METHOD FOR CONDUCTING SITE-SPECIFIC MODIFICATION ON ENTIRE PLANT VIA GENE TRANSIENT EXPRESSION
20220411810 · 2022-12-29
Assignee
Inventors
Cpc classification
C12N2310/20
CHEMISTRY; METALLURGY
C12N9/22
CHEMISTRY; METALLURGY
C12N15/8241
CHEMISTRY; METALLURGY
C12N15/8201
CHEMISTRY; METALLURGY
C12N15/8213
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention discloses a method for site-directed modification of whole plant through gene transient expression. The method as provided for conducting site-directed modification to a target fragment of a target gene in a whole plant comprises the following steps: transiently expressing a sequence-specific nuclease in said plant, wherein a whole plant is used as the subject for transient expression, said sequence-specific nuclease targets and cleaves said target fragment, thereby the site-directed modification is achieved via the self DNA repairing of said plant. In the present invention, tissue culture is omitted by transient expression of the sequence-specific nuclease; mutation is obtained at whole plant level; the method is independent of the genotype and recipient, and thus can be applied to various varieties of various species; T1 mutants can be obtained directly and the mutation can be stable inherited; more importantly, the mutant plant as obtained is free of exogenous genes, and thus have higher bio-safety.
Claims
1-16. (canceled)
17. A method for conducting site-directed modification to a target fragment of a target gene in a whole plant, comprising: transiently expressing a sequence-specific nuclease in said plant, wherein a whole plant is used as the subject for transient expression, said sequence-specific nuclease targets and cleaves said target fragment, whereby the site-directed modification is achieved via the self DNA repairing of said plant, wherein the method for transiently expressing said site-directed nuclease in said plant comprises the following steps: a) delivering the sequence-specific nuclease or a genetic material for expressing the sequence-specific nuclease into a leaf of said plant, and b) growing the plant obtained in step a) in the absence of selection pressure, whereby the sequence-specific nuclease or the genetic material not integrated into the plant chromosome is degraded, wherein step b) does not comprise tissue culture, wherein said genetic material is a recombinant vector or a DNA linear fragment or an in vitro transcribed RNA; wherein when said part of plant is a leaf, the delivery is performed by injecting a solution of Agrobacterium tumefaciens carrying said recombinant vector or DNA linear fragment into the leaf.
18. The method according to claim 17, wherein the delivery is performed by injecting a solution of Agrobacterium tumefaciens carrying said recombinant vector or DNA linear fragment into the leaf.
19. The method of claim 17, wherein said sequence-specific nuclease is a CRISPR/Cas9 nuclease, a TALENs nuclease, a Zinc finger nuclease, or any nuclease that can achieve genome editing, wherein when the sequence-specific nuclease is a CRISPR/Cas9 nuclease, the genetic material is composed of a recombinant vector or DNA fragment capable of transcribing guide RNA and expressing Cas9 protein; a recombinant vector or DNA fragment capable of transcribing guide RNA and a recombinant vector or DNA fragment or RNA capable of expressing Cas9 protein; or a guide RNA and a recombinant vector or DNA fragment or RNA capable of expressing Cas9 protein, wherein the guide RNA is an RNA with a palindromic structure which is formed by partial base-pairing between crRNA and tracrRNA and the crRNA contains an RNA fragment that can complementarily bind to the target fragment; wherein when the sequence-specific nuclease is a TALENs nuclease, the genetic material is a recombinant vector or DNA fragment or RNA capable of expressing paired TALEN proteins; wherein the TALEN protein is composed of a DNA binding domain capable of recognizing and binding to the target site, and a Fok I domain; wherein when the sequence-specific nuclease is a Zinc finger nuclease, the genetic material is a recombinant vector DNA fragment or RNA capable of expressing paired ZFN proteins; and wherein the ZFN protein is composed of a DNA binding domain capable of recognizing and binding to the target site, and a Fok I domain.
20. The method of claim 17, wherein the site-directed modification is an insertion, deletion, and/or replacement mutation in the target fragment.
21. A method for making a transgene-free mutant plant comprising performing site-directed modification to a target fragment of a target gene in a plant of interest according to the method of claim 17 so as to obtain a plant in which a function of the target gene is lost and the genome of the plant is free of integrated exogenous genes.
