WO2023092731A1 - Protéine de fusion mad7-nls, et construction d'acide nucléique pour édition dirigée sur site de génome végétal et application associée - Google Patents

Protéine de fusion mad7-nls, et construction d'acide nucléique pour édition dirigée sur site de génome végétal et application associée Download PDF

Info

Publication number
WO2023092731A1
WO2023092731A1 PCT/CN2021/138184 CN2021138184W WO2023092731A1 WO 2023092731 A1 WO2023092731 A1 WO 2023092731A1 CN 2021138184 W CN2021138184 W CN 2021138184W WO 2023092731 A1 WO2023092731 A1 WO 2023092731A1
Authority
WO
WIPO (PCT)
Prior art keywords
mad7
sequence
nucleic acid
plant
acid construct
Prior art date
Application number
PCT/CN2021/138184
Other languages
English (en)
Chinese (zh)
Inventor
周红菊
李相敢
郑华颖
裴睿丽
刘政
刘子嘉
李莹莹
Original Assignee
科稷达隆(北京)生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 科稷达隆(北京)生物技术有限公司 filed Critical 科稷达隆(北京)生物技术有限公司
Publication of WO2023092731A1 publication Critical patent/WO2023092731A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal

Definitions

  • the present invention relates to the field of biotechnology. It specifically relates to a MAD7-NLS fusion protein, a nucleic acid construct for site-directed editing of plant genomes, and an efficient site-directed gene editing method for plant genomes based on the novel nuclease MAD7 using RNA guidance.
  • CRISPR regularly interspaced short palindromic repeats
  • Cas9 recognizes the 3'G-rich site
  • Cpf1 Cas12a
  • the Cas12a protein has the functions of both DNA shearing enzyme and RNA trimming enzyme, not only can target DNA double-strand cutting, but also can process and shear the corresponding immature crRNA (pre-crRNA) into a mature form.
  • the Cas12a protein molecule is relatively smaller and more specific, and has stronger advantages over Cas9 for multi-target gene editing; in addition, compared with Cas9
  • the blunt end is formed by cutting the genome, and the sticky end formed by Cas12a cutting the genome is more conducive to the directional insertion of foreign genes.
  • MAD7 belongs to the type II V-A Cpf1-like family. It was discovered by Inscripta in the genus Eubacterium and optimized. It is available for free use in scientific research institutes and commercial research. MAD7 has the highest homology with AsCpf1 protein, and the homology is only 31%. The PAM recognition site is YTTN. At present, the MAD7 system has been verified to have high editing activity in bacteria, yeast, zebrafish, mice and human cells. In order to promote its application range in plants, the rice codon was optimized to study its editing efficiency in plants. Although the application of the MAD7 system in rice has also been reported recently, its mutation efficiency is 49-65.6%. To sum up, in order to meet the needs of plant genetic engineering and enrich the toolbox of plant gene editing, it is necessary to develop a more efficient gene editing system with strong advantages.
  • the purpose of the present invention is to provide a set of MAD7-based CRISPR/MAD7 plant genome efficient site-directed editing system, which can simply and efficiently realize single gene knockout, multiple gene knockout and homologous recombination or foreign fragment site-directed knockout in monocotyledonous plants enter.
  • the present invention firstly provides a MAD7-NLS fusion protein, which has the following structure:
  • C is MAD7 protein
  • B1 and B2 are independent nuclear localization signal sequences (NLS).
  • the nuclear localization signal sequence is selected from: SV40, KRP2 (Kiprelated protein gene NO.2), MDM2, CDc25C, DPP9, MTA1, CBP80, AreA, M9, Rev, One or a combination of any two or more of hTAP, MyRF, EBNA-6, TERT or Tfam.
  • the N-terminal of the MAD7-NLS fusion protein further includes a signal peptide and/or protein tag sequence.
  • nucleic acid construct for site-directed editing of plant genomes comprising a first expression cassette comprising a sequentially connected first promoter, the above-mentioned MAD7-NLS fusion protein The coding nucleotide sequence and the first terminator;
  • the first promoter is a Pol II type promoter, preferably Ubi, Actin, CmYLCV, UBQ, 35S, SPL, one of the tissue-specific promoter YAO, CDC45, rbcS and the inducible promoter XEV or a combination of any two.
  • Pol II type promoter preferably Ubi, Actin, CmYLCV, UBQ, 35S, SPL, one of the tissue-specific promoter YAO, CDC45, rbcS and the inducible promoter XEV or a combination of any two.
  • the nucleic acid construct further comprises a second expression cassette, said second expression cassette comprising a second promoter connected in sequence, several tandem repeat sequences;
  • the repeat sequence is one or both of a mature direct repeat sequence (direct repeat, DR) and an immature direct repeat sequence; preferably, the second promoter is a Pol II type or Pol III type promoter; More preferably, the second promoter is selected from one of OsU3, OsU6a, OsU6b, OsU6c, Actin, 35S, Ubi, UBQ, SPL, CmYLCV, tissue-specific promoter YAO, CDC45, rbcS or inducible promoter XEV , two or more.
  • the second expression cassette further comprises a second termination sequence connected to the end of the repeat sequence
  • the second termination sequence is selected from polyT, NOS, polyA or a combination thereof.
  • the repeat sequence further includes a target site guide sequence sg; preferably, the length of the sg sequence is 17-35bp, preferably 19-28bp, more preferably 19-25bp ;
  • the repeat number of the repeat sequence is 2-50, preferably 2-10, more preferably 3-15, further preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.
  • the nucleic acid construct is selected from one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:21 or more.
