Diversified base editing

Abstract

The present invention relates to the field of increasing genetic diversity in a targeted manner. In particular, the present invention relates to providing methods and means for target sequence diversification using a base editor with an extended mutation profile, including providing Cas12a diversification base editing systems and uses thereof.

Description

Technical Field

[0001] The present invention relates to the field of increasing genetic diversity in a targeted manner. In particular, the present invention relates to providing methods and methods for diversifying target sequences using base editors with an expanded mutation spectrum, including providing a Cas12a diversified base editing system and its use. Background Art

[0002] Improving traits is a continuing goal in agriculture and other fields. The classical method used to achieve this is random mutagenesis, usually carried out via UV or EMS-induced mutagenesis. Although these mutagenesis methods allow the discovery of new mutants, they are very time-consuming and laborious.

[0003] Furthermore, these strategies are non-targeted and therefore induce random mutations throughout the genome and therefore do not allow for directed evolution or manipulation of loci of interest without the risk of inducing undesired mutations in the genome of interest.

[0004] In contrast, targeted genetic modification can be achieved by CRISPR-Cas methods. While these methods do allow precise editing of the genetic location of interest, these methods are mostly limited to insertions and deletions, and standard CRISPR-Cas methods do not allow directed evolution. To achieve directed evolution, strategies that rely on the creation of random or semi-random mutagenesis libraries in vitro have been developed. However, since these methods are performed in vitro in the target organism, they do not allow easy phenotypic analysis of the generated mutations.

[0005] With the creation of base editors, the CRISPR-Cas system has been successfully modified to induce targeted point mutations instead of cleaving the target DNA. There are currently two main types: cytidine / cytosine (CBE) and adenine / adenosine base editors (ABE). CBEs are usually generated by fusing the cytidine deaminase domain to a catalytically impaired Cas9 (either inactive (D10A / H840A) or nickase (D10A) Cas9). A variety of cytidine deaminases have been used for base editing, including APOBEC1 (A1), A3A, A3B, PmCDA1, AID, and their derivatives (Rees and Liu, 2018). CBE catalyzes the deamination of cytidine on the non-target DNA strand to uracil, ultimately generating a CG to TA mutation (for CBE, see Komor et al., 2016; Komor et al., 2017). Regarding Cas9 variants suitable for base editors, nCas9 is believed to be more active than dCas9 because nicking of the target strand results in the non-target strand being used as a template in mismatch-mediated repair (e.g., Eid et al., 2018). However, early base editors only allowed a single type of conversion, C to T or A to G, respectively, and were therefore not suitable in cases where full-range mutagenesis with high diversification potential was of interest.

[0006] Although recent developments have shown that these two types of base editors can be combined into so-called dual base editors, the resulting C to T and A to G conversions still only provide limited diversification. Therefore, there is a great need in the art for systems that allow diversification that is closer to random mutagenesis, while being targeted, i.e., inducing modifications specifically in the locus of interest, and allowing targeted diversification to be applied in situ, i.e., in a cell or organism of interest.

[0007] Another limitation is that base editing systems are currently limited to Cas9 base editors. Although Cas12a (also known as Cpf1) has received increasing attention as an alternative to Cas9 in recent years, in addition to other CRISPR-Cas, Cas12a base editor systems remain largely ineffective, especially in plants. In addition, no practical Cas12a dual base editing system has been described to date.

[0008] Therefore, the object of the present invention is to provide novel and specifically optimized base editor systems including Cas12a diversification base editors so as to allow in situ targeted diversification with improved editing range and at the same time have high overall activity and high base editing efficiency, which base editor systems can be used in directed evolution methods. Summary of the invention

[0009] In a first aspect, a method for targeted diversified base editing of at least one target nucleic acid segment is provided, the method comprising (a) providing at least one cell or construct comprising at least one target nucleic acid segment; (b) introducing into the target cell or contacting with the target construct: (i) at least one diversified base editor (DBE) or at least one nucleic acid molecule encoding the at least one diversified base editor; and (ii) at least one suitable guide RNA or at least one nucleic acid molecule encoding the at least one suitable guide RNA; (c) allowing a complex of (i) the at least one diversified base editor and (ii) the at least one suitable guide RNA to form; (d) obtaining at least one cell or construct comprising at least one modified target nucleic acid segment; wherein the total base editing efficiency of introducing at least one substitution of any kind into the at least one target nucleic acid segment is at least 0.2%, 0.5%, 1%, 5%, 10%, 15%, 20%, or at least 25%, wherein the upper limit is 100% or less; and / or wherein the ratio of C to G substitutions is at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the ratio of C to T substitutions and / or the ratio of C to A substitutions is at least 0.1% of the ratio of C to T substitutions. %, 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or at least 90%; and / or wherein at least one modification of the target nucleic acid segment occurs within an extended base editing window; wherein the method does not include treatment of the human or animal body by surgery or therapy, and / or a diagnostic method practiced on the human or animal body, and / or a process for modifying the germline genetic identity of a human.

[0010] In an embodiment of the first aspect, the diversified base editor comprises a CRISPR-Cas portion derived from 2 class II type CRISPR-Cas nucleases (including Cas9 nuclease) or 2 class V type CRISPR-Cas nucleases, preferably wherein the diversified base editor comprises a CRISPR-Cas portion derived from a Cas12a nuclease.

[0011] In another embodiment of the first aspect, the at least one target cell is a prokaryotic cell, including a bacterial cell or an archaeal cell; or a eukaryotic cell, including an insect cell, a mammalian cell, or a plant cell.

[0012] In another embodiment of the first aspect, the at least one target cell is a plant cell, including a plant protoplast.

[0013] In another embodiment of the first aspect, the at least one diversified base editor comprises (i) one or more cytosine deaminase portions, (ii) one or more adenine deaminase portions, (iii) one or more CRISPR-Cas portions, preferably wherein the CRISPR-Cas domain does not cut both strands of double-stranded DNA, (iv) one, two, three or more nuclear localization sequences; and (v) at least one linker region, preferably one or more linker regions between (i) and (ii), and optionally one or more linker regions between (ii) and (iii).

[0014] In another embodiment of the first aspect, at least one diversified base editor in step (bi) is at least one diversified base editor in the form of a fusion protein, preferably wherein parts (i), (ii) and (iii) as defined above are arranged in the order of (i)-(ii)-(iii) in the N-terminal to C-terminal direction, wherein there are one or more linker regions between each segment, and further preferably wherein one, two, three or more nuclear localization sequences (iv) are located at the C-terminal end of the diversified base editor, or wherein one or more nuclear localization sequences (iii) are located at the N-terminal end of the diversified base editor and one or more nuclear localization sequences (iii) are located at the C-terminal end of the diversified base editor.

[0015] In another embodiment of the first aspect, the diversified base editor comprises at least one additional portion, preferably wherein the at least one additional portion is selected from a ssDNA, ssRNA or dsRNA binding protein portion including an MS2 protein portion, an affinity tag binding protein, a uracil glycosylase inhibitor portion and / or a uracil glycosylase portion, or any combination thereof.

[0016] In one embodiment, the at least one additional portion comprises at least one uracil DNA N-glycosylase (UNG), optionally Escherichia coli derived uracil DNA N-glycosylase (eUNG), optionally wherein the at least one UNG is delivered in trans with at least one diversified base editor of the present invention. For the delivery of the at least one UNG (optionally the at least one eUNG), it may be desirable to express the at least one UNG or eUNG from a strong promoter such as the 35S promoter (SEQ ID NO: 59). In a preferred embodiment, the at least one UNG (optionally the at least one eUNG) is delivered in trans with the at least one diversified base editor, wherein the at least one base editor is in the form of a fusion protein as disclosed herein.

[0017] In another embodiment of the first aspect, the one or more adenine deaminase moieties and / or the one or more cytosine deaminase moieties are linked to at least one ssRNA or dsRNA binding protein moiety, preferably at least one MS2 protein moiety, and the at least one suitable guide RNA is adjusted to allow interaction with the at least one ssRNA or dsRNA binding protein moiety, preferably wherein the one or more adenine base editor moieties and / or the one or more cytosine base editor moieties are linked to at least one MS2 protein moiety, and the suitable guide RNA is adjusted to comprise two MS2 stem-loops, optionally wherein the suitable guide RNA comprises a sequence selected from SEQ ID NO: 38 to SEQ ID NO: NO:41, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity thereto.

[0018] In another embodiment of the first aspect, the diversified base editor comprises an amino acid molecule selected from any one of SEQ ID NOs: 1-27, 52, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the corresponding reference sequence.

[0019] In a second aspect, there is provided an edited cell, tissue, organ, material or whole organism obtained or obtainable by a method according to the first aspect.

[0020] In a third aspect, a diversified base editor or a diversified base editor complex further comprising at least one suitable guide RNA, or at least one nucleic acid molecule encoding the diversified base editor or the diversified base editor complex is provided, wherein the diversified base editor is as defined in the first aspect.

[0021] In a fourth aspect, a vector or expression construct, or more than one vector and expression construct, is provided, each vector and / or expression construct comprising at least one nucleic acid molecule of the third aspect, wherein different parts of the diversified base editor are encoded on the same vector or expression construct or on different vectors or expression constructs, and / or wherein the diversified base editor or part thereof and the at least one suitable guide RNA are encoded on the same vector or expression construct or on different vectors or expression constructs.

[0022] In a fifth aspect, a cell is provided, comprising at least one diversified base editor or at least one diversified base editor complex of the third aspect or at least one nucleic acid molecule encoding the at least one diversified base editor or at least one diversified base editor complex; or at least one vector or expression construct of the fourth aspect; wherein the cell is a prokaryotic cell, including a bacterial cell or an archaeal cell; or a eukaryotic cell, including an insect cell, a mammalian cell including a human cell, or a plant cell including a plant protoplast, preferably wherein the cell is a plant cell including a plant protoplast, optionally wherein the plant cell including a plant protoplast is a cell belonging to or derived from a plant selected from the following, wherein the at least one target cell is a plant cell belonging to or derived from the following plant, the plant belongs to the Viridiplantae superfamily, in particular monocotyledons and dicotyledons, including forage beans or forage beans, ornamental plants, food crops, trees or shrubs selected from the following list: Acer spp., Actinidia spp., Abelmoschus spp. spp.), sisal (Agave sisalana), Agropyron spp., creeping bentgrass (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 (Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Brazil chestnut (Bertholletia excelsea), Betavulgaris, Brassica spp. (e.g., Brassica napus, Brassica rapa ssp.) [canola, rapeseed, turnip rape]), Cadabafarinosa, 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 nut spp. 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, 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.), willow species (Salix sp.), elderberry species (Sambucus spp.), rye (Secale cereale), sesame species (Sesamum spp.), white mustard species (Sinapis sp.), Solanum species (Solanum spp.) (for example, potato (Solanum tuberosum), red eggplant (Solanum integrifolium) or tomato (Solanum lycopersicum)), sorghum (Sorghum bicolor), spinach species (Spinacia spp.), Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g., Triticum umaestivum, 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.

[0023] In the sixth aspect, a kit is provided, comprising at least one diversified base editor or at least one diversified base editor complex of the third aspect or at least one nucleic acid molecule encoding the at least one diversified base editor or at least one diversified base editor complex; or at least one vector or expression construct of the fourth aspect; or at least one cell of the fifth aspect.

[0024] In the seventh aspect, the use of at least one diversified base editor or at least one diversified base editor complex or at least one nucleic acid molecule encoding the at least one diversified base editor or at least one diversified base editor complex of the third aspect; or the use of at least one vector or expression construct of the fourth aspect; or the use of at least one cell of the fifth aspect; or the use of at least one kit of the sixth aspect is provided; they are used for the targeted directed evolution of at least one target nucleic acid segment, preferably for the botanical targeted directed evolution of at least one target nucleic acid segment, the use including the use for optimizing or modifying the traits of the plant, including optimizing or modifying yield-related traits, or disease or pathogen resistance-related traits, or herbicide resistance-related traits, or abiotic stress-related traits including salinity or drought stress-related traits, wherein the disease is caused by or the pathogen is selected from the following: viruses, bacteria, fungi, nematodes or insects, and the use further includes the use for identifying at least one lead gene.

[0025] definition

[0026] The terms "adenine deaminase" and "adenosine deaminase" are used interchangeably herein. Likewise, the terms "cytidine deaminase" and "cytosine deaminase" are used interchangeably herein.

[0027] As used herein, the term "base editor complex" refers to a complex of at least one base editor and at least one guide RNA suitable for at least one CRISPR-Cas portion of the at least one base editor. Although the present invention includes base editors comprising more than one polypeptide that forms a diversified base editor by non-covalent binding, these base editors are also referred to as diversified base editors or DBEs, and are only referred to as base editor complexes when they also include at least one suitable guide RNA. However, mentioning a diversified base editor or DBE without explicitly mentioning a complex does not exclude that the base editor can be in the form of a complex with at least one suitable guide RNA.

[0028] As used herein, the term "base editing window" refers to the region that generally contains the target nucleic acid segment to be modified in the genomic sequence, wherein the base editing window is such a window in which a diversified base editor as guided by a suitable guide RNA is theoretically capable of inducing at least one target nucleotide exchange as a base editor. This window is defined by the architecture of the diversified base editor, and the physical accessibility of the diversified base editor as guided by a suitable guide RNA, and the region to be modified (particularly a genomic region).

[0029] As used herein, a "diversified base editor" or "DBE" refers to a base editor comprising: at least one cytosine deaminase portion; at least one adenosine deaminase portion; at least one CRISPR-Cas portion, wherein the CRISPR-Cas portion can be modified to cut only one strand of the target DNA, or can be modified to not cut any strand of the target DNA; and at least one nuclear localization sequence, wherein the DBE can further comprise one or more additional portions, such as a ssDNA, ssRNA or dsRNA binding protein portion, a uracil glycosylase inhibitor portion and / or a uracil glycosylase portion, wherein these portions are covalently and / or non-covalently linked to each other, wherein non-covalent linkage can also be achieved by covalently and / or non-covalently attaching one or more portions that are not CRISPR-Cas portions to a suitable guide RNA, which in turn non-covalently interacts with the CRISPR-Cas portion or a group of portions comprising a CRISPR-Cas portion, wherein covalent linkage of the portions can be achieved via at least one linker region.

[0030] The term "guide RNA" may refer to any RNA comprising a Cas protein binding region and a targeting region, and can guide the Cas protein to a target nucleotide sequence that is fully complementary to the targeting region of the guide RNA, as long as the target nucleotide sequence is located next to the protospacer adjacent motif (PAM) suitable for the corresponding Cas protein. As used herein, "suitable guide RNA" refers to a guide RNA suitable for a CRISPR-Cas portion used as a part of a DBE, i.e., a suitable guide RNA can be bound to the CRISPR-Cas portion employed via the Cas protein binding region, and the targeting region has complementarity with the nucleotide sequence immediately upstream of the PAM sequence recognized by the employed CRISPR-Cas portion. As is well known in the art, the Cas12a system typically relies on a single crRNA as a guide RNA, and the Cas9 system typically uses a crRNA::tracrRNA duplex, which can be simulated by a synthetic single guide RNA molecule. It is well known to the technician to design, express / synthesize and adjust guide RNA for the desired purpose.