22. The method of claim 17, wherein said plant is a plant of any genotype.
23. The method of claim 17, wherein the plant is selected from the group consisting of tobacco, Brassica oleracea, Fagopyrum tataricum, or Hevea brasiliensis.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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DETAILED EMBODIMENTS
[0051] The experimental methods used in the following Examples are all conventional methods, unless otherwise indicated.
[0052] The materials, reagents used in the following Examples are all commercially available, unless otherwise indicated.
[0053] Expression vector pZmU3-gRNA was disclosed in “Liang, Z. et al. Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas System. Journal of Genetics and Genomics.41:63-68, (2014)”.
[0054] Expression vectors pJIT163-Ubi-Cas9 was disclosed in “Wang, Y. et al. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology. 32, 947-951 (2014)”.
[0055] Expression vectors pHSN401 and pBUE411 were disclosed in “Xing, H. et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biology.14:327, (2014)”.
[0056] Maize variety HiII was disclosed in “Armstrong, C. L., Green, C. E. & Phillips, R. L. Development and availability of germplasm with high type II culture formation response. Maize Genet. Coop. News Lett. 65,92-93 (1991)”.
[0057] Maize variety B73 was disclosed in “Russell, W. A. Registration of B70 and B73 parental lines of maize. Crop Sci. 12, 721 (1972)”.
[0058] Maize variety Zheng58 was disclosed in “Zhang Falin, Breeding and application of a Maize inbred line Zheng58. Crop Journal, 2001(4):31-31”.
[0059] Arabidopsis thaliana ecotype Columbia was disclosed in “Koorneef, M. et al. Linkage map of Arabidopsis thaliana. Journal of Heredity. 74,265-272 (1983)”.
[0060] MS medium: 4.43 g/L MS salts (Sigma, M5524), 30 g/L sucrose, 3 g/L phytogel, pH 5.7, autoclaved at 121° C. for 20 min.
[0061] LB medium: 10 g/L Tryptone, 5 g/L Yeast Extract, 10 g/L NaCl pH7.0 (for solid LB medium, 15 g agar was added per liter liquid medium), autoclaved at 121° C. for 20 min.
[0062] Solutions used in the preparation and transformation of protoplast are shown in Tables 1-6.
TABLE-US-00001 TABLE 1 50 ml enzymolysis solution for Arabidopsis The amount Final added Concentration Cellulase R10 0.75 g 1.5% Macerozyme R10 0.15 g 0.3% mannitol 3.6434 g 0.4M 2-(N-Morpholino)ethanesulfonic acid 0.2132 g 20 mM KCl 0.07456 g 20 mM made up to 50 ml with double distilled water, pH adjusted to 5.7 with KOH; incubated in 55° C. water bath for 10 min, and cooled at room temperature before adding CaCl.sub.2 0.0735 g 10 mM BSA 0.05 g 0.1% filtered with a 0.45 μm filter
TABLE-US-00002 TABLE 2 50 ml enzymolysis solution for Maize The amount Final added Concentration Cellulase R10 0.75 g 1.5% Macerozyme R10 0.15 g 0.3% mannitol 5.4651 g 0.6M 2-(N-Morpholino)ethanesulfonic acid 0.1066 g 10 mM made up to 50 ml with double distilled water, pH adjusted to 5.7 with KOH; incubated in 55° C. water bath for 10 min, and cooled at room temperature before adding CaCl.sub.2 0.00735 g 1 mM BSA 0.05 g 0.1% filtered with a 0.45 μm filter
TABLE-US-00003 TABLE 3 500 ml W5 The amount Final added Concentration NaCl 4.5 g 154 mM CaCl.sub.2 9.189 g 125 mM KCl 0.1864 g 5 mM 2-(N-Morpholino)ethanesulfonic acid 0.4264 g 4 mM made up to 500 ml with double distilled water, pH adjusted to 5.7 with NaOH
TABLE-US-00004 TABLE 4 250 ml WI solution The amount Final added Concentration mannitol 27.324 g 0.6M KCl 0.07456 g 4 mM 2-(N-Morpholino)ethanesulfonic acid 0.2135 g 4 mM (200 mM) made up to 250 ml with double distilled water, pH adjusted to 5.7 with KOH
TABLE-US-00005 TABLE 5 10 ml MMG solution The amount Final added Concentration mannitol (0.8M) 5 ml 0.4M MgCl.sub.2 (1M) 0.15 ml 15 mM 2-(N-Morpholino)ethanesulfonic acid 0.2 ml 4 mM (200 mM) double distilled water Made up to 10 ml
TABLE-US-00006 TABLE 6 4 ml PEG solution The amount Final added Concentration PEG4000 1.6 g 40% mannitol (0.8M) 1 ml 0.2M CaCl.sub.2 (1M) 0.4 ml 0.1M double distilled water Made up to 4 ml
[0063] % in above Tables 1-6 indicates weight-volume percentage, g/100 ml.
