EP4077652A1

EP4077652A1 - Improving efficiency of base editing using typev crispr enzymes

Info

Publication number: EP4077652A1
Application number: EP20842218.8A
Authority: EP
Inventors: Wyatt Paul; Fabien NOGUÉ; Anouchka GUYON; Pierre-François PERROUD
Original assignee: Limagrain Europe SA
Current assignee: Limagrain Europe SA
Priority date: 2019-12-20
Filing date: 2020-12-18
Publication date: 2022-10-26
Also published as: WO2021123397A1

Abstract

The invention pertains to the field of genome modification and relates to a method to improve efficacy of base editing by cutting a single strand of a nucleotide sequence near the location of the edited base.

Description

IMPROVING EFFICIENCY OF BASE EDITING USING TypeV CRISPR

ENZYMES

Introduction

Base editing has become an important tool in genome engineering. Currently tools exist to target Cytidine residues and more recently Adenine residues (Cytidine base editors (Cytidine BE) and Adenine base editors (Adenine BE)) (Kim (2018), Rees and Liu (2018)). These base editors consist of linking a cytidine deaminase or an adenine deaminase to a DNA targeting system allowing the deaminase activity to be directed to a specific DNA region. Deaminase activity for the cytidine deaminase in majority eventually converts a C residue to a T, although other bases can be introduced, depending on the enzyme and base editing construction used. The Adenine base editing system is more specific, eventually converting an A residue only to a G residue.

The base editing targeting system is most frequently based on the fusion of the deaminase to the type II CRISPR enzyme Cas9 (Cas9-BE). Expression of an appropriate guide sequence (gRNA) will then target the Cas9-BE deaminase fusion protein to its target DNA sequence. First versions of Cas9-BE used a Cas9 that has a double mutation removing its ability to create a double strand DNA break at the target site (deadCas9-BE or dCas9-BE) (Komor et al (2016).

The ability of Cas9 to create a double strand DNA break is mediated by two nuclease activities, a RuvC activity and an HNH activity. If one of these domains is mutated, the Cas9 enzyme loses its ability to cut the double-stranded DNA and can only cut one strand. A Cas9 that is only able to cut one DNA strand is a nickase. Mutation D10A in Cas9 eliminates RuvC activity and H840A eliminates the HNH activity. Although the first base editors with dead Cas9 were active they were significantly improved by the use of a nickase Cas9-BE (nCas9-BE). (Komor et al (2016). Nickase Cas9-BE are now preferred for BE.

The possibility to edit a particular base is limited by the ability to find a targeting system that binds the BE in close proximity to the targeted base. Generally, for a dCas9-BE or nCas9 BE there is a window of base editing that is in the region distal to the PAM (Protospacer adjacent motif) of the gRNA (Rees and Liu (2018).

Type V CRISPR enzymes offer alternative PAMs and thus increase the number and variety of sequences that can be targeted by a base editor.

There are few reports of the use of CRISPR Type V enzymes as a DNA targeting system to direct cytidine or adenine deaminases to target sites. Li et al (2018) demonstrated base editing activity of a dead LbCas12a cytidine BE and Kleinstiver et al (2019) that of an enhanced dead AsCas12a- cytidine BE.

The invention is based on the strategy of a combined use of a nucleotide deaminase to induce deamination of a target nucleotide in a sequence of interest on a first DNA strand and of a nickase that is separately provided to induce a cut in the non-edited (other or second) DNA strand different

In particular and in order to improve the efficiency of Type V base editors, it is proposed to combine a dead TypeV-BE (the companion protein is a dead type V Cas protein associated with a deaminase) with the expression of a nCas9 that can cleave the DNA strand that is complementary to the strand that has been targeted and edited. Such cutting is preferably performed in the vicinity (up to 300 base pairs) of the target binding site of the TypeV-BE.

One can use either version of nCas9 (a RuvC or an HNH mutant), depending on the guide RNA (gRNA) that is the most appropriate and that will be selected; however, as indicated above, the non-edited strand is to be nicked (Figure 1). It is to be noted that a single or multiple nicks can be introduced into the non-edited strand, and that the nickase can perform the nick 5’ or 3’ from the site of action of the deaminase. In figure 1B) and C), the nCas9 mutant is targeted to nick 3’ of the dCas12a-BE on the non-edited strand.

The invention thus relates to a method to modify a target nucleotide into a double-stranded nucleotide sequence of interest in a cell comprising: a) providing a nucleotide deaminase to the target sequence, in conditions so as to induce deamination of the target nucleotide of the target nucleotide sequence on a first strand of the double-stranded nucleotide sequence, b) providing a nickase endonuclease, in conditions so as to introduce a nick (single strand cut) to the second strand of the double-stranded nucleotide sequence within 300 base pairs (bp) of the target nucleotide, c) culturing the cell in adequate conditions so as to allow the modification of the target nucleotide in the double-stranded target sequence.

The above method may be used for edition of multiple targets nucleotides, in a single step. Indeed, when multiple nucleotides are located in the editing window (which corresponds to the region where the deaminase is active after it reaches the sequence of interest), the deaminase will be able to act on one or several nucleotides. When the deaminase is part of a Cas Type V base editor complex (as described below), the editing window corresponds to the nucleotide region of reach of the deaminase after the dead Cas protein has bound the sequence of interest. Consequently, the method makes it possible to modify multiple target nucleotides into a double-stranded nucleotide sequence of interest in a cell, when the nucleotide deaminase is provided to the target sequence, in conditions so as to induce deamination of the target nucleotides of the target nucleotide sequence on a first strand of the double-stranded nucleotide sequence. Steps b) and c) remain the same.

The modification of the target nucleotide into the double-stranded nucleotide sequence of interest can also be described as the edition of the target nucleotide of the double-stranded nucleotide sequence. Thus, the first strand can be designated as the edited strand.

The nick (single strand cut) is performed on the opposite or complementary strand, i.e. the strand which doesn’t contain the nucleotide that has been deaminated, which can be designated as the non-edited strand. It can also be designated as the second strand or the other strand.

The culture of the cell is performed so as to allow repair of the cut introduced by the nickase endonuclease by the cell machinery, and introduction of the mutation (edited nucleotide). Thus, the cell is cultured in such conditions that DNA replication and mitosis are observed. As shown in Figure 1, the strand containing the edited nucleotide is used as the template for the repair machinery and the resulting repaired double-strand nucleotide sequence thus contains the edited target nucleotide in place of the original target nucleotide.

The skilled person knows how to grow cells in adequate conditions and adapt the parameters (culture medium, temperature, hygrometry, oxygen percentage...) for each type of cell (mammalian, fungi, plant...). The invention can be performed on any cell, in particular eukaryotic cell. In particular, it can be performed on plant cells, fungal cells or animal cells. It is particularly interesting to perform the method with mammalian cells, more particularly with human cells.

