WO2021123397A1 - Amélioration de l'efficacité de l'édition de base à l'aide d'enzymes crispr de type v - Google Patents

Amélioration de l'efficacité de l'édition de base à l'aide d'enzymes crispr de type v Download PDF

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WO2021123397A1
WO2021123397A1 PCT/EP2020/087344 EP2020087344W WO2021123397A1 WO 2021123397 A1 WO2021123397 A1 WO 2021123397A1 EP 2020087344 W EP2020087344 W EP 2020087344W WO 2021123397 A1 WO2021123397 A1 WO 2021123397A1
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cas
sequence
plant
type
nucleotide
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Wyatt Paul
Fabien NOGUÉ
Anouchka GUYON
Pierre-François PERROUD
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Biogemma
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    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • 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.
  • 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 (2016), Rees and Liu (2016)).
  • 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).
  • Cas9-BE type II CRISPR enzyme Cas9
  • gRNA guide 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).
  • Cas9 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.
  • a targeting system that binds the BE in close proximity to the targeted base.
  • 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 (2016).
  • Type V CRISPR enzymes offer alternative PAMs and thus increase the number and variety of sequences that can be targeted by a base editor.
  • 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.
  • nCas9 a RuvC or an HNH mutant
  • gRNA guide RNA
  • Figure 1B 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.
  • the editing window corresponds to the nucleotide region of reach of the deaminase after the dead Cas protein has bound the sequence of interest.
  • 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 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.
  • the deaminase is part of a Cas Type V base editor complex comprising a dead Type V Cas protein and the deaminase.
  • a 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.
  • gRNA guide RNA
  • 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.
  • 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.
  • Type V Cas protein is 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).
  • FnCas12a Francisella novicida
  • LbCas12a Lachnospiraceae bacterium
  • AsCas12a Acidaminococcus sp.
  • the deaminase is associated with a dead Type II Cas protein.
  • 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.
  • 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.
  • Adenine deaminases used in base editors can be TadA (Gaudelli. 2017).
  • 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.
  • 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..
  • the dead Cas12a can further comprises a D156R mutation that improves Cas12a action (Schindele and Puchta (2020).
  • 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).
  • NLS Nuclear Localization Signals
  • 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.
  • UMI uracil DNA glycosylase inhibitor
  • a dead type II Cas protein 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.
  • 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.
  • 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.
  • the sequencing can be implemented using NEXT Generation Sequencing (NGS).
  • NGS NEXT Generation Sequencing
  • ddPCRTM BIO RAD droplet digital PCR method
  • KASP method Biosearch Technologies
  • 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.
  • the nick is performed by a type II CRISPR nickase, in particular a Cas9 nickase (nCas9).
  • a Cas9 nickase nickase
  • Such nickase has been modified as compared to the wild type protein to cut only one strand of a double stranded nucleic acid molecule.
  • the nCas9 is a nCas9 RuvC mutant and the guide is designed to bind to the non-edited strand.
  • the nCas9 is a nCas9 HNH mutant and the guide is designed to bind to the edited strand.
  • 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).
  • 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).
  • NLS Nuclear Localization Signals
  • the NLS can be situated at one or both ends of the nickase Cas9 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.
  • the deaminase can be linked to the dead Cas protein using a chemical linker.
  • linkers may comprise reactive moieties including such as aminoxy groups, azido groups, alkyne groups, thiol groups or maleimido groups, either alone or in combination.
  • 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.
  • 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.
  • 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.
  • FKBP FK506 binding protein 12
  • FKBP rapamycin binding domain used to create a split Cas9 in Zetsche et al. (2015).
