds from the non-bagged spike or flower or flowering organ of said selected plant. Said seeds will be used in a subsequent round of intercrossing in a population of which 50% of the plants is male fertile (heterozygous for the edit in the male fertility gene) and 50% of the plants is male sterile (homozygous for the edit in the male fertility gene or having an edit in both alleles of the male fertility gene). BASF Agricultural Solutions US LLC 240242WO02

[0123] 19

[0124] In an alternative aspect, intercrossing can be promoted by including an editing cassette for editing an endogenous single dominant male sterility gene. In case one or two alleles of the single dominant male sterility gene are edited, the plants are male sterile. Thus, in the methods according to the invention, plants comprising the edit in the single dominant male sterility gene are cross-pollinated with plants not comprising the single dominant male sterility gene. When plants not comprising the single dominant male sterility gene are removed after pollination, the resulting seeds will not comprise the edits in the single dominant male sterility gene (25%) which are fertile; comprise the edits in the single dominant male sterility gene in heterozygous form (50%) which are male sterile, and comprise the edits in the single dominant male fertility gene in homozygous form (25%) which are male sterile. Said population is again used for intercrossing, plants not comprising the single dominant male sterility gene are removed after pollination.

[0125] Optionally, after each crossing step, the plants are screened for the desired phenotype and the top 5% of plants are retained. Also suitable to the invention is the retention of the top 10%, or the top 15%, or the top 20%, or the top 25%, or the top 30%, or the top 40%, or the top 50% or more of plants with the desired phenotype. Also suitable to the invention is no selection after the first crossing step, or after the first and second crossing step, and only selection after the subsequent crossing step or crossing steps.

[0126] Recurrent mass selection

[0127] In one embodiment, the optimal combination of multiple edits is selected using recurrent mass selection, or recurrent selection.

[0128] Recurrent mass selection as used in breeding in plant genetics aims at improving the performance and stability of specific traits within a population. This strategy focuses on the continuous enhancement of a plant population by systematically selecting superior individuals over multiple generations. It is particularly effective in developing new varieties that exhibit desirable, multigenic characteristics such as increased yield, enhanced disease resistance, and improved adaptability to varying environmental conditions.

[0129] The fundamental principle behind recurrent mass selection is to exploit the genetic variability present within a population i.e. in the context of the current invention, the variability of combinations of multiple edits in different target sequences, including different genes, different homeologous genes, different genes in a family, or different genes from different families or different genes from one or multiple pathways. By identifying and selecting individuals with the desired phenotype, the frequency of favorable edits will be increased in subsequent generations. The first step involves creating or selecting a diverse plant population with different edits. This genetic diversity is essential, as it provides a reservoir of alleles from which favorable traits can be combined and selected.

[0130] Once the superior individuals are identified, they are allowed to intercross. This step is crucial as it facilitates the recombination of genetic material, leading to the generation of a new population that inherits traits from both of the crossed individuals. As indicated above, intercrossing can be improved using male sterility to avoid self-pollination, especially for (mainly) self-pollinating species.

[0131] The newly formed population then undergoes another round of phenotypic assessment and the process of selection is repeated, focusing on identifying the best-performing individuals within the population once again. This cyclical approach ensures that each generation continues to accumulate favorable edits. BASF Agricultural Solutions US LLC 240242WO02

[0132] 20

[0133] The population size for recurrent mass selection will depend on the number of target edits, the editing efficiency, number of seeds per plant, number of generations and phenotyping throughput. The population can comprise, for example, at least 50 plants, or at least 100 plants, or at least 500 plants, or at least 1000 plants, at least 5000 plants, or at least 10 000 plants.

[0134] In a further aspect, the method according to the invention is provided wherein said plant are crossed for at least two generations, and wherein (i) plants are selected for the absence of the editing machinery; or (ii) plants comprising the editing machinery in the plant genome are selected after one or more initial crossings, and plants comprising the editing machinery are not selected after one or more subsequent crossings, optionally wherein after said one or more subsequent crossings, plants are selected for the absence of the editing machinery.

[0135] It is understood that the method aims at maximizing the number of possible combinations of edits in the genome of individuals of the population.

[0136] In the embodiment where the editing machinery is not stably integrated in the plant genome, or in which plants are selected for the absence of the editing machinery, edits can be generated during a limited time window in which the editing machinery is expressed. Selection for the absence of the editing machinery can be done before or after the first crossing, or after subsequent crossing.

[0137] Alternatively, to increase the number of edits in the genome, the editing machinery can be integrated stably in the plant genome and retained during initial crossing steps. Stable integration of the editing machinery in the plant genome allows the generation of new edits during the different cycles of crossing and selection. It may also be advantageous to allow generation of new edits only during a limited number of cycles of crossing and selection, and no longer in later cycles of crossing and selection. Therefore, in one embodiment, plants comprising the editing machinery are selected after one or more initial crossings, such as after the first crossing, or after the first and second crossing, of after the first, second and third crossing, or after the first, second, third and fourth crossing. After subsequent crossings, the presence of the editing machinery is no longer selected for. In an alternative embodiment, after subsequent crossings, such as after the second crossing, or after the third crossing, or after the fourth crossing, or after the fifth crossing, plants are selected for the absence of the editing machinery.