The method can also be performed with plant cells. One can perform the method on moss. One can perform the method on monocotyledonous plant cells. It is also possible to perform the method on dicotyledonous plant cells. Among monocotyledonous plants, one can cite rice, wheat, barley, sorghum, maize or sugarcane. Among dicotyledonous plants, one can cite soybean, cotton, tomato, beet, sunflower, or rapeseed.

The deaminase may be provided to the target nucleotide using any companion protein known in the art, which can be associated with the deaminase and direct the deaminase to the proper location in the genome. One can cite zinc- finger proteins, TALEN, or proteins of the CRISPR-Cas system.

It is preferred when the deaminase is associated with an RNA-guided DNA endonuclease enzyme from the CRISPR-Cas system (however devoid of endonuclease activity) to be provided to the target nucleotide.

In particular, it is preferred when the deaminase is part of a Cas Type V base editor complex comprising a dead Type V Cas protein and the deaminase. Such Cas Type V base editor can be targeted to the sequence of interest by an appropriate guide RNA (gRNA) associated with the dead Type V Cas protein, which can herein be described as a first guide RNA, and perform deamination of the target nucleotide by way of the deaminase. This first guide RNA may also be referred to as crRNA in this application. The term “Cas Type V base editor” or “Cas Type V base editor complex” thus designs a dead type V Cas protein associated with a deaminase.

As used therein the terms “base editor complex” or “Cas base editor complex” refer to a deaminase associated with a dead Cas (a Cas protein having retained its ability to bind to DNA using a guide RNA but having lost its endonuclease activity (its ability to induce a cut in the DNA sequence)). Depending on the type of the dead Cas protein, one can refer, for instance, to a Cas type V base editor complex or to a Cas type II base editor complex. It is reminded that there are two major classes of CRISPR systems, according to the type of effector nuclease in the class 2 system, the effector nuclease is a monomer and consists of a single polypeptide chain. The type II CRISPR systems have a Cas9 endonuclease, with two separate catalytic domains belonging to the RuvC and HNH catalytic groups as described above, with the HNH domain cutting the strand targeted by the guide (complementary to the guide) target strand, and the RuvC domain cutting the complementary strand of the strand targeted by the guide (non-complementary to the guide) non-target strand. The ability for Cas9 to cleave a DNA sequence depends on the presence of an adequate protospacer adjacent motif (PAM). Type V systems have a Cpf1, C2c1 or C2c3 type endonuclease. In particular, Type II Cas proteins (such as Cas9) cleave double-stranded DNA, Type V Cas proteins (such as Cas12) cleave dsDNA adjacent to protospacer adjacent motif (PAM) sequences and single-stranded DNA (ssDNA) nonspecifically (Type VI Cas nucleases such as Cas13 cleave RNA exclusively).

One of skill in the art thus understands the meaning of Type V Cas protein, from the similarity with the Cpf1 also called Cas12a, C2c1 or C2c3 endonucleases and the nucleic acid cutting properties.

Cas12a have been identified in several bacterial species such as Francisella novicida (FnCas12a), Lachnospiraceae bacterium (LbCas12a) or Acidaminococcus sp. (AsCas12a).

In another embodiment, the deaminase is associated with a dead Type II Cas protein.

As intended herein, a “deaminase” is intended to designate a cytidine deaminase (which has the ability to convert a C nucleotide to an uracil, which will bind to an A, thereby leading to the replacement of a C-G base pair by a T-A base pair) or an adenine deaminase which converts an A nucleotide to hypoxanthine, which will then pair to a cytosine, thus resulting in a post-replicative transition mutation, where the original A-T base pair has been transformed into a G-C base pair. The modification of C to T is favorized by using a cytidine deaminase coupled with a uracil DNA glycosylase inhibitor (UGI) domain. The UGI domain inhibits the Base Excision Repair and avoids the excision of the uracil obtained after the deamination. A cytidine deaminase may also convert a C into G or a C into A thanks to the Base Excision Repair. The cytidine deaminase converts a C into uracil but the Base Excision Repair removes this uracil and allows the incorporation of any base at the position.

Cytidine deaminases used in base editors complexes can be APOBEC1 (SEQ ID NO: 85), APOBEC3-A (SEQ ID NO: 86), pmCDA (SEQ ID NO: 87).

Adenine deaminases used in base editors can be TadA (Gaudelli. 2017).

As intended herein, a “dead Cas protein” is a Cas protein devoid of nucleolytic activity. Such protein is still able to bind to DNA with an appropriate gRNA, but lacks the ability to induce a strand break to the sequence to which it is bound.

As used therein, a “nickase” is a protein that is able to cut only one strand of a double-stranded polynucleotide sequence at a specified location. It is preferred when the nickase is a mutated type II Cas protein, in particular a mutated Cas9 endonuclease. Preferred nickases are the Cas9 endonucleases that contain the D10A mutation (in the RuvC domain) or the H840A mutation (in the HNH domain). Depending on the mutation, the nickase has the ability to cut the strand that is complementary to its guide RNA (D10A nickase) or the strand that corresponds to the guide RNA (H840A nickase).

In a preferred embodiment, the dead Type V Cas protein is the dead Cas12a protein. Such dead Cas12a protein can be obtained by introduction of one or several mutations in the RuvC domain, the only nuclease domain of Cas12a. The RuvC domain can be identified in any Cas12a by homology searches (Shmakov et al. 2015). The relevant positions to be mutated to inactivate the RuvC domains can also be identified by homology searches using known dead Cas12a..

One can introduce one or several modifications in the RuvC domain of FnCas12a (SEQ ID NO: 81), AsCas12a (SEQ ID NO: 82) or LbCas12a (SEQ ID NO: 80).

In a preferred embodiment, the dead Cas12a is a Cas12a modified at one or several of the following positions 832, 1006 and 1125 when aligned with LbCas12a. The preferred substitutions are D832A, E1006A, D1125A.

In a preferred embodiment, the dead Cas12a is a LbCas12a modified at one or several of the following positions 832, 1006 and 1125. The preferred substitutions are D832A, E1006A, D1125A. By way of example, dl_bCas12a (D832A) is represented in SEQ ID NO: 83 and dl_bCas12a (D832A/E1006A/D1125A) is represented in SEQ ID NO: 8.

The dead Cas12a can further comprises a D156R mutation that improves Cas12a action (Schindele and Puchta (2020).

Additionally, the dead Cas12a protein can be associated with Nuclear Localization Signals (NLS) like the SV40 NLS (SEQ ID NO: 88) or the XINucleoplasmin NLS (SEQ ID NO: 89). The NLS can be situated at one or both ends of the dead Cas12a protein.

The dead Cas12a can be further associated with a uracil DNA glycosylase inhibitor (UGI) (Uniprot P14739) to reduce Base Excision repair (BER) and thus improve the predictability and frequency of editing.

Alternatively, one can use a dead Cas12b protein as described by Ming et al. (2020).

When a dead type II Cas protein is used, it is preferred to use a dead Cas9 protein, comprising the D10A and H840A mutations to eliminate the RuvC and HNH nuclease activity.

