Application of Artemisia apiacea AaU6 promoter in CRISPR / Cas9 gene editing and gene editing method

Abstract

The invention belongs to the technical field of gene engineering, and relates to application of an artemisia apiacea AaU6 promoter in CRISPR / Cas9 gene editing and a gene editing method, the promoter comprises AaU6-1, AaU6-4 and AaU6-8, the nucleotide sequence of the AaU6-1 is as shown in SEQ ID NO.1, the nucleotide sequence of the AaU6-4 is as shown in SEQ ID NO.2, and the nucleotide sequence of the AaU6-8 is as shown in SEQ ID NO.3. The invention also relates to an application of the artemisia apiacea AaU6 promoter in CRISPR / Cas9 gene editing. The promoter AaU6-1, the promoter AaU6-4 and the promoter AaU6-8 are utilized, sgRNA expression can be efficiently driven, the editing efficiency of a CRISPR / Cas9 gene editing system is improved, and great significance is achieved for breeding of sweet wormwood.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and relates to an application of an Artemisia annua AaU6 promoter in CRISPR / Cas9 gene editing and a gene editing method. Background Art

[0002] CRISPR / Cas9 gene editing technology uses artificially designed sgRNA (guide RNA) to identify the target gene and guide the Cas9 protease to cut the target gene, forming a double-strand break, and repairing the damage to achieve gene knockout or insertion. However, in the gene editing process of Artemisia annua, it was found that different promoters that start sgRNA transcription have different sgRNA transcription levels, and the search for promoters with higher transcriptional activity has received continuous attention from technicians in this field.

[0003] Artemisinin is a key anti-malarial drug, and its biosynthesis pathway is mainly regulated by multiple genes. Through gene editing technology, especially the CRISPR / Cas9 system, specific genes in the Artemisia annua genome can be precisely modified to optimize the synthesis efficiency of artemisinin. However, in order to improve the expression efficiency of gRNA, the selection of gRNA promoter is crucial.

[0004] In the breeding process, traditional breeding technology still occupies an important position, but traditional breeding methods have the disadvantages of long cycle and low efficiency; although artificial mutagenesis increases the frequency of mutation, the precision of directional breeding is low; although hybrid breeding can make up for the shortcomings of both, it also faces the problem of incompatibility of distant hybridization and difficulty in breaking the reproductive isolation of species. The establishment of CRISPR / Cas9 gene editing system helps to create germplasm resources with specific traits but without exogenous genes, eliminating the safety concerns of genetically modified plants. The rapid development of biotechnology represented by gene editing provides a new research method for the directional breeding of new varieties of Artemisia annua with high content of artemisinin, drought resistance, cold resistance, and disease and pest resistance, and improves the efficiency and precision of directional breeding of Artemisia annua. Summary of the invention

[0005] The present invention provides an application of an Artemisia annua AaU6 promoter in CRISPR / Cas9 gene editing and a gene editing method. Based on the endogenous AaU6-8 promoter of Artemisia annua, a gRNA-driven tool that is more adaptable to the in vivo environment of Artemisia annua is provided, which significantly improves the application efficiency of the CRISPR / Cas9 system in Artemisia annua.

[0006] In a first aspect of the present invention, an application of an Artemisia annua AaU6 promoter is provided for use in a CRISPR / Cas9 gene editing system, wherein the Artemisia annua AaU6 promoter comprises any one of AaU6-1, AaU6-4 and AaU6-8, wherein the nucleotide sequence of AaU6-1 is shown in SEQ ID NO.1, the nucleotide sequence of AaU6-4 is shown in SEQ ID NO.2, and the nucleotide sequence of AaU6-8 is shown in SEQ ID NO.3.

[0007] In a second aspect of the present invention, a CRISPR / Cas9 gene editing system is provided, which comprises any one of the AaU6-1, AaU6-4 and AaU6-8 promoters, wherein the nucleotide sequence of AaU6-1 is shown in SEQ ID NO.1, the nucleotide sequence of AaU6-4 is shown in SEQ ID NO.2, and the nucleotide sequence of AaU6-8 is shown in SEQ ID NO.3.

