Rice deubiquitinating enzyme OsOTU2 gene and application thereof in resisting southern rice black-streaked dwarf virus

Abstract

The invention relates to a rice deubiquitination enzyme OsOTU2 gene which is subjected to targeted editing by utilizing a CRISPR / Cas9 technology and application of the rice deubiquitination enzyme OsOTU2 gene in breeding of southern rice black-streaked dwarf virus resistant crops.

Description

Technical Field

[0001] The present invention relates to the fields of transgenic technology and plant disease prevention and control, and specifically to targeted editing of the rice deubiquitinase OsOTU2 gene using CRISPR / Cas9 technology and its application in resistance to southern rice black-streaked dwarf virus. Background Art

[0002] Southern rice black-streaked dwarf virus (SRBSDV) is a double-stranded RNA virus. The SRBSDV genome consists of ten double-stranded RNA strands, designated S1-S10. SRBSDV encodes 13 open reading frames (ORFs), at least six putative structural proteins (P1, P2, P3, P4, P8, and P10), and five putative nonstructural proteins (P6, P7-1, P7-2, P9-1, and P9-2). P3, P8, and P10 are an inner shell protein, a putative core protein, and a major outer shell protein, respectively. The P6 protein has been identified as a suppressor of RNA silencing.

[0003] SRBSDV is primarily transmitted through the insect vector, the white-backed planthopper (Sogatella furcifera), in a persistent, multiplying manner. In nature, SRBSDV hosts grasses and can infect rice, corn, coix seed, barnyard grass, white grass, and water sedge. Rice plants infected with SRBSDV exhibit stunted growth and dark green leaves early in the growth cycle, followed by small leaf spots on the stems and abnormal tillering in the upper parts of the plant. Infected corn plants exhibit stunted growth, a dark green color, and characteristic white, waxy galls on the veins on the undersides of the leaves. In recent years, climate warming and changes in agricultural management practices have increased the population density of the virus-transmitting planthopper, leading to outbreaks of rice viral diseases. Reducing the density of the insect vector by spraying large amounts of pesticides can lead to serious environmental pollution. Breeding resistant varieties is the most economical and effective way to control SRBSDV. However, there are currently no better resistant varieties. Therefore, in-depth research on the interaction between plant hosts and rice viruses can lay the foundation for us to control the harm of rice virus diseases from the source. Summary of the Invention

[0004] In order to address the deficiencies of the prior art, the present invention relates to a method for improving the resistance of crops (e.g., rice) to southern rice black-streaked dwarf virus by using CRISPR / Cas9 technology to target and edit the rice deubiquitinase OsOTU2.

[0005] In one aspect, the present invention relates to a deubiquitinating enzyme OsOTU2 gene, the nucleotide sequence of which is shown below (SEQ ID NO: 1).

[0006]

[0007] On the other hand, the amino acid sequence of rice deubiquitinase OsOTU2 (SEQ ID NO: 2) is as follows: MTRIFVQRGAAGSSSSSSRSGSQTLQQQQQLQATSAVAREEELPPQPHQHPSELTASDNITDHLVEDADNSSNSNKPLGLDDPTSESSSSAEERAVMEKPPKDDSSVIDPAFLVEELTGLQFSDQFEQENLVQSGIGPSQIAGAASHPPPPPAPPPKPSSGNNGLRRMGSGSSNNARIGSSRRPVAWPPVAVRSSASGSRPSSPRSLADSEGYNSADEQGPCYASNYYDSERERMFEHDLRRVRGFEINKMAEDGNCL FRAVADQVYGDPEAYDMARQMCVDYMERERDHFSQFMTEGFTSYCRRKRRDKVYGNNMEIQAFAEMYNRPIHIYSYSTEPINIFQGSYNTDVPPIRLSYHHGNHYNSVVDPRGLTVGAGLGFSSLRGTNNVDRD QVKAAIKAQQDQQIENALLAEGRLYSDLELTEKEIERMVMEASRAEYLKQQQQQLNFRESSTSGAEPSSSAAISGSSRSAGTADRVGEECFVLPDTVLTRSMQLLLAMGFNYIQVMEAYSIFGEDVDSMIYYLVE MGGTGASAGGSNRRKGKAAE*