In a preferred embodiment, the nick (single strand cut on the non-edited strand) is performed using a Cas protein that has been modified to only cut one strand of a double stranded nucleotide sequence, and is introduced in the vicinity of the edited target nucleotide. In this matter, one uses a second guide RNA (gRNA) to provide the modified Cas protein to the site where the nick is to be introduced.

In particular, it is preferred when the nick is introduced within 300 bases 5’ or 3’ of the edited target nucleotide (orientation as determined with regards to the strand of the edited target nucleotide). More specifically, it is preferred when the nick is introduced within 200 bases 5’ or 3’ of the edited target nucleotide. More specifically, it is it is preferred when the nick is introduced within 150 bases 5’ or 3’ of the edited target nucleotide.

In one embodiment, one can also perform the step of verifying whether the cells have the expected modification. This step allows identifying such cells where the nucleotide of interest has been edited. Such step can be performed by screening cells which have been submitted to adequate conditions for edition of the nucleotide of interest (provision of the deaminase, of the nickase, and further culture) to identify cells in which the nucleotide of interest has been edited (cells in which a C-G base pair has been replaced by a T-A, or a C-G base-pair had been replaced into G-C or a C-G base-pair has been replaced into A-T or a A-T base pair has been replaced by a G-C base pair at the position of the nucleotide of interest).

Such screening can be performed by any method known in the art. By way of example, one can extract the DNA of a cell, or a tissue or an organism, amplify the nucleotide sequence of interest with specific primers by PCR and sequence the sequence of interest to detect the presence of the expected modification. The sequencing can be implemented using NEXT Generation Sequencing (NGS). One can also use the droplet digital PCR method (ddPCR™ BIO RAD) or the KASP method (Biosearch Technologies) based on detection of fluorescence. One can also use phenotypic screening, for example if the base editing creates a mutation allowing the cell to resist to a toxic component, the screening can be made on a medium comprising such toxic component.

In a preferred embodiment, the nick is performed by a type II CRISPR nickase, in particular a Cas9 nickase (nCas9). Such nickase has been modified as compared to the wild type protein to cut only one strand of a double stranded nucleic acid molecule. In a specific embodiment, the nCas9 is a nCas9 RuvC mutant and the guide is designed to bind to the non-edited strand. In another embodiment, the nCas9 is a nCas9 HNH mutant and the guide is designed to bind to the edited strand.

In a specific embodiment, the nCas9 RuvC mutant is the nCas9 D10A (SEQ ID NO: 90) and the nCas9 HNH mutant is preferably the nCas9 H840A (SEQ ID NO: 91).

Additionally, the nickase Cas9 protein can be associated with Nuclear Localization Signals (NLS) like the SV40 NLS (SEQ ID NO: 88) or the Xenopus Nucleoplasmin NLS (SEQ ID NO: 89). The NLS can be situated at one or both ends of the nickase Cas9 protein.

Associating the deaminase and the dead Cas protein

The deaminase can be associated with the dead Cas protein by various ways in the Cas base editor complex. It can be fused to a dead Cas. Such fusion can be a genetic fusion (the ORFs (open reading frames) of each of the proteins can be placed in frame to form a new ORF which codes for a polypeptide containing the amino acids of the two proteins (generally with spacer amino acids between them). The deaminase and the dead Cas can be associated with the deaminase in N-term and the dead Cas in C-term of the fusion or with the deaminase in C-term and the deadCas in N-term in the fusion.

The 16-residue XTEN linker, known in the art, can be used to link the deaminase and the dead Cas in the fusion protein.

Alternatively, the deaminase can be linked to the dead Cas protein using a chemical linker. Such linkers may comprise reactive moieties including such as aminoxy groups, azido groups, alkyne groups, thiol groups or maleimido groups, either alone or in combination. Generally, the linkers comprise two functional moieties, one providing rapid and efficient labeling and another enabling rapid and efficient coupling of the polypeptides, in particular through an amine group or preferably through the thiol group of the cysteine. Preferably, the complex is formed by first reacting one protein with the linker, and subsequently with the thiol group of the other protein.

The dead Cas can also be bound to the deaminase using binding domains, Protein-protein interaction domains, intein. In the protein-protein interaction domains embodiment, each of the dead Cas and of the deaminase are modified such as to contain a protein-protein interaction domain that are complementary to each other. When the two proteins are close to each other (which happens within the cell), the two domains bind to each other thereby bridging the dead Cas and the deaminase.

One can cite the dockerin/cohesin system described in You et al (2012). One can also use the system involving FK506 binding protein 12 (FKBP), and FKBP rapamycin binding (FRB) domain used to create a split Cas9 in Zetsche et al. (2015).

In a preferred embodiment, the deaminase and the dead Cas protein are associated in a fusion protein. In a specific embodiment, the fusion protein is Apobec1::dLbCas12a::UGI (SEQ ID NO:2), Apobec3::dl_bCas12a (SEQ ID NO: 4), dl_bCas12a::PmCDA (SEQ ID NO: 6).

Providinq proteins to the sequence of interest The proteins can be provided to the nucleotide sequence of interest by multiple ways. It is reminded that it is preferred when the proteins can reach the target nucleotide or its vicinity using the CRISPR system and in particular use of a first guide RNA and a Cas base editor complex and a second guide RNA and a nickase Cas, which ensures that the proteins reach the proper sequence of interest and act on the target nucleotide. As the proteins and guide RNAs are directed to the cell nucleus, the main question is to introduce the proteins and guide RNA within the cells. It can be done directly as RiboNucleoprotein (RNP) (the proteins and guide RNA(s) are pre-assembled and directly introduced within the cells) or indirectly (vectors are introduced within the cells and the proteins and guide RNAs are produced inside the cells).

One can deliver the proteins, nucleic acid (DNA, mRNA) coding for the proteins or for the guide RNAs using conjugation Cell Penetrating Peptides (CPP), nanoparticles, or biolistics.

It is preferred when the proteins and guide RNA are introduced within the cells, by the use of vectors, as transgenes, the proteins being produced by the cell machinery (after transcription and translation) and the guide RNAs being transcribed by the cell machinery. Such transgenes can be introduced within the genome of the cells (genomic integration) or present on extrachromosomal vectors (such as plasmids or artificial chromosomes).

The DNA constructs used in these methods are introduced in the genome of the cells by transgenesis, through any method known in the art. In particular, it is possible to cite methods of direct transfer of genes such as direct micro-injection into embryos or nuclei, vacuum infiltration or electroporation, direct precipitation by means of PEG or the bombardment by gun of particles (preferably gold particles) covered with the DNA of interest. When the cells are plant cells, it is preferred to transform them with a bacterial strain, using in particular Agrobacterium bacterial strains, and preferably Agrobacterium tumefaciens. One can also introduce the transgenes by protoplast transformation.