  • the deaminase and the dead Cas protein are associated in a fusion protein.
  • 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).
  • 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.
  • RNP RiboNucleoprotein
  • CPP Cell Penetrating Peptides
  • the DNA constructs used in these methods are introduced in the genome of the cells by transgenesis, through any method known in the art.
  • 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.
  • 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.
  • the sequence encoding the proteins and the gRNA(s) are under the control of adequate promoters.
  • 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.
  • tissue-specific promoter and in particular a promoter that is expressed in embryos, in pollen or in ovarian cells
  • 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.
  • 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.
  • 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.
  • 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.
  • probes appropriate to detect the nature of the nucleotide that is at the location of the nucleotide of interest.
  • the sequencing can be implemented using NEXT Generation Sequencing (NGS).
  • NGS NEXT Generation Sequencing
  • ddPCRTM BIO RAD droplet digital PCR method
  • KASP Biosearch Technologies
  • a plant sample from cultured cells 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.
  • 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.
  • 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.
  • monocotyledonous plants one can cite rice, wheat, barley, sorghum, maize or sugarcane.
  • dicotyledonous plants one can cite soybean, cotton, tomato, beet, sunflower, or rapeseed.
  • the method 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).
  • 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.
  • step c) 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.
  • SAM shoot apical meristem
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the DNA construct is present (integrated) in the genome of the cell.
  • 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.
  • all cells of the organism contain the DNA construct.
  • the DNA construct is integrated in the genome of all cells of the organism.
  • the organism is homozygous for the DNA construct.
  • the organism is a plant.
  • 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.
  • the vicinity is no further than 300 bp from the target nucleotide.
  • Base editing is improved by increasing the frequency of expected editing.
  • 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.
  • 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.
  • the kit contains four vectors, each of the vectors containing one of the sequences mentioned above.
  • Figure 1 Example of the use of the invention.
  • 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.
  • Non-edited DNA strand cleaved favouring incorporation of edit (large arrow).
  • 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.
  • Non-edited DNA strand cleaved favouring incorporation of edit (large arrow).
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • Apobec1::dLbCas12a::UGI 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the Proline in position 75 can be modified into Leucine (L), Arginine (R) or Isoleucine (I).
  • 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)
  • 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).
  • BFP Blue Fluorescent protein
  • 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)).
  • nCas9-cytidine deaminase BEs were 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
  • ZmUbi-BFPmm plasmid was then transformed into maize protoplasts with a ZmUbi-Cas9-BE plasmid and the ZmU6 Cas9-BE gRNA plasmid.
  • 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.
  • Cas12a-cytosine deaminase BE plasmids pBIOS12998 or pBIOS12997 were transformed with pBIOS12786 and the ZmUbi-BFPmm construct into maize and wheat protoplasts ( Figure 4).
  • 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.
  • 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).
  • PSY1 maize Phytoene Synthase
  • 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.
  • 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.
  • 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) .
  • 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.
  • NGS Next Generation Sequencing
  • 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.
  • NGS Next Generation Sequencing
  • 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.
  • 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.
  • ACCase wheat acetyl-CoA carboxylase
  • 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 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 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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Abstract