  • the nucleic acid construct is a vector comprising both the first expression cassette and the second expression cassette, or,
  • a combination of vectors consisting of a first vector each comprising a first expression cassette and a second vector comprising a second expression cassette.
  • the present invention also provides a kit for gene editing in plants, which is characterized in that it includes the above-mentioned nucleic acid construct; preferably, it also includes an auxiliary vector carrying a donor DNA expression cassette.
  • Another aspect of the present invention provides a method for plant gene editing, comprising:
  • step (iii) regenerating or culturing the plant cell, plant tissue or plant body identified in step (ii) as having undergone the gene editing;
  • the gene editing includes one of gene knockout, site-directed insertion or gene replacement, or an optional combination of two or three;
  • the gene editing is single-site or multi-site gene editing
  • the introduction is achieved by any method selected from Agrobacterium transformation, gene gun method, microinjection method, electric shock method, ultrasonic method or polyethylene glycol (PEG)-mediated method;
  • the plant is selected from any one of grasses, leguminous plants, solanaceae or cruciferous plants;
  • the plant is selected from any one of Arabidopsis thaliana, wheat, barley, oat, corn, rice, sorghum, millet, soybean, peanut, tobacco or tomato.
  • the MAD7-based CRISPR/MAD7 plant genome efficient fixed-point editing system of the present invention can simply and efficiently realize single gene knockout, multiple gene knockout and homologous recombination or foreign fragment fixed-point knock-in in monocotyledonous plants.
  • CRISPR/Cas9, CRISPR/Cpf1 gene editing system, the CRISPR/MAD7 system provided by the present invention has higher specificity and is more flexible for the selection of target sites.
  • 1 is a schematic diagram of the structural composition of each element in the related sequence (SEQ ID NO.2) of the rice crRNA expression cassette of MAD7 according to the present invention.
  • Figure 2 is a schematic diagram of the structural composition of each element in the related sequence (SEQ ID NO.3) of the rice multi-gene site editing sg-DR crRNA expression cassette of MAD7 according to the present invention.
  • FIG. 3 is a schematic diagram of the structural composition of each element in the related sequence (SEQ ID NO.4) of the rice multi-gene site editing tRNA-sg-DR crRNA expression cassette of MAD7 according to the present invention.
  • FIG. 4 is a schematic diagram of the structural composition of each element in the related sequence (SEQ ID NO.5) of the rice multi-gene locus editing miniOsU3/U6-DR-sg crRNA expression cassette according to the present invention.
  • FIG. 5 is a schematic diagram of the structural composition of each element in the related sequence (SEQ ID NO.6) of the rice multi-gene site editing HH-DR-sg-HDV crRNA expression cassette of MAD7 according to the present invention.
  • FIG. 6 is a schematic diagram of the structural composition of each element in the related sequence (SEQ ID NO.21) of the maize crRNA expression cassette of MAD7 according to the present invention.
  • Figure 7 is a schematic diagram of the construction of the rice single-site knockout CRISPR-MAD7 (ncNLS) expression vector.
  • Figure 8 is a schematic diagram of the construction of the expression vector under the three placement modes of the NLS design when the OsHD3A site is knocked out at a single site in rice.
  • Figure 9 is a schematic diagram of the construction of the rice multi-site knockout CRISPR-MAD7 expression vector comprising the DR-sg sequence string.
  • Figure 10 is a schematic diagram of the construction of the rice multi-site knockout CRISPR-MAD7 expression vector with tRNA tandem DR-sg sequence.
  • FIG 11 Schematic diagram of the construction of CRISPR-MAD7 expression vectors driven by miniOsU3/miniOsU6 respectively driving the DR-sg sequence.
  • Figure 12 is a schematic diagram of the construction of a CRISPR-MAD7 expression vector driven by a Pol III type promoter and HH-HDV tandem DR-sg sequence.
  • Figure 13 is a schematic diagram of the construction of a CRISPR-MAD7 expression vector driven by a Pol II type promoter and HH-HDV tandem DR-sg sequence.
  • Fig. 14 is a schematic diagram of the construction of a maize single-site knockout CRISPR-MAD7 (ncNLS) expression vector.
  • the inventors After extensive and in-depth research, the inventors have constructed a highly versatile and highly specific CRISPR/MAD7 plant genome-directed and efficient editing system based on MAD7, as well as nucleic acid constructs, vectors or vectors for plant genome-directed editing Combinatorial, and site-directed editing methods for plant genomes. Specifically, based on the method of the present invention, single gene knockout, multiple gene knockout, or homologous recombination and directional insertion of foreign fragments can be easily and efficiently performed at predetermined plant genome sites. On this basis, the present invention has been accomplished.
  • the CRISPR/MAD7 editing system of the present invention uses mature crRNA. Since the mature crRNA is shorter, it is convenient for artificial synthesis and easier to transform into cells, so it will be more advantageous to develop a mature crRNA-based CRISPR-MAD7 system. On this basis, a technical solution for placing the nuclear localization signal sequence (NLS) at the N-terminus and C-terminus of the MAD7 protein was also developed. Experimental results show that the above-mentioned technical scheme has successfully completed the gene editing of rice with an efficiency as high as 95%.