[0031] When used in reference to comparing two or more nucleic acid or amino acid molecules, "identity" means that the sequences of the molecules share a degree of sequence similarity, such that the sequences are partially identical.

[0032] Enzyme variants can be defined by their sequence identity when compared with the parent enzyme.Sequence identity is usually provided in the form of "sequence identity %" or "identity %".In order to determine the identity percentage between two amino acid sequences in the first step, a paired sequence alignment is generated between the two sequences, wherein the two sequences are aligned on their complete length (i.e., paired global alignment).With the program implementing Needleman and Wunsch algorithm (J.Mol.Biol. [Journal of Molecular Biology] (1979) 48, pp. 443-453), preferably by using the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) with program default parameters (gap opening = 10.0, gap extension = 0.5 and matrix = EBLOSUM62) to generate an alignment.Preferred alignment for the purpose of the present invention is the alignment from which the highest sequence identity can be determined.

[0033] The following example is intended to illustrate two nucleotide sequences, but the same calculations apply to protein sequences:

[0034] Sequence A: AAGATACTG, length: 9 bases

[0035] Sequence B: GATCTGA, length: 7 bases

[0036] Therefore, the shorter sequence is sequence B.

[0037] Produces a pairwise global alignment of two sequences showing their full length, resulting in

[0038]

[0039] The "|" symbol in the alignment represents identical residues (this means bases for DNA or amino acids for proteins). The number of identical residues is 6.

[0040] The "-" sign in the alignment indicates a gap. The number of gaps introduced by the alignment within sequence B is 1. The number of gaps introduced by the alignment at the boundaries of sequence B is 2, while the number of gaps introduced at the boundaries of sequence A is 1.

[0041] The alignment length for the full length of aligned sequences is 10.

[0042] Therefore, according to the invention, a pairwise alignment of shorter sequences showing the full length is generated, resulting in:

[0043]

[0044] Thus, according to the invention, a pairwise alignment of sequence A showing the entire length is generated, resulting in:

[0045]

[0046] Thus, according to the invention, a pairwise alignment of sequence B showing the entire length is generated, resulting in:

[0047]

[0048] The alignment length of the shorter sequence showing the full length is 8 (there is a gap which is included in the alignment length of the shorter sequence).

[0049] Therefore, the alignment length showing the full length of sequence A is 9 (meaning that sequence A is a sequence of the present invention).

[0050] Therefore, the alignment length showing the full length of sequence B is 8 (meaning that sequence B is a sequence of the present invention).

[0051] After comparing two sequences, in the second step, the identity value is determined according to the comparison produced. For the purpose of this description, the identity percentage is calculated as follows: identity %=(same residue / length of the comparison area of ​​the corresponding sequence of the present invention showing the complete length)*100. Therefore, the sequence identity related to the comparison of two amino acid sequences according to the present embodiment is calculated by dividing the number of identical residues by the length of the comparison area of ​​the corresponding sequence of the present invention showing the complete length. This value is multiplied by 100 to obtain "identity %". According to the example provided above, identity %: for sequence A as a sequence of the present invention, it is (6 / 9)*100=66.7%; for sequence B as a sequence of the present invention, it is (6 / 8)*100=75%.

[0052] "Indel (indel)" is a term for the random insertion or deletion of bases related to repairing DSB by NHEJ in an organism's genome. It is classified as belonging to a small genetic variation, and the length measured is 1 to 10 000 base pairs. As used herein, it refers to random insertion or deletion of bases in or near the target site (for example, less than 1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp, 50bp, 40bp, 30bp, 25bp, 20bp, 15bp, 10bp or 5bp upstream and / or downstream of the target site).

[0053] As used herein, when referring to materials obtained or obtainable by the methods of the present disclosure, the term "material" refers to any material that can contain at least one target nucleic acid segment. "Material" can refer to cell materials such as those obtained or obtainable directly from an organism or group of organisms or such as those obtained or obtainable by lysis, dissolution and / or other preparation methods. In addition, the material can be self-proliferating (such as reproductive systems and / or seeds) or non-proliferating. In addition, the material can also refer to purified or synthesized materials, such as plasmids, linear polynucleotides or oligonucleotides, etc.

[0054] As used herein, the term "portion" refers to a functional unit of a diversified base editor. A portion may be a single domain having one or more functions (such as enzymatic activity or binding activity), and a portion may also be composed of two or more domains that synergistically have one or more functions. A portion may contain or consist of a complete protein sequence of a given protein (such as a complete Cas12a protein sequence or a complete adenosine or cytidine deaminase protein sequence), or, in the case where a portion of a known sequence is sufficient to have one or more desired functions, may contain or consist of a portion of the sequence of a polypeptide from which the portion is derived. Compared to the wild-type protein sequence from which the portion is derived, the portion may generally contain or consist of a mutant amino acid sequence.

[0055] As used herein, the term "plant" encompasses whole plants, original forms and progeny of plants, and plant parts, including seeds, buds, stems, leaves, roots (including tubers), flowers, and tissues and organs. The term "plant" also encompasses plant cells, suspension cultures, callus, embryos, meristem regions, gametophytes, sporophytes, pollen, and microspores.

[0056] As used herein, the term "targeted diversified base editing" refers to the guidance of a diversified base editor to the target nucleic acid sequence by a suitable guide RNA through hybridization of the guide RNA with the target nucleic acid sequence, thereby resulting in the state, quality, process and / or result of base substitutions in the editing window at the target site. Targeted base editing does not exclude the presence of off-target base substitutions (i.e., base substitutions that do not occur at the target site). Technicians are fully aware of the various factors that affect off-target base substitutions.

[0057] As used herein, "target nucleic acid segment" refers to a stretch of DNA (single-stranded or double-stranded) or even RNA, such as genomic DNA for in vivo applications and / or applications targeting cells in cell culture, or isolated DNA (e.g., plasmid DNA) for in vitro applications outside of living cells, in which DBE-induced base substitutions occur, wherein the target nucleic acid segment is within the target sequence (in applications where the base editing window is smaller than the target sequence), or wherein the target sequence is within the target nucleic acid segment (in applications where the base editing window extends beyond the target sequence). Typically, a "target nucleic acid segment" refers to a stretch of DNA within the target site, and may extend up to 10 bp, up to 20 bp, up to 30 bp, or up to 40 bp beside the target site (including both directions) depending on the base editing window.

[0058] As used herein, "target site" refers to both strands of double-stranded DNA, namely the target strand to which the guide RNA anneals and the complementary non-target strand, wherein the target site is a stretch of DNA where the guide RNA has appropriate complementarity with at least one DNA strand.

[0059] As used herein, "total base editing efficiency" refers to the rate at which at least one nucleobase substitution is introduced into a target nucleic acid segment (i.e., at least one nucleobase in the target nucleic acid segment is replaced by a different nucleobase, regardless of the type of substitution of a naturally occurring nucleobase for another). For example, a total base editing efficiency of 10% means that 10 out of 100 nucleic acid molecules carry at least one nucleobase substitution in the target nucleic acid segment, as determined before and after a DBE as disclosed herein, or without and with a DBE as disclosed herein. Typically, the total base editing efficiency is determined by sequencing, i.e., it can be assumed that the percentage of reads showing nucleobase substitutions in a target nucleic acid segment relative to the total number of reads covering the target nucleic acid segment represents the total base editing efficiency within a reasonable error range for a given sequencing application. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1AThe architecture of the Cas9-based DBE is shown compared to the previous Cas9 base editor (Li et al., 2020). Ubi indicates the maize Ubi-1 promoter, APOBEC3A indicates the human APOBEC3A deaminase, 48aa indicates the 48aa XTEN linker, 32aa indicates the 32aa XTEN linker, ecTadA and ecTadA7.10 indicate dimeric Escherichia coli TadA / Tad7.10 deaminase, Npl indicates nucleoplasmin NLS, UGI indicates uracil glycosylase inhibitor, SV40 indicates SV40 NLS, 3'35S indicates the cauliflower mosaic virus 35S terminator, and enCas9 indicates an "enhanced" Cas9 containing mutations K848A, K1003A, and R1060A (in addition to D10A). The average base editing efficiency of the two base editors at the OsAAT locus is shown in Figure 1B middle.

[0061] Figure 2 Shown by Figure 1A Different types of base substitutions made in rice protoplast cells for the OsAAT locus by the different constructs shown in . The y-axis shows the number of substitutions identified within the target nucleic acid segment. For STEME-1 and DBE-1, the total read counts totaled 590,176 and 659,999, respectively.

[0062] Figure 3 The total base editing efficiency of different LbCas12a-DBE constructs for the OsAAT locus in rice protoplasts as determined by next-generation sequencing is shown. The Y axis shows the percentage of sequencing reads with base substitutions. The different LbCas12a-DBE architectures used are (all dpNLS portions in the constructs shown have the sequence of SEQ ID NO: 49):

[0063] 1: hA3A-48aa-XTEN-Linker-ecTadA-32aa-XTEN-Linker-TadA7.10-32aa-XTEN-Linker-enLbCas12a(D832A)-SV40-NLS(3x) (SEQ ID NO: 17)

[0064] 2: hA3A-48aa-XTEN-Linker-ecTadA-32aa-XTEN-Linker-TadA7.10-32aa-XTEN-Linker-enLbCas12a(D832A / R1138A)-SV40-NLS(3x) (corresponding to SEQ ID NO: 17 with an additional R1138A mutation in enLbCas12a)

[0065] 3: hA3A-48aa-XTEN-Linker-ecTadA-32aa-XTEN-Linker-TadA7.10-32aa-XTEN-Linker-enLbCas12a(D832A / E795L)-SV40-NLS(3x) (SEQ ID NO:21)

[0066] 4: hA3A(R128A)-48aa-XTEN-Linker-ecTadA-32aa-XTEN-Linker-TadA8e(V106W)-32aa-XTEN-Linker-enLbCas12a(D832A)-SV40-NLS(3x) (SEQ ID NO:20)

[0067] 5: hA3A(R128A)-48aa-XTEN-Linker-ecTadA-32aa-XTEN-Linker-TadA8e(V106W)-32aa-XTEN-Linker-enLbCas12a(D832A / R1138A)-SV40-NLS(3x) (corresponding to SEQ ID NO:20 with an additional R1138A mutation in enLbCas12a)

[0068] 6: hA3A(R128A)-48aa-XTEN-Linker-ecTadA-32aa-XTEN-Linker-TadA8e(V106W)-32aa-XTEN-Linker-enLbCas12a(D832A / E795L)-SV40-NLS(3x) (corresponding to SEQ ID NO:20 with an additional E795L mutation in enLbCas12a)

[0069] 7: dpNLS (dual-part nuclear localization signal)-hA3A-48aa-XTEN-linker-monomer TadA8e-32aa-XTEN-linker-LbCas12a (D156R / D832A)-dpNLS (SEQ ID NO: 7)

[0070] 8: dpNLS-hA3A-48aa-XTEN-linker-monomer TadA8e-32aa-XTEN-linker-LbCas12a (D156R / D832A / K932G / N933G)-dpNLS (SEQ ID NO: 8)

[0071] 9: dpNLS-hA3A-48aa-XTEN-linker-monomer TadA8e-32aa-XTEN-linker-LbCas12a (D156R / D832A / E795L)-dpNLS (SEQ ID NO: 9)

[0072] 10: dpNLS-hA3A-48aa-XTEN-linker-monomer TadA8e-GGGGS-linker-(5x)-LbCas12a(D156R / D832A)-dpNLS (SEQ ID NO: 10)

[0073] Figure 4A and Figure 4B The results of base editing with LbCas12a-DBE-10 (SEQ ID NO: 10) in rapeseed (Brassica napus) and soybean (Glycine max) protoplasts are shown. Figure 4A Successful editing of extrachromosomal mBFP or dGFP reporters in Brassica napus is shown. 35S>eGFP represents a positive control, in which cells were transformed with eGFP under the control of the 35S promoter. R represents the red channel, and G represents the green (GFP) channel. The average base editing efficiency of DBE-10 at the FAD2 and ALS3 loci in Brassica napus and at the FAD2 locus in E. coli as determined by next-generation sequencing or droplet digital PCR analysis is shown in Figure 4B middle.

[0074] Figure 5A and Figure 5B Four different Cas12a guide RNA architectures carrying two MS2 stem-loops are shown. Grey shading marks the MS2 stem-loops. crRNA1: SEQ ID NO: 38, crRNA2: SEQ ID NO: 39, crRNA3: SEQ ID NO: 40, crRNA4: SEQ ID NO: 41. The MS2 labeling system is disclosed in Beach DL, Keene JD. Methods Mol. Biol. 2008; 419: 69-91.

[0075] Figure 6 The results of in vitro digestion of double-stranded PCR products with four different Cas12a-MS2 guide RNAs shown in Figure 5 are shown. crRNA-AAT represents a control guide RNA without MS2 stem-loop, and crt1 represents a negative control without guide RNA. U represents uncut DNA, and C represents cut DNA.

[0076] Figure 7The total base editing efficiency of dLbCas12a-guided MS2-hA3A fusions against the OsAAT locus in rice protoplasts with the four different Cas12a-MS2 guide RNAs shown in Figure 5 is shown. The Y axis shows the percentage of sequencing reads with base substitutions. nCas9-DBE1 represents the Cas9 DBE-1 editor shown in Figure 1, and Cas12a-DBE-10 refers to Figure 3 Construct 10 (SEQ ID NO: 10) in.

[0077] Figure 8 The cleavage activity of different Cas12a gRNAs shown in Table 4 as determined by next generation sequencing is shown. The Y axis shows the percentage of NGS reads with indels. The black and white bars represent data from two independent experiments.

[0078] Fig.9A A stereoscopic view of the bispyribac-sodium binding site in AtAHAS is shown (Source: Garcia et al., 2017). The herbicide is shown in a ball-and-stick model, while the key residues for herbicide binding are depicted as stick models. ' indicates that these residues are from adjacent subunits. Binding site residues with identified mutations are circled. Fig. 9B The Connolly surface in AtAHAS and the herbicide blocking the substrate access channel are shown. Bispyribac-sodium is represented by a ball-and-stick model. The binding site residues with the identified mutations are circled. DETAILED DESCRIPTION

[0079] In order to achieve the purpose of the present invention, a new class of Cas9-based diversified base editors has been developed, which enable a wider mutation spectrum. In addition, Cas12a-based diversified base editors have been designed and optimized from scratch.