[0138] Absence of the editing machinery ensures that no further edits can be generated and that the genotype of the plants can be fixed.

[0139] In an alternative embodiment, plants are selected for the absence of the editing machinery in initial crossings, and editing machinery is re-introduced in subsequent crossings.

[0140] In one aspect, the editing machinery consists of two components, such as an RNA-guided nuclease and an RNA, such as a gRNA, wherein one of the components of the editing machinery, such as either the RNA-guided nuclease, or the RNA, is linked to the transgenic dominant male sterility gene, and is stably integrated in the plant genome. Plants comprising the editing machinery including both components, such as the RNA-guided nuclease and the RNA, are selected after one or more initial crossings, whereas after subsequent crossings, plants are selected for the absence of the component of the editing machinery, RNA-guided nuclease or the RNA, which is not linked to the transgenic dominant male sterility gene, and for the presence of the component of the editing machinery, such as the RNA-guided nuclease or the RNA, which is linked to the transgenic dominant male sterility gene.

[0141] It is suitable to the invention that the component of the editing machinery which is not linked to the transgenic dominant male sterility gene contains a first selectable marker and optionally a visual marker, and the component of the editing machinery BASF Agricultural Solutions US LLC 240242WO02

[0142] 21 which is linked to the transgenic dominant male sterility contains a second selectable marker. Selection of plants comprising the editing machinery including the RNA-guided nuclease and the RNA are selected after one or more initial crossings using a selective agent for both first and second selectable marker. In subsequent generation, selection of plants for the absence of the component of the editing machinery which is not linked to the transgenic dominant male sterility gene can be performed using molecular analysis or absence of the first selectable marker using, for example, a non-destructive leaf assay, or for the absence of the visual marker; and for the presence of the component of the editing machinery which is linked to the transgenic dominant male sterility gene can be performed using a selective agent for the second selectable marker only.

[0143] In another aspect, the method according to the invention is provided in which activation of said editing machinery is inducible. Editing machinery can be inducible by operably linking the coding sequences of the editing machinery to an inducible promoter. For example, the sequences coding the Cas proteins can be operably linked to an inducible promoter.

[0144] Suitable inducible promoters, such as chemical inducible promoters, are known in the art and described, for example, in Corrado and Karali (2009) Biotechnol Advances 27: 733.

[0145] Expression of the editing machinery may be induced in one or more initial crossings. After subsequent crossings, the editing machinery may no longer be induced to ensure that no further edits can be generated and the phenotype can be fixed.

[0146] In one embodiment, plants with the desired phenotype are selected and retained after at least one of the crossing steps. Optionally, plants are selected for the desired phenotype after one or more first crossing steps, and plants are selected for the desired phenotype only after one or more subsequent crossing steps. It is understood that, in this scenario, first crossing steps are performed to enrich the diversity of edits in the population, whereas subsequent crossing steps are performed for selection of optimal edit combinations.

[0147] In the selected individuals, the optimal combination of target site edits may comprise homozygous or heterozygous edits. Homozygous edits can be fixed in an individual by selfing or crossing with individuals also comprising the same edit.

[0148] Heterozygous edits can be fixed in an individual by crossing with an individual not comprising the edit. Heterozygous edits are particularly suitable in hybrid breeding, where only one of the parents needs to contain the edit.

[0149] In another aspect, the methods according to the invention are used to produce a plant with an optimal combination of multiple gene edits to confer a desired phenotype.

[0150] In a further aspect, a plant is provided, or cell, tissue, organ, material, or seed of said plant, obtained by or obtainable by methods according to the invention.

[0151] Also suitable to the invention is a method to produce a plant comprising an optimal combination of multiple gene edits for a desired phenotype, said method comprising (a) providing a seed mixture comprising a plurality of combinations of multiple gene edits obtained from a crossing of edited plants in a population for at least one generation, wherein said edited plants in said population are generated from cells to which editing machinery is provided, said editing machinery comprising a library of editing cassettes to create multiple edits, wherein said edited plants comprise one or more of said multiple edits; and (b) selecting a plant with the desired phenotype. BASF Agricultural Solutions US LLC 240242WO02

[0152] 22

[0153] Also suitable to the invention is a method to select an optimal combination of multiple edits to confer a desired phenotype in a plant, said method comprising (a) providing a seed mixture comprising a plurality of combinations of multiple gene edits obtained from a crossing of edited plants in a population for at least one generation, wherein said edited plants in said population are generated from cells to which editing machinery is provided, said editing machinery comprising a library of editing cassettes to create multiple edits, wherein said edited plants comprise one or more of said multiple edits; and (b) selecting a plant with the desired phenotype.