[0064] Transformation of Agrobaterium tumefaciens: [0065] 1) Competent cells (stored at −80° C.) were thawed on ice, then 2 μg plasmid DNA was added and mixed; the mixture was placed on ice for 30 min; [0066] 2) the EP tube was submerged in liquid nitrogen for 1 min, and transferred quickly to a 37° C. water bath for thawing (2 min); [0067] 3) then 1 ml LB liquid medium was added and incubated at 28° C. for 4˜5h with shaking at a low speed (150 rpm); [0068] 4) bacteria cells were harvested by centrifuging at 10000 rpm for 30 s, the supernatant was discarded, and 100 μl resuspended bacteria cells were plated on the selection plates containing corresponding antibiotics. [0069] 5) plates were incubated upside down at 28° C. until white colonies (transformants) emerge.
EXAMPLE 1
Site-directed Editing of Maize Endogenous Gene ZmIPK via the Pollen Tube Approach and the Shoot Apex Regeneration Approach
[0070] I. Design of the Target Fragment: target-C1
TABLE-US-00007 Target-C1: (SEQ ID NO: 11) 5′-CCGAGCTCGACCACGCCGCCGAC-3′; (position 393-415 of the gene ZmIPK as shown in Genbank No. AY172635).
[0071] II. Preparation of pZmU3-gRNA Plasmid and pBUE411 Plasmid Containing C1 Site
[0072] C1 is the DNA sequence for the RNA that can complementarily bind to target-C1.
[0073] The following single-stranded oligonucleotides with sticky ends (underlined) were synthesized:
TABLE-US-00008 C1-1F: (SEQ ID NO: 7) 5′-AGCAGTCGGCGGCGTGGTCGAGCT-3′; C1-2F: (SEQ ID NO: 8) 5′-GGCGGTCGGCGGCGTGGTCGAGCT-3′; C1R: (SEQ ID NO: 12) 5′-AAACAGCTCGACCACGCCGCCGAC-3′.
[0074] Double-stranded DNA with sticky ends was formed through annealing between C1-1F/C1R, and inserted between the two BbsI restriction sites in pZmU3-gRNA plasmid, resulting in a pZmU3-gRNA plasmid containing C1 site. The positive plasmid was verified by sequencing. A recombinant plasmid, which was obtained by inserting the DNA fragment as shown in 5′-AGCAGTCGGCGGCGTGGTCGAGCT-3′ (SEQ ID NO: 7) in forward direction at the BbsI restriction site of pZmU3-gRNA plasmid, was positive, and designated as pZmU3-gRNA-C1.
[0075] Double-stranded DNA with sticky ends was formed through annealing between C1-2F/C1R, and inserted between the two BsaI restriction sites in pBUE411 plasmid, resulting in a pBUE411 plasmid containing C1 site. The positive plasmid was verified by sequencing. A recombinant plasmid, which was obtained by inserting the DNA fragment as shown in 5′-GGCGGTCGGCGGCGTGGTCGAGCT-3′ (SEQ ID NO: 8) in forward direction at the BsaI restriction site of pBUE411 plasmid, was positive, and designated as pBUE411-C1.
[0076] III. Delivering the gRNA:Cas9 System Into Maize Protoplast
[0077] The pJIT163-Ubi-Cas9 vector and the pZmU3-gRNA-C1 plasmid obtained in step II were introduced into the protoplast of maize protoplast. The specific process includes:
[0078] 1. Growth of Maize Seedling
[0079] Seeds of maize hybrid variety HiII and inbred lines B73 and Zheng58 were soaked in water overnight, and transferred to a plate containing absorbent paper (water added), treated under light condition for 3 days for germination. The geminated maize seeds were grown in soil at 24° C. for 10-11 days, resulting in maize seedlings.
