WO2019120310A1 - Système et procédé d'édition de bases reposant sur la protéine cpf1 - Google Patents

Système et procédé d'édition de bases reposant sur la protéine cpf1 Download PDF

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WO2019120310A1
WO2019120310A1 PCT/CN2018/123158 CN2018123158W WO2019120310A1 WO 2019120310 A1 WO2019120310 A1 WO 2019120310A1 CN 2018123158 W CN2018123158 W CN 2018123158W WO 2019120310 A1 WO2019120310 A1 WO 2019120310A1
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deaminase
base
fusion protein
dna
cleavage activity
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Caixia Gao
Yanpeng WANG
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Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences
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    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the invention relates to the field of genetic engineering.
  • the invention relates to a base editing system and method based on CPF1 protein.
  • the present invention relates to a system and method for efficient base editing of a target sequence in the genome of an organism (e.g., a plant) by a guide RNA-directed Cpfl-deaminase fusion protein, and the genetically modified organism (e.g., a plant) produced by the method and progeny thereof.
  • TILLING targeting induced local lesions in genomes
  • Genomic editing techniques particularly those based on the CRISPR/Cas9 system, enable the introduction of specific base substitutions in genomic loci by homologous recombination (HR) -mediated DNA repair pathways.
  • HR homologous recombination
  • the successful use of this method is currently limited, mainly due to the low frequency of HR-mediated double-strand broken chain repair in plants.
  • effectively providing a sufficient amount of DNA repair templates is also a major difficulty.
  • Cas9 and deaminase can be fused to achieve precise conversion of cytosine (C) to thymine (T) and conversion of adenine (A) to guanine (G) in a target gene.
  • the system for C to T transformation mainly includes fusions of SpnCas9-BE3, SpnCas9-AID and Cas9 variants, such as VQR-BE3, EQR-BE3 and VRER-BE3, as well as SaCas9-BE3 and variant SaKKH-BE3.
  • the PAMs for Cas9 and Cas9 variants are generally limited in G/C-rich region, thus the types of PAM for the single-base editing system still needs to be broadened.
  • the single-base editing system still needs to be improved in terms of specificity.
  • nCas9-BE3 and its variants, and nCas9-ABE usually produce single-stranded nicks on the non-targeting strands of the target site, and it tends to generate DNA indels while it generates single-base mutations during the mismatch repair process, therefore there is still room for improvement in the high fidelity of single base editing. Therefore, new systems and methods for base editing of plant genomes are still needed in the art.
  • Cpf1 nuclease Cpf1 protein
  • Cpf1 Cpf1
  • Cpf1 protein Cpf1
  • DSBs DNA double-strand breaks
  • crRNA guide RNA
  • the Cpf1 protein contains a cleavage domain of DNA and an independent RNA cleavage domain.
  • the RNA cleavage domain of the Cpf1 protein is capable of processing pre-crRNA to form a mature crRNA.
  • guide RNA and “gRNA” can be used interchangeably herein.
  • the guide RNA of the Cpf1-mediated genome editing system is typically composed only of mature crRNA molecules, wherein the crRNA comprises a sequence that is sufficiently identical to the target sequence to hybridize to the complement of the target sequence and direct the complex (Cpf1+crRNA) to sequence specifically bind to the target sequence.
  • Deaminase refers to an enzyme that catalyzes a deamination reaction.
  • the deaminase refers to a cytosine deaminase that catalyzes the deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively.
  • the deaminase refers to adenine deaminase which is capable of catalyzing the formation of inosine (I) by adenosine or deoxyadenosine (A) .
  • Gene as used herein encompasses not only chromosomal DNA present in the nucleus, but also organellar DNA present in the subcellular components (e.g., mitochondria, plastids) of the cell.
  • organism includes any organism that is suitable for genome editing, eukaryotes are preferred. Examples of organisms include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, cats; poultry such as chickens, ducks, geese; plants including monocots and dicots such as rice, corn, wheat, sorghum, barley, soybean, peanut, Arabidopsis and the like.
  • a “genetically modified organism” or “genetically modified cell” includes the organism or the cell which comprises within its genome an exogenous polynucleotide or a modified gene or expression regulatory sequence.
  • the exogenous polynucleotide is stably integrated within the genome of the organism or the cell such that the polynucleotide is passed on to successive generations.
  • the exogenous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct.
  • the modified gene or expression regulatory sequence means that, in the organism genome or the cell genome, said sequence comprises one or more nucleotide substitution, deletion, or addition.