[0154] Also suitable to the invention is a method to select a plant with an optimal combination of multiple edits to confer a desired phenotype, said method comprising (a) providing a seed mixture comprising a plurality of combinations of multiple gene edits obtained from a crossing of edited plants in a population for at least one generation, wherein said edited plants in said population are generated from cells to which editing machinery is provided, said editing machinery comprising a library of editing cassettes to create multiple edits, wherein said edited plants comprise one or more of said multiple edits; and (b) selecting a plant with the desired phenotype.

[0155] In one aspect, said method further comprises the step of genotyping said selected plant with the desired phenotype for the target site edits.

[0156] In a further aspect, said editing machinery which is provided to said cells is designed to be stably integrated into the plant genome and, optionally, said edited plants have been selected from the presence of the editing machinery in the plant genome. In a further aspect, said editing machinery is linked to a selectable or screenable marker gene, and cells or plants comprising the editing machinery in the plant genome have been selected based on expression of the selectable or screenable marker gene. In yet another aspect, said editing machinery is linked to a selectable marker gene and to a male sterility gene and, optionally, said edited plants have been crossed in a first crossing to plants not comprising male sterility and not comprising the editing machinery to produce F1 plants, and wherein after each crossing step, the wild-type plants not comprising the selectable marker have been eliminated with a selective agent. In yet another aspect, said library of editing cassettes contains an editing cassette for editing an endogenous male fertility gene, optionally a single dominant male fertility gene, wherein said editing machinery optionally is designed to be stably integrated into the plant genome and linked to a selectable marker gene and, optionally, to a late pollen ablation gene. In another aspect, said library of editing cassettes contains an editing cassette for editing an endogenous male fertility gene, optionally a single dominant male fertility gene, and is designed to be not stably integrated into the plant genome and, optionally, wherein the editing machinery is delivered as ribonucleoprotein (RNP). In another aspect, said seed mixture comprising a plurality of combinations of multiple gene edits obtained from a crossing of edited plants only comprises seeds obtained from male sterile plants. In a further aspect, said seed mixture is obtained from a crossing of edited plants in a population for at least two generations, for at least three generations, for at least four generations, for at least five generations, or for at least six generations, and wherein after each crossing step plants with the desired phenotype have been selected and retained. In a further embodiment, said seed mixture is obtained from from a crossing of edited plants in a population for at least two generations, wherein plants have been selected for the absence of the editing machinery, or wherein plants comprising the editing machinery in the plant genome have been selected after one or more initial crossings, and plants comprising the editing machinery have not been selected after one or more subsequent crossings and, optionally, plants have been selected for the absence of the editing machinery after said one or more subsequent crossings. In a further aspect, activation of said editing machinery is inducible. BASF Agricultural Solutions US LLC 240242WO02

[0157] 23

[0158] In one aspect, the method according to the invention is provided wherein the plant is a Brassica plant, and the desired phenotype is pod shatter reduction.

[0159] A suitable library of editing cassettes to create multiple gene edits can be editing cassettes for creating gene edits in target genes in the pod dehiscence pathway. Target genes in the pod dehiscence pathway can, for example, be IND, ADPG1, ADPG2, FIL, SHP1, PID, SHP2, FUL, ALC, NST1-3, PIN3, JAG, transducing family protein, QRT2, YAB3 and HEC3. Preferably, target genes in the pod dehiscence pathway are target genes of which loss of function results in reduction of pod shattering, such as IND, ALC, NST and SHP. Preferably, all homologs and / or homeologs of said genes are targeted. Suitable gRNAs for targeting genes in the pod dehiscence pathway are gRNAs having the sequences of SEQ ID NO: 1 to 18.