[0008] Preferably, the method further comprises an sgRNA for targeting a target gene and a Cas9 nuclease for knocking out the target gene, wherein the Cas9 nuclease for knocking out the target gene is a Cas9 nuclease encoded by a nucleotide sequence encoding a cas protein in the Arabidopsis genome.

[0009] In a third aspect of the present invention, a method for constructing the above-mentioned CRISPR / Cas9 gene editing system is provided, comprising the following steps:

[0010] Step 1, constructing the AaU6 promoter sequence and tracrRNA sequence into a pUC19 vector, and introducing a BsbI restriction site between the AaU6 promoter sequence and the tracrRNA sequence to construct a pUC19-proAaU6-BsbI-tracrRNA vector;

[0011] Step 2, pro35S, the gene encoding Arabidopsis AtCas9 and the Tnos terminator were respectively recombined into the XbaI restriction site of the pCAMBIA1300 vector to construct the pCAMBIA1300-35S-AtCas9 knockout vector;

[0012] Step 3, design an sgRNA sequence targeting the target gene according to the target gene, synthesize a primer with a BsbI restriction site sequence, anneal to form an sgRNA double strand, connect to the BsbI site of the pUC19-proAaU6-BsbI-tracrRNA vector through the Golden braid system, and design primers to amplify to obtain the pUC19-proAaU6-sgRNA-tracrRNA fragment;

[0013] Step 4: Use recombinase to connect the pUC19-proAaU6-sgRNA-tracrRNA fragments to the pCAMBIA1300-35S-AtCas9 knockout vector to construct the gene editing system.

[0014] A fourth aspect of the present invention provides the use of the above-mentioned CRISPR / Cas9 gene editing system in Artemisia annua gene editing.

[0015] A fifth aspect of the present invention provides a CRISPR / Cas9 gene editing method using the above-mentioned CRISPR / Cas9 gene editing system.

[0016] Specifically, the gene editing method comprises the following steps:

[0017] (1) constructing the gene editing system;

[0018] (2) Obtaining protoplast cells;

[0019] (3) transiently transforming the protoplast cells with the gene editing vector so that the gene editing system is expressed in the protoplast cells to achieve editing of the target gene.

[0020] Preferably, the protoplast cells are Artemisia annua protoplast cells.

[0021] Obtaining Artemisia annua protoplast cells comprises the following steps:

[0022] Take a leaf of Artemisia annua in good growth condition, cut the middle part of the leaf into leaf strips of 0.5-1 mm, and put the leaf strips into an enzymatic hydrolysis solution; vacuum the leaf and the enzymatic hydrolysis solution, and let it stand and digest in the dark, and after the leaf strips basically disappear, an enzymatic digestion solution including protoplast cells is obtained.

[0023] As in the above method, the gene editing vector is transiently transformed into the Artemisia annua protoplast cell, so that the gene editing vector is expressed in the Artemisia annua protoplast cell to achieve editing of the target gene, specifically comprising:

[0024] The enzymatic digestion solution was diluted with an equal volume of W5 solution, centrifuged and filtered, the supernatant was discarded, the protoplasts were collected, and resuspended with W5 solution to control the concentration of protoplast cells to 2×10 4 / mL;

[0025] Mix 10 μL of gene editing vector with 100 μL of protoplast cell resuspension, add 110 μL of PEG solution and mix well, leave at room temperature for 10 minutes, then add 440 μL of W5 solution to dilute the reaction solution, mix well and centrifuge, and discard the supernatant;

[0026] Resuspend the protoplast cells with 1 mL of WI solution and incubate at room temperature in the dark for 12-16 hours;

[0027] The PEG solution includes 40% PEG4000, 0.2M mannitol and 0.1M CaCl 2 ;

[0028] The WI solution included 4 mM MES, 0.4 M mannitol, and 15 mM MgCl 2 .

[0029] In a sixth aspect, the present invention further provides the application of the above-mentioned CRISPR / Cas9 gene editing system for the cultivation of new plant varieties, which provides a new research method for the targeted cultivation of new Artemisia annua varieties with high content of artemisinin, drought resistance, cold resistance, and disease and pest resistance, thereby improving the efficiency and accuracy of targeted breeding of Artemisia annua.