The sequence encoding the proteins and the gRNA(s) are under the control of adequate promoters. One can use, as an illustration, a constitutive promoter, a tissue-specific promoter (and in particular a promoter that is expressed in embryos, in pollen or in ovarian cells), or an inducible promoter. When working on plants, and although some promoters may have the same pattern of regulation when there are used in different species, it is often preferable to use monocotyledonous promoters in monocotyledons and dicotyledonous promoters in dicotyledonous plants.

Examples of constitutive promoters useful for expression include the 35S promoter or the 19S promoter (Kay et al., 1987, Science, 236 : 1299-1302), the rice actin promoter (McElroy et al., 1990, Plant Cell, 2 : 163-171), the pCRV promoter (Depigny-This et al., 1992, Plant Molecular Biology, 20 :467-479), the CsVMV promoter (Verdaguer et al., 1998, Plant Mol Biol. 6:1129-39), the ubiquitin 1 promoter of maize (Christensen et al., 1996, Transgenic. Res., 5 :213) and the ubiquitin promoter from rice or sugarcane.

Other promoters of the invention are the U3 promoter (P. patens U3 promoter SEQ ID NO:20) and the U6 promoter (P. patens U6 promoter SEQ ID NO: 21 ; ZmU6 promoter SEQ ID NO: 45, TaU6 promoter (SEQ ID NO: 69).

The Cas base editor, the nCas and the guides can be cloned in a single expression cassette in a single vector or in several cassettes in the same vector or in several cassettes in several vectors.

Screening for edition of the nucleotide

It is preferred when the cells are exposed to the deaminase and the nickase that they are cultured in conditions appropriate to allow chromosome replication and mitosis (the conditions are similar to that used for classical CRISPR-Cas sequence modification).

Screening can be performed by any method known in the art, in particular as performed for other methods of CRISPR-Cas sequence modification. One can, for instance, isolate the DNA from the part of the cultured cells and sequence the sequence of interest to verify that the target nucleotide has been edited (has been replaced by the expected nucleotide), and that no deletion or insertion of nucleotide is present. Alternatively, one can use probes appropriate to detect the nature of the nucleotide that is at the location of the nucleotide of interest.

By way of example, one can extract the DNA of a cell, of a tissue or an organism, amplify the nucleotide sequence of interest with specific primers by PCR and sequence the sequence of interest to detect the presence of the expected modification. The sequencing can be implemented using NEXT Generation Sequencing (NGS). One can also use the droplet digital PCR method (ddPCR™ BIO RAD) or the KASP (Biosearch Technologies) method based on detection of fluorescence. One can also use phenotypic screening, for example if the base editing creates a mutation allowing the cell to resist to a toxic component, the screening can be made on a medium comprising such toxic component.

In another embodiment, it is possible to use a plant sample from cultured cells to screen for the presence of the edited target nucleotide. If present, the cells can be cultured in vitro and regenerated to whole plants.

In another embodiment, if the edited target nucleotide creates a mutation allowing the plant cell to resist to a toxic component (such as an herbicide), the screening can be made on a medium comprising such toxic component. The plant cell can be regenerated to a whole plant.

The invention can be performed on any cell, in particular eukaryotic cell. In particular, it can be performed on plant cells, fungal cells or animal cells. It is particularly interesting to perform the method with mammalian cells, more particularly with human cells.

The method can be performed with plant cells. One can perform the method on mosses like P. patens. One can perform the method on monocotyledonous plant cells. It is also possible to perform the method on dicotyledonous plant cells. Among monocotyledonous plants, one can cite rice, wheat, barley, sorghum, maize or sugarcane. Among dicotyledonous plants, one can cite soybean, cotton, tomato, beet, sunflower, or rapeseed.

When the method is performed on plant cells, one can use the totipotency property of such plant cells, which makes it possible to regenerate a whole plant from a given cell (for instance after growing the cell and forming a callus from the cultured cells).

Thus, in a specific embodiment, the invention also relates to a method for obtaining a plant in the genome of which a target nucleotide of has been edited in a nucleotide sequence of interest, comprising the steps of: a. providing a plant cell or plant tissue comprising, in its genome, a sequence of interest containing said target nucleotide, b. providing to the target nucleotide in said plant cell or plant tissue a deaminase, that induces deamination of the target nucleotide, and a nickase that cuts the other strand of the sequence of interest c. culturing the plant cell or plant tissue in adequate conditions for multiplication of cells d. screening the cultured plant(s) cell or plant tissue from step c) to determine whether the target nucleotide has been edited e. growing a plant from the cultured plant cell(s) or plant tissuesif the screen performed in d. indicated that the target nucleotide has been edited.

Plant cells can be protoplast plant cells.

Plant tissues can be embryos, shoot apical meristem (SAM), plant parts like pollen, microspores, leaves or plant explants.

The screening of the cultured plant cell(s) or plant tissue can be performed by sampling a part of the cultured plant cells or plant tissues and screening to determine whether the target nucleotide has been edited. The screening can also be a phenotypic screening if the target nucleotide induces a phenotype. This phenotype can be a resistance to an herbicide, an antibiotic, a chemical.

In a preferred embodiment, the nick is introduced in the vicinity of the target nucleotide at the editing site. In a preferred embodiment, the nick is introduced not further than 300 bp from the target nucleotide at the editing site.

The invention also relates to a vector comprising a DNA construct containing a gene (or ORF) coding for a dead Cas protein fused to a deaminase. Preferably, the dead Cas protein is a dead type V Cas protein, in particular a dead Cas12a protein. The part coding for the dead Cas protein can be located 5’ of the gene and the part coding for the deaminase is then located 3’ of the gene. Alternatively, the part coding for the dead Cas protein can be located 3’ of the gene and the part coding for the deaminase is then located 5’ of the gene.

The DNA construct also contains a sequence that is transcribed as the guide RNA needed to direct the fusion protein to the target nucleotide.

The DNA construct also contains a gene coding for the nickase (in particular the mutated Cas9 protein) and, optionally but preferably, a sequence that is transcribed as the guide RNA needed to direct such nickase to the vicinity of the target nucleotide in the sequence of interest {i.e. within 300 bp of the target nucleotide).

The DNA construct can be such as all sequences are under the control of the same promoter (transcription as an operon). Alternatively, the DNA construct may contain multiple expression cassettes (an expression cassette being a DNA sequence that is to be transcribed (such as a sequence coding for a protein, the transcript being then translated, or a RNA guide), with appropriate regulation (promoter, enhancer, terminator) elements to allow the transcription), each sequence (deaminase, nickase, RNA guides) being under the control of its own regulation elements.

The invention also relates to a cell containing a DNA construct as comprised in the vector described above. In a specific embodiment, the DNA construct is present (integrated) in the genome of the cell. In another embodiment, the DNA construct is present on an extrachromosomal vector that is within the cell.

The invention also relates to an organism comprising at least one of such cell. Preferably, all cells of the organism contain the DNA construct. Preferably, the DNA construct is integrated in the genome of all cells of the organism. Preferably, the organism is homozygous for the DNA construct. In a specific example, the organism is a plant. In preferred embodiments, the plant is a moss, a wheat plant or a corn plant.