  • a genetically modified organism obtained by the present invention may comprise one or more substitutions of C to T or A to G relative to a wild type (corresponding organism without such genetic modification) .
  • Exogenous in reference to a sequence means a sequence from a foreign species, or refers to a sequence in which significant changes in composition and /or locus occur from its native form through deliberate human intervention if from the same species.
  • nucleic acid sequence RNA or DNA polymers, optionally containing synthetic, non-natural or altered nucleotide bases.
  • Nucleotides are referred to by their single letter names as follows: “A” is adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively) , “C” means cytidine or deoxycytidine, “G” means guanosine or deoxyguanosine, “U” represents uridine, “T” means deoxythymidine, “R” means purine (A or G) , “Y” means pyrimidine (C or T) , “K” means G or T, “H” means A or C or T, “I” means inosine, and “N” means any nucleotide.
  • Polypeptide, " “peptide, “ and “protein” are used interchangeably in the present invention to refer to a polymer of amino acid residues. The terms apply to an amino acid polymer in which one or more amino acid residues is artificial chemical analogue of corresponding naturally occurring amino acid (s) , as well as to a naturally occurring amino acid polymer.
  • polypeptide, “ “peptide, “ “amino acid sequence, “ and “protein” may also include modified forms including, but not limited to, glycosylation, lipid ligation, sulfation, ⁇ carboxylation of glutamic acid residues, and ADP-ribosylation.
  • expression construct refers to a vector such as a recombinant vector that is suitable for expression of a nucleotide sequence of interest in an organism.
  • “Expression” refers to the production of a functional product.
  • expression of a nucleotide sequence may refer to the transcription of a nucleotide sequence (e.g., transcription to produce mRNA or functional RNA) and/or the translation of an RNA into a precursor or mature protein.
  • the "expression construct" of the present invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector or, in some embodiments, an RNA that is capable of translation (such as mRNA) .
  • the "expression construct" of the present invention may comprise regulatory sequences and nucleotide sequences of interest from different origins, or regulatory sequences and nucleotide sequences of interest from the same source but arranged in a manner different from that normally occurring in nature.
  • regulatory sequence and “regulatory element” are used interchangeably to refer to a nucleotide sequence that is located upstream (5 'non-coding sequence) , middle or downstream (3' non-coding sequence) of a coding sequence and affects the transcription, RNA processing or stability or translation of the relevant coding sequence.
  • Regulatory sequences may include, but are not limited to, promoters, translation leaders, introns and polyadenylation recognition sequences.
  • Promoter refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment.
  • the promoter is a promoter capable of controlling the transcription of a gene in a cell, whether or not it is derived from the cell.
  • the promoter may be a constitutive promoter or tissue-specific promoter or developmentally-regulated promoter or inducible promoter.
  • Constant promoter refers to a promoter that may in general cause the gene to be expressed in most cases in most cell types.
  • tissue-specific promoter and “tissue-preferred promoter” are used interchangeably and mean that they are expressed primarily but not necessarily exclusively in one tissue or organ, but also in a specific cell or cell type.
  • Developmentally-regulated promoter refers to a promoter whose activity is dictated by developmental events.
  • inducible promoter selectively express operably linked DNA sequences in response to an endogenous or exogenous stimulus (environment, hormones, chemical signals, etc. ) .
  • operably linked refers to the linkage of a regulatory element (e.g., but not limited to, a promoter sequence, a transcription termination sequence, etc. ) to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) such that transcription of the nucleotide sequence is controlled and regulated by the transcriptional regulatory element.
  • a regulatory element e.g., but not limited to, a promoter sequence, a transcription termination sequence, etc.
  • nucleic acid sequence e.g., a coding sequence or an open reading frame
  • “Introduction" of a nucleic acid molecule (e.g., plasmid, linear nucleic acid fragment, RNA, etc. ) or protein into an organism means that the nucleic acid or protein is used to transform a cell of the organism such that the nucleic acid or protein is capable of functioning in the cell.
  • “transformation” includes both stable and transient transformations.
  • “Stable transformation” refers to the introduction of exogenous nucleotide sequences into the genome, resulting in the stable inheritance of foreign genes. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any of its successive generations.
  • Transient transformation refers to the introduction of a nucleic acid molecule or protein into a cell, performing a function without the stable inheritance of an exogenous gene. In transient transformation, the exogenous nucleic acid sequences are not integrated into the genome.
  • plant includes a whole plant and any descendant, cell, tissue, or part of a plant.
  • plant parts include any part (s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed) ; a plant cutting; a plant cell; a plant cell culture; a plant organ (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, and explants) .
  • a plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit.