[0080] In a first aspect, a method for targeted diversified base editing of at least one target nucleic acid segment can be provided, the method comprising (a) providing at least one cell or construct comprising at least one target nucleic acid segment; (b) introducing into the target cell or contacting with the target construct: (i) at least one diversified base editor (DBE) or at least one nucleic acid molecule encoding the at least one diversified base editor; and (ii) at least one suitable guide RNA or at least one nucleic acid molecule encoding the at least one suitable guide RNA; (c) allowing a complex of (i) the at least one diversified base editor and (ii) the at least one suitable guide RNA to form; (d) obtaining at least one cell or construct comprising at least one modified target nucleic acid segment;

[0081] wherein the overall base editing efficiency of introducing at least one substitution of any kind into the at least one target nucleic acid segment is at least 0.2%, 0.5%, 1%, 5%, 10%, 15%, 20%, or at least 25%, with an upper limit of 100% or less; or

[0082] wherein the ratio of C to G substitutions is at least 0.1, 0.5, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the ratio of C to T substitutions, optionally wherein the upper limit is 100% or less, 110% or less, 120% or less, 130% or less, 140% or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less; or

[0083] wherein the ratio of C to A substitution is at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the ratio of C to T substitution, optionally wherein the upper limit is 100% or less, 110% or less, 120% or less, 130% or less, 140% or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less; or

[0084] wherein at least one modification of the target nucleic acid segment occurs within an extended base editing window; or

[0085] wherein the overall base editing efficiency of introducing at least one substitution of any kind into the at least one target nucleic acid segment is at least 0.2%, 0.5%, 1%, 5%, 10%, 15%, 20%, or at least 25%, wherein the upper limit is 100% or less; and wherein the ratio of C to G substitutions is at least 0.1, 0.5, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the ratio of C to T substitutions, optionally wherein the upper limit is 100% or less, 110% or less, 120% or less, 130% or less, 140% or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less; or

[0086] wherein the total base editing efficiency of introducing at least one substitution of any kind into the at least one target nucleic acid segment is at least 0.2%, 0.5%, 1%, 5%, 10%, 15%, 20%, or at least 25%, wherein the upper limit is 100% or less; and wherein the ratio of C to G substitutions is at least 0.1, 0.5, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the ratio of C to T substitutions, optionally wherein the upper limit is 100% or less, 110% or less, 120% or less, 130% or less, 140% or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less; and wherein the ratio of C to A substitution is at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the ratio of C to T substitution, optionally wherein the upper limit is 100% or less, 110% or less, 120% or less, 130% or less, 140% or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less; or

[0087] wherein the overall base editing efficiency of introducing at least one substitution of any kind into the at least one target nucleic acid segment is at least 0.2%, 0.5%, 1%, 5%, 10%, 15%, 20%, or at least 25%, wherein the upper limit is 100% or less; and wherein the ratio of C to G substitutions is at least 0.1, 0.5, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the ratio of C to T substitutions, optionally wherein the upper limit is 100% or less, 110% or less, 120% or less, 130% or less, 140% or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less; and wherein at least one modification of the target nucleic acid segment occurs within an extended base editing window; or

[0088] wherein the overall base editing efficiency of introducing at least one substitution of any kind into the at least one target nucleic acid segment is at least 0.2%, 0.5%, 1%, 5%, 10%, 15%, 20%, or at least 25%, wherein the upper limit is 100% or less; and wherein the ratio of C to A substitutions is at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the ratio of C to T substitutions, optionally wherein the upper limit is 100% or less, 110% or less, 120% or less, 130% or less, 140% or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less; or

[0089] wherein the overall base editing efficiency of introducing at least one substitution of any kind into the at least one target nucleic acid segment is at least 0.2%, 0.5%, 1%, 5%, 10%, 15%, 20%, or at least 25%, wherein the upper limit is 100% or less; and wherein the ratio of C to A substitutions is at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the ratio of C to T substitutions, optionally wherein the upper limit is 100% or less, 110% or less, 120% or less, 130% or less, 140% or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less; and wherein at least one modification of the target nucleic acid segment occurs within an extended base editing window; or

[0090] wherein the overall base editing efficiency of introducing at least one substitution of any kind into the at least one target nucleic acid segment is at least 0.2%, 0.5%, 1%, 5%, 10%, 15%, 20%, or at least 25%, with an upper limit of 100% or less; and wherein at least one modification of the target nucleic acid segment occurs within an extended base editing window; or

[0091] wherein the ratio of C to G substitutions is at least 0.1, 0.5, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the ratio of C to T substitutions, optionally wherein the upper limit is 100% or less, 110% or less, 120% or less, 130% or less, 140% or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less; and wherein the ratio of C to A substitution is at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the ratio of C to T substitution, optionally wherein the upper limit is 100% or less, 110% or less, 120% or less, 130% or less, 140% or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less; or

[0092] wherein the ratio of C to G substitutions is at least 0.1, 0.5, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the ratio of C to T substitutions, optionally wherein the upper limit is 100% or less, 110% or less, 120% or less, 130% or less, 140% or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less; and wherein at least one modification of the target nucleic acid segment occurs within an extended base editing window, or

[0093] wherein the ratio of C to G substitutions is at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the ratio of C to T substitutions, optionally wherein the upper limit is 100% or less, 110% or less, 120% or less, 130% or less, 140% or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less; and wherein the ratio of C to A substitutions is at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the ratio of C to T substitutions, optionally wherein the upper limit is 100% or less, 110% or less, 120% or less, 130% or less, 140% or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less. %, 60%, 70%, 80% or at least 90%, optionally wherein the upper limit is 100% or less, 110% or less, 120% or less, 130% or less, 140% or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less; and wherein at least one modification of the target nucleic acid segment occurs within an extended base editing window, or wherein the ratio of C to G substitutions is at least 0.1, 0.5, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or at least 90% of the ratio of C to T substitutions, optionally wherein the upper limit is 100% or more or less, 110% or less, 120% or less, 130% or less, 140% or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less; and wherein at least one modification of the target nucleic acid segment occurs within an extended base editing window, or wherein the ratio of C to A substitutions is at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the ratio of C to T substitutions, optionally wherein the upper limit is 100% or less, 110% or less, 120% or less, 130% or less, 140% or less, or less, 150% or less, 160% or less, 170% or less, 180% or less, 190% or less, or 200% or less; and wherein at least one modification of the target nucleic acid segment occurs within an extended base editing window; preferably, wherein the diversified base editor comprises a CRISPR-Cas portion derived from a Class 2 V-type CRISPR-Cas nuclease, wherein the Class 2 V-type CRISPR-Cas nuclease can be a Cas12a nuclease or a portion thereof, wherein the method does not include treatment of the human or animal body by surgery or therapy, and / or a diagnostic method practiced on the human or animal body, and / or a process for modifying the germline genetic identity of a human.

[0094] In some embodiments, the method is performed outside of a living cell, wherein the at least one target nucleic acid segment is contained in at least one construct, such as in a linear DNA molecule (e.g., a PCR product or a restriction digest product) or a DNA vector (including a plasmid vector). In such embodiments, the at least one DBE is typically used in a purified form. The skilled artisan is well aware of a variety of standard procedures for protein expression and purification. The at least one guide RNA can be purified from, for example, in vitro transcription or synthesized de novo.

[0095] In other embodiments, the method is performed in a living cell, i.e., the at least one DBE, the at least one suitable guide RNA, or at least one nucleic acid encoding the at least one DBE, the at least one suitable guide RNA is introduced into the at least one cell. The at least one DBE and the at least one suitable guide RNA may be introduced separately, together and / or as an RNP complex. In embodiments involving the introduction of at least one nucleic acid molecule encoding at least one DBE and at least one suitable guide RNA, the DBE may be encoded on the same nucleic acid molecule as the at least one suitable guide RNA, or it may be encoded on a different nucleic acid molecule. The nucleic acid molecule may be RNA (typically an mRNA molecule) or DNA, including DNA expression vectors (including expression plasmid vectors). The at least one guide RNA is typically provided directly as a guide RNA molecule or as a DNA encoding the guide RNA molecule. The skilled artisan is fully aware of the design and preparation of different nucleic acid molecules, as well as the various different methods for introducing proteins, nucleic acids and RNPs into living cells.

[0096] In certain embodiments, the overall base editing efficiency of introducing at least one substitution of any kind into at least one target nucleic acid segment is 30% to 100% or 35% to 100%, or 40% to 100% or 45% to 100% or 50% to 100%.

[0097] As used herein, a "modified target nucleic acid segment" refers to the presence of at least one nucleobase substitution of any kind within the target nucleic acid segment, wherein, unless otherwise specified, any kind of substitution refers to a substitution of any of the four natural nucleobases A, C, G or T to any different one of the four natural nucleobases.

[0098] The at least one nucleic acid molecule encoding at least one DBE according to various embodiments and aspects herein can be codon-optimized and can further comprise a nucleic acid sequence encoding at least one compatible guide RNA. In any of the embodiments described herein, the nucleic acid sequence or molecule can be operably linked to a variety of promoters and other regulatory elements for expression in a cell and / or organism of interest.

[0099] Methods according to embodiments and aspects may include the further step of regenerating at least one population of edited cells, tissues, organs, materials or whole organisms from at least one edited cell or construct.

[0100] In one embodiment of the first aspect, the diversified base editor comprises a CRISPR-Cas portion derived from a naturally occurring and subsequently artificially modified Class 2, Type II CRISPR-Cas nuclease (including Cas9 nuclease) or a Class 2, Type V CRISPR-Cas nuclease, preferably wherein the diversified base editor comprises a CRISPR-Cas portion derived from a Cas12a nuclease.

[0101] The CRISPR-Cas portion may comprise or consist of a mutant Cas9 or Cas12a amino acid sequence. Typically, the CRISPR-Cas portion comprises at least one mutation that causes the CRISPR-Cas portion to not cut both strands of double-stranded DNA, thereby converting the CRISPR-Cas portion into a nickase (cutting one strand of double-stranded DNA) or an inactivated (not cutting DNA) CRISPR-Cas portion. The CRISPR-Cas portion may comprise additional mutations that alter PAM specificity, thermotolerance, and / or other characteristics.

[0102] In a preferred embodiment using a CRISPR-Cas9 portion, the CRISPR-Cas portion comprises or consists of SpCas9 with mutations D10A, K848A, K1003A and R1060A (referred to herein as "enCas9" or "enCas9 nickase"). The K848A, K1003A, R1060A mutations have been shown to weaken non-target strand binding by neutralizing positively charged residues in the non-target strand groove, thereby promoting dissociation of nCas9 from DNA after nicking the target locus (Slaymaker et al., 2016).

[0103] Preferred CRISPR-Cas12a parts include or consist of: LbCas12a with mutations D156R and D832A, optionally further with double mutations G532R / K538R and / or mutations E795L. In one embodiment, the CRISPR-Cas part includes or consists of LbCas12a-D156R / G532R. In another embodiment, the CRISPR-Cas part includes or consists of LbCas12a-D156R / G532R / K538R / D832A. In a further embodiment, the CRISPR-Cas part includes or consists of LbCas12a-D156R / D832A / E795L. In yet another embodiment, the CRISPR-Cas part includes or consists of D156R / G532R / K538R / D832A / E795L.

[0104] In one embodiment of the first aspect, at least one target cell is a prokaryotic cell, including a bacterial cell or an archaeal cell; or a eukaryotic cell, including an insect cell, a mammalian cell, or a plant cell.

[0105] In another embodiment according to the first aspect, at least one target cell is a plant cell including a plant protoplast, optionally the plant cell including the plant protoplast is a cell belonging to or derived from a plant belonging to the subkingdom superfamily Viridiplantae, in particular monocots and dicots, including forage or forage legumes, ornamentals, food crops, trees or shrubs selected from the list comprising Acer species, Actinidia species, Okra species, Sisal, Agropyron species, Creeping bentgrass, Allium species, Amaranth species, European seashore grass, pineapple, Annona species, celery, Arachis species, Jackfruit species, Asparagus, Avena species (e.g., Avena, Wild Oats, Byzantine Oats, Wild Oats sativa var., hybrid oats), carambola, spp. of balsam pear, wax gourd, Brazil nut, sugar beet, Brassica species (e.g., Brassica napus, Brassica rapa subsp. [canola, rapeseed, Brassica rapa]), citrus aurantium, tea, canna, cannabis, capsicum species, tall sedge, papaya, Carica oleracea, safflower, castanea species, gibbey, endive, camphor species, watermelon, citrus species, coconut species, coffee species, taro, cola nut species, jute species, cilantro, hawthorn species, saffron, pumpkin species, cucumber species, artichoke species, carrot, Desmodium species, longan, Dioscorea species, Diospyros species, Echinochloa species, oil palm (e.g., oil palm, American oil palm) , millet, teff, saccharum species, loquat, eucalyptus species, red fruit, buckwheat species, beech species, reed fescue, fig, kumquat species, strawberry species, ginkgo, soybean species (e.g., sedge beans, soybeans or soybeans), upland cotton, sunflower species (e.g., sunflower), daylily, hibiscus species, barley species (e.g., barley), sweet potato, walnut species, lettuce, Lathyrus species, lentil, flax, litchi, Lotus species, angular loofah, lupinus species, forest bayberry, tomato species (e.g., common tomato, tomato, pear-shaped tomato), hard-shelled bean species, apple species, slightly concave golden malus, Man honey apple, mango, cassava species, sapodilla, alfalfa, Melilotus species, Mentha species, miscanthus, momordica species, black mulberry, banana species, tobacco species, olive species, cactus species, bird's claw bean species, rice species (e.g., rice, broadleaf rice), millet, switchgrass, passion fruit, parsnip, Pennisetum species, avocado species, parsley, phalaenopsis, Phaseolus species, timothy grass, Eryngium species, reed, Phalaenopsis species, pine species, pistachio, pea species, bluegrass species, poplar species, mesquite species, plum species, guava species, pomegranate, pear, oak species, radish, wavy rhubarb, Ribes species, comb, Rubus species, Saccharum species, Willow species, elderberry species, rye, sesame species, Sinapis species, Solanum species (e.g.,potato, red eggplant or tomato), sorghum, spinach species, syzygium species, marigold species, sour beans, cocoa, clover species, orchard grass, triticale, wheat species (e.g., spelt, durum, cone, common, moga, floating, einkorn or soft wheat), nasturtium, nasturtium, vaccinium species, vetch species, vetch species, cowpea species, sweet violet, grape species, maize, wild rice or jujube species. ,

[0106] Preferred plants are okra species, Allium species, celery, Asparagus, Avena species (e.g., oats, wild oats, Byzantine oats, wild oats var. sativa, hybrid oats), beet, Brassica species (e.g., Brassica napus, rapa subsp. [canola, rape, Brassica napus]), Capsicum species, watermelon, Cucumis species, Cynara species, carrot, Glycine max species (e.g., sedge, soybean or soybean), cotton, Helianthus species (e.g., sunflower), Hordeum species (e.g., barley), lettuce, alfalfa, Oryza species (e.g., rice, broadleaf rice), Pennisetum species, Saccharum species, rye, Solanum species (e.g., potato, red eggplant or tomato), sorghum, Spinach species, Triticum species (e.g., bulgur, durum, cone wheat, common wheat, mocha, floating wheat, einkorn or soft wheat) or maize.