The invention also relates to the combined use of deaminase and of a nickase to improve base editing of a target nucleotide in a sequence of interest, wherein the deaminase induces deamination of the target nucleotide of one strand of the sequence of interest and the nickase introduces a strand cut in the other strand, in the vicinity of the target nucleotide. As indicated above, the vicinity (where the cut in the other strand is introduced) is no further than 300 bp from the target nucleotide.

Base editing is improved by increasing the frequency of expected editing. In a specific embodiment, base editing with a dead Cas Type V base editor and a nickase Cas Type II is improved compared to base editing with a dead Cas Type V base editor used alone. In a preferred embodiment, base editing with a dead Cas12a base editor and a nickase Cas9 is improved compared to base editing with a dead Cas12a base editor used alone.

The invention also relates to a kit to perform the methods herein disclosed, comprising a base editor complex, a nickase and RNA guides appropriate to direct the Cas Base editor and nickase to a target nucleotide in a sequence of interest.

In particular, one can consider a kit to perform the method comprising one or multiple vector wherein the one or multiple vector comprises sequences coding for a Cas Type V Base editor, a type II Cas nickase, a first RNA guide for directing the Cas Type V Base editor to a target nucleotide and a second RNA guide for directing the type II Cas nickase to a sequence that is within 300 bp of the target nucleotide.

In one embodiment, the kit contains one unique vector comprising all four sequences.

In another embodiment, the kit contains two vectors. Preferably, one vector comprises the Cas Type V Base editor and its guide and the other vector comprises the type II Cas nickase and its guide. It is also preferred when one vector comprises the Cas Type V Base editor and the type II Cas nickase and the the other vector comprises the two RNA guides.

In another embodiment, the kit contains three vectors.

In another embodiment, the kit contains four vectors, each of the vectors containing one of the sequences mentioned above.

FIGURES

Figure 1: Example of the use of the invention.

A. Use of a dead Cas12a (TypeV CRISPR) Base Editor alone. The desired edit is frequently repaired to recreate the existing sequence.. Consequently, repair to introduce edit is not favoured (large arrow).

In B. and C. Introduction of a nick in the non-edited strand in the vicinity of the edit increases the frequency of introduction of the desired edit.

B. The nick is introduced by a gRNA targeting the non-edited strand and a Cas9 nickase with a D10A (RuvC) mutation. The Cas9 gRNA binds to non-edited strand. Nick induced in vicinity of edit by nCas9 RuvC mutant on non-edited strand. Non-edited DNA strand cleaved favouring incorporation of edit (large arrow). C. The nick is introduced by a gRNA targeting the edited strand and a Cas9 nickase with a H840A (HNH) mutation. Cas9 gRNA binds to edited strand. Nick induced in vicinity of edit by nCas9 NHN mutant on non-edited strand. Non-edited DNA strand cleaved favouring incorporation of edit (large arrow).

The system of B) and C) will favour the introduction of the desired edit.

This method can be applied to prokaryotic and eukaryotic cells and organisms.

Figure 2: Base Editing experiments in APT in P. patens with crRNA#15. Each column of the table indicates a combination of plasmids (p) that are transformed into P. patens protoplasts. C= control transformation, no N = the Base-Editor is used without a nick. N = transformation designed to create an DNA nick adjacent to the Base-Editor target. N1 = nick using nCas9(D10A), N2 = nick using nCas9(H840A). * = distance of nick to base editing site C11.

Figure 3: Base Editing experiments in APT in P. patens with crRNA#20. Each column of the table indicates a combination of plasmids (p) that are transformed into P. patens protoplasts. C= control transformation, no N = the Base-Editor is used without a nick. N = transformation designed to create an DNA nick adjacent to the Base-Editor target. N1 = nick using nCas9(D10A), N2 = nick using nCas9(H840A). * = distance of nick to base editing site C9.

Figure 4: Base Editing experiments in Maize and Wheat protoplasts with BFPmm. Each column of the table indicates a combination of plasmids (p) that are transformed into protoplasts. M = transformation of Maize protoplasts. MW = transformation of Maize and Wheat protoplasts. C= control transformation, no N = the Base-Editor is used without a nick, N = transformation designed to create an DNA nick adjacent to the Base-Editor target. N1 = nick using nCas9(D10A), N2 = nick using nCas9(H840A).

Figure 5: Base Editing experiments of ZmPSYI in Maize protoplasts. Each column of the table indicates a combination of plasmids (p) that are transformed into protoplasts. No N = the Base-Editor is used without a nick. N = transformation designed to create an DNA nick adjacent to the Base-Editor target. N1 = nick using nCas9(D10A), N2 = nick using nCas9(H840A).

Figure 6: Base Editing experiments of TaACCase in Wheat protoplasts. Each column of the table indicates a combination of plasmids (p) that are transformed into protoplasts. No N = the Base-Editor is used without a nick. N = transformation designed to create an DNA nick adjacent to the Base-Editor target. N1 = nick using nCas9(D10A), N2 = nick using nCas9(H840A).

Figure 7: Base Editing outcomes for crRNA#15. 6 and 11 represent the possible targeted positions in the APT#15 target. CGT encodes R in position 54 in APT in wild-type moss and TAC encodes Y in position 55 in APT in wild-type moss. The left column represents the different possible modifications at position 6 and 11 in the target. The two columns on the right represent the possible amino-acid modifications in the APT protein in position 54 and 55. The star represents a STOP codon. In black, the preferred outcomes to obtain 2-FA resistance. 2FA^R signifies resistance to 2-FA chemical.

Figure 8: Base Editing outcomes for crRNA#20. 9 and 10 represent the targeted positions in the APT#20 target. CCA encodes P in position 75 in APT in wild-type moss. The two columns on the left represents the different possible modifications/combinations of modifications at position 9 and 10 in the target. The column on the right represents the possible amino-acid modifications in the APT protein in position 75. 2FA^R signifies resistance to 2-FA chemical.

EXAMPLES

Example 1: Cas12a base-editors

In order to test Cas12a base editing and improvements based on the creation of DNA nicks on the non-base edited DNA strand adjacent to the targeted base edit (Figure 1) three dead Lachnospiraceae bacterium (Lb) Cas12a -cytidine- deaminase fusions were constructed.

The first, termed Apobec1::dLbCas12a::UGI (SEC ID NO: 1-2), is a maize-codon optimized version of dCpf1-BE (Li et al. 2018). This has the following elements; N and C-terminal SV40 nuclear localization sequences (NLS) plus an internal SV40 NLS, a human Apobed cytidine deaminase domain, an XTEN linker, LbCas12a with three amino acid changes (D832A, E1006A, D1125A) to prevent nuclease activity (dLbCas12a) and uracil DNA glycosylase inhibitor (UGI) to reduce Base Excision repair (BER) and thus improve the predictability and frequency of editing. (SV40NLS-Apobec1-XTEN-dLbCpf1(D832A/E1006A/D1125A)-SV40NLSSGGS- UGI-SV40NLS).