[0030] By implementing the above technical solution, the present invention has the following beneficial effects:

[0031] The present invention provides an endogenous AaU6 promoter of Artemisia annua that can be used in the Artemisia annua CRISPR / Cas9 gene editing system, which can drive the efficient expression of sgRNA and improve the editing efficiency of the CRISPR / Cas9 gene editing system, and is of great significance for the breeding of Artemisia annua. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 For promoter sequence analysis of Artemisia annua AaU6-1, AaU6-4 and AaU6-8;

[0033] Figure 2 This is a diagram of the construction of the pUC19-proAaU6-1-BsbI-tracrRNA vector;

[0034] Figure 3 This is a diagram of the construction of the pUC19-proAaU6-4-BsbI-tracrRNA vector;

[0035] Figure 4 This is a diagram of the construction of the pUC19-proAaU6-8-BsbI-tracrRNA vector;

[0036] Figure 5 This is a diagram of the construction of the pCAMBIA1300-35S-AtCas9 vector;

[0037] Figure 6 The diagram shows the construction of the pDBG1-Ω1-proNOS-Fluc-pro35S-NanoLUC vector;

[0038] Figure 7To verify the editing efficiency of the CRISPR / Cas9 gene editing system in protoplasts. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] The experimental methods for which specific conditions are not indicated in the following examples are usually performed under conventional conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, or under conditions recommended by the manufacturer.

[0041] Agrobacterium tumefaciens GV3101 and plasmid pCAMBIA1300 can be obtained through publicly available commercial channels, such as from CAMBIA Company in Australia.

[0042] Example 1 Cloning of the Artemisia annua AaU6 promoter

[0043] 1. Extraction of Artemisia annua genomic DNA

[0044] The plant genomic DNA extraction kit (CW0553) provided by Beijing Kangwei Century Biotechnology Co., Ltd. was used to extract the genomic DNA of Artemisia annua, which specifically includes the following steps:

[0045] Take about 100 mg of fresh Artemisia annua leaves and add liquid nitrogen to grind them thoroughly. Collect the ground powder into a centrifuge tube, add 400 μL Buffer LP1 and 6 μL RNase A (10 mg / ml), vortex for 1 minute, and leave at room temperature for 10 minutes to fully lyse. Add 130 μL Buffer LP2, mix well, and vortex for 1 minute. Centrifuge at 12,000 rpm (~13,400 × g) for 5 minutes and transfer the supernatant to a new centrifuge tube. Add 1.5 times the volume of Buffer LP3 (add anhydrous ethanol before use) and mix thoroughly (e.g., add 750 μL Buffer LP3 to 500 μL filtrate). Add all the resulting solution and precipitate to the adsorption column (Spin Columns DM) that has been loaded into the collection tube, centrifuge at 12,000 rpm for 1 minute, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube. Add 500μL Buffer GW2 to the adsorption column (check whether anhydrous ethanol has been added before use), centrifuge at 12,000rpm for 1 minute, discard the waste liquid in the collection tube, put the adsorption column back into the collection tube, and repeat once. Centrifuge at 12,000rpm for 2 minutes, discard the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to dry thoroughly. Place the adsorption column in a new centrifuge tube, add 100μL of sterilized water to the middle part of the adsorption membrane, leave it at room temperature for 2-5 minutes, centrifuge at 12,000rpm for 1 minute, and collect the DNA solution.

[0046] 2. Obtaining the Artemisia annua U6 promoter sequence

[0047] The Arabidopsis AtU6 sequence was aligned with the Artemisia annua genome database, and multiple Artemisia annua U6 promoter sequences were obtained, of which three sequences had conserved elements of the U6 promoter, named AaU6-1, AaU6-4 and AaU6-8. The U6 promoter is a DNA sequence that RNA polymerase recognizes, binds to and starts transcription. Aa is the Latin abbreviation for Artemisia annua, and AaU6-1, AaU6-4 and AaU6-8 represent the U6 promoter of Artemisia annua.

