CN106367435B - A method for targeted knockout of miRNA in rice - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, in particular to a method for directional knockout of rice miRNA. The technical problem to be solved by the present invention is to provide a method and detection means for directional modification of plant miRNA sites. The invention discloses a method for obtaining a rice micoRNA mutant. The specific operations are as follows: using the CRISPR-Cas9 method to knock out a single miRNA, knock out multiple miRNAs or knock out large fragments of miRNA. The method of the invention is suitable for the creation and screening of rice miRNA directional knockout mutants.

Description

A method for targeted knockout of miRNA in rice

technical field

The invention belongs to the technical field of plant genetic engineering, in particular to a method for directional knockout of rice miRNA.

Background technique

miRNA (microRNA) is a kind of non-coding small RNA that is ubiquitous in eukaryotes and is about 19-24 nucleotides in length. It is encoded by endogenous miRNA genes and transcribed by RNA polymerase II. It mainly regulates the expression level of its target gene mRNA by splicing degradation, inhibiting translation, and chromosomal remodeling (methylation). miRNAs play important roles in plant growth and development, metabolism, epigenetics, and response to abiotic stress.

The traditional methods to study the function of miRNA mainly include: (1) The indirect method of inhibiting the function of the target gene by transgenic expression of miRNA to study the function of miRNA. However, because miRNAs often regulate multiple target genes with the same or different functions, this method can only reflect the functions of some miRNAs. (2) Interfering (blocking) miRNA by target gene analogs (Target Mimicry, TM) or short tandem target analogs (Short Tandem Target Mimic, STTM), so as to reflect the effect of target gene derepression and study the function of miRNA. However, the interference efficiency of this mode for many miRNAs is low, or even completely inactive. Therefore, it is of great significance to find a more suitable method to study the function of miRNA.

Traditional methods for elucidating gene function mainly rely on the creation of genetic mutants, such as physicochemical mutagenesis, T-DNA or transposon tag insertion mutagenesis, etc. However, these methods are time-consuming, labor-intensive, blind, and very difficult to achieve in the study of miRNA function, mainly because the miRNA is very small (about 21bp), and it is difficult to obtain mutants.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR-Cas9) technology was developed in early 2013, following the first generation genome editing technology-zinc finger nucleases (Zinc finger nucleases, ZFN) and the second generation genome editing Technology—a new third-generation genome editing technology developed after transcription activator-like effector nucleases (TALEN). It is mainly based on the transformation of an acquired immune system of bacteria. Compared with ZFN and TALEN, it has the advantages of simple production, low cost and high efficiency. The working principle of CRISPR-Cas9 is that crRNA (CRISPR-derived RNA) combines with tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA/crRNA complex, which guides the nuclease Cas9 protein at the sequence target position paired with crRNA. Dot cut double-stranded DNA. By artificially designing these two RNAs, sgRNA (single guideRNA) can be transformed into a guiding role, which is sufficient to guide the site-directed cleavage of DNA by Cas9. At present, CRISPR-Cas9 technology has been successfully applied not only to the targeted modification of animal and microbial genomes, but also to the creation of plant gene mutants such as Arabidopsis, tobacco, rice, wheat, and maize.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide an efficient method for creating rice miRNA mutants.

The technical solution of the present invention to solve the above technical problem provides a method for directional knockout of rice miRNA, and a rice miRNA mutant can be obtained by using the method. The steps of this method are: in rice, the CRISPR-Cas9 method is used to knock out a single miRNA, knock out multiple miRNAs, or knock out large fragments of miRNA.

Specifically, the method includes the following steps:

a. Construct a vector for knocking out a single miRNA, knocking out multiple miRNAs or knocking out large fragments of miRNA;

b. Transform the CRISPR-Cas9 vector obtained in step a into rice callus, and screen to obtain rice miRNA mutants.

Specifically, the vector backbone described in step a is pZHY988, and the core region between the left and right borders of the vector T-DNA includes the following elements: a hygromycin-resistant gene expression element, a Cas9 expression element, and a gRNA transcription unit;

When knocking out a single miRNA, the gRNA transcription unit is one gRNA transcription element, which recognizes the PAM site near the target miRNA; when multiple miRNAs are knocked out, the gRNA transcription unit is two or more gRNA transcription elements in series, which recognize respectively PAM sites near different target miRNAs; when knocking out large fragments of the same miRNA, the gRNA transcription unit is two gRNA transcription elements in series, which respectively recognize the nearest PAM sites on both sides of the target miRNA; the PAM recognized by the gRNA transcription elements The site is 5'-N _X -NGG-3', N represents any one of A, G, C and T, and x=20;

Described gRNA transcription element comprises rice U6 promoter, ccdB and gRNA (guide RNA) scaffold, U6 terminator, each end of ccdB fragment has a BsaI restriction site, has the nucleoside as described in SEQ ID No.1 When constructing the vector, the backbone of the vector is cut with BsaI enzyme, and the sgRNA (single-guide RNA, single guide RNA) targeting the target miRNA is inserted.