The second, termed Apobec3::dLbCas12a (SEC ID NO: 3-4), is maize codon- optimized and has the human Apobec3 cytidine deaminase domain, LbCas12a with the D832A mutation preventing nuclease activity plus the D156R mutation that improves LbCas12a action (Schindele and Puchta (2020)). This version has a Xenopus Nucleoplasmin NLS after LbCas12a, an SV40 NLS at the C-terminus and lacks UGI.

(Apobec3_XTEN_syndl_bCpf1_D156R_D832A_XINucleoplasmin_NLS_HA_tag_S

V40NLS)

The third version, termed dl_bCas12a::PmCDA (SEQ ID NO: 5-6), is maize optimized and has the PmCDA cytidine deaminase domain at the C-terminus. LbCas12a has the D156R and D832A mutations (dl_bCas12a). This version also has a Xenopus Laevis Nucleoplasmin NLS after LbCas12a, an SV40 NLS at the C- terminus and lacks UGI.

(syndLbCpf1_D156R_D832A_XINucleoplasmin_NLS_HA_tag_SV40NLS_SH3_3x

Flag_PmCDA_Sv40NLS)

These three versions were used in the experiments described in the examples below. They are referred as dLbCas12a-BE.

Example 2: Improved Cas12a base-editing in the moss Physcomitrium patens IP. patens)

Disruption of the P. patens adenine phosphoribosyltransferase (APT) (SEQ ID NO: 9 encoding SEQ ID NO: 10) gene function leads to resistance of P. patens protoplasts to the chemical 2-Fluoroadenine (2-FA) which is present at 10uM in the media, since APT active metabolises 2-FA to the cytotoxic 2-FluoroAMP. This 2-FA resistance has been used as a powerful screen to identify APT mutations since only loss of function in APT leads to development of plants from the protoplasts (Trouiller et al. , (2006)). This positive selection screen can be used for optimizing GE tools and was adapted in this example to optimize Cas12a cytosine deaminase base editing.

Three different dLbCas12a-cytidine deaminase base editors; Apobec1::dLbCas12a::UGI (SEQ ID NO: 1), Apobec3::dLbCas12a (SEQ ID NO: 3) and dLbCas12a::PmCDA (SEQ ID NO: 5) were cloned in between the maize ubiquitin promoter and first intron (SEQ ID NO: 7) and an AtNos polyadenylation sequence (SEQ ID NO: 8) forming the plasmids pBIOS12007, pBIOS12998 and pBIOS12997 respectively. The LbCas12a contains amino acid changes preventing nuclease activity, creating a dead or dLbCas12a sequence. The dl_bCas12a-BE plasmids were each transformed according to Trouiller et al. 2006 into P. patens protoplasts with a crRNA specific to APT expressed from a Rice Actin promoter SEQ ID NO: 11 or ZmUbiquitin promoter SEQ ID NO: 7, such that the dl_bCas12a-BE is positioned for base-editing of APT. The LbCas12a crRNA was cloned between hammerhead and HDV ribozymes such that the crRNA is liberated by ribozyme cleavage from the transcript. Two crRNA constructs were made. pAct-crRNA-APT#15 SEQ ID NO: 13 (target sequence in P. patens APT SEQ ID NO: 12) can disrupt APT function upon base editing by amino acid changes at position C6 (modification of C to T (Arginine (R) in position 54 is modified into Cysteine (C) in the APT protein) or C to G (Arginine (R) in position 54 is modified into Glycine (G) in the APT protein)) or via the introduction of a stop codon at position C11 (modification of C to G or C to A) (Figure 7). pZmUbi-crRNA- APT#20 SEQ ID NO: 15 (target sequence in P. patens APT SEQ ID NO: 14) can disrupt APT function upon base editing by amino acid changes at position P75 (Figure 8). According to the modifications or combinations of modifications in position C9 and C10, the Proline in position 75 can be modified into Leucine (L), Arginine (R) or Isoleucine (I).

Some transformations in addition comprised a plasmid encoding a nickase Cas9 associated with NLS (either nCas9 (D10A) (SEQ ID NO: 16 encoding SEQ ID NO: 17) or nCas9 (H840A) (SEQ ID NO: 18 encoding SEQ ID NO: 19)) expressed from the maize Ubiquitin promoter (SEQ ID NO: 7) (plasmids pBIOS12870 and pBIOS12871 respectively) and a Cas9 gRNA designed so that nCas9 introduces a DNA nick on the non-base edited DNA strand in the vicinity of the target site for the dLbCas12a-BE. These Cas9gRNA (SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, respectively targeting the targets in the P. patens APT gene SEQ ID NO: 22 (APT#2), 24 (APT#22), 26 (APT#9), 28 (APT#21), 30 (APT#23), 32 (APT#5), 34 (APT#27)) were either expressed from a P. patens U3 promoter SEQ ID NO: 20 or from a P. patens U6 promoter (SEQ ID NO: 21)

The transformation experiments are outlined in Figure 2 (crRNA#15) and Figure 3 (crRNA#20). The distances of nCas9-induced DNA nicks from the base editing sites are indicated in Figures 2 and 3. The number of plants developing on 2-FA containing media was recorded and is a measure of dLbCas12a base editing levels. Example 3: BFP system fortesting improved Cas12a base-editing in plants

A mutation changing Tyrosine (Tyr) 67 to a Histidine (His) in GFP protein changes the fluorescence spectrum of GFP such that it moves from green to blue forming a Blue Fluorescent protein (BFP). Zong et al., (2017) made A to G base change in a BFP gene at 218bp (altering Serine 73 to Glycine in the BFP protein) creating a Cas9 NGG PAM site and forming BFPm. This added Cas9 PAM allows the positioning of a gRNA in the BFPm sequence permitting an nCas9-cytidine deaminase Base editor to revert the His CAC codon to the Tyr TAC codon, hence reverting BFP to GFP. This BFPm gene was used to optimize nCas9-BE performance in rice and wheat protoplasts (Zong et al., (2017)).

A BFPm gene was further modified by the change of sequence CG at 183- 184bp to TT to form a Cas12a PAM (TTTV) positioned so as to allow editing of His67 to Tyr67 by a Cas12a Cytidine deaminase base editor. The change of sequence CG at 183-184bp to TT to create the Cas12a PAM in the BFPmm gene also causes a change of Valine 62 to Leucine in the BFPmm protein. This remodified BFPmm (BFPmm) SEQ ID NO: 36 (encoding SEQ ID NO:37) can thus be edited by Cas9 or Cas12a cytidine base editors to restore green fluorescence by targeting the amino acid at position 67.