[0107] In certain embodiments, the preferred plant may also be selected from Brassica species (e.g., Brassica rapa, Brassica rapa subspecies [canola, rapeseed, Brassica rapa]), Capsicum species, Glycine max species (e.g., sedge, soybean or soybean), Gossypium hirsutum, Helianthus annuus species (e.g., sunflower), Oryza sativa species (e.g., rice, broad-leaved rice), Solanum species (e.g., potato, red eggplant or tomato), Triticum species (e.g., bulgur, durum, strychnine, common wheat, mocha, floating wheat, einkorn or soft wheat), or Zea mays.

[0108] In another embodiment of the first aspect, the at least one diversified base editor of step (bi) comprises (i) one or more cytosine deaminase portions, (ii) one or more adenine deaminase portions, (iii) one or more CRISPR-Cas portions, preferably wherein the CRISPR-Cas domain does not cut both strands of double-stranded DNA, (iv) one, two, three or more nuclear localization sequences; and (v) at least one linker region, preferably one or more linker regions between (i) and (ii), and optionally one or more linker regions between (ii) and (iii).

[0109] A variety of adenine deaminases and cytosine deaminases are known to the skilled person (e.g., Fan et al., 2021; Jeong et al., 2020; Yan et al., 2021). Any adenine deaminase and / or cytosine deaminase (including variants of known deaminases) can be used in the diversified base editors of the present invention if combined with other building blocks in a suitable manner according to the construction details disclosed herein.

[0110] In some embodiments, the cytosine deaminase may be an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the cytosine deaminase may be an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase, an APOBEC4 deaminase, an activation-induced deaminase (AID) (e.g., hAID or AICDA), rAPOBEC1, PpAPOBEC1, AmAPOBEC1, SsAPOBEC3B, RrA3F, FERNY, a cytosine deaminase (e.g., CDA1, CDA2, pmCDA1, or atCDA1), or a cytosine deaminase acting on rRNA (CDAT) or a variant thereof.

[0111] In a preferred embodiment, the one or more cytosine deaminase parts comprise or consist of human apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3A (hA3A). In one embodiment, the one or more cytosine deaminase parts comprise or consist of hA3A with mutation R128A, or mutation Y130F or double mutation W104A / P134Y.

[0112] The adenosine deaminase portion can comprise or consist of a monomeric adenosine deaminase or a dimeric adenosine deaminase, wherein the monomers of the dimeric adenosine deaminase are preferably linked via at least one linker region, preferably via a 32 aa XTEN linker.

[0113] In some embodiments, the adenine deaminase moiety can be a tRNA-specific adenosine deaminase, such as TadA (Gaudelli et al., 2017) or adenosine deaminase 1 (ADA1), ADA2; adenosine deaminase 1 acting on RNA (ADAR1), ADAR2, ADAR3 (e.g., Savva et al., 2012); or adenosine deaminase 1 acting on tRNA (ADAT1), ADAT2, ADAT3 or variants thereof.

[0114] In some embodiments, TadA can be from Escherichia coli (ecTadA). In some embodiments, TadA can be modified and / or truncated. In certain embodiments, TadA does not contain an N-terminal methionine. The TadA deaminase that can be used as a base editor or a part of a base editor complex according to the present invention can be, for example, TadA8, TadA8e, TadA8s, TadA7.9, TadA7.10, TadA7.10d, TadA8.17, TadA8.20, TadA9, or a variant thereof.

[0115] In preferred embodiments, the one or more adenosine deaminase moieties comprise or consist of dimer ecTadA / ecTadA7.10, or dimer ecTadA / TadA8e-V106W, or monomer TadA8e or monomer TadA9, preferably the one or more adenosine deaminase moieties comprise or consist of monomer TadA9.

[0116] In one embodiment, the DBE comprises at least one monopartite or bipartite nuclear localization signal (NLS), preferably at least one NLS comprising or consisting of the sequence of SEQ ID NO: 49. Any other NLS and NLS combinations or combinations thereof specifically tested for the DBE core structure as disclosed herein may also be used. Suitable NLSs are disclosed in, for example, Lange et al., 2010.

[0117] The terms "nuclear localization signal," "nuclear localization sequence," and "NLS" are used interchangeably herein.

[0118] In certain embodiments, at least two or at least three (eg, three) repeats of the SV40 NLS may be used.

[0119] In certain embodiments, a two-part NLS (dpNLLS) may be used, at least one of which is located at the C-terminus or N-terminus of the DBE and at another position within the DBE, preferably at the C-terminus and at the N-terminus. At least one part or both parts of the dpNLS may be a bipartite NLS, such as a sequence of SEQ ID NO: 49 or a sequence having at least 99% identity thereto. In certain embodiments, only one part of the dpNLS will be a bipartite NLS, including SEQ ID NO: 49 or a sequence having at least 99% identity thereto, and the second part will be, for example, a three-part SV40 NLS as disclosed herein.

[0120] In a preferred embodiment, the DBE comprises an NLS at the N-terminus and / or at the C-terminus (preferably at the N-terminus and at the C-terminus), the NLS comprising or consisting of the sequence of SEQ ID NO: 49 or a sequence having at least 99% identity thereto.

[0121] In all embodiments using non-covalent linkage of moieties to form a DBE, each polypeptide forming part of the DBE preferably comprises at least one NLS, more preferably wherein each polypeptide forming part of the DBE comprises an SV40 NLS, preferably three repeats of an SV40 NLS, or more preferably a bipartite NLS (dpNLS); the NLS being located at the C-terminus and / or N-terminus of the DBE and at a second position within the DBE, preferably at the C-terminus and the N-terminus, preferably wherein at least one or even both of the dpNLS sequences is SEQ ID NO: 49 or a sequence having at least 99% identity thereto.

[0122] Non-covalent binding can be achieved by any binding pair that allows specific interaction, such as an affinity tag, a biotin-streptavidin interaction or, for example, FRB-FKBP (Inobe and Nukina, 2016). Non-covalent binding can be achieved by non-covalent protein-protein interactions and / or by non-covalent protein-RNA interactions with a guide RNA. If the DBE is formed by non-covalent association with a guide RNA, the binding pair can be an RNA binding moiety fused to a portion of the DBE and a modification of the guide RNA, such as comprising a stem-loop and / or a binding sequence that allows specific interaction with the RNA binding moiety. Thus, any part or group of parts can be non-covalently linked to a CRISPR-Cas part or a group of parts comprising a CRISPR-Cas part via a guide RNA.

[0123] In certain embodiments using non-covalent linkage, the DBE comprises or consists of a first group of moieties, each of which is covalently linked to one another, and a second group of moieties, each of which is covalently linked to one another, wherein one moiety in the first group can be fused to another moiety via at least one linker region, and one moiety in the second group of moieties can be fused to another moiety via at least one linker region, wherein the first group of moieties and the second group of moieties each comprise a moiety that allows non-covalent linkage of a moiety in the first group to a moiety in the second group.

[0124] In some embodiments of non-covalent linkage, the first set of moieties comprises a CRISPR-Cas moiety and the second set of moieties comprises a ssRNA and / or dsRNA binding moiety, and the appropriate guide RNA is modified to allow binding to the ssRNA and / or dsRNA binding moiety.

[0125] In certain embodiments of non-covalent linkage, the first group of parts comprises or consists of: one or more CRISPR-Cas parts; optionally one or more additional parts, such as a uracil glycosylase inhibitor part, a uracil glycosylase part and / or a ssDNA binding part; and one, two, three or more nuclear localization signals at the C-terminus and / or N-terminus of the first group of parts, wherein one of the parts can be fused to another part via at least one linker region; and wherein the second group of parts comprises or consists of: one or more adenosine deaminase parts; and / or one or more cytosine deaminase parts; and one or more ssRNA and / or dsRNA binding parts, preferably MS2 protein parts; and one, two, three or more nuclear localization signals at the C-terminus and / or N-terminus of the second group of parts, wherein one of the parts can be fused to another part via at least one linker region.

[0126] As used herein, a linker region refers to a polypeptide linker wherein the first portion is fused to the N-terminus of the polypeptide linker and the second portion is fused to the C-terminus of the polypeptide linker.There are a variety of available polypeptide linker regions recognized and used in the art.

[0127] The polypeptide linker can be a GS linker, such as a polypeptide linker comprising or consisting of an amino acid sequence of (GGS)n, S(GGS)n, or SGGS, wherein n is a number from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). The polypeptide linker can also comprise or consist of the amino acid sequence: SEQ ID NO:45. The polypeptide linker can also comprise or consist of the amino acid sequence: SEQ ID NO:46, also referred to as an XTEN linker. In addition, the polypeptide linker can comprise or consist of the amino acid sequence: SEQ ID NO:47, also referred to as a GS-XTEN-GS linker and referred to herein as a "32aaXTEN linker." In addition, the polypeptide linker can comprise or consist of the amino acid sequence SEQ ID NO:48, referred to herein as a "48aa XTEN linker."

[0128] In one embodiment, the one or more linker regions between moieties (i) and (ii) as defined above comprise or consist of a 48aa XTEN linker.

[0129] In one embodiment, the one or more linker regions between parts (ii) and (iii) as defined above comprise or consist of a 32aa XTEN linker, or preferably a GS linker consisting of three, five, or six repeats of the amino acid sequence GGGGS (see SEQ ID NO: 51), ((GGGGS)3, (GGGGS)5, and (GGGGS)6, respectively). In another embodiment, the linker between part (ii) and part (iii) is replaced with a non-sequence-specific ssDNA binding moiety, preferably a Rad51 ssDNA binding domain (Rad51 ssDBD), or a non-sequence-specific ssDNA binding moiety, preferably a Rad51 ssDBD, is added to the linker region between part (ii) and part (iii), preferably to the (GGGGS)5 linker region. In yet another embodiment, the linker between part (ii) and part (iii) is replaced by a non-covalent linkage as described above.

[0130] In a preferred embodiment, the linker between part (ii) and part (iii) as defined above comprises or consists of a (GGGGS)5 linker.

[0131] In certain embodiments, the different moieties may be linked via bioconjugation, for example using the SNAP tag system (Hussain et al., 2013); the Halo tag system (Los et al., 2008); the CLIP tag system (Gautier et al., 2008) or any linkage of specific biomolecules (collectively referred to in the art as "click chemistry").

[0132] In another embodiment of the first aspect, at least one diversified base editor in step (bi) is at least one diversified base editor in the form of a fusion protein, preferably wherein parts (i), (ii) and (iii) as defined above are arranged in the order of (i)-(ii)-(iii) in the N-terminal to C-terminal direction, wherein there are one or more linker regions between each segment, and further preferably wherein one, two, three or more nuclear localization sequences (iv) are located at the C-terminal end of the diversified base editor, or wherein one or more nuclear localization sequences (iii) are located at the N-terminal end of the diversified base editor and one or more nuclear localization sequences (iii) are located at the C-terminal end of the diversified base editor.

[0133] In one embodiment, three repeats of the SV40 NLS are located at the C-terminus of the DBE.

[0134] In another embodiment, the two parts (dpNLS) are fused to the N-terminus or C-terminus of a DBE as disclosed herein, preferably at the N-terminus and the C-terminus, preferably wherein at least one of the dpNLS sequences is SEQ ID NO: 49 or a sequence having at least 99% identity thereto. In other embodiments, the two parts of the dpNLS have the sequence of SEQ ID NO: 49 or a sequence having at least 99% identity thereto.

[0135] In another embodiment of the first aspect, the diversified base editor comprises at least one additional portion, preferably wherein the at least one additional portion is selected from a ssDNA, ssRNA or dsRNA binding protein portion including an MS2 protein portion, an affinity tag binding protein, a uracil glycosylase inhibitor portion and / or a uracil glycosylase portion, or any combination thereof.

[0136] In one embodiment, at least one additional moiety comprises or consists of a uracil glycosylase inhibitor (UGI).

[0137] In one embodiment, the DBE comprises uracil DNA glycosylase (UDG), including uracil-n-glycosylase (UNG).

[0138] In certain embodiments, the DBE does not comprise a uracil glycosylase inhibitor moiety and / or does not comprise a uracil glycosylase moiety.

[0139] In certain embodiments, the DBE comprises a non-specific ssDNA binding portion, preferably a ssDNA binding domain of Rad51 (Rad51ssDBD). For Cas9 cytosine base editors, it has been previously shown that the RAD51ssDBD between Cas9 and cytosine deaminase can increase base editing efficiency and extend the base editing window in cell lines and mouse embryos (Zhang et al., 2020). In certain embodiments, Rad51ssDBD is used to replace the linker region between part (ii) and part (iii), or Rad51ssDBD is added to the linker region between part (ii) and part (iii), preferably to the (GGGGS)5 linker region.

[0140] In another embodiment of the first aspect, one or more adenine deaminase moieties and / or one or more cytosine deaminase moieties are linked to at least one ssRNA or dsRNA binding protein moiety, preferably at least one MS2 protein moiety, and at least one suitable guide RNA is adjusted to allow interaction with at least one ssRNA or dsRNA binding protein moiety, preferably wherein one or more adenine base editor moieties and / or one or more cytosine base editor moieties are linked to at least one MS2 protein moiety and the suitable guide RNA is adjusted to comprise two MS2 stem-loops.

[0141] The MS2 tagging strategy relies on the binding of the MS2 bacteriophage coat protein (referred to as the "MS2 protein" or, in the context of DBE, herein referred to as the "MS2 protein portion") to a hairpin structure from the phage genome (referred to herein as the "MS2 (stem) loop").

[0142] In an embodiment, the one or more CRISPR-Cas portions are one or more Cas12a portions, and the one or more adenine deaminase portions and / or the one or more cytosine deaminase portions are linked to the one or more MS2 protein portions, and the guide RNA comprises two MS2 loops, optionally wherein the guide RNA comprises the sequence of SEQ ID NO: 38 or SEQ ID NO: 39 or SEQ ID NO: 40 or SEQ ID NO: 41, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity thereto.

[0143] In certain embodiments, (ii) one or more adenosine deaminase portions are fused to one or more MS2 protein portions and one or more NLS portions, optionally as a fusion protein having the amino acid sequence of SEQ ID NO:42, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity thereto; and (i) one or more cytosine deaminase portions are fused to portions (iii) and (iv) as a second fusion protein, preferably with one or more linker regions between (i) and (iii), optionally a 32aa-XTEN linker, a 48aa-XTEN linker, a (GGGGS)5, or a (GGGGS)6 linker.

[0144] In certain embodiments, (i) one or more cytosine deaminase portions are fused to one or more MS2 protein portions and one or more NLS portions, optionally as a fusion protein having the amino acid sequence of SEQ ID NO:43 or SEQ ID NO:44, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity thereto; and (ii) one or more adenosine deaminase portions are fused to portions (iii) and (iv), preferably with one or more linker regions between (ii) and (iii).