Among them, the Cas9 expression element includes the maize ubiquitin Ubiquitin-1 promoter Ubi, the Cas9 protein gene and the heat shock protein terminator HSP.

The hygromycin expression element includes the cauliflower mosaic virus promoter 35S, the hygromycin-resistant gene Hygromycin, and the cauliflower mosaic virus terminator 35ST.

Wherein, in step b, the method used for the screening of knockout mutants of a single miRNA and multiple miRNAs is a combination of PCR-SSCP single-strand conformation polymorphism and sequencing; for large fragments of miRNA knockout mutants The screening method was a combination of PCR-agarose electrophoresis and sequencing.

Further, in step a, a knockout vector pMIR528-1 for a single miRNA was constructed for the rice OsmiR528 gene, and the core region between the left and right borders of the vector T-DNA contained hygromycin expression elements, Cas9 expression elements and The gRNA transcription unit has the nucleotide sequence as set forth in SEQ ID No.2.

Further, in step a, a vector pZJP028 for knocking out two miRNAs was constructed for OsmiR397a and OsmiR97b, members of the rice OsmiR397 gene family. The core region between the left and right borders of the vector T-DNA contains a hygromycin expression element, Cas9 Expression element and gRNA transcription unit, the gRNA transcription unit has the nucleotide sequence as set forth in SEQ ID No.3.

Further, in step a, a large fragment knockout vector pZJP025 of miRNA was constructed for the rice OsmiR408 gene, and the core region between the left and right borders of the vector T-DNA contained a hygromycin expression element, a Cas9 expression element and a gRNA transcription unit. , the gRNA transcription unit has the nucleotide sequence as described in SEQ ID No.4.

Specifically, the transformation of the rice callus in step b adopts the fixed transformation mediated by Agrobacterium.

The present invention also provides the use of the CRISPR-Cas9 method in obtaining rice micoRNA mutants.

The method for realizing targeted knockout and detection of rice miRNA described in the present invention utilizes the basic principle of CRISPR-Cas9. The basic principle is that under the combined action of guide RNA and Cas9 nuclease, the double-stranded target site on the miRNA precursor of the target gene is cleaved, and then through the cell's own DNA repair function, the target in the miRNA precursor of the target gene is finally realized. Random deletions and/or insertions at sites. In the CRISPR/Cas9 genome editing system, the specific cleavage of the genome target sequence by CRISPR/Cas9 mainly depends on the guide RNA (guided RNA, gRNA, a single guide RNA single guide RNA fused by transactivating crRNA tracrRNA and short palindromic repeat RNA crRNA). The ribonucleoprotein complex formed by crRNA and Cas9 protein in the chain) recognizes the PAM (protospacer adjacent motif) on the target sequence and its adjacent 20bp specific target sequence (protospacer).

When targeting multiple genes in a gene family, a strategy of simultaneous deletion of multiple miRNAs can be employed. When it is difficult to find suitable PAM sites near the precursors of some mature miRNAs, or when all or most of the precursor miRNAs are to be knocked out, it is necessary to consider selecting the PAM sites at the far points on both sides of the miRNA, and using double-site knockout Strategies that result in large fragment deletions.

The beneficial effects of the present invention are as follows: the mutant obtaining method of the present invention is simple and programmed, and can be knocked out at a fixed point, with a high knockout rate; among the detected T0 generation individual plants, the mutant type accounts for 90% or more. This method is suitable for the creation of targeted modification mutants of rice miRNAs.

Description of drawings

Figure 1. Schematic diagram of the core unit of pZHY988 vector

Among them, "35S" is the cauliflower mosaic virus promoter, "Hygromycin" is the hygromycin resistance gene, "Ubi" is the maize ubiquitin promoter, "Cas9" is the Cas9 protein gene, "U6" is the rice U6 promoter, "ccdB" is the gene whose expression product can inhibit the growth of common E. coli, "gRNA scaffold" is the fusion of crRNA and tracRNA into a chimeric RNA chain, "35ST" is the 35S terminator, and "HSP" is the heat shock protein terminator son, "U6T" is the rice U6 terminator.

Figure 2 PCR-SSCP screening of OsmiR528-01 knockout mutants

Among them, "M" represents marker, "WT" represents the T0 generation plant transformed by the backbone vector, and "1-34" represents the number of the T0 individual plant stably transformed by Cas9-directed modification OsmiR528.

Figure 3. Sequencing result of PCR amplification product of OsmiR528 mutation site

Among them, "WT" indicates the wild-type gene sequence, "-" indicates the sequence with deletion mutation, "+" indicates the sequence with insertion mutation, and the number after "-/+" indicates the deletion or insertion nucleotide sequence. Quantity, "#" indicates the number of the mutant individual plant, "△" indicates the sequence of the base mutation, "Reference" indicates the DNA sequence of the wild-type OsmiR528 gene at this position, and other numbers indicate the individual plant number of the mutant, " Locus 1, 2" indicates the DNA sequence of the mutant OsmiR528 gene at this position on the two homologous chromosomes, and the black underline is the guide sgRNA of the OsmiR528 site.