[0080] 2. Isolation of Protoplast
[0081] 1) Tender leaves of maize were taken, and the middle part thereof was cut into 0.5-1 mm threads using a cutter blade, placed into 50 ml enzymolysis solution for 5 h of digestion (0.5 h enzymolysis in vacuum, then 4.5 h slow shaking at 10 rpm).
[0082] Note: The temperature during enzymolysis should be kept between 20-25° C., the reaction should be carried out in the dark; and the solution should be gently shaken after the reaction so as to release the protoplasts.
[0083] 2) the enzymolysis product was diluted by adding 30 ml of W5, and filtrated into a 50 ml round bottom centrifuge tube using a 75 μm Nylon filter membrane.
[0084] Note: The Nylon filter membrane should be submerged in 75% (volume percentage) ethanol, washed with water and then soaked in W5 for 2 min before use.
[0085] 3) 23° C., 150 g centrifugation for 3 min, and the supernatant was discarded.
[0086] 4) the pellet was suspended with 10 ml W5, centrifuged at 150 g for 3 min, and the supernatant was discarded.
[0087] 5) the protoplasts were suspended by adding a proper amount of MMG solution, placed on ice until transformation.
[0088] Note: The concentration of the protoplasts needs to be determined by microscopy (×100). The amount of protoplasts was 2×10.sup.5/ml to 1×10.sup.6/ml.
[0089] 3. Transformation of maize protoplast [0090] 1) 10 μg pJIT163-2NLSCas9 vector and 10 μg pZmU3-gRNA-C1 plasmid were added into a 2 ml centrifuge tube. 200 μl of the protoplast was added using a pipette and then mixed by gentle patting, kept still for 3-5 min. Then 220 μl of PEG4000 solution was added and mixed by gentle patting. Transformation was performed in dark for 15 min; [0091] 2) 880 μl W5 (room temperature) was added and mixed by reversing, 100 g centrifugation for 3 min, and the supernatant was discarded; [0092] 3) 1 ml WI solution was added and mixed by reversing, the content was gently transferred to a 6-well plate (with pre-added 1 ml WI solution), and then cultured at 23° C. overnight.
[0093] IV. Using PCR/RE Experiments to Analyze the Mutagenesis of Maize Endogenous Gene ZmIPK Using gRNA:Cas9 System
[0094] 48 hours after the transformation of maize protoplast, genome DNA was extracted, which was used as template for PCR/RE (Polymerase Chain Reaction/Restriction digestion) experiment analysis. At the same time, the protoplasts of wild-type maize variety Hi II were used as a control. PCR/RE analysis method is based on Shan, Q. et al. Rapid and efficient gene modification in rice and Brachypodium using TALENs. Molecular Plant (2013). Since the target fragment (positions 393-415 of Genbank No. AY172635) of maize endogenous gene ZmIPK (Genbank No. AY172635) contains the recognition sequence (5′-GAGCTC-3′) of restriction endonuclease SacI, and thus the restriction endonuclease SacI was used in the experiment for conducting the PCR/RE test. Primers used in the PCR amplification were:
TABLE-US-00009 ZmIPK-1F: (SEQ ID NO: 13) 5′-TCGCAGCCCCTGGCAGAGCAA-3′; ZmIPK-1R: (SEQ ID NO: 14) 5′-GAGACCTGGGAGAAGGAGACGGATCC-3′
[0095] The results of PCR/RE experiments can be seen in
[0096] V. Site-directed Editing of Maize Endogenous Gene ZmIpk via the Pollen Tube Approach
[0097] Cell-penetrating peptides (CPPs) are a class of short peptides which can carry macromolecules (including protein and nucleic acid) into the cells. Recent study shows that cell-penetrating peptides, when binding to DNA, can protect the DNA against enzymatic degradation. Therefore, cell-penetrating peptides are commonly used in the pollen tube approach so as to improve the efficiency. [0098] 1) Preparation of the DNA solution containing CPPs: solid powder CPPs (amino acid sequence: RKKRRQRRRRKKRRQRRR (SEQ ID NO: 15), synthesized by Shanghai Bio-engineering Co., Ltd.) were formulated into a 30 mg/ml stock solution with sterile water. CPPs were added into a mixture of pZmU3-gRNA-C1 plasmid and pJIT163-Ubi-Cas9 plasmid (the weight ratio of pZmU3-gRNA-C1 and pJIT163-Ubi-Cas9 