  • NLS nuclear localization signal sequence
  • operably linked refers to the condition that some portion of a linear DNA sequence is capable of modulating or controlling the activity of other portions of the same linear DNA sequence.
  • a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.
  • the present invention provides a nucleic acid construct for high-efficiency site-directed editing of plant genomes, the nucleic acid construct comprising a first expression cassette and an optional second expression cassette;
  • the first expression cassette is a MAD7-NLS fusion protein expression cassette, wherein the MAD7-NLS fusion protein has a formula I structure:
  • P1 is the first promoter
  • A is none, signal peptide, and/or protein tag sequence
  • B1 is no or nuclear localization signal sequence NLS
  • B2 is no or nuclear localization signal sequence NLS
  • the additional condition is that at most one of B1 and B2 is none;
  • C is MAD7 protein
  • E1 is the first terminator
  • the second expression cassette is a crRNA expression cassette, and the crRNA expression cassette contains coding sequences corresponding to mature crRNA or immature pre-crRNA.
  • the above-mentioned elements can be prepared by conventional methods (such as PCR method, artificial total synthesis), and then connected by conventional methods to form the nucleic acid construct of the present invention.
  • An enzyme cleavage reaction may optionally be performed before the ligation reaction, if desired.
  • nucleic acid construct of the present invention can be linear or circular.
  • the nucleic acid construct of the present invention can be single-stranded or double-stranded.
  • the nucleic acid construct of the present invention can be DNA, RNA, or DNA/RNA hybrid.
  • nNLS refers to the nuclear localization signal sequence NLS located at the 5' end of the MAD7 protein coding sequence
  • cNLS refers to the nuclear localization signal sequence NLS located at the 3' end of the MAD7 protein coding sequence
  • ncNLS refers to the 5' end of the MAD7 protein coding sequence.
  • the nuclear localization signal sequence NLS can be connected to both the end and the 3' end.
  • exogenous gene refers to an exogenous DNA molecule that acts in stages.
  • the exogenous genes that can be used in this application are not particularly limited, and include various exogenous genes commonly used in the field of transgenic animals. Representative examples include (but are not limited to): ⁇ -glucuronidase gene, red fluorescent protein gene, green fluorescent protein gene, lysozyme gene, salmon calcitonin gene, lactoferrin, or serum albumin gene and the like.
  • selectable marker gene refers to a gene used to screen transgenic cells or transgenic animals during the transgenic process.
  • the selectable marker gene that can be used in this application is not particularly limited, including various commonly used in the field of transgenic. Representative examples Including, but not limited to: hygromycin resistance gene (Hyg), kanamycin resistance gene (NPTII), neomycin, or puromycin resistance gene.
  • the term "expression cassette” refers to a polynucleotide sequence that contains the sequence components of the gene to be expressed and the elements required for expression.
  • the term “selectable marker expression cassette” refers to a polynucleotide sequence comprising a sequence encoding a selectable marker and sequence modules of elements required for expression. Components required for expression include a promoter and polyadenylation signal sequence.
  • the screening marker expression cassette may or may not contain other sequences, including (but not limited to): enhancers, secretion signal peptide sequences, and the like.
  • plant promoter refers to a nucleic acid sequence capable of initiating transcription of a nucleic acid in a plant cell.
  • the plant promoter may be derived from plants, microorganisms (such as bacteria, viruses) or animals, or artificially synthesized or modified.
  • plant terminator refers to a terminator capable of stopping transcription in a plant cell.
  • the plant transcription terminator may be derived from plants, microorganisms (such as bacteria, viruses) or animals, or artificially synthesized or modified terminators. Representative examples include (but are not limited to): Nos terminators.
  • MAD7 protein refers to a nuclease. Typical MAD7 proteins include (but are not limited to):
  • the term "coding sequence of MAD7 protein” refers to a nucleotide sequence encoding MAD7 protein having cleavage activity.
  • the skilled artisan will recognize that, because of codon degeneracy, there are a large number of polynucleotide sequences that can encode the same polypeptide .
  • technicians will also recognize that different species have certain preferences for codons, and may optimize the codons of the MAD7 protein according to the needs of expression in different species. These variants are all referred to by the term “MAD7 protein Coding sequences" are specifically covered.
  • the term specifically includes a full-length sequence that is substantially identical to the MAD7 gene sequence, as well as a sequence that encodes a protein that retains the function of the MAD7 protein.
  • said C corresponds to the full-length or fusion protein of MAD7 protein
  • said second expression cassette has a crRNA expression cassette with formula II structure:
  • P2 is the second promoter
  • Each R is independently corresponding to a mature or immature direct repeat sequence (direct repeat, DR)
  • each S is independently none or a target site leader sequence sg;