Figure 4 Schematic diagram of OsmiR397a and OsmiR397b double-site knockout mutant vector pZJP028

Among them, "35S" is the cauliflower mosaic virus promoter, "Hygromycin" is the hygromycin resistance gene, "Ubi" is the maize ubiquitin promoter, "Cas9" is the Cas9 protein gene, "U6" is the rice U6 promoter, "gRNA scaffold" is the fusion of crRNA and tracRNA into a chimeric RNA strand, "35ST" is the 35S terminator, "HSP" is the heat shock protein terminator, "U6T" is the rice U6 terminator, and "OsmiR397a" is the OsmiR397a terminator. OsmiR397b is the guide sgRNA of the OsmiR397b site.

Figure 5 PCR-SSCP screening of pZJP028 double-site knockout mutants

Among them, a is the PCR-SSCP screening of the OsmiR397a-gRNA1 site, and b is the PCR-SSCP screening of the OsmiR397b-gRNA1 site. "M" stands for marker, "WT" stands for backbone vector transformed T0 generation plants, numbers "2-1, ... 25-4" stand for Cas9 directional modification OsmiR397 gene locus stably transformed T0 individual plant number. From Figure 5, among the 17 individual plants, 12 individual plants (ie 2-1, 7-1, 8-1, 13-1, 15-1, 16-1, 20-1, 21-1, 22-1, 23-1, 24-1, 24-2) had successful deletion/insertion mutations.

Figure 6 Schematic diagram of OsmiR408 large fragment deletion mutation double-site knockout mutation vector pZJP025

Among them, "35S" is the cauliflower mosaic virus promoter, "Hygromycin" is the hygromycin resistance gene, "Ubi" is the maize ubiquitin promoter, "Cas9" is the Cas9 protein gene, "U6" is the rice U6 promoter, "gRNA scaffold" is crRNA and tracRNA fused into a chimeric RNA strand, "35ST" is 35S terminator, "HSP" is heat shock protein terminator, "U6T" is rice U6 terminator, "OsmiR408-sgRNA1, OsmiR408" -sgRNA2" is the guide sgRNAs for the deletion site of OsmiR408.

Figure 7 OsmiR408 large fragment deletion mutation PCR amplification product agarose electrophoresis detection results

Among them, "WT" represents the transformation of the backbone vector into the T0 generation plant, and the horizontal line and number behind pZJP025 represent the number of the T0 individual plant stably transformed by the Cas9-directed modification of the OsmiR408 gene locus. From Figure 7, among the 10 individual plants, there are 6 strains pZJP025-01-01, pZJP025-02-01, pZJP025-03-01, pZJP025-04-01, pZJP025-09-02, and pZJP025-10-01 There are large fragments missing.

Detailed ways

Example 1 Using the method of the present invention to create a single miRNA mutant (taking rice OsmiR528 as an example)

1. Vector construction of CRISPR-Cas9-miRNA mutants

(1) Primer design

Primers were designed according to the recognition and cleavage rules of the target site by CRISPR/Cas9. For the precursor genome sequence of rice OsamiR528 (GenBank number: GQ419957.2), primers were designed as OsmiR528-sgRNA1-F: 5'-gtgtGAAGGGGCATGCAGAGGAGC-3'; OsmiR528-sgRNA1-R: 5'-aaacGCTCCTCTGCA TGCCCCTTC-3'.

(2) Primer annealing

The upstream and downstream primers of each target site were diluted 10 times, 10 μL of each was taken, denatured at 98° C. for 5 min, cooled naturally, and the annealed product was diluted 20 times for use.

(3) Enzyme digestion, gel recovery, and ligation

The backbone vector used in the experiment is pZHY988 (Fig. 1), which was constructed by our laboratory. The construction process is as follows: First, the basic fragment pZHY998-01:SbfI-Cas9 orf-Hsp terminator was synthesized (synthesized by Shanghai Yingjun Biotechnology Co., Ltd.) -BamH Ⅰ and pZHY998-02:BamH Ⅰ-OsU6 promoter-Bsa Ⅰ-ccdB unit-Bsa Ⅰ-sgRNA backbone unit-Hsp terminator-Sac Ⅰ; then carry out the enzyme cleavage reaction respectively: A: Sbf Ⅰ+BamH Ⅰ enzyme Cut pZHY998-01, B: BamH Ⅰ+Sac Ⅰ digest pZHY998-02, C: Sbf Ⅰ+Sac Ⅰ digest vector pTX172; then recover and purify the digested product, and ligate the three fragments with T4 DNA ligase.