[0048] 3. PCR amplification

[0049] Using genomic DNA as a template, the promoter sequences of Artemisia annua AaU6-1, AaU6-4 and AaU6-8 were amplified by PCR. Primers were designed according to the promoter sequences of AaU6-1, AaU6-4 and AaU6-8 (the nucleotide sequences of the primers are shown in SEQ ID NO.4-7, respectively, wherein SEQ ID NO.4 is the upstream primer corresponding to AaU6-1, SEQ ID NO.6 is the upstream primer corresponding to AaU6-4, SEQ ID NO.7 is the upstream primer corresponding to AaU6-8, and SEQ ID NO.5 is the downstream primer of the three promoters).

[0050] The PCR reaction system is shown in Table 1. The PCR conditions are: 94°C pre-denaturation for 10 min; 94°C for 40 s, 55°C

[0051] 40s, 68℃2min, 34 cycles; 68℃ extension 10min. PCR products were detected by electrophoresis in 1% agarose gel, and specific bands were recovered and connected to pLB vector for sequencing to obtain AaU6-1, AaU6-4 and AaU6-8 promoter sequences, which correspond to SEQ ID NO.1-3 respectively.

[0052] Table 1 PCR reaction system

[0053]

[0054]

[0055] The cloned AaU6-1, AaU6-4 and AaU6-8 promoters were compared with Arabidopsis AtU6 and cotton GhU6.9 promoter sequences. Figure 1 As shown, the results showed that the sequences of AaU6-1, AaU6-4 and AaU6-8 promoters of Artemisia annua, AtU6 promoter of Arabidopsis thaliana and GhU6.9 promoter of cotton all contained TATA box and USE (upstream sequence element) elements, and the sequences and positions of the two upstream cis-acting elements were relatively conserved.

[0056] Example 2 Construction of Artemisia annua CRISPR / Cas9 gene editing vector

[0057] 1. The AaU6-1, AaU6-4 and AaU6-8 promoter sequences were connected to the tracrRNA sequence, and the BsbI restriction site was introduced and constructed into the pUC19 vector to obtain pUC19-proAaU6-1-BsbI-tracrRNA, pUC19-proAaU6-4-BsbI-tracrRNA and pUC19-proAaU6-8-BsbI-tracrRNA vectors. The schematic diagrams of the vector construction are as follows: Figure 2-4 shown.

[0058] tracrRNA is the abbreviation of tracr-activating crRNA. tracrRNA and pre-crRNA are combined through base pairing, trimmed by RNaseIII, and combined with Cas9 to become a complex with DNA cutting ability.

[0059] 2. Using multi-fragment recombinase, pro35S (promoter), AtCas9 (AtCAS9 is the Arabidopsis Cas9 gene) and Tnos (terminator) were recombined into the XbaI restriction site of the pCAMBIA1300 vector to construct the pCAMBIA1300-35S-AtCas9 vector. The construction diagram of the pCAMBIA1300-35S-AtCas9 vector is shown in the figure. Figure 4 shown.

[0060] 3. The Golden braid system was used to construct proNOS (promoter), Fluc (internal standard gene, which is to allow NanoLUC and fluc to be expressed simultaneously in protoplast cells. The reporter gene NanoLUC was edited by the gene editing vector, resulting in NanoLUC being unable to encode normal proteins and catalyze substrates. The catalytic value was compared with that of the internal standard gene to prove the knockout efficiency of the vector) and Tnos (terminator), as well as pro35S (promoter), NanoLUC (reporter gene) and T35S (terminator) into the pDBG1-Ω1 vector, and the pDBG1-Ω1-proNOS-Fluc-pro35S-NanoLUC vector was constructed. The construction diagram of the vector is shown in the figure. Figure 5 shown.

[0061] 4. Design sgRNA targeting the reporter gene NanoLUC, synthesize primers, anneal to form sgRNA double strands, and connect to pUC19-proAaU6-1-BsbI-tracrRNA, pUC19-proAaU6-4-BsbI-tracrRNA, and pUC19-proAaU6-8-BsbI-tracrRNA vectors.