[0145] In certain embodiments, (i) one or more cytosine deaminase portions are fused to (ii) one or more adenosine deaminase portions (preferably via one or more linker regions), to one or more MS2 protein portions, and to one or more NLS portions; and (iii) one or more CRISPR-Cas portions are fused to portion (iv) as a second fusion protein.

[0146] In certain embodiments, (i) one or more cytosine deaminase portions are fused to one or more MS2 protein portions and one or more NLS portions, optionally as a fusion protein having an amino acid sequence of SEQ ID NO:43 or SEQ ID NO:44, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity thereto, and (ii) one or more adenosine deaminase portions are fused to one or more MS2 protein portions and one or more NLS portions, optionally as a fusion protein having an amino acid sequence of SEQ ID NO:42, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity thereto; and (iii) one or more CRISPR-Cas portions are fused to portion (iv) as a third fusion protein.

[0147] In another embodiment of the first aspect, the diversified base editor comprises an amino acid molecule selected from any one of SEQ ID NOs: 1-27, 52, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity thereto.

[0148] SEQ ID NO: 1 is a Cas9-based DBE comprising hA3A, dimer ecTadA / ecTadA7.10, enCas9 with an additional D10A nickase mutation, and three repeats of SV40 NLS: hA3A 48aa-XTEN-linker-ecTadA-32aa-XTEN-linker-ecTadA7.10-32aa-XTEN-linker-enCas9(D10A)-SV40 NLS (3x).

[0149] SEQ ID NO:2 has the same architecture as SEQ ID NO:1, but contains LbCas12a(D156R / D832A) instead of Cas9: hA3A-48aa-XTEN-linker-ecTadA-32aa-XTEN-linker-ecTadA7.10-32aa-XTEN-linker-LbCas12a(D156R / D832A)-SV40 NLS(3x).

[0150] SEQ ID NO: 3 has an additional E795L mutation: hA3A-48aa-XTEN-linker-ecTadA-32aa-XTEN-linker-ecTadA7.10-32aa-XTEN-linker-LbCas12a(D156R / E795L / D832A)-SV40 NLS (3x).

[0151] SEQ ID NO:4 has the same architecture as SEQ ID NO:2, but comprises a hA3A(R128A) cytosine deaminase mutant: hA3A(R128A)-48aa-XTEN-linker-ecTadA-32aa-XTEN-linker-ecTadA7.10-32aa-XTEN-linker-LbCas12a(D156R / D832A)-SV40 NLS(3x).

[0152] SEQ ID NO:5 has the same architecture as SEQ ID NO:2, but comprises a dimeric TadA8e(V106W) adenine deaminase mutant: hA3A-48aa-XTEN-linker-ecTadA-32aa-XTEN-linker-ecTadA8e(V106W)-32aa-XTEN-linker-LbCas12a(D156R / D832A)-SV40 NLS(3x).

[0153] SEQ ID NO: 6 comprises both hA3A(R128A) and dimer TadA8e(V106W): hA3A(R128A)-48aa-XTEN-linker-ecTadA-32aa-XTEN-linker-ecTadA8e(V106W)-32aa-XTEN-linker-LbCas12a(D156R / D832A)-SV40 NLS(3x).

[0154] SEQ ID NO: 7 comprises an N-terminal and C-terminal dpNLS and a monomeric TadA8e adenine deaminase: dpNLS-hA3A-48aa-XTEN-Linker-TadA8e-32aa-XTEN-Linker-LbCas12a(D156R / D832A)-dpNLS.

[0155] SEQ ID NO:8 has the same architecture as SEQ ID NO:7, but contains additional K932 / N933 mutations in LbCas12a: dpNLS-hA3A-48aa-XTEN-Linker-TadA8e-32aa-XTEN-Linker-LbCas12a(D156R / D832A / K932G / N933G)-dpNLS.

[0156] SEQ ID NO:9 has the same architecture as SEQ ID NO:7, but contains an additional E795L mutation in LbCas12a: dpNLS-hA3A-48aa-XTEN-Linker-TadA8e-32aa-XTEN-Linker-LbCas12a(D156R / E795L / D832A)-dpNLS.

[0157] SEQ ID NO: 10 has the same structure as SEQ ID NO: 7, but the linker region between part (ii) and part (iii) is a (GGGGS)5 linker: dpNLS-hA3A-48aa-XTEN-linker-TadA8e-(GGGGS)5-LbCas12a(D156R / D832A)-dpNLS.

[0158] SEQ ID NO: 11 has the same architecture as SEQ ID NO: 7, but comprises a monomeric TadA9 adenine deaminase: dpNLS-hA3A-48aa-XTEN-Linker-TadA9-32aa-XTEN-Linker-LbCas12a(D156R / D832A)-bdpNLS.

[0159] SEQ ID NO: 12 has the same structure as SEQ ID NO: 11, but the linker region between part (ii) and part (iii) is a (GGGGS)6 linker: dpNLS-hA3A-48aa-XTEN-linker-TadA9-(GGGGS)6-LbCas12a(D156R / D832A)-dpNLS.

[0160] SEQ ID NO: 13 has the same structure as SEQ ID NO: 12, but comprises a hA3A(W104A / P134Y) mutant: dpNLS-hA3A(W104A / P134Y)-48aa-XTEN-Linker-TadA9-(GGGGS)6-LbCas12a(D156R / D832A)-dpNLS.

[0161] SEQ ID NO: 14 has the same architecture as SEQ ID NO: 12, but comprises a hA3A(Y130F) mutant: dpNLS-hA3A(Y130F)-48aa-XTEN-Linker-TadA9-(GGGGS)6-LbCas12a(D156R / D832A)-dpNLS.

[0162] SEQ ID NO: 15 has the same structure as SEQ ID NO: 10, but comprises a uracil glycosylase inhibitor portion and (GGGGS)6: dpNLS-hA3A-48aa-XTEN-Linker-TadA8e-(GGGGS)6-LbCas12a(D156R / D832A)-UGI-dpNLS.

[0163] SEQ ID NO:16 has the same architecture as SEQ ID NO:10, but the linker region between part (ii) and part (iii) is a (GGGGS)6 linker and it comprises the E. coli uracil-N-glycosylase portion: dpNLS-eUNG-hA3A-48aa-XTEN-linker-TadA8e-(GGGGS)6-LbCas12a(D156R / D832A)-dpNLS.

[0164] SEQ ID NO: 17 has the same architecture as SEQ ID NO: 2, but comprises a LbCas12a (D832A / D156R / G532R / K538R) mutant (enCas12a (D832)): hA3A-48aa-XTEN-linker-ecTadA-32aa-XTEN-linker-ecTadA7.10-32aa-XTEN-linker-enCas12a (D832)-SV40 NLS (3x).

[0165] SEQ ID NO: 18 has the same structure as SEQ ID NO: 17, but comprises dimer TadA8e(V106W): hA3A-48aa-XTEN-linker-ecTadA-32aa-XTEN-linker-TadA8e(V106W)-32aa-XTEN-linker-enCas12a(D832)-SV40NLS(3x).

[0166] SEQ ID NO: 19 has the same structure as SEQ ID NO: 17, but comprises hA3A(R128A): hA3A(R128A)-48aa-XTEN-linker-ecTadA-32aa-XTEN-linker-ecTadA7.10-32aa-XTEN-linker-enCas12a(D832)-SV40 NLS(3x).

[0167] SEQ ID NO: 20 comprises both hA3A(R128A) and dimer TadA8e(V106W): hA3A(R128A)-48aa-XTEN-linker-ecTadA-32aa-XTEN-linker-TadA8e(V106W)-32aa-XTEN-linker-enCas12a(D832)-SV40 NLS(3x).

[0168] SEQ ID NO:21 has the same architecture as SEQ ID NO:17, but contains an additional E795L mutation in enLbCas12a: hA3A(R128A): hA3A-48aa-XTEN-linker-ecTadA-32aa-XTEN-linker-ecTadA7.10-32aa-XTEN-linker-enCas12a(E795L / D832)-SV40 NLS(3x).

[0169] SEQ ID NO: 22 comprises dpNLS, monomeric TadA9, (GGGGS)5 linker region, and Rad51ssDBD: dpNLS-hA3A-48aa-XTEN-Linker-TadA9-(GGGGS)5-Rad51ssDBD-LbCas12a(D156R / D832A)-dpNLS.

[0170] SEQ ID NO:23 comprises dpNLS, monomeric TadA9, and a (GGGGS)5 linker region: dpNLS-hA3A-48aa-XTEN-Linker-TadA9-(GGGGS)5-LbCas12a(D156R / D832A)-dpNLS.

[0171] SEQ ID NO:24 comprises dpNLS, hA3A(W104A / P134Y), monomeric TadA9, and a (GGGGS)5 linker region: dpNLS-hA3A(W104A / P134Y)-48aa-XTEN-Linker-TadA9-(GGGGS)5-LbCas12a(D156R / D832A)-dpNLS.

[0172] SEQ ID NO:25 comprises dpNLS, hA3A(Y130F), monomeric TadA9, and a (GGGGS)5 linker region: dpNLS-hA3A(Y130F)-48aa-XTEN-Linker-TadA9-(GGGGS)5-LbCas12a(D156R / D832A)-dpNLS.

[0173] SEQ ID NO:26 has the same structure as SEQ ID NO:10, but includes a uracil glycosylase inhibitor portion: dpNLS-hA3A-48aa-XTEN-Linker-TadA8e-(GGGGS)5-LbCas12a(D156R / D832A)-UGI-dpNLS.

[0174] SEQ ID NO:27 has the same structure as SEQ ID NO:10, but includes the E. coli uracil-N-glycosylase portion: dpNLS-eUNG-hA3A-48aa-XTEN-linker-TadA8e-(GGGGS)5-LbCas12a(D156R / D832A)-dpNLS.

[0175] SEQ ID NO:52 comprises dpNLS, hA3A, monomeric TadA9, and a (GGGGS)3 linker region: dpNLS-hA3A-48aa-XTEN-Linker-Monomeric TadA9-(GGGGS)3-LbCas12a(D156R / D832A)-dpNLS (SEQ ID NO:52).

[0176] In a second aspect, there may be provided an edited cell, tissue, organ, material or whole organism obtained or obtainable by a method according to the first aspect.

[0177] In a third aspect, a diversified base editor or a diversified base editor complex further comprising at least one suitable guide RNA, or at least one nucleic acid molecule encoding the diversified base editor or the diversified base editor complex can be provided, wherein the diversified base editor is as defined in the first aspect.

[0178] As known to the skilled person, there are guide RNA scaffolds for different types of CRISPR nucleases, and these can be individually designed to interact with the PAM motif at / near the target base to be edited / exchanged.

[0179] In a fourth aspect, a vector or expression construct, or more than one vector and expression construct, may be provided, each vector and / or expression construct comprising at least one nucleic acid molecule of the third aspect, wherein different parts of the diversified base editor are encoded on the same vector or expression construct or on different vectors or expression constructs, and / or wherein the diversified base editor or part thereof and the at least one suitable guide RNA are encoded on the same vector or expression construct or on different vectors or expression constructs.

[0180] In the fifth aspect, a cell can be provided, which comprises at least one diversified base editor or at least one diversified base editor complex of the third aspect or at least one nucleic acid molecule encoding the at least one diversified base editor or at least one diversified base editor complex; or at least one vector or expression construct of the fourth aspect; wherein the cell is a prokaryotic cell, including a bacterial cell or an archaeal cell; or a eukaryotic cell, including an insect cell, a mammalian cell, or a plant cell including a plant protoplast, preferably wherein the cell is a plant cell including a plant protoplast, optionally wherein the plant cell including a plant protoplast is a cell belonging to or derived from a plant selected from the following, wherein the at least one target cell is A plant cell belonging to or derived from a plant belonging to the subkingdom Viridiplantae superfamily, in particular monocots and dicots, including forage or forage legumes, ornamentals, food crops, trees or shrubs selected from the list comprising Acer species, Actinidia species, Okra species, Sisal, Agropyron species, Creeping bentgrass, Allium species, Amaranth species, European seashore grass, pineapple, Annona species, celery, Arachis species, Jackfruit species, Asparagus, Avena species (e.g., Avena, Oats, Oats Byzantine, Avena sativa var., Oats hybrid), Carambola, Balsam pear species, wax gourd, Brazil nut, Sugar beet, Brassica species (e.g., Brassica napus, Rapeseed subsp. [canola, oilseed ... cabbage, Brassica rapa]), pink-leaved leech orange, tea, canna, cannabis, capsicum species, tall sedge, papaya, large-flowered caryophyllaceae, pecan species, safflower, castanea species, gibbey, endive, camphor species, watermelon, citrus species, coconut species, coffee species, taro, cola nut species, jute species, coriander, hazel species, hawthorn species, saffron, pumpkin species, cucumber species, cardoon species, carrot, beech species, longan, Dioscorea species, Diospyros species, Echinochloa species, oil palm (e.g., oil palm, American oil palm), millet, teff, sugarcane species, loquat, eucalyptus species, red fruit, buckwheat species, Fagus species, reed fescue, fig, kumquat species, strawberry species, Ginkgo, Glycine max species (e.g., sedge beans, soybeans or soybeans), Upland cotton, Helianthus annuus species (e.g., sunflower), Hemerocallis, Hibiscus spp., Hordeum spp. (e.g., barley), Sweet potato, Juglans spp., Lettuce, Lathyrus spp., Lentil, Flax, Litchi, Lotus truncatula species, angular loofah, Lupinus spp., Forest waxberry, Tomato spp. (e.g., Common tomato, Tomato, Pear-shaped tomato), Hard-shelled bean species, Malus spp., Concave golden malus, Mango, Cassava spp., Sapodilla, Medicago sativa, Mentha spp., Miscanthus spp., Momordica charantia species, Black mulberry, Musa spp., Nicotiana spp., Olea europaea species, Opuntia spp., Opuntia spp., Rice spp. (e.g.,rice, broadleaf rice), millet, switchgrass, passionflower, parsnip, Pennisetum spp., avocado spp., parsley, phalaenopsis, Phaseolus spp., timothy grass, eryngium spp., reed, Physalis spp., pine spp., pistachio, pea spp., bluegrass spp., poplar spp., mesquite spp., prunus spp., guava spp., pomegranate, pear, oak spp., radish, rhubarb, ribes spp., comb, Rubus spp., Saccharum spp., willow spp., elderberry spp., rye, sesame species, Sinapis species, Solanum species (e.g., potato, red eggplant, or tomato), Sorghum, Spinacia species, Syzygium species, Tagetes species, Tamarillo species, Cacao, Trifolium species, Dactylum foetida, Triticale, Triticum species (e.g., Triticum durum, Triticum paniculate, Triticum truncatum, Triticum moga, Triticum flounder, Triticum einkorn, or Triticum ulmoides), Nasturtium, Nasturtium, Vaccinium species, Vicia species, Vigna species, Vigna species, Viola species, Viola oleracea, Vitis species, Zea mays, Ziziphus spp., or Ziziphus species.