The core unit of pZHY988 is the Cas9 protein expression element promoted by the maize ubiquitin (Ubiquitin-1) promoter, the gRNA clone and transcription unit (including the Bsa I-ccdB-Bsa I unit, that is, two parts of the ccdB gene) promoted by the rice U6 promoter. The side contains two BsaI enzyme cleavage sites, which can be used to load the corresponding target sites after single enzyme cleavage of the vector) (with the nucleotide sequence as described in SEQ ID No. 1). The backbone vector also includes: left and right border sequences of T-DNA, and the vector contains the hygromycin gene (Hyg) promoted by the cauliflower mosaic virus promoter (CaMV35S). The described hygromycin-resistant gene expression element sequence is consistent with the corresponding hygromycin-resistant gene expression element sequence of plasmid pTX172 (see document Tang X et al., A SingleTranscript CRISPR-Cas9 System for Efficient Genome Editing in Plants.Molecular Plant 9(7) : 1088-1091), the Cas9 expression element sequence is consistent with the corresponding Cas9 expression element sequence of plasmid pTX172 (refer to the literature Tang X et al., A Single Transcript CRISPR-Cas9 System for Efficient Genome Editing in Plants.Molecular Plant 9(7): 1088 –1091).

pZHY988 was single-enzyme digested with BsaI, and the establishment of the restriction enzyme system and restriction enzyme restriction conditions were carried out with reference to the restriction endonuclease instructions of Thermo Scientific Company. The specific digestion system is as follows: 10×Fast digest buffer 5 μL, plasmid DNA or PCR product 10 μL (1-1.5 μg), restriction endonuclease 1 μL, ddH ₂ O supplemented to 50 μL.

The reaction was carried out in a constant temperature incubator at 37° C. for 2 hours. After the reaction, 10 μL of 6×loading bufer was added, 1% agarose gel electrophoresis was performed, and the gel was cut and recovered. The gel recovery method was performed according to the AXYGEN AxyPrep ^™ DNA Gel Extraction Kit method.

The digested recovery products (large fragments) of pZHY988 were ligated with the annealed products of each target site respectively. The establishment of the ligation system and the ligation conditions were carried out with reference to the instructions of New England Biolabs' T4 DNA ligase. The specific ligation system is as follows: 10×T4DNA Ligase reaction buffer 2 μL, T4 DNA ligase 1 μL, pZHY988 digestion product 5 μL, annealing product 5 μL, ddH ₂ O supplemented by 20 μL.

(4) Escherichia coli transformation

1) The ligation product was transformed into E. coli DH5α competent.

The preparation method of Escherichia coli DH5α competent competence is as follows: picking a single clone of Escherichia coli DH5α, and activating and culturing in liquid LB medium. To prepare 1.5M MgCl ₂ , 3.0495g of MgCl ₂ ·6H ₂ O was weighed, and ddH ₂ O was adjusted to 10 mL. To prepare Solution A, dissolve 0.098955g MnCl ₂ ·4H ₂ O (10mM), 0.277450g CaCl ₂ (50mM), 0.097615g 2-morpholinoethanesulfonic acid (10mM) in an appropriate amount of ddH ₂ O, and then add 7.5mL Glycerol, and finally made up to 50 mL with _ddH2O . Take 1 mL of activated bacterial solution in 50 mL of liquid LB (containing 15 mM MgCl ₂ ), incubate at 37° C. with a shaker to OD600=0.6-0.85, and centrifuge at 1500 rpm-3000 rpm to collect bacterial cells. Discard the supernatant, add 3 mL of frozen Solution A to resuspend, and place on ice for 20 min. Dispense 50 μL per tube into 1.5 mL centrifuge tubes, snap-frozen in liquid nitrogen, and store at -80°C.

2) Operation steps of Escherichia coli DH5α transformation:

Melt the competent cells on ice slowly, add 5-10 μL ligation product or 1 μg plasmid, and place on ice for 20 min. Heat shock at 42°C for 1 min and place on ice for 1-2 min. Add 350 μL of liquid LB, mix well, and shake at 37°C for 45 min. Centrifuge at 12000 rpm for 1 min, remove 200 μL of supernatant, and resuspend the remaining 200 μL of bacterial liquid. All the resuspended bacterial liquids were spread on LB plates containing corresponding antibiotics (50 mg/L Kan), and cultured upside down at 37°C for 18-22 h.

(5) Colony PCR

Pick the single clone on the LB plate with a sterilized toothpick, rinse it in 50 μL of ddH ₂ O water, and use this bacterial solution as a template for PCR amplification. A 25uL system was used, and the system was as follows: 10×PCR Buffer 2.5μL, dNTP 0.5μL, OsmiR528-sgRNA1-F 0.5μL, ZY065RB (5'-ttctaataaacgctcttttctct-3') 0.5μL, Taq DNA enzyme 0.2μL, Template 1μL, ddH ₂ O 19.8 μL. The PCR program is: 94℃, 5min→(94℃, 30s→56℃, 30s→72℃, 10-60s) for 32 cycles→72℃, 5min→10℃, 5min; (Taq DNA enzyme, dNTP, etc. were purchased from Tiangen Biological Company).