  • q is a positive integer ⁇ 1;
  • T is none or polyT or Nos or polyA sequence.
  • the present invention also provides a vector or a combination of vectors, which contains the nucleic acid construct of the present invention.
  • the vector combination of the present invention further includes an auxiliary vector carrying a donor DNA expression cassette.
  • some elements are operably linked.
  • a promoter when operably linked to a coding sequence, it means that the promoter is capable of initiating the transcription of the coding sequence.
  • the present invention also provides a reagent combination and a kit containing the above-mentioned vector or vector combination, which can be used in the plant gene editing method of the present invention.
  • the present invention also provides a method for gene editing of plants, comprising the steps of:
  • the gene editing includes gene knockout, site-directed insertion, gene replacement, or a combination thereof.
  • the plant gene editing method of the present invention can be used to improve various plants, especially crops.
  • plant includes whole plants, plant organs (eg, leaves, stems, roots, etc.), seeds and plant cells, as well as their progeny.
  • plant organs eg, leaves, stems, roots, etc.
  • the types of plants that can be used in the method of the present invention are not particularly limited, and generally include any type of higher plants that can be subjected to transformation techniques, including monocots, dicots and gymnosperms.
  • the invention can be used in the field of plant genetic engineering, such as the study of plant gene function and crop genetic improvement.
  • the coding sequence of the MAD7 protein is a codon-optimized coding sequence for rice, and the specific sequence is shown in SEQ ID NO.1.
  • the related sequence (SEQ ID NO.2) of the rice crRNA expression cassette of sequence 2 MAD7, the element structure in this expression cassette is as shown in Figure 1, and the underline before and after this sequence marks respectively AvrII, AfeI enzyme cutting site, for the convenience of cloning into
  • the pCAMBIA expression vector is set; the black shading marks the mature DR sequence corresponding to MAD7; the sg site is inside the box, and the sequence is artificially synthesized complementary double strands when constructing a gene knockout vector, and the PCR product with the OsU3 promoter is passed through Overlapping PCR
  • the full length was amplified by the method; the bold letter is the transcription terminator sequence; the rest is the sequence of the OsU3 promoter.
  • Sequence 3 The related sequence (SEQ ID NO.3) of the rice multi-gene site editing sg-DR crRNA expression cassette of MAD7, the element structure in the expression cassette is shown in Figure 2, wherein the underline indicates the BsaI restriction site, Set for the convenience of cloning into the pCAMBIA expression vector; the black shading marks the mature DR sequence corresponding to MAD7; the sg sequences of the four targeting sites are in the box, and they are OsDEP1-g6, OsDEP1-g6, OsBEL260-g1, OsRoc5-g1 and OsHD3A; bold letters are transcription terminator sequences; the rest are sequences of OsU3 promoter.
  • Sequence 4 The related sequence (SEQ ID NO.4) of the rice multi-gene locus editing tRNA-sg-DR crRNA expression cassette of MAD7, the element structure in the expression cassette is shown in Figure 3, wherein the underlines before and after indicate AvrII, The BsaI restriction site is set for the convenience of cloning into the pCAMBIA expression vector; the black shading marks the mature DR sequence corresponding to MAD7; the sg sequences of the four targeting sites are in the box, in order from 5' to 3' They are OsDEP1-g6, OsBEL260-g1, OsRoc5-g1 and OsHD3A respectively; double underlines are tRNA sequences; bold letters are transcription terminator sequences; the rest are sequences of OsU3 promoter.
  • Sequence 5 The relevant sequence of the rice multi-gene locus editing miniOsU3/U6-DR-sg crRNA expression cassette (SEQ ID NO.5) of MAD7, the structure of the elements in the expression cassette is shown in Figure 4, where the underlines before and after are respectively marked AvrII and BsaI restriction sites are set for convenient cloning into the pCAMBIA expression vector; the black shading marks the mature DR sequence corresponding to MAD7; the sg sequences of the four targeting sites are in the box, according to 5' to 3' The sequences are OsDEP1-g6, OsBEL260-g1, OsRoc5-g1 and OsHD3A respectively; bold letters are transcription terminator sequences; the rest are sequences of miniOsU3/U6 promoters.
  • Sequence 6 The related sequence (SEQ ID NO.6) of the rice multi-gene site editing HH-DR-sg-HDV crRNA expression cassette of MAD7, the element structure in the expression cassette is shown in Figure 5, wherein the underline indicates the BsaI enzyme
  • the cutting site is set for the convenience of cloning into the pCAMBIA expression vector; the black shading marks the mature DR sequence corresponding to MAD7; the sg sequences of the four targeting sites are in the box, and they are respectively in the order of 5' to 3' OsDEP1-g6, OsBEL260-g1, OsRoc5-g1, and OsHD3A; bold letters are transcription terminator sequences; double underlines and dots are hammerhead (HH) and Hepatitis deltavirus (HDV) ribozyme sequences, respectively.
  • HH hammerhead
  • HDV Hepatitis deltavirus
  • the 5' in the HH nucleic acid sequence Complementary to first 6 bases of DR.