L'invention concerne le domaine de la modification du génome et concerne un procédé pour améliorer l'efficacité de l'édition de base par découpe d'un simple brin d'une séquence nucléotidique à proximité de l'emplacement de la base éditée.
PCT/EP2020/087344 2019-12-20 2020-12-18 Amélioration de l'efficacité de l'édition de base à l'aide d'enzymes crispr de type v WO2021123397A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022171736A1 (fr) 2021-02-10 2022-08-18 Limagrain Europe Recombinaisons ciblées multiplex pour des applications d'introgression de caractères
WO2023199198A1 (fr) * 2022-04-12 2023-10-19 John Innes Centre Compositions et procédés pour augmenter l'efficacité d'édition du génome
WO2024096790A1 (fr) * 2022-10-31 2024-05-10 Soledits Ab Système crispr modulaire

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018202199A1 (fr) * 2017-05-05 2018-11-08 Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences Procédés pour isoler des cellules sans utiliser de séquences de marqueurs transgéniques
WO2018213708A1 (fr) * 2017-05-18 2018-11-22 The Broad Institute, Inc. Systèmes, procédés et compositions d'édition ciblée d'acides nucléiques
WO2018213726A1 (fr) * 2017-05-18 2018-11-22 The Broad Institute, Inc. Systèmes, procédés et compositions d'édition ciblée d'acides nucléiques
WO2019126762A2 (fr) * 2017-12-22 2019-06-27 The Broad Institute, Inc. Systèmes cas12a, procédés et compositions d'édition ciblée de bases d'arn
WO2019178427A1 (fr) * 2018-03-14 2019-09-19 Arbor Biotechnologies, Inc. Nouveaux systèmes et enzymes de ciblage d'adn crispr

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018202199A1 (fr) * 2017-05-05 2018-11-08 Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences Procédés pour isoler des cellules sans utiliser de séquences de marqueurs transgéniques
WO2018213708A1 (fr) * 2017-05-18 2018-11-22 The Broad Institute, Inc. Systèmes, procédés et compositions d'édition ciblée d'acides nucléiques
WO2018213726A1 (fr) * 2017-05-18 2018-11-22 The Broad Institute, Inc. Systèmes, procédés et compositions d'édition ciblée d'acides nucléiques
WO2019126762A2 (fr) * 2017-12-22 2019-06-27 The Broad Institute, Inc. Systèmes cas12a, procédés et compositions d'édition ciblée de bases d'arn
WO2019178427A1 (fr) * 2018-03-14 2019-09-19 Arbor Biotechnologies, Inc. Nouveaux systèmes et enzymes de ciblage d'adn crispr

Non-Patent Citations (27)