  • a plant cell or tissue culture may be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant. In contrast, some plant cells are not capable of being regenerated to produce plants.
  • Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, or stalks.
  • Plant parts include harvestable parts and parts useful for propagation of progeny plants.
  • Plant parts useful for propagation include, for example and without limitation: seed; fruit; a cutting; a seedling; a tuber; and a rootstock.
  • a harvestable part of a plant may be any useful part of a plant, including, for example and without limitation: flower; pollen; seedling; tuber; leaf; stem; fruit; seed; and root.
  • a plant cell is the structural and physiological unit of the plant, and includes protoplast cells without a cell wall and plant cells with a cell wall.
  • a plant cell may be in the form of an isolated single cell, or an aggregate of cells (e.g., a friable callus and a cultured cell) , and may be part of a higher organized unit (e.g., a plant tissue, plant organ, and plant) .
  • a plant cell may be a protoplast, a gamete producing cell, or a cell or collection of cells that can regenerate into a whole plant.
  • a seed which comprises multiple plant cells and is capable of regenerating into a whole plant, is considered a "plant part" in embodiments herein.
  • protoplast refers to a plant cell that had its cell wall completely or partially removed, with the lipid bilayer membrane thereof naked.
  • a protoplast is an isolated plant cell without cell walls which has the potency for regeneration into cell culture or a whole plant.
  • Plant of a plant comprises any subsequent generation of the plant.
  • Trait refers to the physiological, morphological, biochemical, or physical characteristics of a plant or a particular plant material or cell.
  • the characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g. by measuring tolerance to water deprivation or particular salt or sugar concentrations, or by the observation of the expression level of a gene or genes, or by agricultural observations such as osmotic stress tolerance or yield.
  • trait also includes ploidy of a plant, such as haploidy which is important for plant breeding.
  • trait also includes resistance of a plant to herbicides.
  • “Agronomic trait” is a measurable parameter including but not limited to, leaf greenness, yield, growth rate, biomass, fresh weight at maturation, dry weight at maturation, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in a vegetative tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in a vegetative tissue, total plant protein content, fruit protein content, seed protein content, protein content in a vegetative tissue, drought tolerance, nitrogen uptake, root lodging, harvest index, stalk lodging, plant height, ear height, ear length, disease resistance, cold resistance, salt tolerance, and tiller number and so on.
  • the present invention provides a system for base editing of a target sequence in the genome of an organism, comprising at least one of the following i) to v) :
  • an expression construct comprising a nucleotide sequence encoding a base-editing fusion protein, and a guide RNA;
  • a base-editing fusion protein a base-editing fusion protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA
  • an expression construct comprising a nucleotide sequence encoding a base-editing fusion protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
  • the base-editing fusion protein comprises a Cpf1 lacking DNA cleavage activity, and a deaminase, the guide RNA being capable of targeting the base-editing fusion protein to a target sequence in the genome, resulting in one or more C to T or A to G substitution (s) in the target sequence.
  • Cpf1 contains a DNA cleavage domain (RuvC) , which can be mutated to delete the DNA cleavage activity of Cpf1 to form a "Cpf1 lacking DNA cleavage activity" .
  • the Cpf1 lacking DNA cleavage activity still retains gRNA-directed DNA binding ability.
  • the Cpfl lack of DNA cleavage activity can readily target the additional protein to almost any DNA sequence simply by co-expression with a suitable guide RNA.
  • the Cpf1 lacking DNA cleavage activity of the present invention may be derived from Cpf1 of different species, for example, Cpf1 proteins derived from Francisella novicida U112, Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006, designated FnCpf1 (amino acid sequence of the wild type is set forth in SEQ ID NO: 19) , AsCpf1 (amino acid sequence of the wild type is set forth in SEQ ID NO: 18) and LbCpf1 (amino acid sequence of the wild type is set forth in SEQ ID NO: 20) , respectively.
  • FnCpf1 amino acid sequence of the wild type is set forth in SEQ ID NO: 19
  • AsCpf1 amino acid sequence of the wild type is set forth in SEQ ID NO: 18
  • LbCpf1 amino acid sequence of the wild type is set forth in SEQ ID NO: 20
  • the Cpf1 lacking DNA cleavage activity is the FnCpfl lacking DNA cleavage activity. In some embodiments, the FnCpfl lacking DNA cleavage activity comprises a D917A mutation relative to wild-type FnCpfl.
  • the Cpf1 lacking DNA cleavage activity is the AsCpfl lacking DNA cleavage activity. In some embodiments, the AsCpfl lacking DNA cleavage activity comprises a D908A mutation relative to wild-type AsCpfl.