After PCR, 5 μL of 6× bromophenol blue was added and detected by agarose gel electrophoresis.

(6) Plasmid extraction and sequencing verification

The colony PCR confirmed the correct single clone, shake the bacteria in LB containing 50mg/LKan, and extract the plasmid in the bacterial solution. The extraction of plasmid DNA is carried out according to the instructions of AXYGEN AxyPrepTM Plasmid Miniprep Kit. The extracted plasmids were sent to Qingke Biotechnology Co., Ltd. for sequencing verification. The plasmid was named pMIR528-1.

2. Rice genetic transformation mediated by Agrobacterium tumefaciens

(1) Preparation method of Agrobacterium tumefaciens EHA105 competent

The competent preparation of Agrobacterium tumefaciens EHA105 needs to be carried out under low temperature conditions, and the specific methods are as follows:

A sterilized toothpick was used to pick a single clone of Agrobacterium tumefaciens EHA105 strain in 10 mL of LB liquid medium (50 mg/LKan, 50 mg/LRif), and incubated at 28°C with constant shaking for 36-48 hours to activate the strain. Take 2 mL of the activated bacterial liquid in 100 mL of LB liquid medium (50 mg/LKan, 50 mg/LRif) for expansion culture, and shake it on a constant temperature shaker at 28°C until OD ₆₀₀ =0.7. Place on ice for 30min, centrifuge at 6000rpm for 10min at 4°C, and collect the cells. Add 1 mL of pre-chilled and sterilized 20 mM CaCl ₂ to resuspend the bacteria. The resuspended bacterial solution was dispensed into 1.5 mL centrifuge tubes in an amount of 100 μL per tube, quick-frozen in liquid nitrogen, and stored at -80 °C for later use.

(2) Transformation of Agrobacterium tumefaciens

Transform the extracted plasmid DNA into the competent Agrobacterium tumefaciens EHA105. The specific transformation method is as follows: put the competent cells on ice and slowly melt them, add 0.5-1 μg plasmid DNA to the melted competent cells, mix them gently, and put them on ice. placed on the 30min. 3) Quick-freeze in liquid nitrogen for 5 minutes, incubate at 37°C for 5 minutes, and ice bath for 2 minutes. Add 800 μL of liquid LB, and shake at 200 rpm for 2 to 3 hours at 28°C. Centrifuge at 6000 rpm for 2 min, collect the cells, remove 600 μL of supernatant, resuspend the remaining 200 μL, and spread evenly on LB plates containing 50 mg/L Kan and 50 mg/L Rif. 28°C, invert and incubate in the dark for 36-48h.

(3) Rice genetic transformation mediated by Agrobacterium tumefaciens

Agrobacterium-mediated transformation of rice refers to the method disclosed in the document Toki S, et al. Early infection of scutellumtissue with Agrobacterium allows high-speed transformation of rice. The Plant Journal, 2006, 47(6): 969-976. The specific steps of genetic transformation of rice are as follows (the following operations are all carried out under sterile conditions):

Peel and sterilize the mature seeds of rice (Nihonbare); inoculate the sterilized seeds on the N-6-D solid medium containing 0.4% gellan gum, and cultivate them in continuous light at 32°C for 1 to 5 days; The CRISPR-Cas9 expression plasmid pMIR528-1 was transformed into rice by the mediated transformation method, and the transformed rice seeds were cultured in the induction selective medium at 32°C for 2 weeks; the callus produced by proliferation was transformed into RE-III culture medium; young plants arising from callus were transferred to HF medium to induce root production. When the obtained resistant regenerated seedlings grow to about 15 cm, the root medium is washed with clean water, transplanted into nutrient soil, and cultivated in a greenhouse.

3. Positive detection of transgenic rice regenerated seedlings

(1) Extraction of genomic DNA from rice seedlings

The DNA extraction of rice seedlings adopts CTAB method, and the specific operation steps are as follows:

The CTAB extract was preheated in a 65°C water bath. Take 0.2 g of the material and put it in a mortar, add liquid nitrogen for quick freezing, quickly grind it into powder, and transfer it into an EP tube. Add 600 μL of pre-warmed CTAB extract solution, take a water bath at 65°C for 30 to 50 minutes, and mix thoroughly during this period. Add 600 μL of chloroform:isoamyl alcohol (24:1), invert and mix thoroughly, and centrifuge at 12000 rpm for 10 min at 4°C. Take the supernatant, add an equal volume of isopropanol for precipitation, and precipitate at -20 °C for 30 min to 2 h. Centrifuge at 10,000 rpm for 10 min at room temperature, and collect the precipitate. The supernatant was removed, rinsed with 75% ethanol, and centrifuged at 10,000 rpm for 2 min. Remove the supernatant, and dry the DNA naturally or blow dry until the smell of ethanol can no longer be smelled. Generally, wait for 10 to 20 minutes. Add 30-50 μL ddH ₂ O to dissolve the DNA, and store it in a -20°C refrigerator until use.