in the mixture is 1:1) at a weight ratio of 1:1, such that the final concentrations of DNA and CPPs are 25-30 μg/ml (the final concentration of sum of the two plasmid is 25-30 μg/ml, the final concentration of CPPs is 25-30 μg/ml). [0099] 2) Strong maize plants (HiII, B73 and Zheng 58) in the field were selected as the recipient materials. After flowering, the stigmas of these plants were bagged to avoid cross or self-fertilization. The hand-pollinate was conducted at the right time. 18-21 hr post pollination, bags were removed, and filaments and bracts were cut, with a length of 2-3 cm from the top of the cob retained. The cut section of filaments is slightly lower than that of bracts, forming a small groove between filaments and bracts, in which 300-400 ul DNA solution from step 1) was dripped quickly with pipette. The filaments were immersed by DNA solution and the stigmas were bagged again. Each experiment was carried out in 40-50 corn cobs. After the grains mature, the corn cobs were harvested and dry individually. [0100] 3) The dried seeds were grown, and ZmIPK gene mutants were detected with the PCR/RE method (specific steps and the primers as used can be seen in IV) after germination.
[0101] Mutants were obtained via the pollen tube approach for maize plants of different genotypes. Detection results of some mutants are shown in
[0102] VI. Site-directed Editing of Maize Endogenous Gene ZmIPK via the Shoot Apex Regeneration Approach
[0103] 1. Preparation of the Maize Materials [0104] 1) Seeds of maize inbred line HiII were placed into a triangular flask, sterilized with 70% (v/v) alcohol for 5 min and 5% (v/v) sodium hypochlorite for 30 min, then washed in sterile water for 5 times. 1.5 volume of water was added and the flask was sealed and incubated at 28° C. for 4-6 h. [0105] 2) Second sterilization. The seeds were sterilized with 5% (v/v) sodium hypochlorite for 30 min, and then washed in sterile water for 5 times. [0106] 3) The sterilized seeds were placed on a sterilized plate with filter paper, incubated at 28° C. in dark for 3-4 days for germination. Germinated seeds with synchronous growth were transferred onto MS medium and cultured at 28° C. in dark for 3-4 days until the seedlings reached 4-5 cm.
[0107] 2. Regeneration of Maize Shoot Apex [0108] 1) Cutting the buds: the stem was cut transversely at 1.5-2 mm above the joints, exposing the bud inside the stem. Then the bud was cut in the middle longitudinally to 0.2 mm below the joints (or just through the joints). About 0.8mm root was retained. [0109] 2) pBUE411-C1 plasmid containing Cl was transformed into Agrobacterium competent cell AGL1. After verification by PCR and restriction digestion, a positive strain was used for infecting the plants. [0110] 3) Positive strain was plated onto LB solid medium, cultured at 28° C. in dark for 2 days. A few bacteria were scraped into 20 ml MS liquid medium, cultured at 28° C. to about OD.sub.600=0.8. Then 200 μM Acetosyringone was added. [0111] 4) The incised plants were placed in to a plate, with the incisions downward. The plate was placed slantingly (30-45° C.) into a Vacuum device; Agrobacterium solution was added to submerge the incisions so as to allow an infection of 20 min. During infection, evacuation was set for 10 min, with a pressure of 0.05 MP. [0112] 5) After infection, the plants were taken out from the Agrobacterium solution (excess Agrobacterium solution on the plants was removed using filter paper) and inserted into MS medium, cultured at 23° C. in the dark for 3 days. [0113] 6) After the co-culture, the materials were taken out and washed to remove the medium, and then grown into a pot (⅘ common soil, ⅕ vermiculite on top). After transplant, seedlings were cultured at 28° C. in dark for 2 days and then 7-10 days in light, and then grown under normal conditions until fructification. Maize seeds as obtained were grown and tested for the ZmIPK gene mutation via the PCR/RE method after germination.