  • the Cpf1 lacking DNA cleavage activity is the LbCpfl lacking DNA cleavage activity. In some embodiments, the LbCpfl lacking DNA cleavage activity comprises a D832A mutation relative to wild type LbCpfl.
  • an expression construct comprising a nucleotide sequence encoding a guide RNA in a system of the invention may comprise a sequence encoding a plurality of different guide RNA (crRNA) precursors in tandem, which may be processed by the Cpf1 lacking DNA cleavage activity to form a plurality of different guide RNAs (crRNAs) upon transcription to simultaneously target a plurality of different target sequences.
  • crRNA guide RNA
  • the deaminase in the fusion protein is a cytidine deaminase, such as the apolipoprotein B mRNA editing complex (APOBEC) family deaminase.
  • APOBEC apolipoprotein B mRNA editing complex
  • Cytidine deaminase catalyzes the deamination of cytidine (C) in the DNA to form uracil (U) .
  • the present inventors have surprisingly found that the fusion of a Cpf1 lacking DNA cleavage activity and a cytidine deaminase, under the guidance of a guide RNA, can target a target sequence in the genome.
  • the DNA double strands are not cleaved, and the cytidine deaminase in the fusion protein can deamination of the cytidine of the single-stranded DNA produced during the formation of the Cpf1-guide RNA-DNA complex into U, and then C to T replacement is achieved through the base mismatch repairs.
  • the cytidine deaminase of the present invention is particularly a cytidine deaminase which can accept single-stranded DNA as a substrate.
  • cytidine deaminase useful in the present invention include, but are not limited to, APOBEC1 deaminase, activation-induced cytidine deaminase (AID) , APOBEC3G or CDA1.
  • the cytidine deaminase comprises the amino acid sequence set forth in SEQ ID NO: 1.
  • the base editing system of the present invention can mutate one or more C (s) to T (s) in the genomic target sequence, thus also referred to as the Cpf1-PBE system.
  • uracil DNA glycosylase catalyzes the removal of U from DNA and initiates base excision repair (BER) , resulting in the repair of U: G to C: G.
  • BER base excision repair
  • the base-editing fusion protein further comprises a uracil DNA glycosylase inhibitor (UGI) .
  • the uracil DNA glycosylase inhibitor comprises the amino acid sequence set forth in SEQ ID NO: 2.
  • the deaminase is an adenine deaminase.
  • the naturally occurring adenine deaminase converts adenosine in single-stranded RNA into inosine (I) by deamination using RNA as a substrate.
  • DNA-dependent adenine deaminase that convert deoxyguanosine in single-stranded DNA to inosine (I) using single-stranded DNA as a substrate has been obtained based on tRNA adenine deaminase TadA of E. coli by means of directed evolution. See Nicloe M. Gaudelli et al., doi: 10.1038/nature 24644, 2017.
  • the present inventors have surprisingly found that when Cpf1 lacking DNA cleavage activity is fused to a DNA-dependent adenine deaminase, under the guidance of a guide RNA, the fusion protein can target a target sequence in the plant genome. Due to the deficient of the DNA cleavage activity in Cpf1, the DNA double strands are not cleaved, and the DNA-dependent adenine deaminase in the fusion protein is capable of deaminating the adenosine of the single-stranded DNA produced during the formation of the Cpf1-guide RNA-DNA complex into a inosine (I) .
  • the base editing system of the present invention can mutate one or more A in the genomic target sequence to G, and thus is also called Cpf1-ABE system.
  • the DNA-dependent adenine deaminase is a variant of the E. coli tRNA adenine deaminase TadA (ecTadA) , in particular a variant which can accept single-stranded DNA as a substrate.
  • the variant comprises, relative to wild-type ecTadA, one or more sets of mutations selected from the group consisting of:
  • the DNA-dependent adenine deaminase (ABE version 7.9) comprises the following mutations relative to wild-type ecTadA: W23R, H36L, R51L, S146C, K157N, A106V, D108N, P48A, L84F, H123Y, I156F, A142N, D147Y, E155V and R152P.
  • the DNA-dependent adenine deaminase (ABE version 7.10) comprises the following mutations relative to wild-type ecTadA: W23R, H36L, R51L, S146C, K157N, A106V, D108N, P48A, L84F, H123Y, I156F, D147Y, E155V and R152P.
  • the amino acid sequence of preferred ecTadA derived DNA-dependent adenosine deaminase is shown below: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNR AIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIH SRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALL CYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 4) .
  • the initiating methionine may be absent.
