WO2018103686A1 - 叶绿体基因组编辑方法 - Google Patents

叶绿体基因组编辑方法 Download PDF

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WO2018103686A1
WO2018103686A1 PCT/CN2017/114962 CN2017114962W WO2018103686A1 WO 2018103686 A1 WO2018103686 A1 WO 2018103686A1 CN 2017114962 W CN2017114962 W CN 2017114962W WO 2018103686 A1 WO2018103686 A1 WO 2018103686A1
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nucleic acid
acid construct
formula
vector
chloroplast
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French (fr)
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朱健康
王东
华凯
刘志红
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中国科学院上海生命科学研究院
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    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • 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)

Definitions

  • the present invention relates to the field of biotechnology, and in particular to RNA-directed plant chloroplast genome editing.
  • the existing chloroplast genome editing technology mainly relies on gene gun technology. Exogenous DNA is inserted into the chloroplast genome of plants by homologous recombination, and stable genetic chloroplast transgenic plants can only be obtained in a few plants. In addition, chloroplast transformation based on homologous recombination does not cause double-stranded DNA disruption on the chloroplast genome.
  • CRISPR clustered regular interspaced short palindromic repeats
  • nucleic acid construct is provided, the nucleic acid construct being selected from the group consisting of:
  • X1 is a promoter element
  • X2 is a chloroplast localization signal peptide element
  • X3 is a nuclease element
  • X4 is a no- or marker gene element
  • X5 is a terminator
  • Y1 is a promoter element
  • Y2 is an ncRNA element
  • Y3 is a no- or marker gene element
  • Y4 is a no- or RNA-cleaving enzyme element
  • Ya and Yb are each independently a no- or RNA-cleaving enzyme recognition element
  • Y5 is a sgRNA element
  • Y6 is a terminator
  • a construct comprising a construct of formula I and a construct of formula II.
  • the structures of Formula I and Formula II are in the 5' to 3' direction.
  • the X1 is selected from the group consisting of: 35S, UBQ.
  • the X2 is selected from the group consisting of: infA, RbcS.
  • the X4 is selected from the group consisting of GFP, YFP, RFP.
  • the X5 is a Nos terminator.
  • said Y1 is a 35S promoter.
  • the Y3 is selected from the group consisting of GFP, YFP, RFP.
  • the Ya is a Csy4 recognition sequence.
  • the Yb is a Csy4 recognition sequence.
  • the Y6 is a Nos terminator.
  • nuclease element X3 of the nucleic acid construct of Formula I is selected from the group consisting of:
  • ZFN zinc finger nuclease
  • TALENS transcriptional activator-like nuclease
  • the chloroplast localization signal peptide element X2 in the nucleic acid construct of Formula I is a chloroplast signal peptide infA.
  • the ncRNA element Y2 of the nucleic acid construct of Formula II is from a viroid, or a virus.
  • ncRNA element sequence of the nucleic acid construct of Formula II is as set forth in SEQ ID No.: 5.
  • RNA cleavage enzyme element Y4 of the nucleic acid construct of Formula II is Csy4.
  • the Csy4 sequence is set forth in SEQ ID No.: 6.
  • the sgRNA element Y5 of the nucleic acid construct of Formula II is spCas9 sgRNA.
  • the Y5 is as shown in SEQ ID No.: 8 or 9.
  • a vector or combination of vectors comprising the nucleic acid construct of the first aspect of the invention.
  • nucleic acid construct of Formula I and the nucleic acid construct of Formula II are located on different vectors.
  • nucleic acid construct of Formula I and the nucleic acid construct of Formula II are on the same vector.
  • a reagent combination comprising:
  • a method for editing a plant chloroplast gene comprising the steps of:
  • the method further includes:
  • step (iii) regenerating or cultivating the plant cells, plant tissues or plants in which the gene editing has been identified in step (ii).
  • the gene editing comprises gene knockout, site-specific insertion, gene replacement, or a combination thereof.
  • the targeted insertion comprises a site-directed insertion based on homologous recombination or non-homologous recombination end joining.
  • the gene editing comprises gene editing at one or more sites.
  • the introduction is introduced by Agrobacterium.
  • the introduction is by a gene gun.
  • the introduction is by microinjection, electroporation, sonication, and polyethylene glycol (PEG) mediated introduction.
  • PEG polyethylene glycol
  • the plant is selected from the group consisting of crops, trees, and flowers.
  • the plant is selected from the group consisting of a gramineous plant, a leguminous plant, and a cruciferous plant.
  • the plant comprises: Arabidopsis thaliana, wheat, barley, oats, corn, rice, sorghum, millet, soybean, peanut, tobacco, and tomato.
  • FIG. 1 shows GFP in mutant chloroplasts using CRISPR/Cas9
  • A shows the plasmid map used.
  • the vector used was the 35S promoter (P35S) and the nos terminator (Tnos).
  • a signal peptide of infA (TPinfA) and a non-coding RNA (ncRNA) on a viroid are used to bring Cas9 and sgRNA from Streptococcus pyogenes to the chloroplast.
  • FIG. B shows that the Cas9-GFP fusion protein (top panel) and GFP mRNA (lower panel) were transported into the chloroplast.
  • C shows mutation of the aadA16gfp gene on the chloroplast genome by CRISPR/Cas9.
  • Targets for two sgRNAs used in this experiment sgRNA1 and sgRNA2; driving the non-coding RNA-guided Csy4 and 2 with the Cas9 and 35S promoters driving the chloroplast signal peptide infA with two vectors: 35S promoter
  • the sgRNA2 of the Csy4 recognition site was transiently transformed into pMSK56 plants (bottom panel) and without transformation vector (top panel), and then a fluorescence microscope image was taken.
  • Plant transformation with sgRNA1 and sgRNA2 was named Cas9_PT1 and Cas9_PT2.
  • Protein extracted from the chloroplast of Nicotiana tabacum (tobacco) was used as a wild type control (WT).
  • the BiP protein located on the endoplasmic reticulum was used as a cytoplasmic marker; the Toc75 protein (position indicated by the arrow) located on the outer membrane of the chloroplast was used as a chloroplast marker.
  • the sample load per sample was 30 micrograms of protein. (Scale: 10 microns).
  • FIG. 2 shows the extraction of intact chloroplasts from 2 model plants.
  • the arrow marks the location of the Toc75-specific protein. Proteins were extracted from leaves and intact chloroplasts, respectively. For the leaves, the loading was 20 micrograms of protein; for the chloroplast, the loading was 30 micrograms of protein.
  • Figure 3 shows sequencing confirmed cyclized reverse transcription PCR products of sgRNA1 and sgRNA2. Sequence chromatography of cyclized reverse transcription PCR products of sgRNA1 and sgRNA2, each of which tested 3 independent clones.
  • Figure 4 shows the results of sequencing revealed that the inserted DNA fragment was found in aadA16GFP of pMSK56 plants transformed with sgRNA2 (Fig. 4A); PCR confirmed that only the chloroplast DNA of pMSK56 plants transformed with sgRNA2 was able to amplify the band (Fig. 4B).
  • Figure 5 shows the reduction of the expression of the aadA16gfp gene on the chloroplast genome using CRISPR/Cas9.
  • the amount of Cas9 (dCas9) expression without nuclease activity was detected in a CRISPR/Cas9 T1 transgenic plant of pMSK56 (aadA16gfpT1-1 to aadA16gfpT1-5).
  • the expression level of GFP was low in plants with high dCas9 protein expression.
  • the pMSK56 plant without CRISPR/Cas9 was used as a control, ie control.
  • Figure 6 shows the targeted reduction of the expression level of rpl33 in Arabidopsis.
  • A Plasmid map used in the experiment. PUBQ and TUBQ represent the promoter and terminator of AtUBQ1, respectively.
  • B The knockdown of the rpl33 gene on the chloroplast genome of Arabidopsis var2 plants was carried out by the CRISPR/Cas9 system. The two sgRNAs used in this experiment were targeted to the template strand (T) and the non-template strand (NT) of the rpl33 gene, respectively.
  • CK represents a var2 plant from a CRISPR/Cas9 system that has only been transformed without sgRNA.
  • Arabidopsis var2 T1 plants containing sgRNA targeting the rpl33 template strand and sgRNA targeting the rpl33 non-template strand CRISPR/Cas9 vector were designated 33T and 33NT, respectively.
  • the variegated phenotype caused by var2 was recovered and the plants that were not recovered were labeled as S and NS, respectively.
  • Figure 7 shows that targeted reduction of expression of rpl33 restores the var2 mutation-mediated leaf variegation phenotype. From left to right are Arabidopsis var2 plants transformed with no sgRNA, sgRNA targeting rpl33 template strand and sgRNA targeting rpl33 non-template strand CRISPR/Cas9 vector. The grey arrow indicates the plant that has restored the leaf variegated phenotype by targeting to reduce the expression of rpl33.
  • the inventors have constructed a chloroplast genome fixed-point editing system based on CRISPR technology, and a nucleic acid construct, vector or vector combination for chloroplast genome fixed-point editing, and a chloroplast genome fixed-point editing method.