(2) Positive detection of transgenic rice seedlings

The positive detection of transgenic rice seedlings was carried out by PCR. PCR amplification was performed using the upstream primer TX067-ZmUbi-F: 5'-CATATGCAGCAGCTATATGTGGA-3' of the maize ubiquitin promoter and the downstream primer ZY295-Cas9-HP-2: 5'-TCTTCTCACCAGGGAGCTGAGCA-3' of the Cas9 protein. The fragment length is 838bp. The PCR amplification system and reaction procedures were the same as before.

4. PCR-SSCP mutant detection

Since the mature miRNA is only about 20bp, it is difficult to find a suitable restriction site when designing the target site, so it is difficult to detect the mutant by PCR-RFLP. Therefore, the single-strand conformation polymorphism SSCP (singlestrand conformation polymorphism, SSCP) was used for preliminary screening of mutants. Firstly, the target fragment was amplified by PCR, and then the PCR product was denatured, and the preliminary screening of the mutant was carried out by polyacrylamide gel electrophoresis. If the target site is mutated and denatured into single-stranded DNA, it will form a three-dimensional conformation different from normal DNA during polyacrylamide gel electrophoresis, thereby affecting its mobility and forming different bands. The specific operation steps are as follows:

(1) PCR amplification of CRISPR-Cas9 action site and denaturation of PCR products

The positive plants obtained from the detection were amplified by PCR with primers OsmiR528-SSCP-F: 5'-CACCAATGGATGCATCAGCAG-3' and OsmiR528-SSCP-R: 5'-TGAGAGTTTGGTGCAATAACAG-3', and the length of the amplified fragment was 298 bp. The PCR amplification system and reaction procedures were the same as before.

Add 5 μL of PCR product to 5 μL of SSCP denaturant, mix well, and denature at 95°C for 5 min; after the denaturation is completed, quickly put it in an ice box and cool for 10 min.

(2) Preparation of polyacrylamide gel

The preparation method of 15% PAGE (29:1) gel is as follows: 21 mL of Acr/Bis (29:1) gel solution, 150 μL of 10% Aps, 10 μL of TEMED, quickly stirred and mixed, and used for perfusion of the gel. Acrylamide Acr, methylenebisacrylamide Bis, ammonium persulfate Aps and tetramethylethylenediamine TEMED were purchased from AMRESCO Company.

(3) SSCP electrophoresis

After the gel was solidified, pre-electrophoresis was carried out at 45mA constant current electrophoresis for about 20min. Then disconnect the power supply and load 5 μL of denatured samples in sequence. Then at 4°C, 45mA constant current electrophoresis was performed for 4-5h.

(4) Staining and observation of PAGE gel

Remove the glue and rinse with water for 2-3 times; Dyeing: Add AgNO ₃ dye solution and place on a shaker for 10 minutes; Color development: Add NaOH color developer and place on a shaker for about 5 minutes until clear bands are seen; Dyeing is over , immediately rinsed with water to terminate the reaction. Observation: Spread the glue on the light box, observe and photograph.

The SSCP results (Fig. 2) showed that except for the individual plant No. 34, each individual plant was significantly different from the wild type, indicating that the CRISPR-Cas9 system can efficiently and directionally modify OsmiR528 to obtain mutants.

5. Sequencing verification of knockout mutants

The individual plants obtained by SSCP that were different from the wild type were amplified by PCR with primers OsmiR528-SSCP-F and OsmiR528-SSCP-R. The PCR amplification system and reaction procedure were the same as before. The PCR product was recovered for TA cloning, the TA cloning vector used was pMD19-T, and the operation procedure was according to the kit instructions. The positive clones were sent to Chengdu Qingke Biological Company for sequencing. The sequencing results (Fig. 3) showed that except for single plant No. 34, the rest of the single plants had mutations, and the mutation efficiency reached 93%, indicating that the CRISPR-Cas9 system can efficiently target OsmiR528 knockout.

Example 2 Using the method of the present invention to create multiple miRNA mutants (taking rice OsmiR397a and OsmiR97b as examples)

1. Vector construction of CRISPR-Cas9-miRNAs mutants

(1) Construction method of OsmiR397a and OsmiR397b double-site knockout vector

The OsmiR397 family has two members, OsmiR397a (GenBank number: AP014962:28489785...28489898) and OsmiR97b (GenBank number: XM_015769221). In order to obtain OsmiR397 knockout mutants, a double-site knockout mutant vector with simultaneous knockout of OsmiR397a and OsmiR97b was constructed. Based on the precursor sequences of OsmiR397a and OsmiR97b, we designed and synthesized OsmiR397a-sgRNA1, gRNA scaffold, U6 terminator, U6 promoter, OsmiR397b-sgRNA1 sequences OsmiR397a-gRNA1-OsmiR397b-gRNA1-F and OsmiR397a-gRNA1-OsmiR397b-gRNA1- R (synthesized by Shanghai Yingjun Biotechnology Co., Ltd.). OsmiR397a-gRNA1-OsmiR397b-gRNA1-F:

gtgtGAGTGCAGCGTTGATGAACAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTACTGCGAGCTCAAACTATCAGTGTTTGACAGGATATATTGGCGAGGATCCGCGGATCATGAACCAACGGCCTGGCTGTATTTGGTGGTTGTGTAGGGAGATGGGGAGAAGAAAAGCCCGATTCTCTTCGCTGTGATGGGCTGGATGCATGCGGGGGAGCGGGAGGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAGTGTGAGCGCGAGAGGCGGGAGGAACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTATAAACCCGCGCGCTGTCGCTTGTGTGTGCAGCGTTGATGAACCTGC