[0181] Preferred plants are okra species, Allium species, celery, Asparagus, Avena species (e.g., oats, wild oats, Byzantine oats, wild oats var. sativa, hybrid oats), beet, Brassica species (e.g., Brassica napus, rapa subsp. [canola, rape, Brassica napus]), Capsicum species, watermelon, Cucumis species, Cynara species, carrot, Glycine max species (e.g., sedge, soybean or soybean), cotton, Helianthus species (e.g., sunflower), Hordeum species (e.g., barley), lettuce, alfalfa, Oryza species (e.g., rice, broadleaf rice), Pennisetum species, Saccharum species, rye, Solanum species (e.g., potato, red eggplant or tomato), sorghum, Spinach species, Triticum species (e.g., bulgur, durum, cone wheat, common wheat, mocha, floating wheat, einkorn or soft wheat) or maize.

[0182] In certain embodiments, the preferred plant may also be selected from Brassica species (e.g., Brassica rapa, Brassica rapa subspecies [canola, rapeseed, Brassica rapa]), Capsicum species, Glycine max species (e.g., sedge, soybean or soybean), Gossypium hirsutum, Helianthus annuus species (e.g., sunflower), Oryza sativa species (e.g., rice, broad-leaved rice), Solanum species (e.g., potato, red eggplant or tomato), Triticum species (e.g., bulgur, durum, strychnine, common wheat, mocha, floating wheat, einkorn or soft wheat), or Zea mays.

[0183] In a sixth aspect, a kit may be provided, comprising at least one diversified base editor or at least one diversified base editor complex of the third aspect or at least one nucleic acid molecule encoding the at least one diversified base editor or at least one diversified base editor complex; or at least one vector or expression construct of the fourth aspect; or at least one cell of the fifth aspect, and optionally instructions for use and necessary buffers, equipment and reagents.

[0184] The diversified base editor, the diversified base editor complex comprising a guide RNA, the nucleic acid molecule encoding the diversified base editor, the diversified base editor complex, and / or the vector or expression construct are provided in a functional form, for example, including stabilizers, cofactors, methods for introducing them into target cells or tissues, etc.

[0185] In the seventh aspect, the use of at least one diversified base editor or at least one diversified base editor complex of the third aspect or at least one nucleic acid molecule encoding the at least one diversified base editor or at least one diversified base editor complex can be provided; or the use of at least one vector or expression construct of the fourth aspect; or the use of at least one cell of the fifth aspect; or the use of at least one kit of the sixth aspect; they are used for the targeted directed evolution of at least one target nucleic acid segment, preferably for the botanical targeted directed evolution of at least one target nucleic acid segment, the use including the use for optimizing or modifying the traits of the plant, including optimizing or modifying yield-related traits, or disease or pathogen resistance-related traits, or herbicide resistance-related traits, or abiotic stress-related traits including salinity or drought stress-related traits, wherein the disease is caused by or the pathogen is selected from the following: viruses, bacteria, fungi, nematodes or insects, and the use further includes the use for identifying at least one gene and / or genomic locus associated with at least one trait of interest.

[0186] Targeted directed evolution refers to any strategy for diversifying target nucleic acid segments followed by genotypic and / or phenotypic screening and / or selection, optionally including the application of selective pressure, typically in the form of repeated rounds of mutagenesis, wherein each round of mutagenesis may include a step of regenerating organisms (including plants) from the cells, tissues and / or material (including plant protoplasts or callus) used for mutagenesis and / or a step of regenerating plant material, for example via callus culture or by direct rooting / sprouting, and / or for hybridization (including backcrossing).

[0187] In an eighth aspect, provided is a Brassica napus acetolactate synthase (ALS) 3 protein comprising D358N and R359H mutations or an Arabidopsis thaliana acetohydroxyacid synthase (AHAS) protein comprising D376N and R377H mutations.

[0188] In one embodiment, the Brassica napus ALS3 protein comprises or consists of the amino acid sequence of SEQ ID NO:77, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity thereto.

[0189] In the ninth aspect, a nucleic acid molecule encoding the ALS3 or AHAS protein of the eighth aspect is provided.

[0190] In one embodiment, the nucleic acid molecule comprises or consists of a sequence of SEQ ID NO:76, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity thereto.

[0191] In the tenth aspect, a plant or plant cell is provided, which comprises and / or encodes the ALS3 protein or AHAS protein of the eighth aspect or the nucleic acid molecule of the ninth aspect.

[0192] In one embodiment, the plant or plant cell is a Brassica napus or Arabidopsis thaliana plant or plant cell.

[0193] Examples:

[0194] Example 1: Cloning methods and plasmid construction

[0195] Unless otherwise indicated, the cloning procedures for the purposes of the present invention are carried out as described in the literature available to technicians for a long time (see Sambrook, Fritsch and Maniatis, 1989), including restriction digestion, agarose gel electrophoresis, purification and connection of nucleic acids, transformation, selection and cultivation of bacterial cells. Sequence analysis of recombinant DNA is carried out using Sanger technology by LGC Genomics (LGC Genomics, Berlin, Germany). Restriction endonucleases and Gibson Assembly reagents for constructing various expression vectors are from New England Biolabs (New England Biolabs, Ipswich, Massachusetts, USA). Oligonucleotides are synthesized by Integrated DNA Technologies (Integrated DNA Technologies, Coralville, Iowa, USA). Codon-optimized genes are from Genewiz (Genewiz, South Plainfield, New Jersey, USA).

[0196] All base editors were optimized for expression in plant cells and for codon usage of highly expressed genes in wheat.

[0197] All expression vectors included the maize polyubiquitin (Ubi) promoter (Seq ID NO: 28) for constitutive expression upstream of the coding sequence, and a fragment of the 3' untranslated region of gene 7 from the octopine-type Ti plasmid of Agrobacterium tumefaciens (Seq ID NO: 29) or the 35S gene of cauliflower mosaic virus (Seq ID NO: 30) at the 3' end. A gRNA expression cassette containing Cas12a guide RNA was customized as a synthetic fragment consisting of a truncated glycine-tRNA (Seq ID NO: 31), a 21 bp direct repeat sequence (Seq ID NO: 32), a 23 bp prototype spacer site targeting the rice OsAAT gene (LOC_Os01g55540.1) (Seq ID NO: 33), and a rice polymerase III terminator sequence (containing eight consecutive "Ts"), and these synthetic fragments were cloned into a standard E. coli vector (pUC derivative) via EcoRV blunt-end ligation. The expression of gRNA was driven by the polymerase III type promoter of the rice U6 snRNA gene (Seq ID NO: 35).

[0198] All plasmids were transformed into E. coli for propagation and isolated using the ZymoPure II Plasmid Gigaprep Kit for DNA purification (Zymo Research, Irvine, CA, USA).

[0199] Example 2: Design of Cas9 diversified base editors

[0200] To design a Cas9-based diversified base editor (Cas9 DBE), the existing Cas9 dual base editor STEME-1 (Li et al., 2020) was optimized to allow greater sequence diversification. To generate construct DBE-1, the Cas9 (D10A) nickase in STEME-1 was exchanged with a variant of Cas9 (D10A) with enhanced DNA dissociation, enCas9 (Slaymaker et al., 2016), the UGI domain was removed, and the nucleoplasmin and single SV40 NLS were replaced with three C-terminal repeats of the SV40 NLS (see Figure 1A Both STEME-1 and DBE-1 were optimized for expression in monocots, and their respective activities were determined in rice protoplasts by measuring the total number of base edits at the AAT target site.

[0201] Transformation of rice protoplast cells was performed as described by Shan et al., 2014, with minor modifications. Protoplasts were isolated from the sheaths of 3-week-old sterile-grown rice seedlings. Healthy stems and sheaths were bundled into 20 piles and cut into thin strips with a sharp razor blade. The thin strips were then infiltrated with a cell wall lytic enzyme solution (1.5% cellulase R10 and 0.75% macerozyme R10 in 10 mM KCl and 0.6 M mannitol, pH 7.5) and incubated overnight at 24 ° C in the dark with gentle shaking (40 rpm). After enzyme digestion, the released protoplasts were collected by filtering the mixture through a 40 μm nylon mesh and resuspended in a W5 solution. The resuspended protoplasts were washed with a W5 solution, and the cell pellet was then suspended in an MMG solution at a density of 2.5 million cells / ml. For transformation, 200 μl of cells (5 x 105) were mixed with 20 μg of plasmid DNA and 220 μl of freshly prepared polyethylene glycol (PEG) solution. The mixture was incubated in the dark for 15-20 min. After removing the PEG solution, the protoplasts were resuspended in 2 ml of WI solution, transferred to a six-well plate, and incubated at 24°C for at least 48 h.

[0202] Both STEME-1 and DBE-1 were co-transfected with a Cas9 guide RNA construct targeting AAT, which contained from 5' to 3' end: truncated tRNA, the first mature direct repeat sequence, a spacer RNA, a second mature direct repeat sequence, and a poly-T tail (T extension terminator). Three days after transfection, protoplasts were harvested by centrifugation and genomic DNA was extracted using Phire tissue direct PCR extraction buffer (Thermo Fisher Scientific) or Qiagen DNeasy plant kit. The AAT target region was amplified by PCR using primers SEQ ID NO: 36 and SEQ ID NO: 37, and subjected to amplicon deep sequencing.

[0203] Although the overall base editing efficiencies of STEME-1 and DBE-1 were very similar (49.43% vs. 50.19%), DBE-1 showed an overall broader mutation spectrum with significantly increased C to A and C to G substitutions (see Figure 1B and Figure 2 ). In addition, DBE-1 exhibited a slightly expanded C to T base editing window spanning positions C1 to C16 compared to C7-C16 of STEME-1 (the distal end of the PAM was counted as position 1, data not shown). Consistent with the broader mutation spectrum and expanded editing window of DBE-1, it was also found that a greater number of alleles were identified around the AAT target site compared to cells transfected with STEME-1. Together, these results suggest that DBE-1 can increase mutational diversity at the target site using a single guide RNA.

[0204] Example 3: Development and optimization of Cas12a diversified base editors

[0205] Based on the Cas9 DBE-1 architecture, a series of expression vectors encoding different Cas12a diversified base editors (Cas12a DBE) were constructed. Each of these expression constructs contained different modifications with respect to the NLS configuration, one or more adenosine deaminase moieties, cytosine deaminase moieties; Cas12a moieties and / or protein linkers connecting the adenosine moiety to Cas12a (see Figure 3 ).

[0206] All Cas12a DBEs are optimized for expression in monocots and are transcribed from the constitutive maize Ubi promoter. To examine their base editing activity, each of the Cas12a DBE constructs was transfected into rice protoplasts along with a guide RNA expression construct containing a truncated glycine-tRNA and two mature direct repeats at 5' and 3' of the spacer. Some base editor constructs were tested in combination with the LbCas12a R1138A mutation, which is expected to disrupt base editing via nicking of non-target strands or by residual DSB nuclease activity (Yamano et al., 2016). The total base editing efficiency of the selected Cas12A DBE architecture, as measured by amplicon deep sequencing, is shown in Figure 3 and Table 1.

[0207] Table 1

[0208]

[0209] Table 1 shows the results of different LbCas12a DBE constructs at the OsAAT target site in rice protoplasts. The editing efficiency of the different constructs is expressed relative to the editing efficiency shown for LbCas12a-DBE-10 (see Figure 3 , SEQ ID NO: 10). The different LbCas12a-DBE architectures used are:

[0210] DBE-7: dpNLS (dual-part nuclear localization signal)-hA3A-48aa-XTEN-linker-monomer TadA8e-32aa-XTEN-linker-LbCas12a (D156R / D832A)-dpNLS (SEQ ID NO: 7)

[0211] DBE-10: dpNLS-hA3A-48aa-XTEN-Linker-Monomer TadA8e-GGGGS-Linker-(5x)-LbCas12a(D156R / D832A)-dpNLS (SEQ ID NO: 10)

[0212] DBE-11: dpNLS-hA3A-48aa-XTEN-Linker-Monomer TadA9-GGGGS-Linker-(5x)-LbCas12a(D156R / D832A)-dpNLS (SEQ ID NO: 23)

[0213] DBE-12: dpNLS-hA3A(Y130F)-48aa-XTEN-Linker-Monomer TadA9-GGGGS-Linker-(5x)-LbCas12a(D156R / D832A)-dpNLS (SEQ ID NO: 25)

[0214] DBE-13: dpNLS-hA3A-48aa-XTEN-Linker-Monomer TadA9-GGGGS-Linker-(3x)-LbCas12a(D156R / D832A)-dpNLS (SEQ ID NO: 52)

[0215] DBE-14: dpNLS-eUNG-hA3A-48aa-XTEN-Linker-Monomer TadA8e-GGGGS-Linker-(5x)-LbCas12a(D156R / D832A)-dpNLS (SEQ ID NO: 27)

[0216] Several rounds of optimization have shown that the following modifications can increase base diversity:

[0217] - Use monomeric TadA adenine deaminase instead of dimeric TadA adenine deaminase

[0218] - Use monomeric TadA9 deaminase instead of TadA8e deaminase

[0219] - Use (GGGGS)5 as a linker between part (ii) and part (iii)

[0220] - Use the bipartite NLS SEQ ID NO:49 to replace the three repeats of SV40 NLS

[0221] - Use dpNLS at both the N-terminus and the C-terminus

[0222] The highest base editing level was determined for construct 11 (SEQ ID NO: 23; see Table 1), which contained a bipartite SV40 NLS (SEQ ID NO: 49) at both the 5' and 3' ends, an hA3A cytosine deaminase domain, a monomeric TadA9 as an adenosine deaminase domain, and a (GGGGS)5 linker connecting TadA9 to a catalytically inactive LbCas12a carrying the D156R mutation.

[0223] For construct 10 (SEQ ID NO: 10; see Figure 3) determined the second highest base editing level (average 16.4%), which contained a bipartite NLS (SEQ ID NO:49) at both the 5' and 3' ends, an hA3A cytosine deaminase domain, a monomeric TadA8e as an adenosine deaminase domain, and a (GGGGS)5 linker connecting TadA8e to a catalytically inactive LbCas12a carrying the D156R mutation.

[0224] Interestingly, introduction of the K932G / N933G mutations in the Cas12a domain, which were previously hypothesized to enhance base editing by nicking the target strand (Paul et al., 2021), reduced the efficiency of base substitution by significantly increasing indel formation (Construct 8; see Figure 3 ). In addition, substitution of glutamic acid with leucine at position 795 in Cas12a (E795L, an amino acid change found to enhance Cas12a activity in mammalian cells (WO2020 / 172502A1)) failed to significantly increase the base substitution rate in rice protoplasts (constructs 3, 6, and 9; see Figure 3 ), while the introduction of the Y130F mutation in the hA3A domain or the use of a three-GGGGS linker between the adenosine deaminase domain and Cas12a reduced the editing rate (see Table 1). Interestingly, the efficiency of DBE-10 can be further enhanced by co-delivery with Escherichia coli-derived uracil DNA N-glycosylase (eUNG) expressed in trans from a strong 35S promoter (see Table 1), suggesting that the generation of abasic sites and subsequent induction of base excision DNA repair promote target diversification of DBE. However, in contrast to the findings for Cas9 (Kurt et al., 2021), the addition of eUNG to the N-terminus of Cas12a DBE-10 had a strong negative effect on editing activity (see Table 1).