  • the method of the present invention allows for simple and efficient gene knockout or homologous recombination and directed insertion of exogenous fragments at a predetermined plant genomic locus.
  • the present invention has been completed on this basis.
  • the nuclease of the CRISPR editing system of the present invention employs the Cas9 protein, which carries the Cas9 protein into the chloroplast through the chloroplast signal peptide infA; the sgRNA (eg, spCas9 sgRNA) is brought into the ncRNA using a non-coding RNA (ncRNA), such as a virus-like ncRNA.
  • ncRNA non-coding RNA
  • Chloroplasts thereby enabling knockout of genes on the chloroplast genome.
  • the method of the invention not only reduces the operation difficulty, but also improves the efficiency and accuracy of the chloroplast gene fixed-point editing, and reduces the operation cost.
  • the method of the invention is also effective for plant varieties that are unable to perform gene gun transformation.
  • containing includes “including”, “consisting essentially of”, “consisting essentially of”, and “consisting of.”
  • operably linked refers to a condition in which portions of a linear DNA sequence are capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, if the promoter controls the transcription of a sequence, then it is operably linked to the coding sequence.
  • the invention provides a nucleic acid construct selected from the group consisting of:
  • X1 is a promoter element
  • X2 is a chloroplast localization signal peptide element
  • X3 is a nuclease element
  • X4 is a no- or marker gene element
  • X5 is a terminator
  • Y1 is a promoter element
  • Y2 is an ncRNA element
  • Y3 is a no- or marker gene element
  • Y4 is a no- or RNA-cleaving enzyme element
  • Ya, Yb are no or RNA cleavage enzyme recognition elements
  • Y5 is a sgRNA element
  • Y6 is a terminator
  • a construct comprising a construct of formula I and a construct of formula II.
  • each of the above elements may be prepared by a conventional method (e.g., PCR method, artificial total synthesis), and then ligated by a conventional method to thereby form a nucleic acid construct of the present invention.
  • the enzymatic cleavage reaction can optionally be carried out, if necessary, prior to the ligation reaction.
  • nucleic acid constructs of the invention may be linear or cyclic.
  • the nucleic acid constructs of the invention may be single-stranded or double-stranded.
  • the nucleic acid construct of the present invention may be DNA, RNA, or DNA/RNA hybrid.
  • a "marker gene” refers to a gene used in a gene editing process to screen a cell for successful gene editing.
  • the marker gene to be used in the present application is not particularly limited, and includes various marker genes commonly used in the field of gene editing, and representative examples include (but not limited to): green fluorescent protein (GFP), yellow fluorescent protein (YFP), hygromycin resistance gene (Hyg), kanamycin resistance gene (NPTII), neomycin gene, or sputum Gene resistance gene.
  • 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 a plant, a microorganism (such as a bacterium, a virus) or an animal, or a synthetic or engineered promoter. Representative examples include, but are not limited to, the 35S promoter.
  • plant terminator refers to a terminator capable of stopping transcription in a plant cell.
  • the plant transcription terminator may be derived from a plant, a microorganism (such as a bacterium, a virus) or an animal, or a synthetic or engineered terminator. Representative examples include (but are not limited to): Nos terminator.
  • signal peptide refers to a short peptide chain that a newly synthesized protein transfers to a secretory pathway. Representative examples include, but are not limited to, the infA signal peptide.
  • nuclease element X3 of the nucleic acid construct of Formula I is selected from the group consisting of:
  • ZFN zinc finger nuclease
  • TALENS transcriptional activator-like nuclease
  • nuclease element refers to a nucleotide sequence that encodes a nuclease having cleavage activity.
  • the skilled artisan will recognize that because of the degeneracy of the codon, a large number of polynucleotide sequences can encode the same polypeptide. .
  • the skilled person will also recognize that different species have a certain preference for codons, may optimize the nuclease codons according to the needs of expression in different species, and these variants are all termed “nuclease elements”. "Specific coverage.
  • nuclease element specifically includes sequences of full length, substantially identical to the sequence of the Cas9 and/or Cpfl gene, and sequences encoding proteins that retain the function of the Cas9 and/or Cpfl protein.
  • nuclease element is the coding sequence for the Cas9 protein from Streptococcus pyogenes.
  • the chloroplast localization signal peptide element X2 of the nucleic acid construct of the formula I of the invention is a chloroplast signal peptide infA.
  • the ncRNA element Y2 of the nucleic acid construct of formula II of the invention is derived from a viroid.
  • sequence of the ncRNA element in the nucleic acid construct of the formula II of the present invention is shown in SEQ ID No.: 5.
  • RNA cleavage enzyme element Y4 of the nucleic acid construct of formula II of the invention is Csy4.
  • the Csy4 sequence of the present invention is shown as SEQ ID No.: 6.
  • the sgRNA element Y5 of the nucleic acid construct of Formula II of the invention is a spCas9 sgRNA.
  • the sgRNA sequence of the invention is set forth in SEQ ID No.: 8 or 9.
  • the invention also provides a vector or combination of vectors comprising a nucleic acid construct of the invention.
  • nucleic acid construct of Formula I of the invention and the nucleic acid construct of Formula II are on the same vector.
  • some elements are operably linked.
  • a promoter when operably linked to a coding sequence, it is meant that the promoter is capable of initiating transcription of the coding sequence.
  • the present invention also provides a reagent combination and kit comprising the above vector or carrier combination, which can be used in the plant chloroplast gene editing method of the present invention.
  • the invention also provides a method for editing a plant chloroplast gene, 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 invention can be used for improving various plants, especially for crops.
  • plant as used herein includes whole plants, plant organs (such as leaves, stems, roots, etc.), seeds and plant cells, and progeny thereof.
  • the type of plant which can be used in the method of the present invention is not particularly limited and generally includes any higher plant type which can be subjected to transformation techniques, including monocots, dicots and gymnosperms.
  • the Arabidopsis material used in the experiment was wild type Col-0. Col-0 seeds were sterilized with 5% sodium hypochlorite and sown on 1/2MS solid medium. The plates were treated at 4 ° C for three days and then placed in a light incubator (22 degrees, 16 hrs light / 8 hrs dark) for 10-14 days, then transplanted. In the nutrient soil, the greenhouse continues to be cultivated.
  • the tobacco used in the experiment was wild-type Nicotiana tabacum (tobacco) and chloroplast transgenic plant pMSK56 with Nicotiana tabacum as a receptor (pMSK56 chloroplast genome encoded aadAGFP fusion gene).
  • the pMSK56 transgenic plant is a conventional tobacco plant (Khan and Maliga, 1999). Seeds of Nicotiana tabacum and pMSK56 were sown in nutrient soil, placed in a greenhouse (26 degrees, 16 hrs light / 8 hrs) for about 10 days, and individual seedlings were transferred to nutrient soil. The same conditions were continued for about 3 weeks for transient transformation. experiment.
  • ncRNA capable of introducing RNA into the chloroplast in the experiment was referenced to the sequence (Gómez and Pallás, 2010), and was artificially synthesized and ligated into a commercially available pUC57 vector to obtain pUC57-ncRNA.
  • the ncRNA sequence, GFP coding sequence, and spsgRNA backbone were amplified from pUC57-ncRNA, pGWB505, and pCas9 (AtU6) vectors, respectively.
  • ncRNA-GFP and ncRNA-GFP-spsgRNA were ligated together by PCR, and PCR products were recovered.
  • the vector pCam1300-35S-ncRNA-GFP was obtained by digesting with XmaI, BamHI and ligating into the pCam1300-35S vector between the 35S promoter (SEQ ID No.: 1) and the NOS terminator (SEQ ID No.: 2). , pCam1300-35S-ncRNA-GFP-sgRNA.
  • the chloroplast localization signal infA (SEQ ID No.: 3) was amplified from the Arabidopsis cDNA library.
  • Cas9, GFP (SEQ ID No.: 4) coding sequences were amplified from vector pCas9 (AtU6), pGWB505, respectively.
  • InfA, Cas9, GFP three fragments were ligated into the 35S promoter of pCam1300-35S vector by Gibson assembly method.
  • the pCam1300-35S-infA-Cas9-GFP vector was obtained between the NOS terminator and the NOS terminator.
  • the NOS terminator was amplified from the pCam1300-35S vector using a primer with a stop codon at the 5' end of the upstream primer, and the PCR product was recovered and double digested with BamHI and EcoRI.