  • the related sequence (SEQ ID NO.21) of the maize crRNA expression cassette of sequence 21 MAD7, the element structure in this expression cassette is as shown in Figure 6, wherein, the underline before and after marks the ApaI restriction site respectively, for the convenience of cloning into The pCAMBIA expression vector is set; the black shading marks the mature DR sequence corresponding to MAD7; the sg site is inside the box, and the complementary double-stranded sequence is artificially synthesized when constructing the gene knockout vector, and the PCR product with the ZmU3 promoter is passed through Overlapping PCR The full length was amplified by the method; the bold letter is the transcription terminator sequence; the rest is the sequence of the ZmU3 promoter.
  • the rice codon was optimized for MAD7, two expression cassettes of MAD7 and crRNA were constructed and cloned into the pCAMBIA expression vector.
  • the crRNA expression cassette (SEQ ID NO: 2) has the following four elements from 5' to 3': OsU6 or OsU3 promoter, mature direct repeat (DR) corresponding to MAD7, sg sequence , Transcription terminator sequence (TTTTTTT).
  • the MAD7 expression cassette has the following elements from 5'-3': Ubi promoter from maize, NLS nuclear localization signal sequence (nNLS), coding sequence of MAD7, second NLS nuclear localization signal sequence (cNLS), NOS transcription termination Subsequence ( Figure 7).
  • Another design of the present invention is to change the length of sg, or remove the NLS nuclear localization signal sequence at the 5' or 3' end of the MAD7 coding sequence on the basis of the above-mentioned construction, and keep other elements (Figure 8) to explore the presence of MAD7 in different combinations. Cleavage activity in vivo.
  • MAD7 itself has RNase activity, it can cut and process the transcribed precursor crRNA sequence by itself. It is speculated that if a crRNA expression cassette is connected in series with multiple DR-sg sequences, it can be cut into a single DR-sg sequence by MAD7 after transcription. The realization of multi-site knockout. This design requires the construction of a multi-site crRNA expression cassette.
  • RNA sequence corresponding to MAD7 (including DR sequence), sg1 corresponding to target site 1 sequence, crRNA sequence, sg2 sequence corresponding to target site 2, crRNA sequence, sg3 sequence corresponding to target site 3, crRNA sequence, sgN sequence corresponding to target site N, transcription terminator sequence (TTTTTTTT).
  • TTTTTTTT transcription terminator sequence
  • Another design of the present invention is that multiple DR-sgs are respectively driven by miniOsU3/U6 promoters, or different DR-sg sequences are connected with tRNA processing recognition sequences, or HH-HDV spacers, and the MAD7 expression cassette and single-site knockout The same build.
  • the above two expression cassettes of crRNA and MAD7 nuclease were cloned into the pCAMBIA expression vector (Fig. 9-13).
  • MAD7 cuts like Cpf1 to produce sticky ends, which is theoretically more suitable for directional insertion of foreign fragments.
  • CRISPR-MAD7 to create a DSB near the target site, and using gene gun bombardment or DNA virus replication to introduce a large number of foreign fragments can efficiently achieve gene homologous recombination or directional insertion in plant cells.
  • Example 1 Using the CRISPR-MAD7 (ncNLS) system to perform single-site knockout of endogenous genes in rice
  • LbCpf1 was used to efficiently edit the OsHD3A promoter targeting site (LOCOs06g06320, editing efficiency 81.5%), and was connected to the pCAMBIA-CRISPR-MAD7 expression vector through AvrII and Afel restriction sites, respectively.
  • the constructed vector was transformed into Agrobacterium EHA105, and then the callus of rice variety Nanjing 46 (Oryza sativa ssp japonica cv. The transformed callus was transferred to the selection medium containing hygromycin, and after 28-30 days of culture, it was transferred to the differentiation medium containing hygromycin to regenerate plants.
  • the regenerated plants were sampled to extract DNA, and Taqman detected MAD7 positive individual plants, and the The target sites of positive individual plants were amplified and sequenced. The results show that the system can target mutations in rice cells and generate mutant plants.
  • the editing efficiency of the OsHD3A site is 89.9% and 94.9%, respectively, and the two alleles are edited at the same time
  • the frequency (including homo) of the gene is as high as 77.5% and 78.1%, which is comparable to the editing efficiency of LbCpf1, and there is no significant difference between the editing efficiency under two different temperature treatments (Table 1).
  • Table 1 The PAM-sg sequence of rice endogenous gene single-site knockout in CRISPR/MAD7(ncNLS) system and the identification results of T0 generation plants
  • Example 2 Using the CRISPR-MAD7 (ncNLS) system to perform single-site knockout of multiple endogenous genes in rice
  • CRISPR-MAD7 ncNLS
  • five Cpf1 editing sites in rice reported in the paper were selected (OsRLK-799-g1, LOC_Os02g07960; OsDEP1-g6, LOC_Os09g26999; OsALS- g7, LOC_Os02g30630; OsBEL260-g1, LOC_Os03g55260; OsRoc5-g1, LOC_Os02g45250), and newly designed CRISPR-MAD7 targeting sites (OsDEP1-g7; OsPDS1-g3; LOC_Os03g08570; OsRoc5- g2 and OsBEL260-g4 ), artificially synthesized DR-sgRNA, and the overlap PCR amplification product was connected to the pCAMBIA-CRISPR-MAD7 expression vector through restriction sites.
  • the constructed vector was transformed into Agrobacterium EHA105, and then the callus of rice variety Nanjing 46 was infected by this strain. After 3 days of co-cultivation, the culture was resumed at 30°C for 4 days, and the transformed callus was transferred to a culture medium containing hygromycin. Screen the culture medium, transfer to the differentiation medium containing hygromycin to regenerate plants after 28-30 days of culture, take samples from the regenerated plants to extract DNA, detect MAD7 positive individual plants by Taqman, and amplify and sequence the target sites of positive individual plants.