[0062] 5. Amplify proAaU6-1-BsbI-tracrRNA, proAaU6-4-BsbI-tracrRNA and proAaU6-8-BsbI-tracrRNA vector fragments, connect them to the KpnI restriction site of pCAMBIA1300-35S-AtCas9 vector, and construct NanoLUC knockout vectors (pCAMBIA1300-proAaU6-1-sgRNA-35S-AtCas9, pCAMBIA1300-proAaU6-4-sgRNA-35S-AtCas9,

[0063] pCAMBIA1300-proAaU6-8-sgRNA-35S-AtCas9), and a control vector pCAMBIA1300-proAtU6-sgRNA-35S-AtCas9 was also constructed and sequenced.

[0064] Table 2 Nucleotide sequences involved in Example 2

[0065]

[0066]

[0067] Example 3 Extraction of Endotoxin-free Plasmid

[0068] The OMEGA Endotoxin-free Plasmid Extraction Kit was used to extract pCAMBIA1300, pDBG1-Ω1-proNOS-Fluc-pro35S-NanoLUC, pCAMBIA1300-proAaU6-1-sgRNA-35S-AtCas9, pCAMBIA1300-proAaU6-4-sgRNA-35S-AtCas9,

[0069] pCAMBIA1300-proAaU6-8-sgRNA-35S-AtCas9 and control vector

[0070] pCAMBIA1300-proAtU6-sgRNA-35S-AtCas9 plasmid.

[0071] 1. Add 200 mL of LB medium containing screening antibiotics to a 500 mL culture flask, add the bacteria containing the target plasmid, and then culture at 37°C with shaking for 12-16 hours.

[0072] 2. Centrifuge at 3,500-5,000 x g for 10 min at room temperature to collect the bacterial precipitate.

[0073] 3. Add 10 ml of Solution Ⅰ / RNase A and vortex or pipette up and down to resuspend the cells.

[0074] 4. Add 10 ml of Solution II and gently mix by inverting 10-15 times to obtain a clear lysate.

[0075] 5. Add 5 mL of Buffer N3 and mix thoroughly by gently inverting several times until a white flocculent precipitate appears. Leave at room temperature for 2-3 minutes.

[0076] 6. Prepare the binding column: add 5 mL of Buffer GPS to the binding column, place at room temperature for 3-10 minutes, centrifuge at 3,000-5,000 x g for 5 minutes at room temperature, and discard the filtrate.

[0077] 7. Transfer all the lysate and precipitate to a syringe filter and prepare a clean 50 ml centrifuge tube to collect the filtrate.

[0078] 8. Add an equal volume of ETR Binding Buffer (~25 mL) to the binding column and invert it upside down 7-10 times.

[0079] 9. Carefully transfer the lysate to the HiBind binding column and centrifuge at 3,000-5,000xg for 3-5 minutes to allow the liquid to filter through the column and discard the filtrate. Repeat this step until all the lysate is filtered and discard the filtrate.

[0080] 10. Add 10 mL of ETR Wash Buffer to the binding column and centrifuge at 3,000-5,000 x g for 3-5 min to allow all the liquid to filter through the column and discard the filtrate.

[0081] 11. Add 10 mL of Buffer EHB to the binding column, centrifuge at 3,000-5,000 x g for 3-5 min to allow all the liquid to filter through the column, and discard the filtrate.

[0082] 12. Add 15 mL DNA Wash Buffer to the binding column, centrifuge at 3,000-5,000 x g for 3-5 min to allow all the liquid to filter through the column, discard the filtrate, and repeat this step.

[0083] 13. Put the empty column back into the centrifuge tube and centrifuge at maximum speed for 10-15 minutes to fully dry the column.

[0084] 14. Put the binding column in a new 50mL centrifuge tube, add 1-3mL Endotoxin-Free Elution Buffer (or deionized water) to the membrane of the binding column, leave it at room temperature for 2-5 minutes, and centrifuge at the highest speed for 5 minutes to elute the plasmid DNA.