  • the GFP gene and the NOS terminator were further excised from the pCam1300-35S-infA-Cas9-GFP vector with BamHI and EcoRI, and the fragment was recovered and ligated to the recovered NOS terminator to obtain a pCam1300-35S-infA-Cas9 vector.
  • the Csy4 gene (SEQ ID No.: 6) having a 3x Frag tag at the C-terminus was artificially ligated into the pUC57 vector to obtain pUC57-Csy4.
  • the ncRNA sequence (SEQ ID No.: 5), the Csy4-3 ⁇ Frag coding region, and the sgRNA backbone were amplified from pUC57-ncRNA, pUC57-Csy4, pCas9 (AtU6) vectors, respectively.
  • pUC57-ncRNA pUC57-Csy4, pCas9 (AtU6) vectors, respectively.
  • pGas9 AdU6 vectors, respectively.
  • a 20 nt Csy4 recognition site was added downstream (SEQ ID No.: 7). Two additional AarI restriction sites were added downstream of the Csy4-3 ⁇ Frag coding region.
  • ncRNA sequence, Csy4-3 ⁇ Frag coding region, sgRNA backbone was ligated together by multiplex PCR, and the PCR product was recovered, and the 35S promoter and NOS terminator which were digested with XmaI and BamHI and ligated into pCam1300-35S vector were used. between.
  • the Arabidopsis Col-0 genome was used as a template to amplify the UBQ1 gene (AT3G52590) promoter UBQpro and the terminator UBQTer, respectively.
  • the ncRNA-Csy4-sgRNA fragment was amplified from the pCam1300-35S-ncRNA-Csy4-sgRNA vector, and UBQpro, ncRNA-Csy4-spsgRNA and UBQTer were ligated together by PCR, and the product was recovered by PCR. After digestion with XmaI, it was ligated into the pCambia1300 vector to obtain a pCam1300-UBQ-spsgRNA vector.
  • the 35S-infA-Cas9-NOS fragment was amplified from the pCam1300-35S-infA-Cas9 vector, and the PCR product was recovered and ligated into the pCam1300-UBQ-sgRNA vector by Gibson assembly to obtain pCam1300-UBQ-sgRNA-35S-Cas9. .
  • sgRNA is loaded into the corresponding target vector
  • the vector pCam1300-35S-ncRNA-Csy4-sgRNA and pCam1300-UBQ-sgRNA-35S-Cas9 were digested with AarI enzyme for 4 hours, electrophoresed, and the gel was recovered, and then ligated to the short-stranded DNA fragment formed by annealing.
  • the vector pCam1300-35S-ncRNA-GFP, pCam1300-35S-ncRNA-GFP-sgRNA, pCam1300-35S-infA-Cas9-GFP was transferred into the Agrobacterium GV3101 competent state by freeze-thaw method, and the Agrobacterium was cultured in the dark at 28 degrees for two days. The monoclonal was picked up in 5 ml of LB resistant medium (50 mg/L kanamycin, 25 mg/L rifampicin), incubated at 28 ° C, 240 rpm for 16 hours, and transferred to a new 5 ml LB at a ratio of 1:100.
  • LB resistant medium 50 mg/L kanamycin, 25 mg/L rifampicin
  • Sites in the aadA-GFP reporter gene that meet the 5'-N 20 NGG-3' sequence requirements were selected.
  • the corresponding sgRNA sequence was synthesized, ligated into AarI-digested pCam1300-35S-ncRNA-Csy4-spsgRNA vector.
  • the constructed vector was transferred into the Agrobacterium GV3101 competent state along with the empty pCam1300-35S-infA-Cas9 vector.
  • the vector expressing the p19 protein was also transferred into the Agrobacterium GV3101 competent state.
  • the cells were suspended with 10 mM MES pH 5.6, 10 mM MgCl 2 , 10 ⁇ m acetosyringone solution, and the OD 600 was adjusted to 1.5.
  • the Agrobacterium OD 600 expressing p19 was adjusted to 1.0.
  • the three Agrobacterium containing pCam1300-35S-ncRNA-Csy4-sgRNA, pCam1300-35S-infA-Cas9, p19 vector were mixed at a ratio of 1:1:1, and allowed to stand at room temperature for 2-3 hours, without a needle
  • a 1 ml medical syringe injects Agrobacterium into the back of a tobacco leaf that grows well for about 4 weeks. After 60-72 hours of culture, samples were taken to observe changes in GFP signal.
  • the appropriate sgRNA sequence was selected and loaded into the pCam1300-UBQ-sgRNA-35S-Cas9 vector as described above.
  • the corresponding vector was transferred into Agrobacterium GV3101.
  • the CO-0 of the robust flowering stage was selected for genetic transformation by the dip flower method, and the T 0 generation seeds were harvested after one month of normal care.
  • the T 0 seeds were sterilized with 5% sodium hypochlorite and screened on 1/2 MS plates containing 50 mg/L hygromycin.
  • the positive seedlings were transplanted into nutrient soil and placed in a greenhouse for further cultivation.
  • the suspension was placed on a 40/100% (v/v) gradient of Percoll (sigma) for 4 minutes at 40°C (40% (v/v) Percoll solution: 0.33 M sorbitol, 20 mM HEPES (pH 7.9), 5 mM MgCl 2 , 2.5 mM EDTA, 10 mM NaHCO 3 , 0.2% (w/v) BSA, 2 mM ascorbate, 40% (v/v) Percoll; 100% (v/v) Percoll solution: 0.33 M sorbitol, 20 mM HEPES (pH 7.9 ), 5 mM MgCl 2 , 2.5 mM EDTA, 10 mM NaHCO 3 , 0.2% (w/v) BSA, 2 mM ascorbate, 100% (v/v).
  • the protein was separated with 8% SDS-PAGE gel, and the separated protein was transferred to a PVDF (Millipore) membrane using a Bio-Rad instrument at a voltage of 105V. Blocked for 1 hour in 20 mM Tris-HCl (pH 8), 150 mM NaCl, 0.1% (v/v) Tween 20, 5% SKIM MILK POWDER.
  • the PVDF membrane was incubated for 1 hour. It was washed 4 times with 20 mM Tris-HCl (pH 8), 150 mM NaCl, 0.1% (v/v) Tween 20 for 15 minutes, 5 minutes, 5 minutes, 5 minutes, respectively. Tanon TM High-sig ECL Western Blotting Substrate after color developing X-ray film used.
  • the chloroplast DNA was extracted with the DNeasy Plant Maxi Kit (QIAGEN), and the extracted chloroplast DNA was subjected to mass detection to determine the absence of RNA contamination and the approximate molecular weight of the obtained DNA. Then use COVARIS S220 to interrupt the chloroplast DNA fragment selection to 450-600 bp, volume to 60 ul, add 40ul End Repair Mix using Illumina DNA Sample Preparation Kit, 30-degree treatment for 30 minutes, add 160ul AMPure XP Beads, and dilute to 17.5ul added 12.5ul A-Tailing Mix, 37 degrees 30 minutes, add 2ul DNA Adapter Index, 3ul Resuspension Buffer and 2.5ul Ligation Mix, 30 degree reaction for 30 minutes, add 5ul Stop Ligation Buffer and add 42ul AMPure XP Beads for purification. Then, the volume was adjusted to 10 ul, and the fragment size was measured with Qubit and the Agilent 2100 Bioanalyzer. Finally, it was sequenced using the Illumina HiSeq2500
  • Validation of chloroplast signal peptide infA and non-coding RNA in tobacco can bring Cas9-GFP fusion protein and GFP mRNA into chloroplast, respectively
  • the 35S promoter driven Cas9 and GFP fusion proteins with the chloroplast signal peptide infA were constructed (Fig. 1A); the 35S promoter driven GFP ligated with non-coding RNA was constructed.
  • the transient transformation of these two vectors in tobacco showed that the green fluorescence emitted by GFP and the red fluorescence emitted by chlorophyll co-localized in the chloroplast, indicating that the chloroplast signal peptide infA and non-coding RNA can respectively bind the Cas9-GFP fusion protein and GFP mRNA is introduced into the chloroplast (Fig. 1B).
  • the 35S promoter was constructed to drive non-coding RNA-directed sgRNAs with Csy4 and two Csy4 recognition sites, and then transiently transformed into tobacco. After Agrobacterium tumefaciens was transformed into tobacco for 3 days and a half, the chloroplast of tobacco was extracted. The chloroplasts of tobacco were well purified by Western blot (Fig. 2), and BiP and Toc75 were used as markers for cytoplasm and chloroplast, respectively.
  • sgRNA1 SEQ ID No.: 8
  • sgRNA2 SEQ ID No.: 9
  • the aadA16GFP gene is expressed in the chloroplast genome of pMSK56.
  • the aadA-GFP fusion protein encoded by this gene emits green fluorescence in the chloroplast, so each chloroplast has green fluorescence from aadA-GFP and red fluorescence from chlorophyll (Fig. 1C).
  • Fig. 1C red fluorescence from chlorophyll
  • the chloroplasts were extracted from the leaves of pMSK56 plants transformed with sgRNA2 in Example 3, and then chloroplast DNA was extracted with DNeasy Plant Maxi Kit (Qiagen), and then the mutation of the reporter gene aadA16GFP was detected by a second generation sequencing technique.
  • the sequencing results revealed that the inserted DNA fragment was inserted into aadA16GFP of pMSK56 plant transformed with sgRNA2 (Fig. 4A, the sequence in the box is the inserted DNA fragment), and then the primer was designed according to the corresponding sequence to verify the result, only in the transformation with sgRNA2
  • the chloroplast DNA of the pMSK56 plant was able to amplify the band (Fig. 4B).
  • the 35S promoter-driven Cas9 (dCas9:D10A and H840A) without nuclease activity with the chloroplast signal peptide infA, and the 35S promoter driving non-coding RNA-directed sgRNAs with Csy4 and two Csy4 recognition sites (
  • the aadA16gfp gene was targeted to transform pMSK56 plants.
  • the inventors used western blot and real-time quantitative PCR to detect the expression levels of dCas9 and gfp genes, respectively, in five T1 transgenic plants.
  • the inventors found that the expression level of dCas9 protein was negatively correlated with the expression level of GFP (Fig. 5), that is, the expression level of GFP was low in plants with high expression of dCas9 protein.
  • Targeted knockdown was performed on the chloroplast genome of Arabidopsis var2 plants using CRISPR, thereby restoring its leaf variegated phenotype.