OsmiR397a-gRNA1-OsmiR397b-gRNA1-R:

aaaGCAGGTTCATCAACGCTGCACACACAAGCGACAGCGCGCGGGTTTATAAGTTGGTCGCGTTCGAGTTAGCTGGGCAATGTGGTACTAAACTGTTCCTCCCGCCTCTCGCGCTCACACTCGCCCTGTGGGCCGCTCACCGTGCACGTACTTGGGCCTCCCGCTCCCCCGCATGCATCCAGCCCATCACAGCGAAGAGAATCGGGCTTTTCTTCTCCCCATCTCCCTACACAACCACCAAATACAGCCAGGCCGTTGGTTCATGATCCGCGGATCCTCGCCAATATATCCTGTCAAACACTGATAGTTTGAGCTCGCAGTAATGCCAACTTTGTACAAGAAAGCTGGGTCTAGAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACTTGTTCATCAACGCTGCACTC

After annealing, the two DNA sequences were ligated with the BsaI digested recovery product (large fragment) of pZHY988. Then carry out transformation, colony PCR, plasmid extraction, and sequencing (methods are the same as those in Example 1).

After two PCR amplifications and one fusion PCR, the two target sites of OsmiR397a-gRNA1 and OsmiR397b-gRNA1 were loaded into pZHY988, and the simultaneous knockout of OsmiR397a and OsmiR397b pZJP028 was constructed (Figure 4).

After the construction of the vector is completed, the positive detection of Agrobacterium for transformation, genetic transformation of rice, and transgenic rice regenerated seedlings is the same as the corresponding part in Example 1.

2. PCR-SSCP mutant detection

The positive plants obtained by detection were used with primers OsmiR397a-SSCP-F: 5'-GCAGCTGTAGTACAGTAGTAC-3', OsmiR397a-SSCP-R: 5'-TTCTAACCATAACTGAGT TGCT-3' and OsmiR397b-SSCP-F: 5'-GCAGCTACACACGCACAAGCA- 3', OsmiR397b-SSCP-R:5'-GGATATGGATATGGATTGTGT-3' was subjected to PCR amplification. The PCR amplification system and reaction procedures were the same as before. The steps thereafter are the same as in Example 1.

The SSCP results (Fig. 5) showed that each individual plant was significantly different from the wild type, indicating that the CRISPR-Cas9 system could simultaneously perform targeted knockout of the OsmiR397a and OsmiR397b double sites to obtain double mutants. From Figure 5, among the 17 individual plants, 12 individual plants (ie 2-1, 7-1, 8-1, 13-1, 15-1, 16-1, 20-1, 21-1, 22-1, 23-1, 24-1, 24-2) have mutations.

3. Sequencing verification of knockout mutants

Select some of the individual plants with differences between SSCP and wild type in Figure 5 to carry out PCR amplification with primers OsmiR397a-SSCP-F, OsmiR397a-SSCP-R and OsmiR397b-SSCP-F, OsmiR397b-SSCP-, PCR amplification system and reaction The procedure is the same as before. The PCR product was recovered for TA cloning, the TA cloning vector used was pMD19-T, and the operation procedure was according to the kit instructions. Some positive clones were sent to Chengdu Qingke Biological Company for sequencing. The sequencing results showed that both OsmiR397a and OsmiR397b were mutated in the selected single plant, indicating that the CRISPR-Cas9 system can simultaneously knock out miRNAs and obtain multiple mutants.

Example 3 Using the method of the present invention to create miRNA large fragment deletion mutants (taking rice OsmiR408 as an example)

1. Vector construction of CRISPR-Cas9-miRNAs mutants

(1) Construction method of OsmiR408-sgRNA1 and OsmiR408-sgRNA2 double-site knockout vector

When considering all or most of the precursor miRNAs or when suitable PAM sites are difficult to find near some mature miRNA precursors, strategies for creating large deletions using two-site knockouts need to be considered.

According to the precursor sequence of OsmiR408, the sequences OsmiR408-gRNA1-gRNA2-F and OsmiR408-gRNA1-gRNA2-R containing sgRNA1, gRNA scaffold, U6 terminator, U6 promoter, sgRNA2 were designed and synthesized (delivered by Shanghai Yingjun Biotechnology). Ltd. Synthesis).