[0225] Additional modifications are currently being tested, including the effect of fusing the C-terminal UGI and UNG domains to the Cas12a DBE, the effect of the (GGGGS)6 linker between part (ii) and part (iii) on the mutagenesis efficiency, the introduction of the W104A / P134Y mutations in the hA3A domain, and the effect of the non-sequence-specific ssDNA binding domain between part (ii) and part (iii) on the base substitution rate.

[0226] Example 4: LbCas12a-DBE activity in soybean and rapeseed

[0227] To determine the activity of LbCas12a-DBE in dicots, additional experiments were performed using rapeseed (Brassica napus) and soybean (soybean) protoplasts. Rapeseed protoplasts were isolated from leaves of 4 to 7 week-old sterile growth strains. Healthy leaves were cut into thin strips with a sharp razor blade. These thin strips were infiltrated with cell wall lytic enzyme solution (0.25% cellulase R10 and 0.25% analyte R10) and incubated overnight at 24 ° C in the dark with gentle shaking (40 rpm). After enzyme digestion, the released protoplasts were collected by filtering the mixture through a 40 μm nylon mesh and resuspended in W5 solution. The resuspended protoplasts were kept on ice and allowed to settle by gravity, and the cell pellets were then resuspended in MMG. For transformation, 200 μl cells (2.5 x 105) were mixed with 20 μg plasmid DNA and 220 μl freshly prepared polyethylene glycol (PEG) solution. The mixture was incubated in the dark for 15-20 min. After removing the PEG solution, the protoplasts were resuspended in 2 ml W5 solution and incubated at 24 ° C. Soybean protoplasts were isolated from single leaf blades of 6-day-old seedlings and transfected essentially as described for rapeseed. After removing the PEG solution, the protoplasts were resuspended in 2 ml WI solution.

[0228] First, Cas12a-DBE activity was evaluated using two different reporter systems. The first reporter was activated after C to T editing to convert blue fluorescent protein (BFP) into green fluorescent protein (GFP), which requires codon 66 to be changed from CAC (histidine) to TAC (tyrosine; see SEQ ID NO: 53). The second assay detects the A to G editing of the inactivated GFP reporter, which carries an early stop codon (see SEQ ID NO: 54) produced by changing codon 110 from CGA (arginine) to TAG. The editing of BFP or inactivated GFP reporters will restore the GFP coding sequence and result in GFP fluorescence.

[0229] Rapeseed protoplasts were co-transfected with the following three vectors: (1) a vector encoding BFP (SEQ ID NO: 53) or an inactivated GFP (SEQ ID NO: 54), both of which contain an engineered TTTC Cas12a PAM site (due to a T62S substitution in BFP and a silent AAG to AAA mutation at K114 in GFP), (2) a Cas12a-DBE expression construct comprising hA3A as the cytosine deaminase domain and TadA9 as the adenosine deaminase domain and a five-GGGGS linker connecting TadA8e to the dLbCas12a (D156R) module located 3' of TadA8e (i.e., DBE-10; SEQ ID NO: 10), and (3) a vector encoding a Cas12a gRNA targeting a BFP or GFP reporter and containing two mature direct repeats located 5' and 3' of the spacer (SEQ ID NO: 55; SEQ ID NO:56). The DBE-10 vector includes the Arabidopsis ubiquitin 10 promoter (SEQ ID NO:57) for constitutive expression, while expression of the gRNA is driven by the polymerase III type promoter of the Arabidopsis U6 snRNA gene (SEQ ID NO:58). As a positive control, protoplasts were transfected with a construct expressing wild-type eGFP under the control of the strong cauliflower mosaic virus (CaMV) 35S promoter (SEQ ID NO:59). As a negative control, the Cas12a-DBE-10 fusion protein was tested in the absence of gRNA. Fluorescence imaging 2 days after transfection revealed approximately 35% GFP-fluorescent cells in the positive control, and 3.5% and 2.1% with dCas12a-DBE-10 and BFP and dGFP reporters, respectively (see Figure 4A Importantly, in the absence of gRNA, no GFP-positive cells were observed (data not shown).

[0230] To confirm the activity of Cas12a-DBE at endogenous sites, the pAtUbi10>DBE-10 expression construct was co-transfected into rapeseed or soybean protoplasts together with Cas12a gRNA targeting BnFAD2 (gRNA: SEQ ID NO: 60), BnALS3 (gRNA: SEQ ID NO: 61), or GmFAD2 (gRNA: SEQ ID NO: 62) genes. The transfected rapeseed protoplasts were cultured in alginate, and the editing efficiency was determined by deep amplicon sequencing 14 days after transfection. In contrast, soybean protoplasts were incubated in WI solution for 72 hours and analyzed via droplet digital PCR. Figure 4BAs shown, transfection of DBE-10 resulted in successful editing of all 3 genes tested, with up to 4.5% of NGS or ddPCR reads showing C to T and / or A to G base changes (averages of 1.51%, 2.66%, and 1.92% for BnFAD2, BnALS3, and GmFAD2, respectively). Together with the data in rice protoplasts, these results show that Cas12a-DBE is active in both monocots and dicots.

[0231] Example 5: MS2 Tagging for Diversified Base Editors

[0232] To develop an MS2 labeling strategy for Cas12a-DBE, four different Cas12a guide RNAs were designed that possessed two MS2 stem-loops at the 5′ end of the guide (see Figure 5A and Figure 5B ; SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41). To test the effect of additional MS2 stem-loops on the activity of the Cas12a-crRNA complex, purified OsAAT PCR products targeted by different guide RNA designs were digested in vitro. The sequence of the gRNA target site is listed as SEQ ID NO:33.

[0233] A 25 μl reaction was prepared by mixing 500 ng of purified OsAAT PCR substrate, 2 μl of preassembled Cas12a RNP (including 29 pmoles of crRNA and 22 pmoles of protein), and 2.5 μl of 10x NEB buffer 2.1. The reaction was incubated at 37°C for 60 minutes, heat inactivated at 85°C for 2 minutes, and separated on a 1% agarose gel containing 1 / 100 (v / v) SYBR-Safe (Invitrogen). The positional shift of the OsAAT PCR product indicated successful cleavage. Figure 6 As shown, all four MS2-modified guide RNA designs produced bands indicating substrate cleavage that were similar to those seen in the positive control sample (i.e., unmodified guide RNA). Comparable levels of indel formation were also found in rice protoplasts co-transfected with LbCas12a and unlabeled gRNA or one of the four MS2-modified variants (see Table 2).

[0234] Table 2

[0235]

[0236] Table 2 shows the indel frequencies induced by the four different Cas12a-MS2 guide RNAs shown in FIG. 5 for the OsAAT target site in rice protoplasts compared to those induced by an unlabeled crRNA control.

[0237] It has been confirmed that the addition of MS2 stem-loop does not affect the cleavage activity of Cas12a gRNA, and the effect of MS2 tagging on base editing level is evaluated next. To this end, rice protoplasts were co-transfected with the following: an expression construct containing catalytically inactive LbCas12a (D832A; SEQ ID NO: 63) together with one of the four gRNAs carrying two MS2 hairpin binding sites, and a third vector encoding a fusion of bacteriophage MS2 N55K coat protein (MCP) and hA3A cytosine deaminase domain (SEQ ID NO: 43). The MCP coding sequence contains an N55K mutation that increases protein affinity for MS2 stem-loops (Peabody, 1993). The base editing activity of different dCas12a-guided MS2-hA3A fusions was determined by amplicon deep sequencing three days after transfection, and compared with the base editing activity of Cas12a-DBE-10. Although different MS2-gRNA designs exhibited different mutation efficiencies depending on the target gene, recruitment of hA3A by dCas12a generally improved editing activity relative to that of the DBE-10 fusion protein (see Figure 7 and Table 3). The largest increase in editing was observed for the OsDEP1 target, with MS2-gRNA designs 3 and 4 (see Figure 5) resulting in an average increase in editing efficiency of 8.25-fold and 7.42-fold, respectively, compared to DBE-10. In summary, these results demonstrate that targeted recruitment of deaminases via MS2-modified gRNAs and catalytically inactive Cas12a can be used to increase the level of targeted random mutagenesis in plants.

[0238] Table 3

[0239]

[0240]

[0241] Table 3 shows the overall base editing efficiency of dLbCas12a-guided MS2-hA3A fusions at OsDEP1 and OsACC target sites in rice protoplasts with four different Cas12a-MS2 guide RNA architectures shown in Figure 5. Figure 3 Construct 10 (SEQ ID NO: 10) in . The mutation efficiency is expressed as the percentage of NGS reads with base changes.

[0242] Example 6: Use of Cas12a-DBE for directed evolution of novel herbicide tolerance in rapeseed

[0243] Diversified base editors are expected to rapidly improve agronomic traits via directed protein evolution. In order to test the potential of Cas12a-DBE to evolve new herbicide tolerance, DBE-10 (SEQ ID NO: 10) was used for the directed evolution of acetohydroxyacid synthase (AHAS, EC 2.2.1.6) in rapeseed plants. AHAS (also known as acetolactate synthase (ALS)) is the first enzyme in the biosynthesis pathway of branched essential amino acids valine, leucine and isoleucine. AHAS inhibitor herbicides have been widely used since their first introduction in the early 1980s, due to their very low rate of achieving broad-spectrum weed control, low mammalian toxicity and wide crop selectivity. Design of twelve Cas12 agRNAs targeting the ALS3 gene of rapeseed (Brassica napus), including 6 gRNAs with TTTV-3'PAM sites and 6 gRNAs with TYTC-3'PAM (see Table 4). To test the activity of the designed gRNAs, the guides alone were transfected into rapeseed protoplasts along with LbCas12a (for targeting of TTTV PAM) or LbCas12a-G532R / K595R (for targeting of TYTC PAM). Figure 8), so proof-of-concept experiments were initiated in which rapeseed protoplasts were transformed with a variety of gRNAs targeting ALS3 together with LbCas12a-DBE-10 or LbCas12a (G532R / K595R) -DBE-10. The transfected protoplasts were embedded in a 1% alginate layer and cultured at 24 ° C for at least two weeks before plating on modified MS medium containing a selective concentration of the AHAS inhibitor bispyribac-sodium salt. Approximately 3-4 weeks after plating, the developing structures were transferred to MS regeneration medium, and individual shoots were sequenced to retrieve mutations that conferred resistance. Screening of protoplast-derived shoots transformed with a combined gRNA library together with DBE-10 identified a resistant rapeseed line that survived treatment with 1 nM bispyribac-sodium (see Table 5). Sanger sequencing of this line revealed two monoallelic D358N and R359H mutations, the former resulting from a single C to T transition and the latter resulting from both a C to T transition and an A to G transition, indicating simultaneous deaminase activity from DBE-10 (BnALS3_D358N / R359H coding sequence: SEQ ID NO: 76; BnALS3_D376N / R377H amino acid sequence: SEQ ID NO: 77). While the D358N mutation (corresponding to D376N in Arabidopsis AHAS, i.e., AtAHAS) is a known artificially generated amino acid substitution that confers resistance, R359H (corresponding to R377H in AtAHAS) has previously been documented in resistant weeds (Yu and Powles, 2014). Both amino acid substitutions are predicted to result in changes in protein structure that reduce the binding affinity of AHAS to bispyribac-sodium. As shown in Figure 9, bispyribac-butyl has three aromatic rings, which were found to adopt a distorted "S" conformation when bound to AtAHAS, with the pyrimidine group inserted deepest into the herbicide binding site (Garcia et al., 2017). One of the bispyribac-butyl methoxy groups forms a contact with D376 of AtAHAS, while the carboxylic acid group of bispyribac-butyl forms a salt bridge with the side chain of R377. Together, these results demonstrate the ability of the DBE of the present invention to evolve novel herbicide resistance alleles under selection pressure.

[0244] Table 4

[0245]

[0246]

[0247] Table 4 shows an overview of different Cas12a gRNAs used for targeted evolution of the BnALS3 gene of rapeseed (Brassica napus).

[0248] Table 5

[0249]

[0250] Table 5 shows the results of a Cas12a DBE-mediated directed evolution experiment in rapeseed (Brassica napus) designed to develop resistance to the AHAS-inhibiting herbicide bispyribac-sodium. Screening of protoplasts transformed with a combined gRNA library (G1 to G6 of Table 4) and Cas12a DBE-10 (SEQ ID NO: 10) identified herbicide-resistant lines carrying two amino acid substitutions in the BnALS3 gene.

[0251] References:

[0252] Eid A, Alshareef S, Mahfouz MM. CRISPR base editors: genome editing without double-stranded breaks. Biochem J. 2018Jun11;475(11):1955-1964.doi:10.1042 / BCJ20170793.

[0253] Fan J,Ding Y,Ren C,Song Z,Yuan J,Chen Q,Du C,Li C,Wang X,ShuW.Cytosine and adenine deaminase base-editors induce broad and nonspecific changes in gene expression and splicing.Commun Biol.2021Jul16;4(1):882.doi:10.1038 / s42003-021-02406-5.

[0254] Garcia,MD et al.Comprehensive understanding of acetohydroxyacidsynthase inhibition by different herbicide families.Proceedings of the National Academy of Sciences of the United States of America vol.114,7(2017):E1091-E1100.doi:10.1073 / pnas.1616142114.

[0255] Gaudelli NM,Komor AC,Rees HA,Packer MS,Badran AH,Bryson DI,LiuDR.Programmable base editing of A·T to G·C in genomic DNA without DNAcleavage.Nature.2017Nov 23;551(7681):464-471.doi:10.1038 / nature24644.

[0256] Gautier A,Juillerat A,Heinis C,Corrêa IR Jr,Kindermann M,Beaufils F,Johnsson K.An engineered protein tag for multiprotein labeling in livingcells.Chem Biol.2008Feb;15(2):128-36.doi:10.1016 / j.chembiol.2008.01.007.

[0257] Hussain AF,Amoury M,Barth S.SNAP-tag technology:a powerful tool forsite specific conjugation of therapeutic and imaging agents.Curr PharmDes.2013;19(30):5437-42.doi:10.2174 / 1381612811319300014.

[0258] Inobe T,Nukina N.Rapamycin-induced oligomer formation system ofFRB-FKBP fusion proteins.J Biosci Bioeng.2016 Jul;122(1):40-6.doi:10.1016 / j.jbiosc.2015.12.004.