Abstract

提供了叶绿体基因组编辑方法。具体地，提供了基于CRISPR技术的植物基因组定点编辑的核酸构建物、载体或载体组合、以及植物基因组定点编辑方法。所述核酸构建物包括式I核酸构建物和/或式II核酸构建物，所述式I核酸构建物包含叶绿体定位信号肽-核酸酶表达盒，所述的式II核酸构建物包含ncRNA-sgRNA表达盒。利用该方法，可以将核酸酶和对应的sgRNA导入叶绿体，从而在预定的叶绿体基因组位点，简便而高效地进行基因敲除、或者同源重组和外源片段的定向插入。该方法可用于从叶绿体基因组水平改良作物的性状。

Description

叶绿体基因组编辑方法 技术领域
本发明涉及生物技术领域，具体地，涉及RNA引导的植物叶绿体基因组编辑。
背景技术
现有的叶绿体基因组编辑技术主要依赖于基因枪技术，外源DNA通过同源重组的方法插入到植物的叶绿体基因组上，并且只能在少数的植物里获得稳定遗传的叶绿体转基因植株。另外基于同源重组的叶绿体转化没法在叶绿体基因组上造成双链DNA破坏。最新出现的CRISPR(clustered regularly interspaced short palindromic repeats)技术被广泛的应用于植物细胞核基因组编辑，它通过RNA把DNA核酸内切酶例如Cas9带到与RNA序列匹配的DNA区域进行切割。
迄今为止还没有基于CRISPR技术对植物的叶绿体基因组进行编辑，所以本领域迫切需要开发简便高效的叶绿体基因组编辑方法。
发明内容
本发明的目的在于提供一种高通用性、高特异性、高效地对植物叶绿体进行基因编辑的方法。
在本发明的第一方面，提供了一种核酸构建物，所述的核酸构建物选自下组：
(1)式I核酸构建物：
X1-X2-X3-X4-X5      (I)
式中，
X1为启动子元件；
X2为叶绿体定位信号肽元件；
X3为核酸酶元件；
X4为无或标记基因元件；
X5为终止子；
(2)式II核酸构建物：
Y1-Y2-Y3-Y4-Ya-Y5-Yb-Y6       (II)
式中，
Y1为启动子元件；
Y2为ncRNA元件；
Y3为无或标记基因元件；
Y4为无或RNA切割酶元件；
Ya和Yb各自独立地为无或RNA切割酶识别元件；
Y5为sgRNA元件；
Y6为终止子；
(3)包括式I构建物和式II构建物的构建物。
在另一优选例中，所述的式I和式II结构为5’至3’方向。
在另一优选例中，所述X1选自：35S、UBQ。
在另一优选例中，所述X2选自：infA、RbcS。
在另一优选例中，所述X4选自下组：GFP、YFP、RFP。
在另一优选例中，所述X5为Nos终止子。
在另一优选例中，所述Y1为35S启动子。
在另一优选例中，所述Y3选自下组：GFP、YFP、RFP。
在另一优选例中，所述Ya为Csy4识别序列。
在另一优选例中，所述Yb为Csy4识别序列。
在另一优选例中，所述Y6为Nos终止子。
在另一优选例中，所述式I核酸构建物中核酸酶元件X3选自下组：
(1)Cas9；
(2)Cpf1；
(3)锌指核酸酶(ZFN)；
(3)转录活化剂样核酸酶(TALENS)；
(4)巨核酸酶(meganuclease)；
或其组合。
在另一优选例中，所述式I核酸构建物中叶绿体定位信号肽元件X2为叶绿体信号肽infA。
在另一优选例中，所述式II核酸构建物中ncRNA元件Y2来自类病毒、或病毒。
在另一优选例中，所述式II核酸构建物中ncRNA元件序列如SEQ ID No.:5所示。
在另一优选例中，所述式II核酸构建物中RNA切割酶元件Y4为Csy4。
在另一优选例中，所述Csy4序列如SEQ ID No.:6所示。
在另一优选例中，所述式II核酸构建物中sgRNA元件Y5为spCas9 sgRNA。
在另一优选例中，所述Y5如SEQ ID No.:8或9所示。
在本发明的第二方面，提供了一种载体或载体组合，所述载体或载体组合含有本发明的第一方面所述的核酸构建物。
在另一优选例中，所述的式I核酸构建物和式II核酸构建物位于不同载体上。
在另一优选例中，所述的式I核酸构建物和式II核酸构建物位于同一载体上。
在本发明的第三方面，提供了一种试剂组合，包括：
(i)本发明的第二方面所述的载体或载体组合。
在本发明的第四方面，提供了一种植物叶绿体基因编辑方法，包括步骤：
(i)将(a)本发明的第二方面所述的载体或载体组合以及(b)任选的供体核酸片段，导入植物细胞、植物组织或植物(植株)，从而在所述植物细胞、植物组织或植物中产生基因编辑；和
(ii)任选地，对发生所述基因编辑的植物细胞或植物进行检测、筛选或鉴定。
在另一优选例中，所述的方法还包括：
(iii)对步骤(ii)中经鉴定发生了所述基因编辑的植物细胞、植物组织或植物进行再生或培养。
在另一优选例中，所述的基因编辑包括基因敲除、定点插入、基因置换、或其组合。
在另一优选例中，所述的定向插入包括基于同源重组或非同源重组末端连接的定点插入。
在另一优选例中，所述的基因编辑包括单位点或多位点的基因编辑。
在另一优选例中，所述导入为通过农杆菌导入。
在另一优选例中，所述导入为通过基因枪导入。
在另一优选例中，所述导入为通过显微注射法、电击法、超声波法和聚乙二 醇(PEG)介导法导入。
在另一优选例中，所述的植物选自下组：农作物、树木、花卉。
在另一优选例中，所述的植物选自下组：禾本科植物、豆科植物和十字花科植物。
在另一优选例中，所述的植物包括：拟南芥、小麦、大麦、燕麦、玉米、水稻、高粱、粟、大豆、花生、烟草和番茄。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示用CRISPR/Cas9突变叶绿体里的GFP；
(A)显示使用的质粒图谱。所述载体用的是35S启动子(P35S)和nos终止子(Tnos)。infA的信号肽(TPinfA)和类病毒上的一段非编码RNA(ncRNA)被用来将来自Streptococcus pyogenes(化脓链球菌)的Cas9和sgRNA带到叶绿体。
(B)显示Cas9-GFP融合蛋白(上图)和GFP的mRNA(下图)被运进叶绿体。
(C)显示通过CRISPR/Cas9对叶绿体基因组上的aadA16gfp基因进行突变。本实验用到的2个sgRNA的靶点(sgRNA1和sgRNA2)；用2个载体：35S启动子驱动带有叶绿体信号肽infA的Cas9和35S启动子驱动非编码RNA引导的带有Csy4和2个Csy4识别位点的sgRNA2瞬时转化pMSK56植株(下图)和没有转化载体的(上图)，然后拍摄荧光显微镜图片。
(D)显示转化有CRISPR/Cas9 pMSK56植株的叶绿体里GFP蛋白数量比没有转化的植株少。植物转化有sgRNA1和sgRNA2被命名为Cas9_PT1和Cas9_PT2。从Nicotiana tabacum(烟草)叶绿体里提取的蛋白被用作野生型对照(WT)。位于内质网上的BiP蛋白被当作细胞质标记；位于叶绿体外膜上的Toc75蛋白(用箭头指出的位置)被当作叶绿体标记。每个样本的上样量为30微克蛋白。(标尺:10微米)。
图2显示从2个模式植物里提取完整的叶绿体。烟草(A)和拟南芥(B)的Western blot结果。箭头标记出Toc75特异蛋白的位置。分别从叶片和完整的叶绿体里提取蛋白。对于叶片，上样量为20微克蛋白；对于叶绿体，上样量 为30微克蛋白。
图3显示测序验证sgRNA1和sgRNA2的环化逆转录PCR产物。sgRNA1和sgRNA2的环化逆转录PCR产物的序列色谱，每个测了3个独立的克隆。
图4显示测序结果发现在转化有sgRNA2的pMSK56植物的aadA16GFP里有插入DNA片段(图4A)；PCR验证只有在转化有sgRNA2的pMSK56植物的叶绿体DNA能够扩增到条带(图4B)。
图5显示运用CRISPR/Cas9降低叶绿体基因组上aadA16gfp基因的表达。在pMSK56的CRISPR/Cas9 T1代转基因植株里(aadA16gfpT1-1～aadA16gfpT1-5)检测没有核酸酶活性的Cas9(dCas9)表达量。dCas9蛋白表达量高的植株里GFP的表达量低。没有CRISPR/Cas9的pMSK56植株作为对照，即control。
图6显示在拟南芥里靶向降低rpl33的表达量。(A)实验里的用到的质粒图谱。PUBQ和TUBQ分别代表AtUBQ1的启动子和终止子。(B)通过CRISPR/Cas9系统对拟南芥var2植株叶绿体基因组上的rpl33基因进行knockdown。本实验用到的2个sgRNAs分别靶向rpl33基因的模板链(T)和非模板链(NT)。CK代表得是从只转化了没有sgRNA的CRISPR/Cas9系统的var2植株。含有sgRNA靶向rpl33模板链和sgRNA靶向rpl33非模板链CRISPR/Cas9载体的拟南芥var2 T1代植株被分别标记为33T和33NT。var2引起的叶杂色表型被恢复和没有被恢复的植株被分别标记为S和NS。
图7显示靶向降低rpl33的表达可以恢复var2突变介导的叶杂色表型。从左到右分别是是转化不含有sgRNA，sgRNA靶向rpl33模板链和sgRNA靶向rpl33非模板链CRISPR/Cas9载体的拟南芥var2植株。灰色箭头指出得是通过靶向降低rpl33的表达已经恢复叶杂色表型的植株。