[0160] "Reduction of pod shattering", "pod shatter reduction", “increase of pod shatter resistance”, "increase of pod strength", and “reduction of seed shattering”, as used herein, refers to a decreased seed shatter tendency and / or a delay in the timing of seed shattering, in particular until after harvest, of Brassica plants, the fruits of which normally do not mature synchronously, but sequentially, so that some pods burst open and shatter their seeds before or during harvest. The level of pod shattering is positively correlated with and can, for example, be measured by determining the force needed to break pods in the 'tensile separation test’ (Davies and Bruce, 1997, J Mat Sci 32: 5895-5899; Morgan et al., 1998, Fields Crop Research 58, 153- 165), the number of intact pods remaining after e.g. 20 sec (‘IP20’; Morgan et al., 1998, supra), 9.7 or 17 sec (Bruce et al., 2002, Biosystems Eng 81 (2): 179-184) in a 'random impact test’, the pod sample half-life (hereinafter also referred to as ‘LD50’) in a random impact test, i.e. the treatment time needed to cause the opening of 50% of the pods in tested pod samples, and the 'field score for shattering’ (Morgan et al., 1998, supra). Random impact tests (RITs) and algorithms to define the pod sample half-lives in such RITs have been described in Bruce et al., 2002 {supra), Morgan et al., 1998 {supra) and the Examples below. Both publications are hereby incorporated by reference. Briefly, a sample of intact mature pods is placed in a closed drum together with steel balls and the drum is then vigorously agitated for increasing periods of times (e.g. 10 s, 20 s, 40 s, 80 s). After each period, the drum is opened and the number of broken and damaged pods is counted. The most accurate estimation of the level of shattering resistance for each line is calculated by fitting a linear x linear curve to all the available data and estimating the time taken for half of the pods within a sample to be broken (“pod sample halflife” or “LD50”). It is important however that pods open mainly along the dehiscence zone, and are not simply pulverized, as may occur with indehiscent pods. Such a reduction in pod shattering can be a reduction by at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%. A reduction in pod shattering can thus be an increase in pod shatter resistance or an increase in pod strength, or an increase in LD50, by at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 120%, or at least 150%, or at least 200%, or at least 250%, or at least 300%, or at least 400%, or at least 500%, or can be at least a 1 .2-fold increase, at least a 1 .3-fold increase, at least a 1 .4-fold increase, at least a 1 .5-fold increase, at least a 1 .6 fold increase, at least a 1 .7-fold increase, at least a 1 .8-fold increase, at least a 1 .9-fold increase, at least a 2-fold increase, at least a 2.2-fold increase, at least a 2.5-fold increase, at least a 3-fold increase, at least a 3.5-fold increase, at least a 4-fold increase, at least a 5-fold increase, or at least a 6-fold increase.

[0161] As used herein, “pod shattering" or "seed shattering” or “fruit or pod dehiscence” refers to a process that takes place in a fruit after seed maturation, whereby the valves detach from the central septum freeing the seeds. The region that breaks (i.e. the “dehiscence zone”) runs the entire length of the fruit between the valves and the replum (external septum). At maturity, the “dehiscence zone” is essentially a non-lignified layer of cells between a region of lignified cells in the valve and BASF Agricultural Solutions US LLC 240242WO02

[0162] 24 the replum. Shattering occurs due to the combination of cell wall loosening in the dehiscence zone and the tensions established by the differential mechanical properties of the drying cells in the silique.

[0163] The Brassica plant can be a Brassica oilseed plant, such as Brassica napus, Brassica juncea, or Brassica rapa.

[0164] Whenever reference to a “plant” or “plants” according to the invention is made, it is understood that also plant parts (cells, tissues or organs, seed pods, seeds, severed parts such as roots, leaves, flowers, pollen, etc.), progeny of the plants which retain the distinguishing characteristics of the parents (especially the fruit dehiscence properties), such as seed obtained by selfing or crossing, e.g. hybrid seed (obtained by crossing two inbred parental lines), hybrid plants and plant parts derived there from are encompassed herein, unless otherwise indicated.

[0165] All patents, patent applications, and publications or public disclosures (including publications on internet) referred to or cited herein are incorporated by reference in their entirety.

[0166] Sequence listing

[0167] The sequence listing contained in the file named “240242_SEQLISTING_St26.xml“, which is 23 kilobytes (size as measured in Microsoft Windows®), contains 18 sequences SEQ ID NO: 1 through SEQ ID NO: 18 is filed herewith by electronic submission and is incorporated by reference herein.

[0168] In the description and examples, reference is made to the following sequences:

[0169] SEQ ID No. 1 : IND-BnA03 gRNA

[0170] SEQ ID No. 2: IND-Bn-C03 gRNA

[0171] SEQ ID No. 3: ALC1-BnA07 gRNA

[0172] SEQ ID No. 4: ALC1-BnC07 gRNA

[0173] SEQ ID No. 5: ALC2-BnA04 gRNA

[0174] SEQ ID No. 6: ALC2-BnC04 gRNA

[0175] SEQ ID No. 7: NST1-1-BnA05 gRNA

[0176] SEQ ID No. 8: NST1-1-BnC04 gRNA

[0177] SEQ ID No. 9: NST1-2-BnA04 gRNA

[0178] SEQ ID No. 10: NST1-2-BnC04 gRNA

[0179] SEQ ID No. 11 : SHP1a-BnA04 gRNA

[0180] SEQ ID No. 12: SHP1 a-BnC04 gRNA

[0181] SEQ ID No. 13: SHP1 b-BnA09 gRNA

[0182] SEQ ID No. 14: SHP1 b-BnC08 gRNA

[0183] SEQ ID No. 15: SHP1c-BnA07 gRNA

[0184] SEQ ID No. 16: SHP1c-BnC03 gRNA

[0185] SEQ ID No. 17: SHP2-BnA05 gRNA

[0186] SEQ ID No. 18: SHP2-BnC04 gRNA BASF Agricultural Solutions US LLC 240242WO02

[0187] 25

[0188] Examples

[0189] Example 1. Multiplex editing of podshatter reduction genes in oilseed rape

[0190] Seed dehiscence is regulated by many genes. Examples of Brassica and Arabidopsis genes involved in seed dehiscence are Indehiscent, ADPG1, ADPG2, FIL, SHP1, PID, SHP2, FUL, ALC, NST1-3, PIN3, JAG, transducing family protein, QRT2, YAB3 and HEC3. See, for example, Jaradat et al., (2014) GM Crops & Food 5;4, 302-320.