As a control to ensure that the amino acid change V62L does not affect fluorescence a version of BFPmm but with the His67 restored to Tyr67 (ie the desired based editing event) was synthesized (GFPmm SEQ ID NO: 38 encoding SEQ ID NO: 39). Both BFPmm and GFPmm were linked to the strong constitutive Maize ubiquitin promoter (SEQ ID NO: 7) and transformed into maize and wheat protoplasts using a standard PEG-method (Wolter et al. 2017). Only GFPmm- transformed protoplasts exhibited green fluorescence.

As a further control base editing with two nCas9-cytidine deaminase BEs was tested. Each nCas9-BE (nCas9-CDA SEQ ID NO: 40 and Apobed- nCas9_UGI SEQ ID NO: 42) was cloned between the maize ubiquitin promoter SEQ ID NO: 7 and nos polyadenylation sequence SEQ ID NO: 8. The Cas9 BE gRNA (target in BFPmm SEQ ID NO: 44) was cloned behind a maize U6 promoter SEQ ID NO: 45 forming SEQ ID NO: 46. ZmUbi-BFPmm plasmid was then transformed into maize protoplasts with a ZmUbi-Cas9-BE plasmid and the ZmU6 Cas9-BE gRNA plasmid. As a negative control nCas9(D10A) SEQ ID NO: 16 was transformed with Zmllbi-BFPmm and Zmll6 Cas9-BE gRNA. Green fluorescent protoplasts were observed only in transformations with an nCas9-BE.

A LbCas12a cRNA construct, designed to target the H67Y change in BFPmm, was cloned between hammerhead and HDV ribozymes forming SEQ ID NO: 48 targeting SEQ ID NO: 47 and thus cloned between the maize ubiquitin promoter (SEQ ID NO: 7) and nos polyadenylation sequence (SEQ ID NO: 8) forming plasmid pBIOS12786. Cas12a-cytosine deaminase BE plasmids pBIOS12998 or pBIOS12997 were transformed with pBIOS12786 and the ZmUbi-BFPmm construct into maize and wheat protoplasts (Figure 4). In addition, transformations were performed where the LbCas12a_BE BFP_crRNA-BE and ZmUbi-BFPmm were transformed with the ZmUbi-nCas9(H840A) plasmid pBIOS12871 and a Cas9 gRNA plasmid designed to nick the BFPmm DNA adjacent to the H67 target site and on the DNA strand that is not base-edited (Figure 4). The Cas9 gRNAs BFP_SpCas9_gRNA_RZ_R2 (target in BFPmm SEQ ID NO: 49) and BFP_SpCas9_gRNA_RZ_R3 (target in BFPmm SEQ ID NO: 51 ) were cloned between hammerhead and HDV ribozymes forming SEQ ID NO: 50 and SEQ ID NO: 52 respectively. These sequences were cloned between the maize ubiquitin promoter (SEQ ID NO: 7) and nos polyadenylation sequence (SEQ ID NO: 8) forming plasmids pBIOS12891 and pBIOS12892. nCas9(H840A) SEQ ID NO: 18 and BFP_SpCas9_gRNA_RZ_R2 should give a DNA nick at +67bp from the base to be edited and BFP_SpCas9_gRNA_RZ_R2 -63bp.

The proportion of green-fluorescent protoplasts was determined after 24h to 36h after transformations.

Example 4: Improved Cas12a base-editing in maize

LbCas12a-BEs with and without adjacent nCas9-induced DNA non-edited strand nicks are tested in maize by targeting the maize Phytoene Synthase (PSY1) gene ((SEQ ID NO: 53). A region of ZmPSYI for base editing was selected in the intron 2 upstream of Exon 3 (SEQ ID NO: 54 A188 line and SEQ ID NO: 55 BMS line). The two targets for base editing have an identical sequence in the maize variety A188 and in the maize Black Mexican Sweet (BMS) cell suspension.

An LbCas12a cRNA (PSY1_LbCpf1_v9_gRNA_73r) designed to direct base editing to the first target (first target in ZmPSYI SEQ ID NO: 56)) was cloned behind a Maize U6 promoter SEQ ID NO: 45 forming SEQ ID NO: 57 in plasmid cdsBGA_12074. An LbCas12a cRNA (PSY1_LbCpf1_v9_gRNA_149f) designed to direct base editing to the second target (second target in ZmPSYI SEQ ID NO: 58)) was cloned behind a Maize U6 promoter SEQ ID NO: 45 forming SEQ ID NO: 59 in plasmid cdsBGA_12075. A Cas9 gRNA, PSY1_SpCas9_gRNA_nCas9_F1 (target in ZmPSYI SEQ ID NO: 60) was cloned behind the maize U6 promoter forming SEQ ID NO: 61 and plasmid geBGA_12416. This gRNA is designed, in conjunction with nCas9(D10A), to create a DNA nick on the non-base-edited DNA strand at -158bp from the first editing site (73r) and at -63bp to the second site (149f) . Similarly, a second Cas9 gRNA, (target in ZmPSYI SEQ ID NO: 62) was cloned behind the maize U6 promoter forming SEQ ID NO: 63 and plasmid geBGA_12417. This gRNA is designed, in conjunction with nCas9(H840A), to create a DNA nick on the non-base-edited DNA strand at -55bp from the first editing site (73r) and at +44bp to the second site (149f). Combinations of LbCas12a-BE, nCas9 and guides as shown in Figure 5 are transformed into maize protoplasts (var. A188) according to Wolter et al. 2017 and DNA extracted 24h to 36h after transformations.

The ZmPSYI target sites in ZmPSYI are amplified from the extracted DNA using primers PP-03452F (SEQ ID NO: 64) and PP-03452R (SEQ ID NO: 65). Amplicons are sequenced using Next Generation Sequencing (NGS) technology. The number of sequences with the desired C to T edits is assessed in each sample.

The combinations of Cas12a-BE, nCas9 and guides in Figure 5 are also bombarded into BMS cells in combination with a plasmid encoding the Bar gene under the control of a rice Actin promoter (SEQ ID NO: 11) for selection of transformation events. BASTA-resistant calli are harvested and DNA extracted. The ZmPSYI target sites in ZmPSYI are amplified from the extracted DNA using primers PP-03452F (SEQ ID NO: 64) and PP-03452R (SEQ ID NO: 65). Amplicons are sequenced using Next Generation Sequencing (NGS) technology. The number of sequences with the desired C to T edits is assessed in each sample.

The Cas12a-BE, nCas9 and guides are combined by cloning in combinations used for testing in protoplasts and BMS cells (Figure 5) into plant binary vectors for agrobacterial-mediated transformation of the maize variety A188. The plant binary vector contains a Bar gene under the control of a rice Actin promoter for selection of transformation events. Transformation is via a standard technique based on Ishida et al (1996). DNA from leaves of transformants and progeny is amplified using primers PP-03452F (SEQ ID NO: 64) and PP-03452R (SEQ ID NO: 65) and analysed by NGS for the desired C to T edits.