  • Example 1 the CRISPR-MAD7 system PCR amplification products were respectively connected to the pCAMBIA-CRISPR-MAD7 expression vector through the BsrGI and AvrII restriction sites, or the ApaI restriction site, to construct pCAMBIA-CRISPR-nNLS-MAD7 (remove MAD7 Nucleic acid editing enzyme C-terminal NLS nuclear localization sequence), pCAMBIA-CRISPR-MAD7-cNLS (remove MAD7 nucleic acid editing enzyme N-terminal NLS nuclear localization sequence) system.
  • the constructed vector was transformed into Agrobacterium EHA105, and then the callus of rice variety Nanjing 46 was infected by this strain.
  • the culture was resumed at 30°C for 4 days, and the transformed callus was transferred to a culture medium containing hygromycin.
  • the medium was selected, and after 28-30 days of culture, it was transferred to a differentiation medium containing hygromycin to regenerate plants.
  • the regenerated plants were sampled to extract DNA, Taqman detected MAD7-positive individual plants, and the target sites of positive individual plants were amplified and sequenced.
  • Table 3 The identification results of PAM-sg sequence and T0 generation plants of rice endogenous gene single site knockout in CRISPR-MAD7 system with different NLS positions and numbers
  • the constructed vector was transformed into Agrobacterium EHA105, and then the callus of rice variety Nanjing 46 was infected by this strain. After 3 days of co-cultivation, the culture was resumed at 30°C for 4 days, and the transformed callus was transferred to a culture medium containing hygromycin. The medium was selected, and after 28-30 days of culture, it was transferred to a differentiation medium containing hygromycin to regenerate plants.
  • the regenerated plants were sampled to extract DNA, Taqman detected MAD7-positive individual plants, and the target sites of positive individual plants were amplified and sequenced.
  • the results showed that as the length of sg increased from 19bp, 21bp, 23bp to 25bp, the editing frequency and biallelic (including homo) frequency in rice cells decreased from 95.7% and 89.2% to 85.9% and 56.5%, respectively. Except for the significant reduction in the biallelic frequency of the length, there was no significant difference in the editing rate and biallelic frequency between sg vectors of different lengths (Table 4).
  • Example 5 The CRISPR-MAD7 vector comprising the DR-sg sequence string is used for multi-site knockout in rice
  • MAD7 has the ability to autonomously cut and process pre-crRNA
  • this example selects the editing sites of Cpf1 reported in 4 articles in Example 2 (OsDEP1-g6, OsBEL260-g1, OsRoc5-g1, OsHD3A-g22), Interspaced by the mature DR sequence of MAD7 and under the control of the same OsU3 promoter (SEQ ID NO.3).
  • This expression cassette was then ligated to the MAD7 expression cassette and placed within the LB and RB sequences of pCAMBIA.
  • the constructed vector was transformed into Agrobacterium EHA105, and then the callus of rice variety Nanjing 46 was infected by this strain.
  • the culture was resumed at 30°C for 4 days, and the transformed callus was transferred to a culture medium containing hygromycin.
  • the medium was selected, and after 28-30 days of culture, it was transferred to a differentiation medium containing hygromycin to regenerate plants.
  • the regenerated plants were sampled to extract DNA, Taqman detected MAD7-positive individual plants, and the target sites of positive individual plants were amplified and sequenced.
  • the results showed that the mutation frequencies of the four genes in this system were 34.0%, 80.9%, 3.2% and 3.2%, respectively, and the simultaneous editing efficiencies of two alleles were 11.7%, 59.6%, 0% and 0%, respectively.
  • the first and second genes of this system can be efficiently edited in rice cells.
  • the DR-sg sequence string in Example 5 is connected at intervals through the recognition site of RNAase in the endogenous tRNA processing system, and is under the control of the same OsU3 promoter (sequence 4 SEQ ID NO.4).
  • This expression cassette was then ligated to the MAD7 expression cassette and placed within the LB and RB sequences of pCAMBIA.
  • the constructed vector was transformed into Agrobacterium EHA105, and then the callus of rice variety Nanjing 46 was infected by this strain. After 3 days of co-cultivation, the culture was resumed at 30°C for 4 days, and the transformed callus was transferred to a culture medium containing hygromycin.
  • the medium was selected, and after 28-30 days of culture, it was transferred to a differentiation medium containing hygromycin to regenerate plants.
  • the regenerated plants were sampled to extract DNA, Taqman detected MAD7-positive individual plants, and the target sites of positive individual plants were amplified and sequenced.
  • the results showed that the mutation frequencies of the four genes in this system were 49.4%, 91.0%, 71.9% and 68.2%, respectively, and the simultaneous editing efficiencies of two alleles were 14.6%, 78.7%, 58.4% and 47.7%, respectively.
  • the frequency of simultaneous mutations accounted for 38.2% of positive individual plants, and the system can efficiently edit multiple genes simultaneously in rice cells (Table 6).
  • Example 7 The use of miniOsU3/miniOsU6 to drive the CRISPR-MAD7 vector of the DR-sg sequence for multi-site knockout in rice