[0114] The results indicate that mutations occurred in the target site of ZmIPK gene. Uncut bands were recovered for sequencing. The sequencing results indicate that insertion/deletion (indel) occurred in the ZmIPK gene.
[0115] VII. Determining whether pZmU3-gRNA-C1 and pJIT163-Ubi-Cas9 are present in the maize mutants obtained via the pollen tube approach
[0116] Two primer sets were designed according to the sequences of pZmU3-gRNA-C1 plasmid and pJIT163-Ubi-Cas9 plasmid, for amplifying the two plasmids respectively.
[0117] ZmU3-F/C1R located between ZmU3 and the target fragment:
TABLE-US-00010 ZmU3-F: (SEQ ID NO: 16) 5′-CTGCCAAGATCAACAGCAACCA-3′; C1R: (SEQ ID NO. 12) 5′-AAACAGCTCGACCACGCCGCCGAC-3′.
[0118] Theoretically, the amplified fragment should be about 322 bp, and the sequence should be positions 467-788 of SEQ ID NO:1. SEQ ID NO:1 is the sequence of pZmU3-gRNA-C1.
[0119] Cas9-1F/Cas9-1R located on the pJIT163-Ubi-Cas9 vector:
TABLE-US-00011 Cas9-1F: (SEQ ID NO: 17) 5′-CTTCCCAAGCATTCCCTCCTGT-3′; Cas9-1R: (SEQ ID NO: 18) 5′-CTTATGCCGTCCCATGACCTTC-3′
[0120] Theoretically, the amplified fragment should be about 744 bp, and the sequence should be positions 1573-2316 of SEQ ID NO:2. SEQ ID NO:2 is the sequence of Cas9 in pJIT163-Ubi-Cas9.
[0121] No target bands were amplified for all the plants (
[0122] VIII. Determining whether pBUE411-C1 is present in the maize mutants obtained via the shoot apex regeneration approach
[0123] Two primer sets were designed according to the sequence of pBUE411-C1 plasmid, for amplifying OsU3p and Cas9 respectively.
[0124] pBUE411-1F/C1R locate between OsU3p and the target fragment:
TABLE-US-00012 pBUE411-1F: (SEQ ID NO: 19) 5′-GACAGGCGTCTTCTACTGGTGCTAC-3′; C1R: (SEQ ID NO: 12) 5′-AAACAGCTCGACCACGCCGCCGAC-3′.
[0125] Theoretically, the amplified fragment should be about 289 bp, and the sequence should be positions174-462 of SEQ ID NO:3. SEQ ID NO:3 is the gRNA sequence of pBUE411-C1.
[0126] CAS9-2F/CAS9-2R locate in Cas9 region on the pBUE411-C1 vector:
TABLE-US-00013 CAS9-2F: (SEQ ID NO: 20) 5′-CTCCCTAAGCACTCGCTCCTGT-3′; CAS9-2R: (SEQ ID NO: 21) 5′-TTCTGCGTGGTCTGATTCTCCC-3′.
[0127] Theoretically, the amplified fragment should be about 794 bp, and the sequence should be positions 1639-2432 of SEQ ID NO:4. SEQ ID NO:4 is the Cas9 sequence of pHSN411-C1.
[0128] No target bands were amplified for all the plants, indicating that the present invention prevents the insertion or carrying of a transgene when performing site-directed modification to a plant, and the mutant as obtained have relatively high bio-safety.
EXAMPLE 2
Site-directed Editing of Arabidopsis endogenous Gene AtPTPA via the Inflorescence-dipping Approach
[0129] I. Design of the Target Fragment: target-C2
TABLE-US-00014 Target-C2: (SEQ ID NO: 22) 5′-CCGACGATATCCGCCGATTTCAC-3′; (position 351-373 of the gene AtPTPA as shown in Genbank No. AF360133).
[0130] II. Preparation of pHSN401 Plasmid Containing C2 Fragment
[0131] C2 is the DNA sequence for the RNA that can complementarily bind to target-C2.