具体实施方式
本发明人经过广泛而深入的研究、首次构建了一种基于CRISPR技术的叶绿体基因组定点编辑系统、以及用于叶绿体基因组定点编辑的核酸构建物、载体或载体组合、以及叶绿体基因组定点编辑方法。本发明的方法可以在预定的植物基因组位点、简便而高效地进行基因敲除或者同源重组和外源片段的定向插入。在此基础上完成了本发明。
典型地，本发明CRISPR编辑系统中核酸酶采用Cas9蛋白,通过叶绿体信号肽infA将Cas9蛋白带进叶绿体；采用非编码RNA(ncRNA),例如来自类病毒的 ncRNA将sgRNA(例如spCas9 sgRNA)带进叶绿体，从而实现对叶绿体基因组上基因的敲除。与传统的基因枪技术相比，本发明方法不仅降低了操作难度，提高了叶绿体基因定点编辑的效率和精确性，而且降低了操作成本。此外，本发明方法还可有效用于那些无法进行基因枪转化的植物品种。
术语
除非另外定义、本文使用的所有技术和科学术语的意义与本发明所属领域普通技术人员通常所理解的相同。本文中述及的所有出版物和其他参考文献都通过引用纳入本文。
如本文所用、所述的“含有”、“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”。
如本文所用、术语“操作性相连”或“可操作地连于”指这样一种状况、即线性DNA序列的某些部分能够调节或控制同一线性DNA序列其它部分的活性。例如、如果启动子控制序列的转录、那么它就是可操作地连于编码序列。
基于核酸酶的植物基因组定点编辑的核酸构建物和方法
本发明提供了一种核酸构建物，所述的核酸构建物选自：
(1)式I核酸构建物：
X1-X2-X3-X4-X5       (I)
式中，
X1为启动子元件；
X2为叶绿体定位信号肽元件；
X3为核酸酶元件；
X4为无或标记基因元件；
X5为终止子；
(2)式II核酸构建物：
Y1-Y2-Y3-Y4-Ya-Y5-Yb-Y6       (II)
式中，
Y1为启动子元件；
Y2为ncRNA元件；
Y3为无或标记基因元件；
Y4为无或RNA切割酶元件；
Ya、Yb为无或RNA切割酶识别元件；
Y5为sgRNA元件；
Y6为终止子；
(3)包括式I构建物和式II构建物的构建物。
在上述结构式中，“-”表示键。
在本发明中、上述的各元件可用常规方法(如PCR法、人工全合成)制备、然后用常规方法进行连接、从而形成本发明所述的核酸构建物。如需要、在连接反应之前、可以任选地进行酶切反应。
此外、本发明的所述核酸构建物可以是线性的、也可以是环状的。本发明的所述核酸构建物可以是单链的、也可以是双链的。本发明的所述核酸构建物可以是DNA、也可以是RNA、或DNA/RNA杂合。
如本文所用、“标记基因”指基因编辑过程中用来筛选基因编辑成功的细胞的基因，可用于本申请的标记基因没有特别限制、包括基因编辑领域常用的各种标记基因，代表性例子包括(但并不限于)：绿色荧光蛋白(GFP)、黄色荧光蛋白(YFP)、潮霉素抗性基因(Hyg)、卡那霉素抗性基因(NPTII)、新霉素基因、或嘌呤霉素抗性基因。
如本文所用，术语“植物启动子”指能够在植物细胞中启动核酸转录的核酸序列。该植物启动子可以是来源于植物、微生物(如细菌、病毒)或动物等，或者是人工合成或改造过的启动子。代表性的例子包括(但并不限于)：35S启动子。
如本文所用，术语“植物终止子”指能够在植物细胞中可使转录停止的终止子。该植物转录终止子可以是来源于植物、微生物(如细菌、病毒)或动物等，或者是人工合成或改造过的终止子。代表性的例子包括(但并不限于)：Nos终止子。
如本文所用，术语“信号肽”是指新合成的蛋白质向分泌通路转移的短肽链。代表性的例子包括(但并不限于)：infA信号肽。
如本文所用，典型地，所述式I核酸构建物中核酸酶元件X3选自下组：
(1)Cas9；
(2)Cpf1；
(3)锌指核酸酶(ZFN)；
(3)转录活化剂样核酸酶(TALENS)；
(4)巨核酸酶(meganuclease)；
或其组合。
如本文所用、术语“核酸酶元件”指编码具有切割活性的核酸酶的核苷酸序列。在插入的多聚核苷酸序列被转录和翻译从而产生功能性核酸酶的情况下、技术人员会认识到、因为密码子的简并性、有大量多聚核苷酸序列可以编码相同的多肽。另外、技术人员也会认识到不同物种对于密码子具有一定的偏好性、可能会根据在不同物种中表达的需要、会对核酸酶的密码子进行优化、这些变异体都被术语“核酸酶元件”所具体涵盖。
此外，术语“核酸酶元件”特定地包括了全长的、与Cas9和/或Cpf1基因序列基本相同的序列、以及编码出保留Cas9和/或Cpf1蛋白功能的蛋白质的序列。
典型地，术语“核酸酶元件”为来自化脓链球菌(Streptococcus pyogenes)的Cas9蛋白的编码序列。
优选地，本发明所述式I核酸构建物中叶绿体定位信号肽元件X2为叶绿体信号肽infA。
典型地，本发明所述式II核酸构建物中ncRNA元件Y2来自类病毒。
优选地，本发明所述式II核酸构建物中ncRNA元件序列如SEQ ID No.:5所示。
典型地，本发明所述式II核酸构建物中RNA切割酶元件Y4为Csy4。
优选地，本发明所述Csy4序列如SEQ ID No.:6所示。
典型地，本发明所述式II核酸构建物中sgRNA元件Y5为spCas9 sgRNA。
优选地，本发明所述sgRNA序列如SEQ ID No.:8或9所示。
本发明还提供了一种载体或载体组合，所述载体或载体组合含有本发明所述的核酸构建物。
优选地，本发明所述的式I核酸构建物和式II核酸构建物位于同一载体上。
在本发明的核酸构建物和/或载体中、一些元件之间是可操作连接的。例如当启动子与编码序列可操作连接时、指所述启动子能够启动所述编码序列的转录。
本发明还提供了含有上述载体或载体组合的试剂组合以及试剂盒，它们可用于本发明的植物叶绿体基因编辑方法。
本发明还提供了一种植物叶绿体基因编辑方法，包括步骤：
(i)将(a)本发明所述的载体或载体组合以及(b)任选的供体核酸片段，导入植物细胞、植物组织或植物，从而在所述植物细胞、植物组织或植物中产生基因编辑； 和
(ii)任选地，对发生所述基因编辑的植物细胞或植物进行检测、筛选或鉴定。
在本发明中、所述的基因编辑包括基因敲除、定点插入、基因置换、或其组合。
本发明的植物基因编辑方法、可用于改良各类植物、尤其是对农作物进行改良。
如本文所用、术语“植物”包括全植株、植物器官(如叶、茎、根等)、种子和植物细胞以及它们的子代。可用于本发明方法的植物的种类没有特别限制、一般包括任何可进行转化技术的高等植物类型、包括单子叶、双子叶植物和裸子植物。
本发明的主要优点在于：
(1)首次利用CRISPR技术在叶绿体基因组上造成了有效的双链DNA的切割以及基因编辑；
(2)有效降低了叶绿体基因编辑的成本和难度；
(3)大幅拓宽了叶绿体基因编辑物种的广度。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。本发明中所涉及的实验材料如无特殊说明均可从市售渠道获得。
材料
实验中所用的拟南芥材料为野生型Col-0。Col-0种子用5％次氯酸钠消毒后播种于1/2MS固体培养基上，平板4℃处理三天后放入光照培养箱(22度，16hrs光照/8hrs黑暗)培养10-14天，然后移苗到营养土中，温室继续培养。
实验所用烟草为野生型Nicotiana tabacum(烟草)和以Nicotiana tabacum为受体的叶绿体转基因植株pMSK56(pMSK56叶绿体基因组上编码有aadAGFP融合基因)。pMSK56转基因植株为一种常规的烟草植株(Khan and Maliga,1999)。 Nicotiana tabacum及pMSK56的种子播于营养土中，放置于温室(26度，16hrs光照/8hrs)培养10天左右，单株幼苗移载到营养土中，相同条件继续培养3周左右用于瞬时转化实验。
方法
目标位点设计
对于SpCas9蛋白，其PAM序列要求为5’-NGG-3’，因此叶绿体基因组上合适的靶位点为5’-N 20NGG-3’。
载体构建
pCam1300-35S-ncRNA-GFP，pCam1300-35S-ncRNA-GFP-spsgRNA载体的构建
实验中能将RNA导入到叶绿体中的ncRNA参照了(Gómez and Pallás,2010)文中的序列，人工合成后连入到市售的pUC57载体中得到pUC57-ncRNA。ncRNA序列，GFP编码序列，spsgRNA骨架分别从pUC57-ncRNA，pGWB505，pCas9(AtU6)载体扩增得到，ncRNA-GFP，ncRNA-GFP-spsgRNA可以通过叠连PCR的方法依次连接在一起，PCR产物回收，用XmaI，BamHI消化后连入到pCam1300-35S载体中的35S启动子(SEQ ID No.:1)和NOS终止子(SEQ ID No.:2)之间得到载体pCam1300-35S-ncRNA-GFP，pCam1300-35S-ncRNA-GFP-sgRNA。
核苷酸序列SEQ ID No.:1
Figure PCTCN2017114962-appb-000001