  • the DR-sg sequence strings in Example 5 were respectively driven by miniOsU3/miniOsU6 (SEQ ID NO.5).
  • This expression cassette was then ligated to the MAD7 expression cassette and placed within the LB and RB sequences of pCAMBIA.
  • the constructed vector was transformed into Agrobacterium EHA105, and then the callus of rice variety Nanjing 46 was infected by this strain. After 3 days of co-cultivation, the culture was resumed at 30°C for 4 days, and the transformed callus was transferred to a culture medium containing hygromycin. The medium was selected, and after 28-30 days of culture, it was transferred to a differentiation medium containing hygromycin to regenerate plants.
  • the regenerated plants were sampled to extract DNA, Taqman detected MAD7-positive individual plants, and the target sites of positive individual plants were amplified and sequenced.
  • the results show that the system can efficiently edit multiple genes simultaneously in rice cells.
  • the mutation frequencies of the four genes are 44.4%, 94.4%, 92.2% and 90.0%, respectively, and the efficiency of simultaneous editing of two alleles is 22.2%, respectively. %, 93.3%, 86.7% and 87.8%, the frequency of simultaneous mutation of the four genes accounted for 42.2% of the positive individual plants.
  • This system can efficiently perform multi-gene editing in rice cells simultaneously (Table 7).
  • Example 8 The CRISPR-MAD7 vector driven by the Pol II type promoter and the HH-HDV tandem DR-sg sequence is used for multi-site knockout in rice
  • the DR-guide array driven by the OsU3 or miniOsU3/miniOsU6 promoter can achieve efficient knockout of four gene loci in rice, but both U3/U6 belong to Pol III type promoters, driving long chains The capacity is limited, and Pol III type promoters do not have condition-specific or tissue-specific activation capabilities, but Pol II-like promoters can effectively overcome the above defects.
  • the promoter maize Ubiquitin of Pol II type was constructed to drive the crRNA expression cassette, and two ribozymes hammerhead ribozyme (HH) and Hepatitis deltavirus ribozyme (HDV) with RNA self-cleavage activity were used to convert the transcribed DR-
  • the guide target sequence was isolated (sequence 6 SEQ ID NO.6), and the control of the OsU3 promoter-driven crRNA expression cassette was constructed, and the above two crRNA expression cassettes were cloned into the pCAMBIA expression vector with the original MAD7 expression cassette.
  • the constructed vector was transformed into Agrobacterium EHA105, and then the callus of rice variety Nanjing 46 was infected by this strain.
  • the culture was resumed at 30°C for 4 days, and the transformed callus was transferred to a culture medium containing hygromycin.
  • the medium was selected, and after 28-30 days of culture, it was transferred to a differentiation medium containing hygromycin to regenerate plants.
  • the regenerated plants were sampled to extract DNA, Taqman detected MAD7-positive individual plants, and the target sites of positive individual plants were amplified and sequenced.
  • the mutation frequencies of the four genes in the ZmUbi-HH-HDV system were 82.6%, 92.1%, 88.8% and 93.3%, respectively, and the simultaneous editing efficiencies of the two alleles were 62.8%, 84.3%, 84.3% and 92.1% (Table 8), the use effect even exceeds the editing efficiency of single point knockout, and the frequency of simultaneous mutation of four genes accounts for 77.5% of the positive individual plants.
  • This system can perform multi-gene editing in rice cells at the same time with very high efficiency (Table 8).
  • Example 9 Using the CRISPR-MAD7 (ncNLS) system to perform single-site knockout of endogenous genes in maize
  • CRISPR-MAD7 ncNLS
  • the editing site of Cpf1 in maize glossy2, Zm00001d002353
  • DR-sgRNA was artificially synthesized
  • the overlap PCR amplification product was passed through ApaI
  • the restriction site was connected to the pCAMBIA-CRISPR-MAD7 expression vector.
  • the constructed vector was transformed into Agrobacterium LBA4404, and then the immature embryos of maize variety B104 were infected by this strain. After 7 days of co-cultivation, the culture was resumed at 28°C for two weeks. The transformed immature embryos were transferred to the selection medium containing mannose and cultured.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une protéine de fusion MAD7-NLS ayant la structure suivante : B1-C-B2, B1-C, ou C-B2, C étant une protéine MAD7, et B1 et B2 étant indépendamment des séquences de signal de localisation nucléaire (NLS). L'invention concerne une construction d'acide nucléique pour l'édition dirigée sur site d'un génome végétal, la construction comprenant une première cassette d'expression, la première cassette d'expression comprenant un premier promoteur, une séquence nucléotidique de codage de la protéine de fusion MAD7-NLS, et un premier terminateur qui sont reliés en séquence. L'invention concerne également une application de la construction d'acide nucléique dans l'édition d'un gène végétal. L'inactivation d'un gène unique, l'inactivation de plusieurs gènes, ou la recombinaison homologue et l'insertion directionnelle d'un fragment exogène peuvent être effectuées simplement, commodément et efficacement au niveau d'un site de génome végétal prédéterminé à l'aide de la protéine MAD7.
PCT/CN2021/138184 2021-11-29 2021-12-15 Protéine de fusion mad7-nls, et construction d'acide nucléique pour édition dirigée sur site de génome végétal et application associée WO2023092731A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111427444.X 2021-11-29
CN202111427444.XA CN113846075A (zh) 2021-11-29 2021-11-29 Mad7-nls融合蛋白、用于植物基因组定点编辑的核酸构建物及其应用