[0191] Modifications in those genes can be used to reduce premature seed shattering in crops, such as Brassica napus. Advantages of reduced seed shattering are increased yield, reduced premature seed loss, and increased window for straight cutting, which reduces the need for swathing.

[0192] To obtain these benefits, pod dehiscence should not be inhibited completely, as this would result in unharvestable seeds. Rather, in order to obtain agronomically relevant podshatter reduction, dehiscence should be reduced but not completely abolished, so that pods can still be opened along the dehiscence zone by applying limited physical forces.

[0193] 1.1 T arget genes for multiplex editing for podshatter resistance in Brassica napus

[0194] The genes as shown in Table 1 are targets for multiplex editing.

[0195] Table 1. Thirty-one Brassica napus genes target for multiplex editing. Start and end position refers to the position in the Brassica napus Darmor-bzh genome assembly as described in Rousseau-Gueutin et al., 2020, GigaScience, Volume 9, Issue 12, giaal 37. The last column indicates whether a downregulation (Down) or upregulation (Up) is required to achieve podshatter reduction. BASF Agricultural Solutions US LLC 240242WO02

[0196] 26

[0197] 1 .2 Design of multiplex editing of pod dehiscence genes in Brassica napus targeting individual genes

[0198] 18 gRNAs were designed for knock-out of different copies of the IND, ALC, NST and SHP genes as shown in Table 2.

[0199] Table 2. gRNAs for knock-out of different copies of the IND, ALC, NST and SHP genes. RR refers to the Cas12a variant having the S542R / K607R mutations as described by Gao et al. (2017). Nat Biotechnol 35, 789. BASF Agricultural Solutions US LLC 240242WO02

[0200] 27

[0201] 1 .3 Transformation of Brassica napus.

[0202] A T-DNA construct is generated containing wild-type Cas12a under control of the constitutive Ubiquitin promoter, and with epsps under control of the constitutive 35S promoter. Another T-DNA construct is generated containing the Cas12a variant having the S542R / K607R mutations as described by Gao et al. (2017). Nat Biotechnol 35, 789 (Cas12a RR variant) under control of the constitutive Ubiquitin promoter, and with epsps under control of the constitutive 35S promoter.

[0203] A T-DNA construct is generated, with the gRNAs applicable to wild-type Cas12a as described above. Another T-DNA construct is generated, with the gRNAs applicable to the Cas12a RR variant as described above. The gRNA expression is driven by the U6 promoter. The T-DNAs with the gRNAs further contain the bar gene under control of a Rubisco small subunit promoter, and the Barnase gene under control of the anther-specific TA29 promoter.

[0204] The above-mentioned T-DNA vectors are introduced into Agrobacterium pGV4000 and transformed into Brassica napus plants according to the hypocotyl explant inoculation method (essentially as described in De Block et al, 1989, Plant Physiol., 91 : 64 or in WO 00 / 04173). BASF Agricultural Solutions US LLC 240242WO02

[0205] 28

[0206] The T-DNA construct with the wild-type Cas12a and the T-DNA construct with the gRNAs applicable to wild-type Cas12a are transformed together into one an Agrobacterium strain; the T-DNA construct with the Cas12a RR variant and the T-DNA construct with the gRNAs applicable to the Cas12a RR variant are transformed into one Agrobacterium strain.

[0207] Shoots are selected on PPT. Shoots are analyzed for presence of the respective Cas12a variants and the gRNA expression cassettes, and plants are grown from shoots having low copy insertion of the transgenes.

[0208] 1 .4 Design of multiplex editing of pod dehiscence genes in Brassica napus targeting gene families

[0209] In addition to the gRNAs specifically targeting individual genes, gRNAs are designed to target multiple genes in the same family. All gRNAs are applicable to wild-type Cas12a. T-DNA constructs are generated and Brassica napus is transformed as described above.

[0210] 1 .5 Recurrent selection for optimal combination of edits

[0211] TO stable transformants containing the transgene(s) are pollinated with wild-type plants not containing the Cas12a and gRNA cassettes. After pollination, the wild type plants are eliminated by spraying with glufosinate.

[0212] Resulting F1 seeds are sown out, plants are grown, and pollinated under conditions that allow for cross-pollination of different plants in the F1 population. Plants are rouged for pleiotropic effects on plant development and flowering time. After pollination, plants not containing the bar gene (and the barnase gene) are eliminated by spraying with glufosinate.