[0259] Jeong YK,Song B,Bae S.Current Status and Challenges of DNABaseEditing Tools.Mol Ther.2020 Sep 2;28(9):1938-1952.doi:10.1016 / j.ymthe.2020.07.021.

[0260] Komor,A.,Kim,Y.,Packer,M.et al.Programmable editing of a targetbasein genomic DNA without double-stranded DNA cleavage.Nature 533,420–424(2016).https: / / doi.org / 10.1038 / nature17946.

[0261] Komor AC,Zhao KT,Packer MS,Gaudelli NM,Waterbury AL,Koblan LW,Kim YB,Badran AH,Liu DR.Improved base excision repairinhibition and bacteriophage MuGam protein yields C:G-to-T:A base editorswith higher efficiency and productpurity.Sci Adv.2017 Aug30;3(8):eaao4774.doi:10.1126 / sciadv.aao4774.

[0262] Kurt,IC et al.“CRISPR C-to-G base editors for inducing targetedDNAtransversions in human cells.”Nature biotechnology vol.39,1(2021):41-46.doi:10.1038 / s41587-020-0609-x.

[0263] Lange A,McLane LM,Mills RE,Devine SE,Corbett AH.Expandingthedefinition of the classical bipartite nuclear localizationsignal.Traffic.2010 Mar;11(3):311-23.doi:10.1111 / j.1600-0854.2009.01028.x.

[0264] Li,C.,Zhang,R.,Meng,X.et al.Targeted,random mutagenesis ofplant geneswith dual cytosine and adenine base editors.Nat Biotechnol 38,875–882(2020).https: / / doi.org / 10.1038 / s41587-019-0393-7.

[0265] Los GV,Encell LP,McDougall MG,Hartzell DD,Karassina N,Zimprich C,WoodMG,Learish R,Ohana RF,Urh M,Simpson D,Mendez J,Zimmerman K,Otto P,VidugirisG,Zhu J,Darzins A,Klaubert DH,BulleitRF,Wood KV.HaloTag:a novel proteinlabeling technology for cellimaging and protein analysis.ACS Chem Biol.2008Jun 20;3(6):373-82.doi:10.1021 / cb800025k.

[0266] Paul B,Chaubet L,Verver DE,Montoya G.Mechanics ofCRISPR-Cas12a andengineered variants onλ-DNA.Nucleic Acids Res.2021 Dec 24:gkab1272.doi:10.1093 / nar / gkab1272.

[0267] Peabody,DS.The RNA binding site of bacteriophage MS2 coat protein.TheEMBO journal vol.12,2(1993):595-600.doi:10.1002 / j.1460-2075.1993.tb05691.x.

[0268] Rees HA,Liu DR.Base editing:precision chemistry on the genomeandtranscriptome of living cells.Nat Rev Genet.2018 Dec;19(12):770-788.doi:10.1038 / s41576-018-0059-1.Erratum in:Nat Rev Genet.2018 Oct 19.

[0269] Sambrook,J.,Fritsch,E.R.,&Maniatis,T.(1989).Molecular Cloning:ALaboratory Manual(2nd ed.).Cold Spring Harbor,NY:Cold SpringHarbor LaboratoryPress.

[0270] Savva YA,Rieder LE,Reenan RA.The ADAR protein family.GenomeBiol.2012Dec 28;13(12):252.doi:10.1186 / gb-2012-13-12-252.

[0271] Shan Q,Wang Y,Li J,Gao C.Genome editing in rice and wheat usingtheCRISPR / Cas system.Nat Protoc.2014 Oct;9(10):2395-410.doi:10.1038 / nprot.2014.157.Epub 2014 Sep 18.PMID:25232936.

[0272] Slaymaker IM,Gao L,Zetsche B,Scott DA,Yan WX,Zhang F.Rationallyengineered Cas9 nucleases with improved specificity.Science.2016 Jan 1;351(6268):84-8.doi:10.1126 / science.aad5227.

[0273] Yamano T,Nishimasu H,Zetsche B,Hirano H,Slaymaker IM,Li Y,Fedorova I,Nakane T,Makarova KS,Koonin EV,Ishitani R,Zhang F,Nureki O.Crystal Structureof Cpf1 in Complex with Guide RNA and TargetDNA.Cell.2016 May 5;165(4):949-62.doi:10.1016 / j.cell.2016.04.003.

[0274] Yan D,Ren B,Liu L,Yan F,Li S,Wang G,Sun W,Zhou X,Zhou H.High-efficiency and multiplex adenine base editing in plants using newTadAvariants.Mol Plant.2021 May 3;14(5):722-731.doi:10.1016 / j.molp.2021.02.007.

[0275] Yu Q,Powles SB.Resistance to AHAS inhibitor herbicides:currentunderstanding.Pest management science vol.70,9(2014):1340-50.doi:10.1002 / ps.3710.Zhang X,Chen L,Zhu B,Wang L,Chen C,Hong M,Huang Y,Li H,Han H,Cai B,Yu W,Yin S,Yang L,Yang Z,Liu M,ZhangY,Mao Z,Wu Y,Liu M,Li D.Increasingthe efficiency and targeting rangeof cytidine base editors through fusion ofa single-stranded DNA-bindingprotein domain.Nat Cell Biol.2020 Jun;22(6):740-750.doi:10.1038 / s41556-020-0518-8.

Claims

1. A method for targeted diversified base editing of at least one target nucleic acid segment, the method comprising (a) providing at least one cell or construct comprising at least one target nucleic acid segment; (b) introducing into the target cell or contacting with the target construct: (i) at least one diversified base editor (DBE) or at least one nucleic acid molecule encoding the at least one diversified base editor; and (ii) at least one suitable guide RNA or at least one nucleic acid molecule encoding the at least one suitable guide RNA; (c) allowing formation of a complex of (i) the at least one diversified base editor and (ii) the at least one suitable guide RNA; (d) obtaining at least one cell or construct comprising at least one modified target nucleic acid segment; wherein the overall base editing efficiency of introducing at least one substitution of any kind into the at least one target nucleic acid segment is at least 0.2%, 0.5%, 1%, 5%, 10%, 15%, 20% or at least 25%, with an upper limit of 100% or less; and / or wherein at least one modification of the target nucleic acid segment occurs within an extended base editing window; and wherein the method does not include treatment of the human or animal body by surgery or therapy, and / or diagnostic methods practiced on the human or animal body, and / or processes for modifying the germline genetic identity of a human, And wherein the diversified base editor comprises a CRISPR-Cas portion derived from a class 2 type V CRISPR-Cas nuclease.

2. The method of claim 1, wherein the diversified base editor comprises a CRISPR-Cas portion derived from a Cas12a endonuclease.

3. The method of claim 1 or 2, wherein the at least one target cell is a prokaryotic cell, including a bacterial cell or an archaeal cell; or a eukaryotic cell, including an insect cell, a mammalian cell or a plant cell.

4. The method of any one of the preceding claims, wherein the at least one target cell is a plant cell, including a plant protoplast.

5. The method of any one of the preceding claims, wherein the at least one diversified base editor comprises (i) one or more cytosine deaminase moieties, (ii) one or more adenine deaminase moieties, (iii) one or more CRISPR-Cas parts, preferably wherein the CRISPR-Cas domain does not cleave both strands of double-stranded DNA, (iv) one, two, three or more nuclear localization sequences; and (v) at least one linker region, preferably one or more linker regions between (i) and (ii), and optionally one or more linker regions between (ii) and (iii).

6. A method as claimed in claim 5, wherein the at least one diversified base editor in step (bi) is at least one diversified base editor in the form of a fusion protein, preferably wherein the parts (i), (ii) and (iii) as defined in claim 5 are arranged in the order of (i)-(ii)-(iii) in the N-terminal to C-terminal direction, wherein there are one or more linker regions between each segment, and further preferably wherein one, two, three or more nuclear localization sequences (iv) are located at the C-terminus of the diversified base editor, or wherein one or more nuclear localization sequences (iii) are located at the N-terminus of the diversified base editor and one or more nuclear localization sequences (iii) are located at the C-terminus of the diversified base editor.

7. A method as described in any of the preceding claims, wherein the diversified base editor comprises at least one additional part, preferably wherein the at least one additional part is selected from a ssDNA, ssRNA or dsRNA binding protein part including an MS2 protein part, an affinity tag binding protein, a uracil glycosylase inhibitor part and / or a uracil glycosylase part, or any combination thereof.

8. The method of any one of claims 1 to 5 and 7, wherein the one or more adenine deaminase moieties and / or the one or more cytosine deaminase moieties are linked to at least one ssRNA or dsRNA binding protein moiety, preferably at least one MS2 protein moiety, and the at least one suitable guide RNA is adjusted to allow interaction with the at least one ssRNA or dsRNA binding protein moiety, preferably wherein the one or more adenine base editor moieties and / or the one or more cytosine base editor moieties are linked to at least one MS2 protein moiety, and the suitable guide RNA is adjusted to comprise two MS2 stem-loops, optionally wherein the suitable guide RNA comprises a sequence selected from SEQ ID NO: 38 to SEQ ID NO: NO:41, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity thereto.

9. The method of any one of claims 1 to claim 7, wherein the diversified base editor comprises an amino acid molecule selected from any one of SEQ ID NOs: 1-27 or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the corresponding reference sequence.

10. An edited cell, tissue, organ, material or whole organism obtained or obtainable by a method as claimed in any one of the preceding claims.

11. A diversified base editor or a diversified base editor complex further comprising at least one suitable guide RNA, or at least one nucleic acid molecule encoding the diversified base editor or the diversified base editor complex, wherein the diversified base editor is as defined in any one of claims 5 to 9.

12. A vector or expression construct, or more than one vector and expression construct, each vector and / or expression construct comprising at least one nucleic acid molecule as described in claim 11, wherein different parts of the diversified base editor are encoded on the same vector or expression construct or on different vectors or expression constructs, and / or wherein the diversified base editor or part thereof and the at least one suitable guide RNA are encoded on the same vector or expression construct or on different vectors or expression constructs.

13. A cell comprising at least one diversified base editor or at least one diversified base editor complex as described in claim 11 or at least one nucleic acid molecule encoding the at least one diversified base editor or at least one diversified base editor complex; or at least one vector or expression construct as described in claim 12; wherein the cell is a prokaryotic cell, including a bacterial cell or an archaeal cell; or a eukaryotic cell, including an insect cell, a mammalian cell including a human cell, or a plant cell including a plant protoplast, preferably wherein the cell is a plant cell including a plant protoplast, optionally wherein the plant cell including the plant protoplast is a cell belonging to or derived from a plant selected from the following: The at least one target cell is a plant cell belonging to or derived from a plant belonging to the subkingdom Viridiplantae superfamily, in particular monocots and dicots, including forage or forage legumes, ornamentals, food crops, trees or shrubs selected from the list comprising Acer species, Actinidia species, Okra species, Sisal, Agropyron species, Creeping bentgrass, Allium species, Amaranth species, European seashore grass, pineapple, Annona species, celery, Arachis species, Jackfruit species, Asparagus, Avena species (e.g., Avena, Avena, Avena byzantinea, Avena sativa var., Avena hybrida), Carambola, Balsam pear species, Wax gourd, Brazil nut, Sugar beet, Brassica species (e.g., Brassica napus, Rapeseed subsp. [canola, rapeseed, Brassica napus]), citrus aurantium, tea, canna, cannabis, capsicum species, tall sedge, papaya, large-flowered caryophyllaceae, pecan species, safflower, castanea species, gibbey, endive, camphor species, watermelon, citrus species, coconut species, coffee species, taro, cola nut species, jute species, coriander, hazelnut species, hawthorn species, saffron, pumpkin species, cucumber species, cardoon species, carrot, leech species, longan, Diospyros species, Diospyros species, Echinochloa species, oil palm (e.g., oil palm, American oil palm), millet, teff, sugarcane species, loquat, eucalyptus species, red fruit, buckwheat species, Fagus species, reed fescue, fig, kumquat species, strawberry Species, Ginkgo, Glycine max species (e.g., sedge beans, soybeans or soybeans), Upland cotton, Helianthus annuus species (e.g., sunflower), Hemerocallis, Hibiscus spp., Hordeum spp. (e.g., barley), Sweet potato, Juglans spp., Lettuce, Lathyrus spp., Lentil, Flax, Litchi, Lotus truncatula species, angular loofah, Lupinus spp., Forest waxberry, Tomato spp. (e.g., Common tomato, tomato, pear-shaped tomato), Hard-shelled bean species, Malus spp., Concave golden malus, Mango, Cassava spp., Sapodilla, Medicago sativa, Mentha spp., Miscanthus spp., Momordica charantia species, Black mulberry, Musa spp., Nicotiana spp., Olea europaea species, Opuntia spp., Opuntia spp., Rice spp. (e.g.,rice, broadleaf rice), millet, switchgrass, passionflower, parsnip, Pennisetum spp., avocado spp., parsley, phalaenopsis, Phaseolus spp., timothy grass, eryngium spp., reed, Physalis spp., pine spp., pistachio, pea spp., bluegrass spp., poplar spp., mesquite spp., prunus spp., guava spp., pomegranate, pear, oak spp., radish, rhubarb, ribes spp., comb, Rubus spp., Saccharum spp., willow spp., elderberry spp., rye, sesame species, Sinapis species, Solanum species (e.g., potato, red eggplant, or tomato), Sorghum, Spinacia species, Syzygium species, Tagetes species, Tamarillo species, Cacao, Trifolium species, Dactylum foetida, Triticale, Triticum species (e.g., Triticum durum, Triticum paniculate, Triticum truncatum, Triticum moga, Triticum flounder, Triticum einkorn, or Triticum ulmoides), Nasturtium, Nasturtium, Vaccinium species, Vicia species, Vigna species, Vigna species, Viola species, Viola oleracea, Vitis species, Zea mays, Ziziphus spp., or Ziziphus species.

14. A kit comprising at least one diversified base editor or at least one diversified base editor complex as described in claim 11 or at least one nucleic acid molecule encoding the at least one diversified base editor or at least one diversified base editor complex; or at least one vector or expression construct as described in claim 12; or at least one cell as described in claim 13.

15. Use of at least one diversified base editor or at least one diversified base editor complex or at least one nucleic acid molecule encoding the at least one diversified base editor or at least one diversified base editor complex as described in claim 11; or use of at least one vector or expression construct as described in claim 12; or use of at least one cell as described in claim 13; or use of at least one kit as described in claim 14; they are used for the targeted directed evolution of at least one target nucleic acid segment, preferably for the botanical targeted directed evolution of at least one target nucleic acid segment, the use including use for optimizing or modifying the traits of the plant, including optimizing or modifying yield-related traits, or disease or pathogen resistance-related traits, or herbicide resistance-related traits, or abiotic stress-related traits including salinity or drought stress-related traits, wherein the disease is caused by or the pathogen is selected from the following: viruses, bacteria, fungi, nematodes or insects, and the use further includes use for identifying at least one lead gene.

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