Figure PCTCN2017114962-appb-000002
核苷酸序列SEQ ID No.:2
Figure PCTCN2017114962-appb-000003
pCam1300-35S-infA-Cas9-GFP载体的构建
叶绿体定位信号infA(SEQ ID No.:3)从拟南芥cDNA文库中扩增获得。Cas9，GFP(SEQ ID No.:4)编码序列分别从载体pCas9(AtU6)，pGWB505上扩增得到，infA，Cas9,GFP三个片段通过Gibson assembly的方法连入pCam1300-35S载体的35S启动子和NOS终止子之间得到pCam1300-35S-infA-Cas9-GFP载体。
核苷酸序列SEQ ID No.:3
Figure PCTCN2017114962-appb-000004
核苷酸序列SEQ ID No.:4
Figure PCTCN2017114962-appb-000005
Figure PCTCN2017114962-appb-000006
pCam1300-35S-infA-Cas9载体的构建
用上游引物5’端带有终止密码子的引物从pCam1300-35S载体上扩增出NOS终止子，PCR产物回收后用BamHI和EcoRI进行双酶切。再将GFP基因和NOS终止子从pCam1300-35S-infA-Cas9-GFP载体用BamHI和EcoRI切除，片段回收后与上述回收的NOS终止子连接得到pCam1300-35S-infA-Cas9载体。
pCam1300-35S-ncRNA-Csy4-sgRNA载体的构建
C端带有3×Frag标签的Csy4基因(SEQ ID No.:6)由人工合成连入到pUC57载体中得到pUC57-Csy4。ncRNA序列(SEQ ID No.:5)，Csy4-3×Frag编码区，sgRNA骨架分别从pUC57-ncRNA，pUC57-Csy4，pCas9(AtU6)载体扩增得到。sgRNA骨架上，下游加有20nt的Csy4识别位点(SEQ ID No.:7)。另外两个AarI酶切位点加在Csy4-3×Frag编码区下游。ncRNA序列，Csy4-3×Frag编码区，sgRNA骨架用叠连PCR的方法依次连接在一起，PCR产物回收，用XmaI，BamHI消化后连入到pCam1300-35S载体中的35S启动子和NOS终止子之间。
核苷酸序列SEQ ID No.:5
Figure PCTCN2017114962-appb-000007
核苷酸序列SEQ ID No.:6
Figure PCTCN2017114962-appb-000008
Figure PCTCN2017114962-appb-000009
核苷酸序列SEQ ID No.:7
Figure PCTCN2017114962-appb-000010
pCam1300-UBQ-spsgRNA-35S-Cas9载体的构建
以拟南芥Col-0基因组为模板分别扩增出UBQ1基因(AT3G52590)的启动子UBQpro和终止子UBQTer。从pCam1300-35S-ncRNA-Csy4-sgRNA载体中扩增出ncRNA-Csy4-sgRNA片段，以叠连PCR的方法将UBQpro、ncRNA-Csy4-spsgRNA、UBQTer三段依次连接在一起，PCR回收产物用HindIII和XmaI消化后连入pCambia1300载体之中，得到pCam1300-UBQ-spsgRNA载体。从pCam1300-35S-infA-Cas9载体中扩增出35S-infA-Cas9-NOS片段，PCR产物回收后利用Gibson assembly的方法连入pCam1300-UBQ-sgRNA载体中得到pCam1300-UBQ-sgRNA-35S-Cas9。
sgRNA装入相应目标载体
对于载体pCam1300-35S-ncRNA-Csy4-sgRNA和pCam1300-UBQ-sgRNA-35S-Cas9，选取5’-N 20NGG-3’作为靶标序列。分别合成上游引物GCAGN 20和下游引物AAACN 20，上下游引物退火后形成带有4nt接头的短双链DNA片段。载体pCam1300-35S-ncRNA-Csy4-sgRNA和pCam1300-UBQ-sgRNA-35S-Cas9用AarI酶消化4小时后电泳，切胶回收，然后与退火形成的短链DNA片段连接。
蛋白瞬时定位实验
载体pCam1300-35S-ncRNA-GFP，pCam1300-35S-ncRNA-GFP-sgRNA，pCam1300-35S-infA-Cas9-GFP通过冻融法转入农杆菌GV3101感受态中，农杆菌28度黑暗培养两天，挑取单克隆于5ml LB抗性培养基(50mg/L卡那霉素，25mg/L利福平)中，28℃，240rpm培养16小时，以1：100的比例转接到 新的5ml LB抗性培养基中(培养基中另加有2μM的乙酰丁香酮，10mM MES，pH5.6)，28℃，240rpm培养过夜到OD 600＝3。4000rpm，10min收集菌体，用10mM MES pH 5.6，10mM MgCl 2,10μm乙酰丁香酮溶液将菌体悬起，调OD 600到0.6-0.8。室温静置2-3小时后，用不带针头的1ml医用注射器将农杆菌注射到4周左右生长状态良好烟草叶片背面。培养60-72小时后取样观察蛋白定位。
pMSK56报告基因aadA-GFP敲除
选取aadA-GFP报告基因中符合5’-N 20NGG-3’序列要求的位点。合成相应的sgRNA序列，退火后连入AarI酶切过的pCam1300-35S-ncRNA-Csy4-spsgRNA载体中。构建好的载体连同空载pCam1300-35S-infA-Cas9载体转入农杆菌GV3101感受态中。另外，为了抑制RNAi引起的转录后沉默现象，将表达p19蛋白的载体也转入农杆菌GV3101感受态中。农杆菌28℃黑暗培养两天，挑取单克隆于5ml LB抗性培养基(50mg/L卡那霉素，25mg/L利福平)中，28℃，240rpm培养16小时，以1：100的比例转接到新的5ml LB抗性培养基中(培养基中另加有2μM的乙酰丁香酮，10mM MES，pH 5.6)，28度，240rpm培养过夜到OD 600＝3。4000rpm，10min收集菌体，用10mM MES pH 5.6，10mM MgCl 2,10μm乙酰丁香酮溶液将菌体悬起，调OD 600到1.5。表达p19的农杆菌OD 600调到1.0。将含pCam1300-35S-ncRNA-Csy4-sgRNA，pCam1300-35S-infA-Cas9，p19载体的三种农杆菌以1：1：1的比例混合，室温静置2-3小时后，用不带针头的1ml医用注射器将农杆菌注射到4周左右生长状态良好烟草叶片背面。培养60-72小时后取样观察GFP信号的变化。
拟南芥转化与筛选
选取合适的sgRNA序列依前述方法装入pCam1300-UBQ-sgRNA-35S-Cas9载体中。相应载体转入农杆菌GV3101中。选取健壮的盛花期的Col-0以浸花法进行遗传转化，正常护理一个月后收种得T 0代种子。T 0代种子用5％次氯酸钠消毒后在含50mg/L潮霉素的1/2MS平板上筛选，阳性苗移栽到营养土中放置于温室继续培养。
叶绿体提取与Western blot检测
在冷库中将植物组织在预冷的0.33M sorbitol，20mM tricine(pH 8.4)， 5mM EGTA，5mM EDTA，10mM NaHCO 3，0.1％(w/v)BSA中按照4ml每mg的比例用Polytron
Figure PCTCN2017114962-appb-000011
匀浆器打碎。用3层纱布过滤一次，再用一层Miracloth过滤一次。在4度离心机中2000g离心2分钟，用0.33M sorbitol，20mM HEPES(pH 7.9)，5mM MgCl 2，2.5mM EDTA，10mM NaHCO 3，0.1％(w/v)BSA，2mM ascorbate将沉淀悬浮起来。将悬浮液置于40/100％(v/v)的Percoll(sigma)梯度上4度离心30分钟(40％(v/v)Percoll溶液：0.33M sorbitol，20mM HEPES(pH 7.9)，5mM MgCl 2，2.5mM EDTA，10mM NaHCO 3，0.2％(w/v)BSA，2mM ascorbate，40％(v/v)Percoll；100％(v/v)Percoll溶液：0.33M sorbitol，20mM HEPES(pH 7.9)，5mM MgCl 2，2.5mM EDTA，10mM NaHCO 3，0.2％(w/v)BSA，2mM ascorbate，100％(v/v)。收集位于两层Percoll梯度中间的完整叶绿体，加入10ml 0.33M sorbitol，20mM HEPES(pH 7.9)，5mM MgCl 2，2.5mM EDTA，10mM NaHCO 3，0.1％(w/v)BSA，2mM ascorbate，2000g 4度离心2分钟。得到的沉淀即为叶绿体。