[0132] The following single-stranded oligonucleotides with sticky ends (underlined) were synthesized:
TABLE-US-00015 C2F: (SEQ ID NO: 10) 5′-ATTGGTGAAATCGGCGGATATCGT-3′; C2R: (SEQ ID NO: 23) 5′-AAACACGATATCCGCCGATTTCAC-3′.
[0133] Double-stranded DNA with sticky ends was formed through oligonucleotide annealing, and inserted between the two BsaI restriction sites in pHSN401 plasmid, resulting in a pHSN401 plasmid containing C2 site. The positive plasmid was verified by sequencing. A recombinant plasmid, which was obtained by inserting the DNA fragment as shown in 5′-ATTGGTGAAATCGGCGGATATCGT-3′ (SEQ ID NO: 10) in forward direction at the BsaI restriction site of pHSN401 plasmid, was positive, and designated as pHSN401-C2.
[0134] III. Delivering the gRNA:Cas9 System Into Arabidopsis Protoplast
[0135] The pHSN401-C2 plasmid obtained in step II was introduced into the protoplasts of Arabidopsis ecotype Columbia. The specific process includes:
[0136] 1. Growth of Arabidopsis seedling [0137] 1) Seed treatment: Seeds of Arabidopsis ecotype Columbia were placed into a 1.5 mL tube and soaked in 75% (v/v) alcohol for 1 min and 10% (v/v) sodium hypochlorite for 15 min, then washed in sterile water for 5-6 times. [0138] 2) The sterilized seeds were plated individually onto MS medium with a micropipette. The plates were sealed and placed under 4° C., 3-4 days for vernalization. [0139] 3) After vernalization, the plates were transferred into an incubator, cultured under the following conditions: 25±2° C., illuminance 5500±300Lx, 12 h light/d. After 3 week growth, seedlings were transplanted. [0140] 4) The seedlings were transplanted into soil (peat soil: vermiculite: pearlite=1:1:1) carefully, covered by a film for 3-4 days, and then cultured under 21° C., 6300±300Lx.
[0141] 2. Isolation of Protoplast [0142] 1) Tender leaves of Arabidopsis ecotype Columbia (grown for about 1 month) were taken, and cut into 0.5 mm threads using a cutter blade, placed into 50 ml enzymolysis solution for 5 h of digestion (0.5 h enzymolysis in vacuum, then 4.5 h slow shaking at 10 rpm).
[0143] Note: The temperature during enzymolysis should be kept between 20-25° C., the reaction should be carried out in the dark; and the solution should be gently shaken after the reaction so as to release the protoplasts. [0144] 2) the enzymolysis product was diluted by adding 30 ml of W5, and filtrated into a 50 ml round bottom centrifuge tube using a 75 μm Nylon filter membrane. [0145] Note: The Nylon filter membrane should be submerged in 75% (volume percentage) ethanol, washed with water and then soaked in W5 for 2 min before use. [0146] 3) 23° C., 60 g centrifugation for 5 min, and the supernatant was discarded. [0147] 4) the pellet was resuspended with 10 ml W5 by gently shaking; 60 g centrifugation for 5 min, and the supernatant was discarded. [0148] 5) the protoplasts were suspended by adding a proper amount of MMG solution, placed on ice until transformation. [0149] Note: The concentration of the protoplasts needs to be determined by microscopy (×100). The amount of protoplasts was 2×10.sup.5/ml to 1×10.sup.6/ml.
[0150] 3. Transformation of Arabidopsis Protoplast [0151] 1) 20 μg pHSN401-C2 plasmid was added into a 2 ml centrifuge tube. 200 μl of the protoplast obtained in above step 2 was added using a pipette and then mixed by gentle patting. Then 250 μl of PEG4000 was added and mixed by gentle patting. Transformation was performed in dark for 15-30 min; [0152] 2) 880 μl W5 (room temperature) was added and mixed by reversing, 60 g centrifugation for 5 min, and the supernatant was discarded; [0153] 3) 1 ml W5 was added and mixed by reversing, the content was gently transferred to a 6-well plate (with pre-added 1 ml W5), and then cultured at 23° C. overnight.