OsmiR408-gRNA1-gRNA2-F:

gtgtGATGAGGCAGAGCATGGGATGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTACTGCGAGCTCAAACTATCAGTGTTTGACAGGATATATTGGCGAGGATCCGCGGATCATGAACCAACGGCCTGGCTGTATTTGGTGGTTGTGTAGGGAGATGGGGAGAAGAAAAGCCCGATTCTCTTCGCTGTGATGGGCTGGATGCATGCGGGGGAGCGGGAGGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAGTGTGAGCGCGAGAGGCGGGAGGAACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTATAAACCCGCGCGCTGTCGCTTGTGTGGAAGAGGCAGTGCAGGGGA

OsmiR408-gRNA1-gRNA2-R:

cccaTCCCCTGCACTGCCTCTTCCACACAAGCGACAGCGCGCGGGTTTATAAGTTGGTCGCGTTCGAGTTAGCTGGGCAATGTGGTACTAAACTGTTCCTCCCGCCTCTCGCGCTCACACTCGCCCTGTGGGCCGCTCACCGTGCACGTACTTGGGCCTCCCGCTCCCCCGCATGCATCCAGCCCATCACAGCGAAGAGAATCGGGCTTTTCTTCTCCCCATCTCCCTACACAACCACCAAATACAGCCAGGCCGTTGGTTCATGATCCGCGGATCCTCGCCAATATATCCTGTCAAACACTGATAGTTTGAGCTCGCAGTAATGCCAACTTTGTACAAGAAAGCTGGGTCTAGAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACCATCCCATGCTCTGCCTCATC

The subsequent method is the same as the construction method of the double-site knockout vector in Example 2.

The obtained correct OsmiR408-sgRNA1 and OsmiR408-sgRNA2 double-site knockout vector was named pZJP025 (Figure 6), and the subsequent transformation of Agrobacterium, rice genetic transformation, and positive detection of transgenic rice regenerated seedlings were the same as the corresponding parts in Example 1 .

2. Detection of large fragment deletion mutants

The positive plants obtained by detection were amplified by PCR with primers Osa-miR408-SSCP-F1:5'-atcttgacgatgatggcgttg-3', Osa-miR408-SSCP-R1:5'-AGAGAGAGAGAGAGAGGTGTG-3'. The PCR amplification system and reaction procedures were the same as before. The steps thereafter are the same as in Example 1.

The results (Fig. 7) showed that among the 10 individual plants, there were 6 individual plants (pZJP025-01-01, pZJP025-02-01, pZJP025-03-01, pZJP025-04-01, pZJP025-09-02, pZJP025 -10-01) A band containing a small fragment, that is, a large fragment is deleted. It is shown that the CRISPR-Cas9 system can efficiently carry out targeted knockout of large fragments, thereby obtaining deletion mutants of large fragments. The corresponding bands were recovered and sequenced, and the results further proved that the CRISPR-Cas9 system can efficiently knock out large fragments of miRNAs, thereby obtaining deletion mutants of large fragments of miRNAs.

Claims (2)

1. A miRNAs-directed knockout method for OsmiR397a and OsmiR397b miRNAs, members of the rice OsmiR397 gene family, characterized in that: using the CRISPR-Cas9 method to knock out multiple miRNAs in rice, comprising the following steps: a. Construct the vector pZJP028 for knocking out multiple miRNAs for OsmiR397a and OsmiR97b, members of the rice OsmiR397 gene family; the backbone of the vector is pZHY988, and the core region between the left and right borders of the vector T-DNA contains the following elements: hygromycin resistance Gene expression elements, Cas9 expression elements, and gRNA transcription units; When knocking out multiple miRNAs, the gRNA transcription unit consists of two or more gRNA transcription elements in series, which respectively recognize the PAM sites near different target miRNAs; the PAM sites recognized by the gRNA transcription elements are 5'-N _X -NGG -3', N represents any one of A, G, C and T, x=20; Said gRNA transcription element comprises rice U6 promoter, ccdB and gRNA scaffold, U6 terminator, and each end of the ccdB fragment has a BsaI restriction site, and the nucleotide sequence is shown in SEQ ID No. When , the vector backbone is cut with BsaI enzyme, and the sgRNA targeting the target miRNA is inserted; the nucleotide sequence of the gRNA transcription unit is shown in SEQ ID No.3; The Cas9 expression element comprises the maize ubiquitin Ubiquitin-1 promoter Ubi, the Cas9 protein gene and the heat shock protein terminator HSP; The hygromycin expression element comprises cauliflower mosaic virus promoter 35S, hygromycin-resistant gene Hygromycin, and cauliflower mosaic virus terminator 35ST; b. Transform the CRISPR-Cas9 vector obtained in step a into rice callus, and screen to obtain rice miRNA mutants. 2 . The method of claim 1 , wherein in step b, the method used for screening knockout mutants is a combination of PCR-SSCP single-strand conformation polymorphism and sequencing. 3 .