Example 5: Improved Cas12a base-editing in wheat

LbCas12a-BEs with and without adjacent nCas9-induced DNA non-edited strand nicks are tested in wheat by targeting the wheat acetyl-CoA carboxylase (ACCase) gene. A mutation at amino acid 2004 changing Alanine (Ala) to Valine (Val) gives resistance to the herbicide quizalofop (Ostlie et al. (2015). The sequences of the TaACCase targeted exon in genomes A, B and D of wheat variety Fielder are SEQ ID NO: 66-67-68 respectively.

A LbCas12a cRNA, ACCase_LbCpf1_crRNA_A_to_V (target in TaACCase SEQ ID NO: 70) was cloned behind a Wheat U6 promoter SEQ ID NO: 69 forming SEQ ID NO: 71 in plasmid cdsBGA_12019. A Lb Cas12a cRNA, ACCase_LbCpf1_v9_crRNA_RZ_BE Target was also cloned between hammerhead and HDV ribozymes forming SEQ ID NO: 73 (target in TaACCase SEQ ID NO: 72) and this cloned between the maize ubiquitin promoter (SEQ ID NO: 7) and nos polyadenylation sequence (SEQ ID NO: 8) forming plasmid pBIOS12785. A Cas9 gRNA, ACCase_SpCas9_gRNA_nCas9_F1 (target in TaACCase SEQ ID NO: 74) was cloned behind the wheat U6 promoter forming SEQ ID NO: 75 and plasmid geBGA_12415. This gRNA is designed, in conjunction with nCas9(D10A), to create a DNA nick on the non-base-edited DNA strand at - 58bp from the editing site. A Cas9 gRNA, ACCase_SpCas9_gRNA_nCas9_R1 (target in TaACCase SEQ ID NO: 76) was also cloned behind the wheat U6 promoter forming SEQ ID NO: 77 and plasmid geBGA_12414. This gRNA is designed, in conjunction with nCas9(H840A), to create a DNA nick on the non- base-edited DNA strand at +53bp from the editing site . Combinations of LbCas12a-BE, nCas9 and guides as shown in Figure 6 are transformed into wheat protoplasts (var. Fielder) according to Wolter et al. 2017 and DNA extracted 24h to 36h after transformations.

The Ala-2004-Val target site in TaACCase is amplified from the extracted DNA using primers TaACCase_forw (SEQ ID NO: 78) and TaACCase_rev (SEQ ID NO: 79). Amplicons are sequenced using Next Generation Sequencing (NGS) technology. The number of sequences with the desired C to T (Ala-2004-Val) edit is assessed in each sample. The Cas12a-BE, nCas9 and guides are combined by cloning in combinations used for testing in protoplasts (Figure 6) into plant binary vectors for agrobacterial-mediated transformation of the wheat variety Fielder. The plant binary vector contains a Bar gene under the control of a rice Actin promoter for selection of transformation events. Transformation is via a standard technique based on Ishida et al (2015). DNA from leaves of transformants and progeny is amplified using primers TaACCase_forw (SEQ ID NO: 78) and TaACCase_rev (SEQ ID NO: 79) and analysed by NGS for the desired C to T (Ala-2004-Val) edit. Plants are also sprayed with quizalofop to identify herbicide resistant plants.

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Claims

1. A method to modify a target nucleotide into a double-stranded nucleotide sequence of interest in a cell comprising: d) providing a nucleotide deaminase to the sequence targeted to be deaminated, in conditions so as to induce deamination of the target nucleotide of the target nucleotide sequence on a first strand of the double- stranded nucleotide sequence, e) providing a nickase endonuclease, in conditions so as to introduce a nick (single strand cut) to the second strand of the double-stranded nucleotide sequence within 300 base pairs (bp) 5’ or 3’ of the target nucleotide, f) culturing the cell in adequate conditions so as to allow the modification of the target nucleotide in the double-stranded target sequence.

2. The method of claim 1, wherein the deaminase is part of a Cas Type V base editor complex comprising a dead Type V Cas protein associated with the deaminase and wherein the deaminase is targeted to the target sequence by a first guide RNA.

3. The method of claim 2, wherein the dead Type V Cas protein is the dead

Cas12a protein.

4. The method of any one of claims 1 to 3, wherein the nick is introduced by a modified Cas protein targeted within 300 base pairs (bp) 5’ or 3’ of the target nucleotide by a second guide RNA.

5. The method of any one of claims 1 to 4, wherein the nick is introduced within 150 bases 5’ or 3’ of the edited target nucleotide.

6. The method of any one of claims 1 to 5, comprising the step of verifying/screening/identifying if the cells have the expected modification

7. The method of any one of claims 4 to 6, wherein the modified Cas protein is a modified type II CRISPR nickase, in particular a nickase Cas9 (nCas9).

8. The method of claim 7, wherein the nCas9 is a nCas9 RuvC mutant and the guide is designed to bind to the second strand of the nucleotide sequence of interest.

9. The method of claim 7, wherein the nCas9 is a nCas9 HNH mutant and the guide is designed to bind to the first strand of the nucleotide sequence of interest.

10. The method of any one of claims 1 to 9, wherein the cell is a plant cell.

11. The method of claim 10, further comprising a regeneration step of a whole edited plant.

12. A vector comprising a DNA construct comprising a Cas Type V Base editor, a first RNA guide to direct the Cas Type V Base editor to a target nucleotide in an organism genome, a type II Cas nickase and a second RNA guide to direct the type II Cas nickase to a nucleotide sequence that is located within 300 bp of the target nucleotide in the organism genome.

13. A cell comprising the DNA construct defined in claim 12, preferably stably integrated within its genome.

14. The cell of claim 13, which is a plant cell.

15. An organism comprising at least one cell of claim 13.

16. The organism of claim 15, wherein all cells of the organism comprise the DNA construct defined claim 12.

17. The organism of claim 15 or 16, which is a plant.

18. Use of a deaminase associated with a type V dead Cas endonuclease and of a type II Cas nickase to improve base editing of a genome by introducing a nucleotide modification in the genome.

19. A method for obtaining a plant in the genome of which a target nucleotide of has been edited in a nucleotide sequence of interest, comprising the steps of: a. providing a plant cell or plant tissue comprising, in its genome, a sequence of interest containing said target nucleotide, b. providing to the target nucleotide in said plant cell or plant tissue a deaminase, that induces deamination of the target nucleotide, and a nickase that cuts the other strand of the sequence of interest c. culturing the plant cell or plant tissue in adequate conditions for multiplication of cells d. screening the cultured plant(s) cell or plant tissue from step c) to determine whether the target nucleotide has been edited e. growing a plant from the cultured plant cell(s) or plant tissuesif the screen performed in d. indicated that the target nucleotide has been edited.

20. A kit to perform the method comprising one or multiple vector wherein the one or multiple vector comprises sequence coding for a Cas Type V Base editor, a type II Cas nickase, a first RNA guide for directing the Cas Type V Base editor to a target nucleotide and a second RNA guide for directing the type II Cas nickase to a sequence that is within 300 bp of the target nucleotide.