  • the deaminase is fused to the N-terminus of the Cpfl lacking DNA cleavage activity. In some embodiments, the deaminase is fused to the C-terminus of the Cpfl lacking DNA cleavage activity.
  • the N-terminus of the DNA-dependent adenine deaminase is fused with a corresponding wild-type adenine deaminase. It is expected that the formation of heterodimers by DNA-dependent adenine deaminase and wild-type adenine deaminase can significantly increase the A to G editing activity of fusion proteins.
  • the deaminase and the Cpfl lacking DNA cleavage activity are fused via a linker.
  • the linker may be 1-50 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 20-25, 25-50) amino acids in length, or non-functional amino acid sequences with more amino acids and without secondary or higher structures.
  • the linker can be a flexible linker such as GGGGS, GS, GAP, (GGGGS) x 3, GGS and (GGS) x7, and the like.
  • the linker is an XTEN linker.
  • the linker is 32 amino acids in length.
  • the amino acid sequence of the linker is: SGGSSGGSSGSETPGTSESATPESSGGSSGGS.
  • the base-editing fusion proteins of the present invention further comprise a nuclear localization sequence (NLS) .
  • NLS nuclear localization sequence
  • one or more NLSs in the base-editing fusion protein should be of sufficient strength to drive the base-editing fusion protein in the nucleus of a plant cell to achieve an amount accumulation of base editing function.
  • the intensity of nuclear localization activity is determined by the number, location, one or more specific NLSs used of the NLS in the base-editing fusion protein, or a combination of these factors.
  • the NLS of the base-editing fusion protein of the present invention may be located at the N-terminus and/or C-terminus.
  • the base-editing fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs. In some embodiments, the base-editing fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS at or near the N-terminus. In some embodiments, the base-editing fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the C-terminus.
  • the base-editing fusion protein comprises a combination of these, such as comprises one or more NLSs at the N-terminus and one or more NLSs at the C-terminus. When there is more than one NLS, each can be selected to be independent of other NLSs. In some preferred embodiments of the present invention, the base-editing fusion protein comprises two NLSs, for example, the two NLSs are located at the N-terminus and the C-terminus, respectively.
  • NLS consists of one or more short sequences of positively charged lysine or arginine exposed on the surface of the protein, but other types of NLS are also known.
  • Non-limiting examples of NLS include: KKRKV (nucleotide sequence 5'-AAGAAGAGAAAGGTC-3') , PKKKRKV (nucleotide sequence 5'-CCCAAGAAGAAGAGGAAGGTG-3' or CCAAAGAAGAAGAGGAAGGTT) , or SGGSPKKKRKV (nucleotide sequence 5'-TCGGGGGGGAGCCCAAAGAAGAAGCGGAAGGTG -3') .
  • the N-terminus of the base-editing fusion protein comprises the NLS with the amino acid sequence set forth in PKKKRKV.
  • the C-terminus of the base-editing fusion protein comprises the NLS with the amino acid sequence set forth by SGGSPKKKRKV or KRPAATKKAGQAKKKK.
  • the base-editing fusion proteins of the present invention may also include other localization sequences, such as cytoplasmic localization sequences, chloroplast localization sequences, mitochondrial localization sequences, and the like.
  • the base editing fusion protein also contains a uracil DNA glycosylase inhibitor (UGI) , and two NLSs flanking either N-terminal or C-terminal of the UGI.
  • UGI uracil DNA glycosylase inhibitor
  • the base editing fusion protein of the invention comprises an amino acid sequence selected from SEQ ID NO: 24-29.
  • the nucleotide sequence encoding the base-editing fusion protein is codon optimized for the biological species to be base edited.
  • Codon optimization refers to the replacement of at least one codon (eg, about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of a native sequence by a codon that is used more frequently or most frequently in the gene of the host cell, modifying the nucleic acid sequence while maintaining the native amino acid sequence to enhance expression in the host cell of interest.
  • codon preference differ in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • codon usage table can be easily obtained, for example, in the Codon Usage Database available at www. kazusa. orjp/codon/ , and these tables can be adjusted in different ways. See, Nakamura Y. et. al "Codon usage tabulated from the international DNA sequence databases: status for the year2000 Nucl. Acids Res, 28: 292 (2000) .
  • the base editing fusion protein of the invention is encoded by the nucleotide sequence selected from SEQ ID NO: 8-9, 11-12 or 14-15.
  • the nucleotide sequence encoding the base-editing fusion protein and/or the nucleotide sequence encoding the guide RNA is operably linked to an expression regulatory element such as a promoter.