[0213] The remaining plants are analyzed for podshatter using random impact test as described in Bruce ef al., 2001 (J. Agric. Engng Res. 80, 343-350) and Morgan ef al., 1988 (Fields Crop Research 58, 153-165). Seeds from pods with a random impact higher than the wild-type control but which are still opened along the dehiscense zone are sown out and grown (F2), and subjected to another round of intermating as described above.

[0214] Five iterative cycles of crossing and selection are performed. Upon the fifth iterative cycle, the top 15% of individuals (..selected individuals") are genotyped for target site edit frequencies.

[0215] Selected lines are crossed with wild-type plants. Offspring is genotyped for presence of the target site edits and absence of the transgene(s). Lines containing the target site edits and lacking the transgenes are selected. This step is repeated until all target site edits as present in the “selected individuals” are recovered in one plant.

[0216] The target site edits are fixed to homozygosity by repeated selfings and selection for presence of target site edits.

[0217] Target site edits which are heterozygous in the “selected individuals” are introgressed into the male parent for hybrid production, so that the hybrid will contain the target site edit in heterozygous state. BASF Agricultural Solutions US LLC 240242WO02

[0218] 29

[0219] Target site edits which are homozygous in the “selected individuals” are introgressed both into the male and the female parents for hybrid production, so that the hybrid will contain the target site edit in homozygous state.

[0220] Example 2. Multiplex editing of flowering time genes in soybeans

[0221] Soybean is sensitive to photoperiodic conditions, affecting flowering time and maturity. Many genes are known to affect flowering time in soybean. Modification in these genes can reduce flowering time and resulting in earlier maturity.

[0222] 2.1 T arget genes for multiplex editing for reduce flowering time

[0223] The following genes known to modify flowering time in soybean (Luo et al, 2022, Int J Mol Sci 23(1):466) are chosen as target for knock-out in multiplex editing:

[0224] E1

[0225] E1 La

[0226] E1 Lb

[0227] GmGI (E2)

[0228] GmphyA3 (E3) (phytochrome A1

[0229] GmphyA2 (E4) (phytochrome A2

[0230] GmFT4 (E10) Inhibition (FLOWERING LOCUS T4)

[0231] GmFT1 a (FLOWERING LOCUS T1 a)

[0232] GmCOL1 a / 1 b Inhibition (CONSTANS-like 1 a / 1 b) miR156

[0233] GmTOE4a (TARGET OF EAT 4a)

[0234] GmPRR3a (Tof11) (PSEUDO-RESPONSE REGULATOR3a / Time of Flowering 11)

[0235] GmPRR3b (Tof12) (PSEUDO-RESPONSE REGULATOR3b / Time of Flowering 12) gRNAs are designed to create knock-outs in these genes.

[0236] 2.2 Transformation of soybean.

[0237] A T-DNA construct is generated containing the coding sequence of Lachnospiraceae Cas12a under control of a constitutive Ubiquitin promoter, and with the epsps gene under control of a constitutive 35S promoter.

[0238] T-DNA constructs are generated each containing up to 12 gRNAs as described above. The gRNA expression is driven by an U6 promoter. Each T-DNA with the gRNAs further contains the bar gene under control of a constitutive promoter, and the Barnase gene under control of an anther-specific promoter.

[0239] The above T-DNA vectors are introduced into Agrobacterium and transformed into soybean. Briefly, mature seeds of Thorne are used for stable transformation. Seeds are surface sterilized in a desiccator for about 16 hrs using chlorine gas as described by Di et al. 1996. Agrobacterium transformation using half seed explants is essentially as described by Paz et al. BASF Agricultural Solutions US LLC 240242WO02

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[0241] (2006) and Luth et al. (2015). The disarmed A. tumefaciens strain EHA105 (Hood et al. 1993) and the disarmed A. rhizogenes strain SHA017 [K599(pRi2659)] (Mankin et al., 2007; WO 2006 / 024509), both harboring the T-DNA vector are used for co-cultivation of the half seed explants. After 5 to 6 says co-cultivation, glyphosate resistant shoots are selected on a selection medium containing 0.075mM Glyphosate.

[0242] Shoots are analyzed for presence of the transgene(s), and plants are grown from shoots having low copy insertion of the transgene(s).

[0243] 2.3 Recurrent selection for optimal combination of edits

[0244] TO stable transformants containing the transgene(s) are pollinated with wild-type plants not containing the Cas12a and gRNA cassettes. After pollination, the wild type plants are eliminated by spraying with glufosinate.

[0245] Resulting F1 seeds are grown. After pollination, plants not containing the bar gene (and the barnase gene) are eliminated by spraying with glufosinate. The remaining plants are phenotyped and selected based on early flowering, and rouged for possible pleiotropic effects impacting plant development. Seeds from plants with a preferred range of flowering time are grown (F2).