[0085] Example 4 Preparation and transformation of Artemisia annua protoplasts

[0086] 1. Take a leaf of Artemisia annua with good growth status, tear off the cuticle on the surface of the leaf with tape, cut the middle part of the leaf into 0.5-1 mm leaf strips with a blade, put the leaf strips into 10 ml of enzymatic solution (20 mM MES (PH5.7), 1.5% (W / V) cellulase R-10, 0.4% (W / V) cleavage enzyme R-10, 0.4 M mannitol, 20 mM KCl, warm bath at 55°C for 10 minutes to inactivate DNase, add a final concentration of 10 mM CaCl2, 0.1% BSA, 5 mM mercaptoethanol).

[0087] 2. In a vacuum desiccator, evacuate the mixture for 10 minutes in the dark, and then let it stand for more than 3 hours in the dark for digestion. After gently shaking, you should see that the strip-shaped leaves almost disappear. At this time, you can observe the release of protoplasts under a microscope.

[0088] 3. Dilute the enzymatic digestion solution with an equal volume of W5 solution (2mM MES (PH5.7), 154mM NaCl, 125mM CaCl2, 5mM KCl), filter with a 75μm nylon membrane, centrifuge the filtrate at 100g for 2 minutes at room temperature, discard the supernatant, and resuspend the protoplasts with a small amount of W5 solution.

[0089] 4. Count the protoplast cells with a hemocytometer under a microscope, and finally suspend the protoplast solution with W5 solution to a concentration of 2×105 / mL. Place it on ice for 30 minutes, and then remove the W5 solution as much as possible with a pipette.

[0090] 5. Suspend the protoplasts to a concentration of 2×105 / ml in MMG solution (4 mM MES (pH 5.7), 0.4 M mannitol, 15 mM MgCl2) stored at room temperature.

[0091] 6. Add 10 μL of the endotoxin-free plasmid obtained in Example 3 to each group in a 2 mL EP tube (Group 1 as control: pCAMBIA1300 and pDBG1-Ω1-proNOS-Fluc-pro35S-NanoLUC, Group 2: pCAMBIA1300-proAaU6-1-sgRNA-35S-AtCas9 and

[0092] pDBG1-Ω1-proNOS-Fluc-pro35S-NanoLUC, group 3:

[0093] pCAMBIA1300-proAaU6-4-sgRNA-35S-AtCas9 and

[0094] pDBG1-Ω1-proNOS-Fluc-pro35S-NanoLUC, group 4:

[0095] pCAMBIA1300-proAaU6-4-sgRNA-35S-AtCas9 and

[0096] pDBG1-Ω1-proNOS-Fluc-pro35S-NanoLUC, group 5:

[0097] pCAMBIA1300-proAtU6-sgRNA-35S-AtCas9 and

[0098] pDBG1-Ω1-proNOS-Fluc-pro35S-NanoLUC), add 100 μL protoplast cells

[0099] (2×104 / mL), repeat 6 times for each group, mix gently, add 110 μL of freshly prepared PEG solution (40% PEG4000, 0.2 M mannitol, 0.1 M CaCl 2 ) and flick the EP tube to mix, let it stand at room temperature for 10 minutes, slowly add 440 μL of W5 solution to dilute the reaction solution, gently invert to mix, centrifuge at 100g for 2 minutes, and carefully discard the supernatant.

[0100] 7. Add 1 mL of WI solution (4 mM MES (pH 5.7), 0.4 M mannitol, 15 mM MgCl 2 ) The protoplast cells were gently suspended, transferred to a 6-well plate (previously treated with 5% calf serum for 5 seconds), and cultured at room temperature in the dark for 12-16 hours.

[0101] Example 5 Dual luciferase reporter gene detection

[0102] Transfer the protoplast cells cultured in the 6-well plate to a 2 mL round-bottom centrifuge tube. Use a high-speed refrigerated centrifuge with the speed set to level 3. Centrifuge at 200 g for 5 min at room temperature. Remove the supernatant and add 100 μL of lysis buffer. Mix well and lyse at room temperature for 10 min. Pipette 20 μL of the lysate into the centrifuge tube and add 20 μL Luciferase Reagent, mix well and react for 10 minutes, measure fLuc fluorescence value with Promega GloMax; then add 20 μL of the measured liquid Stop& Reagent, mix well and react for 10 minutes, and measure the fluorescence value of NanoLuc with Promega GloMax. The results showed that when the fluorescence value of pDual-Luc was recorded as 100% and fLuc was used as the internal reference, the editing efficiency of pCAMBIA1300-proAaU6-4-sgRNA vector was higher, at 25.4%, and the editing efficiency of pCAMBIA1300-proAaU6-1-sgRNA vector and pCAMBIA1300-proAaU6-8-sgRNA vector was 23.94% and 13.49%, respectively, while the editing efficiency of pCAMBIA1300-proAtU6-sgRNA vector was lower, at 7%.