  • promoters examples include, but are not limited to, the polymerase (pol) I, pol II or pol III promoters.
  • the pol I promoter examples include the gallus RNA pol I promoter.
  • the pol II promoters include, but are not limited to, the immediate-early cytomegalovirus (CMV) promoter, the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, and the immediate-early simian virus 40 (SV40) promoter.
  • pol III promoters include the U6 and H1 promoters.
  • An inducible promoter such as a metallothionein promoter can be used.
  • promoters include the T7 phage promoter, the T3 phage promoter, the ⁇ -galactosidase promoter, and the Sp6 phage promoter, and the like.
  • Promoters that can be used in plants include, but are not limited to, cauliflower mosaic virus 35S promoter, maize Ubi-1 promoter, wheat U6 promoter, rice U3 promoter, maize U3 promoter, rice actin promoter.
  • the guide RNA (crRNA) is expressed using the Ubi-1 promoter and cleaved to become mature with a ribozyme such as HDV ribozyme.
  • the addition of an intron after the Ubi-1 promoter enhances expression of the protein or RNA of interest.
  • the expression construct for expressing the base editing fusion protein of the invention comprises an expression cassette of SEQ ID NO: 10 or 13.
  • the expression construct comprises a expression regulating sequence set forth in SEQ ID NO: 30.
  • the present invention provides a method of producing a genetically modified organism (e.g. a plant) , comprising introducing a system of the present invention for base editing of a target sequence in the genome of an organism into a cell of the organism, whereby the guide RNA targets the base-editing fusion protein to a target sequence in the genome, resulting in one or more C to T or one or more A to G substitutions in the target sequence.
  • a genetically modified organism e.g. a plant
  • target sequences or crRNA coding sequences that can be recognized and targeted by the Cpf1 protein and the guide RNA (i.e., crRNA) complex can be found, for example, in Zhang et al., Cell 163, 1–13, October 22, 2015.
  • the 5'-terminus of the target sequence targeted by the genome editing system of the present invention needs to include a protospacer adjacent motif (PAM) 5'-TTTN or 5'-YTN, wherein N is independently selected from A, G, C and T, Y is selected from C and T.
  • PAM protospacer adjacent motif
  • the target sequence has the following structure: 5'-TTTN-NX-3' or 5'-YTN-NX-3', wherein N is independently selected from A, G, C and T, Y is selected from C and T; X is an integer of 15 ⁇ X ⁇ 35; Nx represents X consecutive nucleotides.
  • the target sequence to be modified may be located at any location in the genome, for example, in a functional gene such as a protein-encoding gene, or may be, for example, located in a gene expression regulatory region such as a promoter region or an enhancer region, thereby the gene functional modification or gene expression modification can be achieved.
  • a to G or C to T base editing in the target sequence of a cell can be detected by T7EI, PCR/RE or sequencing methods.
  • the base editing system can be introduced into cells by a variety of methods well known to those skilled in the art.
  • Methods that can be used to introduce a genome editing system of the present invention into a cell include, but are not limited to, calcium phosphate transfection, protoplast fusion, electroporation, lipofection, microinjection, viral infection (e.g., baculovirus, vaccinia virus, adenovirus, adeno-associated virus, lentivirus and other viruses) , gene gun method, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation.
  • a cell that can be edited by the method of the present invention can be a cell of mammals such as human, mouse, rat, monkey, dog, pig, sheep, cattle, cat; a cell of poultry such as chicken, duck, goose; a cell of plants including monocots and dicots, such as rice, corn, wheat, sorghum, barley, soybean, peanut and Arabidopsis thaliana and so on.
  • the methods of the invention are particularly suitable for producing genetically modified plants, such as crop plants.
  • the base editing system can be introduced into a plant by various methods well known to those skilled in the art. Methods that can be used to introduce a base editing system of the invention into a plant include, but are not limited to, gene gun method, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube pathway and ovary injection method.
  • the modification of the target sequence can be achieved by only introducing or producing the base-editing fusion protein and the guide RNA in the plant cell, and the modification can be stably inherited, without any need to stably transform the base editing system into plants. This avoids the potential off-target effect of the stable base editing system and also avoids the integration of the exogenous nucleotide sequence in the plant genome, thereby providing greater biosafety.
  • the introduction is carried out in the absence of selection pressure to avoid integration of the exogenous nucleotide sequence into the plant genome.
  • the introduction comprises transforming the base editing system of the present invention into an isolated plant cell or tissue and then regenerating the transformed plant cell or tissue into an intact plant.