在提好的叶绿体中加入200μl 50mM Tris-Hcl(pH6.8)，2％(w/v)SDS，10％(v/v)甘油，1％(v/v)巯基乙醇。在95度煮5分钟，用最大的速度在4度离心15分钟，转移上清到一个新管中。每μl上清中加入2μl 150mM Tris-Hcl(pH6.8)，6％(w/v)SDS，0.3％(w/v)溴酚蓝，30％(v/v)甘油，3％(v/v)巯基乙醇。用8％的SDS-PAGE胶分离蛋白，将分离后的蛋白在105V的电压下用Bio-Rad仪器转移到PVDF(Millipore)膜上。在20mM Tris-HCl(pH 8)，150mM NaCl，0.1％(V/V)Tween 20，5％SKIM MILK POWDER，中封闭1小时。在20mM Tris-HCl(pH 8)，150mM NaCl，0.1％(V/V)Tween 20，2％SKIM MILK POWDER中将一抗(anti-TOC75，anti-Bip(agrisera)，anti-GFP(abcam))用合适的比例稀释之后，将PVDF膜孵育1小时。用20mM Tris-HCl(pH 8)，150mM NaCl，0.1％(V/V)Tween 20洗4次，时间分别为15分钟，5分钟，5分钟，5分钟。在20mM Tris-HCl(pH 8)，150mM NaCl，0.1％(V/V)Tween 20，2％SKIM MILK POWDER中将二抗(abmart)用合适的比例稀释之后，将PVDF膜孵育1小时。用20mM Tris-HCl(pH 8)，150mM NaCl，0.1％(V/V)Tween 20洗4次，时间分别为15分钟，5分钟，5分钟，5分钟。用Tanon TM High-sig ECL Western Blotting Substrate显色之后用X光片显影。
叶绿体DNA提取和测序
用DNeasy Plant Maxi Kit(QIAGEN)提取叶绿体DNA，跑胶对提取的叶绿 体DNA进行质量检测确定没有RNA污染和获得的DNA分子量大致范围。然后使用COVARIS S220把叶绿体DNA片段选择打断至450-600bp，定容体积到60ul，使用Illumina DNA Sample Preparation Kit加入40ul End Repair Mix,30度处理30分钟,加入160ul AMPure XP Beads纯化，定容到17.5ul加入12.5ul A-Tailing Mix,37度30分钟，加入2ul DNA Adapter Index,3ul Resuspension Buffer和2.5ul Ligation Mix,30度反应30分钟,加入5ul Stop Ligation Buffer混匀后加入42ul AMPure XP Beads纯化，然后定容至10ul,用Qubit定量与Agilent 2100 Bioanalyzer检测片段大小。最后用illumina HiSeq2500 Rapid模式PE250测序。
实施例1
在烟草里验证叶绿体信号肽infA和非编码RNA能分别把Cas9-GFP融合蛋白和GFP mRNA带进叶绿体
构建35S启动子驱动的带有叶绿体信号肽infA的Cas9和GFP融合蛋白(图1A)；构建35S启动子驱动的连接有非编码RNA的GFP。把这2个载体在烟草里进行瞬时转化验证，发现由GFP发出的绿色荧光和叶绿素发出的红色荧光在叶绿体里共定位，证明叶绿体信号肽infA和非编码RNA能分别把Cas9-GFP融合蛋白和GFP mRNA带进叶绿体(图1B)。
实施例2
在烟草里验证非编码RNA能引导带有Csy4和2个Csy4识别位点的sgRNA到叶绿体里
构建35S启动子驱动非编码RNA引导的带有Csy4和2个Csy4识别位点的sgRNA，然后通过瞬时转化烟草。农杆菌转化烟草3天半后，提取烟草的叶绿体。通过Western blot证明烟草的叶绿体被很好的纯化获得(图2)，BiP和Toc75分别作为细胞质和叶绿体的标记。
接下来对获得的叶绿体提取RNA，然后通过环化逆转录PCR(circularized reverse transcription PCR，cRT-PCR)对sgRNA进行检测。测序结果：在检测的6个克隆中(各3个克隆)，在叶绿体里均分别存在被Csy4切割后形成的成熟sgRNA(图3)。
实施例3
利用CRISPR在pMSK56植株的叶绿体基因组上进行双链DNA破坏
构建35S启动子驱动的带有叶绿体信号肽infA的Cas9，然后把它与实验步骤2里构建的35S启动子驱动非编码RNA引导的带有Csy4和2个Csy4识别位点的sgRNA(图1A)。
在本实施例中，分别选取了2个不同的靶点：sgRNA1(SEQ ID No.:8)和sgRNA2(SEQ ID No.:9)共转化pMSK56植株。在pMSK56植株的叶绿体基因组上有aadA16GFP基因，这个基因编码的aadA-GFP融合蛋白在叶绿体里发绿色荧光，所以每个叶绿体都有来自aadA-GFP的绿色荧光和来自叶绿素的红色荧光(图1C)。而被这2个载体共转化后的pMSK56植株里，观察到了只有红色荧光而没有绿色荧光的叶绿体(图1C)，这就表明叶绿体上aadA16GFP可能被突变了，接下来提取了野生型烟草、pMSK56、以及分别转化了35S启动子驱动的带有叶绿体信号肽infA的Cas9和35S启动子驱动非编码RNA引导的带有Csy4和2个Csy4识别位点的sgRNA的叶绿体。Western blot证明，转化有Cas9和sgRNA的pMSK56植株里的aadA-GFP蛋白的数量减少了。
核苷酸序列SEQ ID No.:8
Figure PCTCN2017114962-appb-000012
核苷酸序列SEQ ID No.:9
Figure PCTCN2017114962-appb-000013
实施例4
利用二代测序技术检测CRISPR在叶绿体基因组上的突变
对实施例3中转化有sgRNA2的pMSK56植物的叶片提取叶绿体，然后用DNeasy Plant Maxi Kit(Qiagen)提取叶绿体DNA，然后用二代测序技术检测在报告基因aadA16GFP的突变。测序结果发现在转化有sgRNA2的pMSK56植物的aadA16GFP里有插入DNA片段(图4A,方框内的序列为插入DNA片段)，然后按照相应的序列设计引物PCR验证了这个结果，只有在转化有sgRNA2的pMSK56 植物的叶绿体DNA能够扩增到条带(图4B)。
实施例5
利用CRISPR在pMSK56植株的叶绿体基因组上进行靶向knockdown
构建35S启动子驱动的带有叶绿体信号肽infA的没有核酸酶活性的Cas9(dCas9:D10A和H840A)，和35S启动子驱动非编码RNA引导的带有Csy4和2个Csy4识别位点的sgRNA(靶向aadA16gfp基因)转化pMSK56植株。发明人在5个T1代转基因单株里分别运用western blot和实时定量PCR分别检测了dCas9和gfp基因的表达量。发明人发现dCas9蛋白的表达量与GFP的表达量呈现负相关(图5)，即dCas9蛋白表达量高的植株里GFP的表达量低。
实施例6
利用CRISPR在拟南芥var2植株的叶绿体基因组上进行靶向knockdown，从而恢复它的叶杂色表型。
构建UBQ启动子驱动的带有叶绿体信号肽infA的没有核酸酶活性的Cas9(dCas9)，和35S启动子驱动非编码RNA引导的带有Csy4和2个Csy4识别位点的sgRNA转化拟南芥var2植株(图6A)。sgRNA靶向的基因为叶绿体基因组上的rpl33(图6B)。发明人在T1代转基因植株里发现靶向降低rpl33的表达可以恢复var2突变介导的叶杂色表型(图7,表1)，然后对这些表型恢复和没有恢复的植物提取RNA进行实时定量PCR检测。发现在rpl33的表达量在这2类植物里都有降低(图6C)，表达量较低的植株可以恢复var2突变介导的叶杂色表型。
表1.var2突变体靶向敲低rpl33后叶杂色表型统计
Figure PCTCN2017114962-appb-000014
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
参考文献
Gómez,G.,and Pallás,V.(2010).Noncoding RNA Mediated Traffic of Foreign mRNA into Chloroplasts Reveals a Novel Signaling Mechanism in Plants.PloS one 5,e12269.
Haurwitz,R.E.,Jinek,M.,Wiedenheft,B.,Zhou,K.,and Doudna,J.A.(2010).Sequence-and Structure-Specific RNA Processing by a CRISPR Endonuclease.Science 329,1355-1358.
Khan,M.S.,and Maliga,P.(1999).Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants.Nat Biotech 17,910-915.