* Cited by examiner, † Cited by third party
Title
ANZALONE ANDREW V ET AL: "Search-and-replace genome editing without double-strand breaks or donor DNA", NATURE, MACMILLAN JOURNALS LTD., ETC, LONDON, vol. 576, no. 7785, 21 October 2019 (2019-10-21), pages 149 - 157, XP036953141, ISSN: 0028-0836, [retrieved on 20191021], DOI: 10.1038/S41586-019-1711-4 *
AYMAN EID ET AL: "CRISPR base editors: genome editing without double-stranded breaks", BIOCHEMICAL JOURNAL, vol. 475, no. 11, 11 June 2018 (2018-06-11), GB, pages 1955 - 1964, XP055638645, ISSN: 0264-6021, DOI: 10.1042/BCJ20170793 *
CHRISTENSEN ET AL., TRANSGENIC. RES., vol. 5, 1996, pages 213
DEPIGNY-THIS ET AL., PLANT MOLECULAR BIOLOGY, vol. 20, 1992, pages 467 - 479
FUQIANG CHEN ET AL: "Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting", NATURE COMMUNICATIONS, vol. 8, no. 14958, 7 April 2017 (2017-04-07), pages 1 - 12, XP055548732, DOI: 10.1038/ncomms14958 *
GAUDELLI ET AL.: "Programmable base editing of A-T to G-C in genomic DNA without DNA cleavage", NATURE, vol. 551, no. 7681, 23 November 2017 (2017-11-23), pages 464 - 471, XP037203026, DOI: 10.1038/nature24644
ISHIDA ET AL., NAT. BIOTECHNOL., vol. 14, 1996, pages 745 - 750
ISHIDA ET AL.: "High Efficiency Wheat Transformation Mediated by Agrobacterium tumefaciens, Advances in Wheat Genetics, from Genome to Field", PROCEEDINGS OF THE 12TH INTERNATIONAL WHEAT GENETICS SYMPOSIUM, 2015
K. NISHIDA ET AL: "Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems", SCIENCE, vol. 353, 4 August 2016 (2016-08-04), US, pages 1 - 14, XP055367833, ISSN: 0036-8075, DOI: 10.1126/science.aaf8729 *
KAY ET AL., SCIENCE, vol. 236, 1987, pages 1299 - 1302
KIM JS: "Precision genome engineering through adenine and cytosine base editing", NAT PLANTS, vol. 4, 2018, pages 148 - 151, XP036442899, DOI: 10.1038/s41477-018-0115-z
KLEINSTIVER ET AL.: "Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing", NAT BIOTECHNOL, vol. 37, 2019, pages 276 - 282, XP036900601, DOI: 10.1038/s41587-018-0011-0
KOMOR ET AL.: "Programmable editing of a target base in genomic DNA without double- stranded DNA cleavage", NATURE, vol. 533, 2016, pages 420 - 424, XP055551781, DOI: 10.1038/nature17946
LI ET AL.: "Base editing with a Cpf1-cytidine deaminase fusion", NAT BIOTECHNOL, vol. 36, 2018, pages 324 - 327, XP055579743, DOI: 10.1038/nbt.4102
MCELROY ET AL., PLANT CELL, vol. 2, 1990, pages 163 - 171
MING ET AL.: "CRISPR-Cas12b enables efficient plant genome engineering", NAT PLANTS, vol. 6, no. 3, March 2020 (2020-03-01), pages 202 - 208, XP037062240, DOI: 10.1038/s41477-020-0614-6
OSTLIE ET AL.: "Development and Characterization of Mutant Winter Wheat (Triticum Aestivum L.) Accessions Resistant to the Herbicide Quizalofop", THEOR APPL GENET, vol. 128, no. 2, 2015, pages 343 - 51, XP035434275, DOI: 10.1007/s00122-014-2434-4
REES HOLLY A ET AL: "Base editing: precision chemistry on the genome and transcriptome of living cells", NATURE REVIEWS GENETICS, NATURE PUBLISHING GROUP, GB, vol. 19, no. 12, 15 October 2018 (2018-10-15), pages 770 - 788, XP036637435, ISSN: 1471-0056, [retrieved on 20181015], DOI: 10.1038/S41576-018-0059-1 *
REESLIU: "Base editing: precision chemistry on the genome and transcriptome of living cells", NAT REV GENET, vol. 19, 2018, pages 770 - 788
SCHINDELE PPUCHTA H: "Engineering CRISPR/LbCas12a for highly efficient, temperature-tolerant plant gene editing", PLANT BIOTECHNOL J, vol. 18, 2020, pages 1118 - 1120
SHMAKOV ET AL.: "Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems", MOL CELL, vol. 60, no. 3, 2015, pages 385 - 97, XP055482679, DOI: 10.1016/j.molcel.2015.10.008
TROUILLER ET AL.: "MSH2 is essential for the preservation of genome integrity and prevents homeologous recombination in the moss Physcomitrella patens", NUCLEIC ACIDS RES, vol. 34, 2006, pages 232 - 42
VERDAGUER ET AL., PLANT MOL BIOL, vol. 6, 1998, pages 1129 - 39
WOLTER ET AL.: "Characterization of paired Cas9 nickases induced mutations in maize mesophyll protoplasts", MAYDICA, vol. 62, no. 2, 2017
YOU ET AL.: "Facilitated Substrate Channeling in a Self-Assembled Trifunctional Enzyme Complex", ANGEW. CHEM. INT. ED., vol. 51, 2012, pages 8787 - 8790
ZETSCHE ET AL.: "A split-Cas9 architecture for inducible genome editing and transcription modulation", NAT. BIOTECH., vol. 33, 2015, pages 139 - 42, XP055227889, DOI: 10.1038/nbt.3149
ZONG ET AL.: "Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion", NAT BIOTECHNOL, vol. 35, 2017, pages 438 - 440, XP055482699, DOI: 10.1038/nbt.3811

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022171736A1 (fr) 2021-02-10 2022-08-18 Limagrain Europe Recombinaisons ciblées multiplex pour des applications d'introgression de caractères
WO2023199198A1 (fr) * 2022-04-12 2023-10-19 John Innes Centre Compositions et procédés pour augmenter l'efficacité d'édition du génome
WO2024096790A1 (fr) * 2022-10-31 2024-05-10 Soledits Ab Système crispr modulaire

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