[0103] Therefore, the proAaU6-1, proAaU6-4 and proAaU6-8 promoters can be used in the Artemisia annua CRISPR / Cas9 gene editing system, among which the proAaU6-4 promoter has the highest efficiency in driving the expression of sgRNA guide sequences.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An application of Artemisia annua AaU6 promoter, characterized in that: For use in the CRISPR / Cas9 gene editing system, the Artemisia annua AaU6 promoter includes any one of AaU6-1, AaU6-4 and AaU6-8, wherein the nucleotide sequence of AaU6-1 is shown as SEQ ID NO.1, the nucleotide sequence of AaU6-4 is shown as SEQ ID NO.2, and the nucleotide sequence of AaU6-8 is shown as SEQ ID NO.

3.

2. A CRISPR / Cas9 gene editing system, characterized in that: It comprises any one of the AaU6-1, AaU6-4 and AaU6-8 promoters, wherein the nucleotide sequence of AaU6-1 is shown as SEQ ID NO.1, the nucleotide sequence of AaU6-4 is shown as SEQ ID NO.2, and the nucleotide sequence of AaU6-8 is shown as SEQ ID NO.

3.

3. A CRISPR / Cas9 gene editing system according to claim 2, characterized in that: It also includes an sgRNA for targeting a target gene and a nucleotide sequence capable of editing a cas protein for knocking out the target gene.

4. A CRISPR / Cas9 gene editing system according to claim 3, characterized in that: The nucleotide sequence capable of editing the cas protein used to knock out the target gene is a nucleotide sequence encoding the cas protein in the Arabidopsis genome.

5. A method for constructing a CRISPR / Cas9 gene editing system as described in any one of claims 2 to 4, characterized in that: The following steps are involved: Step 1, constructing the AaU6 promoter sequence and tracrRNA sequence into a pUC19 vector, and introducing a BsbI restriction site between the AaU6 promoter sequence and the tracrRNA sequence to construct a pUC19-proAaU6-BsbI-tracrRNA vector; Step 2, pro35S, the gene encoding Arabidopsis AtCas9 and the Tnos terminator were respectively recombined into the XbaI restriction site of the pCAMBIA1300 vector to construct the pCAMBIA1300-35S-AtCas9 knockout vector; Step 3, design an sgRNA sequence targeting the target gene according to the target gene, synthesize a primer with a BsbI restriction site sequence, anneal to form an sgRNA double strand, connect to the BsbI site of the pUC19-proAaU6-BsbI-tracrRNA vector through the Golden braid system, and design primers to amplify to obtain the pUC19-proAaU6-sgRNA-tracrRNA fragment; Step 4: Use recombinase to connect the pUC19-proAaU6-sgRNA-tracrRNA fragments to the pCAMBIA1300-35S-AtCas9 knockout vector to construct the gene editing system.

6. Use of a CRISPR / Cas9 gene editing system as described in any one of claims 2 to 4 in gene editing of Artemisia annua.

7. A CRISPR / Cas9 gene editing method, characterized in that: Use a CRISPR / Cas9 gene editing system as described in any one of claims 2-4.

8. A CRISPR / Cas9 gene editing method according to claim 7, characterized in that: The steps include: (1) constructing the gene editing system; (2) Obtaining protoplast cells; (3) transiently transforming the protoplast cells with the gene editing vector so that the gene editing system is expressed in the protoplast cells to achieve editing of the target gene.

9. A CRISPR / Cas9 gene editing method according to claim 8, characterized in that: The protoplast cells are Artemisia annua protoplast cells.

10. The use of a CRISPR / Cas9 gene editing system according to any one of claims 2 to 4, characterized in that: Used for the cultivation of new plant varieties.

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