  • the regeneration is carried out in the absence of selection pressure, i.e., no selection agent for the selection gene on the expression vector is used during tissue culture. Avoiding the use of a selection agent can increase the regeneration efficiency of the plant, obtaining a modified plant free of exogenous nucleotide sequences.
  • the base editing system of the present invention can be transformed into specific parts of an intact plant, such as leaves, shoot tips, pollen tubes, young ears or hypocotyls. This is particularly suitable for the transformation of plants that are difficult to regenerate in tissue culture.
  • the in vitro expressed protein and/or the in vitro transcribed RNA molecule are directly transformed into the plant.
  • the protein and/or RNA molecule is capable of performing base editing in plant cells and is subsequently degraded by the cell, avoiding integration of the exogenous nucleotide sequence in the plant genome.
  • Plants that can be base-edited by the methods of the invention include monocots and dicots.
  • the plant may be a crop plant such as wheat, rice, corn, soybean, sunflower, sorghum, canola, alfalfa, cotton, barley, millet, sugar cane, tomato, tobacco, tapioca or potato.
  • the target sequence is associated with a plant trait, such as an agronomic trait, whereby the base editing results in a plant having altered traits relative to a wild type plant.
  • a plant trait such as an agronomic trait
  • the target sequence to be modified may be located at any position in the genome, for example, in a functional gene such as a protein-encoding gene, or may be, for example, located in a gene expression regulatory region such as a promoter region or an enhancer region, thereby gene functional modification or gene expression modification can be achieved.
  • a functional gene such as a protein-encoding gene
  • a gene expression regulatory region such as a promoter region or an enhancer region
  • the substitution of C to T or A to G results in an amino acid substitution in the target protein or a truncation of the target protein (a stop codon is generated) .
  • the substitution of C to T or A to G results in a change in expression of the target gene.
  • the method further comprises obtaining progeny of the genetically modified plant.
  • the present invention provides a genetically modified plant or a progeny thereof, or a part thereof, wherein the plant is obtained by the method of the invention described above.
  • the present invention provides a method of plant breeding comprising crossing a genetically modified first plant obtained by the above method of the present invention with a second plant not containing the genetic modification, thereby the genetic modification is introduced into the second plant.
  • the ABE, XTEN, dCPF1 sequences were codon optimized for plants and ordered from GenScript (Nanjing) .
  • the full-length dCPF1-ABE fragment was amplified using primer pairs HindIII-F (with HindIII restriction site) and EcoRI (with EcoRI restriction site) .
  • the PCR product was digested with HindIII and EcoRI, and then inserted into the pJIT163-GFP vectors (this vector sequence is shown in SEQ ID NO: 16) digested with the two enzymes to generate the fusion expression vector dCPF1-ABE.
  • the PBE, XTEN, and dCPF1 sequences were codon optimized for plants and ordered from GenScript (Nanjing) .
  • the full length dCPF1-PBE fragment was amplified using primer pairs HindIII-F (with HindIII restriction site) and EcoRI (with EcoRI restriction site) .
  • the PCR product was digested with HindIII and EcoRI, and then inserted into the pJIT163-GFP vectors (this vector sequence is shown in SEQ ID NO: 16) digested with the two enzymes to generate the fusion expression vector dCPF1-PBE.
  • an sgRNA expression vector was constructed based on pTaU6-sgRNA (Addgene ID53062) or pOsU3-sgRNA (Addgene ID53063) or pZmU3-sgRNA (Addgene ID5306) or OsU3/TaU6-tRNA-sgRNA (Zhang et al. 2017. Genome Biology. DOI: 10.1186 /s13059-017-1325-9) .
  • the hammerhead enzyme and crRNA are activated by the type II promoter to generate crRNA (Tang et al. Nature plant, doi: 10.1038/nplants. 2017.18)
  • pUbi-mGFPP-crRNA pUbi-DEP1-sgRNA
  • pUbi-DEP1-crRNA pUbi-DME-crRNA.
  • Protoplast transformation was performed as described below. The average transformation efficiency is 55-70%.
  • Protoplasts transformation is performed as described below. Transformation is carried out with 10 ⁇ g of each plasmid by PEG-mediated method. Protoplasts were collected after 48h and DNA was extracted for T7EI and PCR-RE assay.
  • Protoplasts were suspended by adding an appropriate amount of MMG , placed on ice until tranformation.
  • Leaf sheath of the seedlings were used for protoplasts isolation, and cut into about 0.5 mm wide with a sharp blade.
  • Enzymolysis was performed for 5-6h in darkness with gently shaking (decolorization shaker, speed 10) .