Publications (1)

Publication Number Publication Date
WO2023092731A1 true WO2023092731A1 (fr) 2023-06-01

Family

ID=78982191

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/138184 WO2023092731A1 (fr) 2021-11-29 2021-12-15 Protéine de fusion mad7-nls, et construction d'acide nucléique pour édition dirigée sur site de génome végétal et application associée

Country Status (2)

Country Link
CN (1) CN113846075A (fr)
WO (1) WO2023092731A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113846075A (zh) * 2021-11-29 2021-12-28 科稷达隆(北京)生物技术有限公司 Mad7-nls融合蛋白、用于植物基因组定点编辑的核酸构建物及其应用
CN114438123B (zh) * 2022-03-07 2024-04-02 中量大黄山高质量发展研究院有限公司 一种双子叶植物多基因编辑载体及其构建方法

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105821073A (zh) * 2015-01-27 2016-08-03 中国科学院遗传与发育生物学研究所 一种通过基因瞬时表达对整株植物定点改造的方法
CN108130342A (zh) * 2016-12-01 2018-06-08 中国科学院上海生命科学研究院 基于Cpf1的植物基因组定点编辑方法
CN109750062A (zh) * 2019-03-12 2019-05-14 湖南杂交水稻研究中心 一种水稻育种方法
CN111621515A (zh) * 2020-05-14 2020-09-04 中国计量大学 一种增强CRISPR/Cas9系统基因编辑效率的方法
CN112251419A (zh) * 2019-11-07 2021-01-22 青岛清原化合物有限公司 一种在生物体内产生新突变的方法及应用
WO2021074191A1 (fr) * 2019-10-14 2021-04-22 KWS SAAT SE & Co. KGaA Nucléase mad7 dans des plantes et élargissement de sa capacité de reconnaissance de pam
WO2021081384A1 (fr) * 2019-10-25 2021-04-29 Greenvenus, Llc Nucléases synthétiques
US20210130838A1 (en) * 2019-11-05 2021-05-06 University Of Maryland, College Park SYSTEMS AND METHODS FOR PLANT GENOME EDITING USING CAS 12a ORTHOLOGS
CN113846075A (zh) * 2021-11-29 2021-12-28 科稷达隆(北京)生物技术有限公司 Mad7-nls融合蛋白、用于植物基因组定点编辑的核酸构建物及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2022103603A (ru) * 2017-06-23 2022-03-11 Инскрипта, Инк. Направляемые нуклеиновыми кислотами нуклеазы
US10011849B1 (en) * 2017-06-23 2018-07-03 Inscripta, Inc. Nucleic acid-guided nucleases
CN114096667A (zh) * 2019-07-08 2022-02-25 因思科瑞普特公司 经由LexA-Rad51融合蛋白增加核酸指导的细胞编辑

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105821073A (zh) * 2015-01-27 2016-08-03 中国科学院遗传与发育生物学研究所 一种通过基因瞬时表达对整株植物定点改造的方法
CN108130342A (zh) * 2016-12-01 2018-06-08 中国科学院上海生命科学研究院 基于Cpf1的植物基因组定点编辑方法
CN109750062A (zh) * 2019-03-12 2019-05-14 湖南杂交水稻研究中心 一种水稻育种方法
WO2021074191A1 (fr) * 2019-10-14 2021-04-22 KWS SAAT SE & Co. KGaA Nucléase mad7 dans des plantes et élargissement de sa capacité de reconnaissance de pam
WO2021081384A1 (fr) * 2019-10-25 2021-04-29 Greenvenus, Llc Nucléases synthétiques
US20210130838A1 (en) * 2019-11-05 2021-05-06 University Of Maryland, College Park SYSTEMS AND METHODS FOR PLANT GENOME EDITING USING CAS 12a ORTHOLOGS
CN112251419A (zh) * 2019-11-07 2021-01-22 青岛清原化合物有限公司 一种在生物体内产生新突变的方法及应用
CN111621515A (zh) * 2020-05-14 2020-09-04 中国计量大学 一种增强CRISPR/Cas9系统基因编辑效率的方法
CN113846075A (zh) * 2021-11-29 2021-12-28 科稷达隆(北京)生物技术有限公司 Mad7-nls融合蛋白、用于植物基因组定点编辑的核酸构建物及其应用

Also Published As

Publication number Publication date
CN113846075A (zh) 2021-12-28

Similar Documents

Publication Publication Date Title
CN108130342B (zh) 基于Cpf1的植物基因组定点编辑方法
JP6505599B2 (ja) 遺伝子標的化および形質スタッキングのための特別設計の導入遺伝子組み込みプラットフォーム（ｅｔｉｐ）
EP0131623B1 (fr) Genes chimeriques appropries a l'expression dans des cellules vegetales
WO2019207274A1 (fr) Remplacement de gène dans des plantes
JP2020508046A (ja) ゲノム編集システムおよび方法
JP4611307B2 (ja) Ｄｎａクローニングベクタープラスミドおよびその使用法
CN110157726B (zh) 植物基因组定点替换的方法
EP2796558A1 (fr) Ciblage génique amélioré et molécule porteuse d'acide nucléique, en particulier pour utilisation dans des plantes
JP2018527920A (ja) 部位特異的ヌクレオチド置換によりグリホサート耐性イネを取得するための方法
WO2023092731A1 (fr) Protéine de fusion mad7-nls, et construction d'acide nucléique pour édition dirigée sur site de génome végétal et application associée
JP2015500648A (ja) 所定の標的核酸配列を修飾するための組成物及び方法
WO2021185358A1 (fr) Procédé d'amélioration de la transformation génétique de plantes et de l'efficacité d'édition génomique
US20230348869A1 (en) Mad7 nuclease in plants and expanding its pam recognition capability
WO2018098935A1 (fr) Vecteur pour substitution de base dirigée du génome d'une plante
JPS61502166A (ja) 植物細胞の形質転換のための改良された方法及びベクタ−
WO2021175289A1 (fr) Procédé et système d'édition de génome multiplex
JP2022511508A (ja) ゲノム編集による遺伝子サイレンシング
WO2019205939A1 (fr) Procédé de recombinaison spécifique à un site de plante à médiation par répétition
CN116286742B (zh) CasD蛋白、CRISPR/CasD基因编辑系统及其在植物基因编辑中的应用
US20220033833A1 (en) Compositions and methods for transferring biomolecules to wounded cells
US20050066386A1 (en) Method of modifying genome in higher plant
WO2018082611A1 (fr) Construction d'acide nucléique exprimant un gène exogène dans des cellules végétales et utilisation associée
CN108424911B (zh) 种子特异性双向启动子及其应用
CN112522299A (zh) 一种利用OsTNC1基因突变获得分蘖增加的水稻的方法
WO2019154285A1 (fr) Combinaison de réactifs sans marqueur pour édition génique, et application de celle-ci

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21965452

Country of ref document: EP

Kind code of ref document: A1