Claims (10)

  1. 一种核酸构建物，其特征在于，所述的核酸构建物选自下组：
    (1)式I核酸构建物：
    X1-X2-X3-X4-X5  (I)
    式中，
    X1为启动子元件；
    X2为叶绿体定位信号肽元件；
    X3为核酸酶元件；
    X4为无或标记基因元件；
    X5为终止子；
    (2)式II核酸构建物：
    Y1-Y2-Y3-Y4-Ya-Y5-Yb-Y6  (II)
    式中，
    Y1为启动子元件；
    Y2为ncRNA元件；
    Y3为无或标记基因元件；
    Y4为无或RNA切割酶元件；
    Ya和Yb各自独立地为无或RNA切割酶识别元件；
    Y5为sgRNA元件；
    Y6为终止子；
    (3)包括式I构建物和式II构建物的构建物。
  2. 如权利要求1所述的核酸构建物，其特征在于，所述式I核酸构建物中核酸酶元件X3选自下组：
    (1)Cas9；
    (2)Cpf1；
    (3)锌指核酸酶；
    (3)转录活化剂样核酸酶；
    (4)巨核酸酶；
    或其组合。
  3. 如权利要求1所述的核酸构建物，其特征在于，所述式I核酸构建物中叶 绿体定位信号肽元件X2为叶绿体信号肽infA。
  4. 如权利要求1所述的核酸构建物，其特征在于，所述式II核酸构建物中ncRNA元件Y2来自类病毒。
  5. 如权利要求1所述的核酸构建物，其特征在于，所述式II核酸构建物中RNA切割酶元件Y4为Csy4。
  6. 如权利要求1所述的核酸构建物，其特征在于，所述式II核酸构建物中sgRNA元件Y5为spCas9 sgRNA。
  7. 一种载体或载体组合，其特征在于，所述载体或载体组合含有权利要求1-6中任一所述的核酸构建物。
  8. 如权利要求7所述的载体或载体组合，其特征在于，所述的式I核酸构建物和式II核酸构建物位于同一载体上。
  9. 一种试剂组合，其特征在于，包括：(i)权利要求7所述的载体或载体组合。
  10. 一种植物叶绿体基因编辑方法，其特征在于，包括步骤：
    (i)将(a)权利要求7所述的载体或载体组合以及(b)任选的供体核酸片段，导入植物细胞、植物组织或植物，从而在所述植物细胞、植物组织或植物中产生基因编辑；和
    (ii)任选地，对发生所述基因编辑的植物细胞或植物进行检测、筛选或鉴定。
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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109456990A (zh) * 2018-10-24 2019-03-12 湖南杂交水稻研究中心 一种利用基因组编辑技术提高叶绿体遗传转化效率的方法
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
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US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
US11920140B2 (en) 2017-08-22 2024-03-05 Napigen, Inc. Organelle genome modification using polynucleotide guided endonuclease

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111254159B (zh) * 2018-11-30 2023-03-31 东北农业大学 一种大豆GmST1基因突变体植株及其制备方法
CN112280799B (zh) * 2020-10-22 2023-02-17 中国热带农业科学院橡胶研究所 利用CRISPR/Cas9系统对橡胶草或蒲公英基因定点突变的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103981215A (zh) * 2014-05-23 2014-08-13 安徽省农业科学院水稻研究所 一种用于基因工程的骨干质粒载体及应用
CN103981216A (zh) * 2014-05-23 2014-08-13 安徽省农业科学院水稻研究所 一种骨干质粒载体及应用
CN105602935A (zh) * 2014-10-20 2016-05-25 聂凌云 一种新型线粒体基因组编辑工具

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2796558A1 (en) * 2013-04-23 2014-10-29 Rheinische Friedrich-Wilhelms-Universität Bonn Improved gene targeting and nucleic acid carrier molecule, in particular for use in plants
CA2913404C (en) * 2013-06-14 2023-10-03 Cellectis Methods for non-transgenic genome editing in plants
CN105829536A (zh) * 2013-08-22 2016-08-03 纳幕尔杜邦公司 用于在不掺入选择性转基因标记的情况下，在植物基因组中产生基因修饰的方法，以及用于这种方法的组合物
US20160237455A1 (en) * 2013-09-27 2016-08-18 Editas Medicine, Inc. Crispr-related methods and compositions

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103981215A (zh) * 2014-05-23 2014-08-13 安徽省农业科学院水稻研究所 一种用于基因工程的骨干质粒载体及应用
CN103981216A (zh) * 2014-05-23 2014-08-13 安徽省农业科学院水稻研究所 一种骨干质粒载体及应用
CN105602935A (zh) * 2014-10-20 2016-05-25 聂凌云 一种新型线粒体基因组编辑工具

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHO, S.W.: "Targeted genome engineering in human cells with the Cas9 RNA- guided endonuclease", NATURE BIOTECHNOLOGY, vol. 31, no. 3, 29 January 2013 (2013-01-29), pages 230 - 232, XP002699850 *
CONG, LE: "Multiplex Genome Engineering Using CRISPR/Cas Systems", SCIENCE, vol. 339, 15 February 2013 (2013-02-15), pages 819 - 823, XP055400719 *
FRANCIS QUÉTIER: "The CRISPR-Cas9 technology: Closer to the ultimate toolkit for targeted genome editing", PLANT SCIENCE, vol. 242, 8 September 2015 (2015-09-08), pages 65 - 76, XP055613051 *
JINEK, M.: "RNA-programmed genome editing in human cells", ELIFE, vol. 2, 29 January 2013 (2013-01-29), pages e00471, XP002699851 *
MALI, P.: "RNA-Guided Human Genome Engineering via Cas9", SCIENCE, vol. 339, 15 February 2013 (2013-02-15), pages 823 - 826, XP055469277 *
VEJNAR, C.E.: "Optimization Strategies for the CRISPR -Cas9 Genome-Edi- ting System", COLD SPRING HARBOR PROTOCOLS, 3 October 2016 (2016-10-03) *

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