  • Protoplasts were filtered into a 50 ml round bottom centrifuge tube with a 40 ⁇ m nylon membrane and washed with W5 solution.
  • the general reaction conditions are: denaturation at 94°C for 5min; denaturation at 94°C for 30s; anneal at 58°C for 30s, extension at 72°C for 30s, amplification for 30 to 35 cycles; incubation at 72°C for 5min; incubation at 12°C. 5 ⁇ l PCR products were subjected to electrophoresis.
  • sgRNA expression vectors were transformed respectively into wheat and rice protoplasts with Ubi-CPF1-PBE/ABE expression vector. Protoplasts were collected 48 hours later, and DNA was extracted for deep sequencing. In the first round of PCR, the target region was amplified using site-specific primers. In second round of PCR, forward and reverse tags were added to the end of the PCR product for library construction. Equal amounts of different PCR products were pooled. Samples were then sequenced using the Illumina High-Seq 4000 at the Beijing Genomics Institute.
  • Example 1 Optimization of CPF1 mediated genome cleavage activity in plants.
  • CPF1 The editing activity of CPF1 in plant cells is quite different in different articles, and the cleavage activity between different types of CPF1 is also very different.
  • the nuclear location state of AsCPF1, FnCPF1 and LbCPF1 was optimized, and the promoter for crRNA was also optimized to improve the cleavage activity of CPF1 in plant cells.
  • Vectors of AsCPF1, FnCPF1 and LbCPF1 carrying 1-4 NLSs were constructed, and different vectors for generating crRNA by ribozyme, driven by U3/U6 or UBI promoter were constructed (Fig. 1) .
  • dCPF1-PBE systems were constructed: dAsCPF1-2NLS-PBE, dFnCPF1-2NLS-PBE, dLbCPF1-2NLS-PBE.
  • the NLSs at the C-terminus are placed at one end of the UGI or placed at both ends of the UGI.
  • the expression of crRNA is initiated with UBI1 promoter and cleaved with a ribozyme.
  • dCPF1-PBE SEQ ID NO: 10 shows the dLBCPF1-PBE-2NLS expression cassette that comprise the sequences of the ZmUbi-1 promoter and an intron
  • CPF1-ABE systems are constructed: dAsCPF1-1NLS-ABE, dFnCPF1-NLS-ABE, dLbCPF1-1NLS-ABE, and dAsCPF1-2NLS-ABE, dFnCPF1-2NLS-ABE, dLbCPF1-2NLS-ABE, where ABE can be ABE7 . 9 or ABE7.10.
  • the crRNA is transcribed with UBI1 promoter and cleaved with a ribozyme.
  • the results by the GFP base editing reporter system of Fig. 3E indicate that both dFnCPF1-ABE7.10 (SEQ ID NO: 11) and dLbCPF1-ABE7.9 and dLbCPF1-ABE7.10 (SEQ ID NO: 12) can work, and efficiency of 7.10 is higher than 7.9 (Fig. 3F) .
  • dCPF1-ABE2-X1 SEQ ID NO. 13
  • ABE was also constructed at the C-terminus of CPF1 (dCPF1-ABE2-X2/X3) (SEQ ID NO. 14, 15) .
  • the results by the GFP base editing reporter system of Fig. 3E indicate that: editing activity of dCPF1-ABE2-X2/X3 is higher than that of dLbCPF1-ABE7.10 (Fig. 3G) .
  • CPF1 In order to further improve the editing efficiency of CPF1, we continued to optimize the CPF1 system. Firstly, all expression vectors for CPF1-mediated editing are driven with BdUbi10 promoter, to increase the expression. In addition, crRNA is transcribed using a type II promoter, and the crRNA Array is placed into the 5'UTR or 3'UTR region of a gene to be expressed, to improve the editing efficiency of CPF1 by increasing mRNA expression.
  • SEQ ID NO: 30 nucleotide sequence of promoter+intron

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Abstract

L'invention se rapporte au domaine du génie génétique. En particulier, l'invention concerne un système et un procédé d'édition de bases reposant sur la protéine CPF1. Plus précisément, l'invention concerne un système et un procédé d'édition efficace de bases d'une séquence cible dans le génome d'un organisme (par exemple, une plante) par une protéine de fusion Cpfl-désaminase dirigée par un ARN guide, et l'organisme génétiquement modifié (par exemple, des plantes) produit au moyen du procédé, ainsi que sa descendance.
PCT/CN2018/123158 2017-12-22 2018-12-24 Système et procédé d'édition de bases reposant sur la protéine cpf1 WO2019120310A1 (fr)

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