[0246] Five iterative cycles of crossing, rogueing and selection are performed. Upon the fifth iterative cycle, the top 15% of individuals (..selected individuals") are genotyped for target site edit frequencies.

[0247] Selected lines are crossed with wild-type plants. Offspring are genotyped to test for the presence of the target site edits and the transgene(s). Lines containing the target site edits and lacking the transgenes are selected. This step is repeated until all target site edits as present in the selected individuals are recovered in one plant.

[0248] For target site edits which are homozygous in the selected individuals, the target site edits are fixed to homozygosity by repeated selfings and selection for the presence of target site edits.

[0249] Example 3. Multiplex editing in wheat

[0250] 3.1 Transformation of wheat

[0251] Two DNA constructs are generated:

[0252] A construct comprising 12x multiplexed gRNAs with one gRNA targeting a single dominant male fertility locus (MS1) on chromosome 4BS; the remaining gRNAs target discovery gene leads.

[0253] A construct containing Cas12a under control of a constitutive Ubiquitin promoter, a seed colour marker dsRED under control of the HvLTP2 promoter, allowing sorting of transgenic from non-transgenic regenerants as well as seed; and a post-meiotic gene that ensures any pollen containing the transgene are eliminated. If the transgene is a single copy single locus event in a hemizygous state (ie in TO generation) then 50% of pollen containing transgene will be eliminated leaving 50% fertile pollen. Self-pollinating a hemizygous single locus transgenic plant will ensure transgene remains in a hemizygous state. The transgene can only be inherited through the female germline. This leads to segregation distortion. BASF Agricultural Solutions US LLC 240242WO02

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[0255] The two DNA constructs are assembled in a single binary vector and transformed into wheat plants according to methods known in the art.

[0256] 3.2 Recurrent mass selection for optimal combination of edits

[0257] Transgenic plants comprising the editing machinery are sorted from non-transgenic plants comprising edits based on the presence or absence of the selectable marker present in the binary vector.

[0258] The non-transgenic plants are selected for presence of a hemizygous ms1 edit or visual expression of male sterility and used for recurrent mass selection. The plants are randomly inter-sib mated. After each generation, plants are either genotyped for Ms1 / ms1 or phenotyped for sterility (ms1 / ms1) by bagging heads, with only seeds from ms1 / ms1 plants being retained to ensure that the seed is derived from cross-pollination and not from a selfing. After each generation, the top 5% of male sterility expressing plants phenotypically selected for the trait of interest are retained for seed, and subsequently replanted as a single population for intersib mating. These plants are crossed and selected for 6 or more successive generations.

[0259] After 6 or more rounds of crossing and selection, best performing ms1 / ms1 individuals for the trait of interest are genotyped for target site edits to understand allele frequency change relative to the founder population and to characterize the best performing edited allele combinations.

[0260] 3.3 Recurrent mass selection for optimal combination of edits using a benchmarking allele.

[0261] The method is performed as in section 3.2. In addition, an individual with a benchmarking allele is introduced as random pollinator in the SO population, said individual having the same stature and flowering time as the transformation genotype. The lines will be genotyped for the benchmarking allele after the final generation to understand the change in frequency and individual plant zygosity for the benchmarking allele.

[0262] 3.4 Re-introduction of new edited alleles in the recurrent mass selection process

[0263] The method is performed as in section 3.2. In addition, transgenic lines comprising the gRNA array, the selectable marker, pollen ablation, and Cas12a gene, are intercrossed. Due to the presence of the Cas12a gene and the gRNAs, the plants will be re-edited. Plants of the T2 generation of the transgenic lines, containing new edited alleles, are introduced as random pollinator in the SO population of the non-transgenic lines, late stage pollen ablation activity allows new edits to transfer via the male germline but not the transgene. All cross-pollination progeny is non-transgenic and contains new edits. The resulting offspring are selected as described above in 3.2.

[0264] Selected lines are crossed with wild-type plants. Offspring are genotyped for presence of the target site edits and the transgene(s). Lines containing the target site edits and lacking the transgenes are selected. This step is repeated until all target site edits are recovered in one plant. BASF Agricultural Solutions US LLC 240242WO02

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[0266] The target site edits are fixed to homozygosity by repeated selfings and / or crossings with lines comprising the same edit, and selection for presence of target site edits. Target site edits that are present in heterozygous form in the selected individual are maintained in heterozygous form by crossing with an individual not comprising the target site edits. These target site edits that optimally perform in heterozygous form are fixed to homozygosity in one of the parents to be used in hybrid breeding, and crossed with another parent not comprising said edit to produce hybrid seed.