[0154] IV. Using PCR/RE experiments to analyze the site-directed mutagenesis of Arabidopsis endogenous gene AtPTPA using gRNA:Cas9 system
[0155] 48 hours after the transformation of Arabidopsis protoplast, genomic DNA was extracted, which was used as template for PCR/RE (Polymerase Chain Reaction/Restriction digestion) experiment analysis. PCR/RE analysis method is based on Shan, Q. et al. Rapid and efficient gene modification in rice and Brachypodium using TALENs. Molecular Plant (2013). Since the target fragment (positions 351-373 of Genbank No. AF360133) of Arabidopsis endogenous gene AtPTPA (Genbank No. AF360133) contains the recognition sequence (5′-GATATC-3′) of restriction endonuclease EcoRV, and thus the restriction endonuclease EcoRV was used in the experiment for conducting the PCR/RE test. Primers used in the PCR amplification were:
TABLE-US-00016 PTPA-F: (SEQ ID NO: 24) 5′-GATGCTCCAGCCACCATATC-3′; PTPA-R: (SEQ ID NO: 25) 5′-CAGTTCGGTACACCACTTATATCA-3′
[0156] The results of PCR/RE experiments can be seen in
[0157] V. Site-directed editing of Arabidopsis endogenous gene AtPTPA via the inflorescence-dipping approach [0158] 1) Preparation of the Arabidopsis materials
[0159] The buds of Arabidopsis were removed at the first flowering to facilitate branching. Siliques were cut off before transformation by inflorescence-dipping. [0160] 2) pHSN401-C2 plasmid containing C2 was transformed into Agrobacterium competent cell GV3101. After verification by PCR and restriction digestion, positive strain was used for infecting the plants. [0161] 3) Positive Agrobacterium strain was cultured in a 2 ml tube for 8-10 hr, and then transferred to 200 ml LB medium (inoculated at a ratio of 1:100), cultured overnight to an OD.sub.600 of about 0.8˜1.0. Agrobacterium cells were collected by centrifuging for 15 min, and resuspended in infection buffer (2.16 g/L MgCl.sub.2.Math.6 H.sub.2O, 5% sucrose, 0.02% silwet L-77) for infecting the plants. [0162] 4) The inflorescences of Arabidopsis were dipped into 100 ml infection buffer contained in a big plate for 2 min, continually rotating the plants. After infection, excess Agrobacterium solution on the plants was removed using filter paper. The plants were covered by a black plastic bag or film for 24 hr cultivation in dark. As the flowering period of Arabidopsis is relatively long, it generally requires 2-3 infections. [0163] 5) Plants were grown under normal conditions. T1 seeds were harvested and grown. After germination, AtPTPA gene was tested using PCR/RE (specific steps and the primers as used can be seen in IV). In the 500 plants as obtained, 20 are mutants of AtPTPA gene. Wild type Arabidopsis ecotype Columbia was set as a control.
[0164] The results were shown in
[0166] pHSN401-1F/C2R locate between U6-26p and the target fragment:
TABLE-US-00017 pHSN401-lF: (SEQ ID NO: 26) 5′-TGTCCCAGGATTAGAATGATTAGGC-3′; C2R: (SEQ ID NO: 27) 5′-AAACACGATATCCGCCGATTTCAC-3′.
[0167] Theoretically, the amplified fragment should be about 286 bp, and the sequence should be positions 170-455 of SEQ ID NO:5. SEQ ID NO:5 is the partial sequence of gDNA in pHSN401-C2.
[0168] CAS9-2F/CAS9-2R locate in Cas9 region of pHSN401-C2 vector:
TABLE-US-00018 CAS9-2F: (SEQ ID NO: 20) 5′-CTCCCTAAGCACTCGCTCCTGT-3′; CAS9-2R: (SEQ ID NO: 21) 5′-TTCTGCGTGGTCTGATTCTCCC-3′
[0169] Theoretically, the amplified fragment should be about 794 bp, and the sequence should be positions 1639-2432 of SEQ ID NO:4. SEQ ID NO:4 is the Cas9 sequence in pHSN401-C2.
[0170] The gel electrophoretogram of the amplification of Arabidopsis AtPTPA gene mutant using primers pHSN401-1F/C2R on pHSN401-C2 is shown in
[0171] 7) 9 plants were randomly selected from the progeny of the 20 mutants obtained in 5) for PCR/RE analysis and the results were shown in