Claims

BASF Agricultural Solutions US LLC 240242WO0233Claims1 . A method to select an optimal combination of multiple gene edits to confer a desired phenotype in a plant, said method comprising: a) providing a population of cells; b) providing said cells with editing machinery comprising a library of editing cassettes to create multiple gene edits; c) generating a population of plants from said cells, said population comprising edited plants, wherein said edited plants comprise one or more of said multiple edits; d) crossing edited plants of said population for at least one generation to generate a plurality of combinations of said multiple gene edits, wherein the crossing is configured to promote diversity of edits within the population; and e) selecting plants with the desired phenotype.

2. The method of claim 1 , wherein the edited plants of said population are intercrossed to promote diversity of edits within the population, and wherein a male sterility system is used to promote intercrossing.

3. The method of claim 1 or 2, wherein in step d) only seeds from a male sterile plant are retained in at least one of the generations.

4. The method of any one of the preceding claims, wherein in step d) said plants are intercrossed for at least two generations, or at least three generations, or at least four generations, or at least five generations, or at least six generations, and wherein after at least one of the crossing steps plants with the desired phenotype are selected and retained.

5. The method of any one of the preceding claims, wherein said editing machinery is designed to be stably integrated into the plant genome; the editing machinery or a component thereof is linked to a selectable or screenable marker gene and to a male sterility gene; after step b) or after step c) cells or plants comprising the editing machinery in the plant genome are selected based on expression of the selectable or screenable marker gene; in a first crossing, said edited plants are cross-pollinated with pollen from plants not comprising a male sterility gene and not comprising the editing machinery; male fertile plants not comprising the selectable marker and the male sterility gene are eliminated by application of a selective agent after seeds are produced and before seeds are harvested; and only seeds from male sterile plants comprising the selectable marker and the male sterility gene are retained; in a next crossing, plants derived from said seeds are inter-crossed; male fertile plants not comprising the selectable marker and the male sterility gene are eliminated with a selective agent after seeds are produced and before seeds are harvested; seeds obtained from plants comprising the selectable marker and the male sterility gene are harvested; and only seeds from male sterile plants comprising the selectable marker and the male sterility gene are retained; and optionally, said next crossing step is repeated for one or more generations, wherein after at least one of the crossing steps plants with the desired phenotype are selected and retained.BASF Agricultural Solutions US LLC 240242WO02346. The method of claim 5, wherein the editing machinery consists of two components, such as an RNA-guided nuclease and an RNA, wherein the two components of the editing machinery are delivered separately, wherein only one of the two components of the editing machinery is linked to the male sterility system, and wherein plants comprising the two components of the editing machinery are selected after one or more initial crossings, and wherein after one or more subsequent crossings, plants are selected for the absence of the component of the editing machinery which is not linked to the transgenic dominant male sterility gene, and for the presence of the component of the editing machinery which is linked to the transgenic dominant male sterility gene.

7. The method of claim 1 or 2, wherein said library of editing cassettes contains an editing cassette for editing an endogenous male fertility gene, optionally a single dominant male fertility gene, and wherein in a first crossing, said edited plants of said population are crossed; in a next crossing, progeny plants obtained from said first crossing are inter-crossed; male sterile plants comprising edits in both alleles of said single dominant male fertility gene are selected, and seeds from male sterile plants are retained; and optionally, said next crossing step is repeated for one or more generations wherein after at least one of the crossing steps plants with the desired phenotype are selected and retained.

8. The method of claim 7, wherein said editing machinery is designed to be stably integrated into the plant genome, and wherein the editing machinery is linked to a selectable marker gene and, optionally, to a late pollen ablation gene, and wherein plants comprising the editing machinery in the plant genome are selected after one or more initial crossings, and wherein after one or more subsequent crossings, plants are selected for the absence of the editing machinery.

9. The method of claim 7, wherein said editing machinery is designed to be not stably integrated into the plant genome and, optionally, wherein the editing machinery is delivered as ribonucleoprotein (RNP).

10. The method of any one of claims 1-9, wherein activation of said editing machinery is inducible.11 . The method of any one of the preceding claims, further comprising the step of genotyping the selected plants with the desired phenotype for the target site edits.

12. A method to select an optimal combination of multiple edits to confer a desired phenotype in a plant, said method comprising a) providing a seed mixture comprising a plurality of combinations of multiple gene edits obtained from intercrossing of edited plants in a population for at least one generation, using a male sterility system, wherein said edited plants in said population are generated from cells to which editing machinery is provided, said editing machinery comprising a library of editing cassettes to create multiple edits, wherein said edited plants comprise one or more of said multiple edits; b) selecting a plant with the desired phenotype; and, optionally c) genotyping said selected plant with the desired phenotype for the target site edits.BASF Agricultural Solutions US LLC 240242WO023513. The method of any one of claims 1 to 12, which is used to produce a plant with an optimal combination of multiple gene edits to confer a desired phenotype.

14. A plant, or cell, tissue, organ, material, or seed thereof, obtained by or obtainable by a method according to any one of claims 1 to 12.

15. The method of any one of claims 1 to 13, wherein said plant is a Brassica plant, and wherein said desired phenotype is podshatter reduction.

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