[0008] On the other hand, the present invention relates to the use of the rice deubiquitinase OsOTU2 gene in the breeding of crops resistant to Reoviridae and Fijivirus, especially the breeding of Gramineae grain crops;

[0009] In some embodiments, the Fijivirus genus includes Maize Rough Dwarf Virus (MRDV), Rice black streaked dwarf virus (RBSDV), and Southern rice black streaked dwarf virus (SRBSDV);

[0010] In some embodiments, the Fijivirus is most preferably Southern Rice Black-streaked Dwarf Virus.

[0011] In some embodiments, the gramineous crops are preferably rice, corn, wheat, oats and barley; more preferably rice, and most preferably Nipponbare;

[0012] In some embodiments, the negative regulation of OsOTU2 gene expression is achieved by the Crispr / Cas9 system; the target of the Crispr / Cas9 system is CATGACCTAAGACGAGTGAGAGG (SEQ ID NO: 3).

[0013] In another aspect, the present invention relates to a method for preparing a transgenic plant resistant to southern rice black-streaked dwarf virus, comprising the steps of:

[0014] 1) Construction of rice OsOTU2 knockout vector;

[0015] 2) Rice genetic transformation, Agrobacterium transformation and callus induction;

[0016] 3) Identification of positive transgenic plants;

[0017] In some embodiments, the rice OsOTU2 knockout vector construction step further comprises:

[0018] Primers were designed based on the OsOTU2 sequence shown in SEQ ID NO:1 using the CRISPR-GE website (http: / / skl.scau.edu.cn / ). The sgRNA fragment was cloned from rice and the PCR product was recovered from gel. Using the pYLsgRNA-OsU3 vector as a template, the target sequence was ligated to the U3 promoter and gRNA scaffold, respectively. The two PCR products were mixed and used as templates to connect the U3 promoter, target sequence, and gRNA scaffold together using overlapping PCR to construct the sgRNA expression cassette. The gRNA expression cassette product was mixed with the uncut pYLCRISPR / Cas9Pubi-H plasmid and digested with BsaI at 37°C for 15 minutes. After digestion, 1.5 μl of 10× DNA Ligase Buffer and 35 U of T4 ligase were added, and 15 cycles of temperature cycling were performed: 37°C for 5 minutes, 10°C for 5 minutes, and 20°C for 5 minutes. Positive clones were selected and sequenced to confirm the successful construction of the expression vector YL-Hu-OsOTU2. The recombinant plasmid was obtained and its quality was checked by 1% agarose gel electrophoresis.

[0019] In some embodiments, the steps of rice genetic transformation, Agrobacterium transformation and callus induction culture further include:

[0020] Callus induction: First, select mature rice seeds, peel the husks, pour them into a centrifuge tube, add 75% ethanol to disinfect for 1 minute, then pour out the ethanol, rinse three times with sterile water, then add 15mL of 30% sodium hypochlorite to disinfect for 20 minutes, then pour out the sodium hypochlorite and rinse 5-6 times with sterile water; use a pipette to remove excess water, transfer the seeds to induction medium, and place them in a 28℃ light incubator for 3 weeks. Use pre-sterilized tweezers to clip the grown callus to the subculture medium and subculture in a 28℃ light incubator for 1 week;

[0021] Transformation and culture of Agrobacterium: The plasmid containing the target vector was transformed into Agrobacterium tumefaciens GV3101 using the following steps: 5 μL of plasmid was added to 100 μL of Agrobacterium GV3101 competent medium, and the mixture was mixed by pipetting. The mixture was added to a sterile electrode cup pre-cooled at 4°C, and the transformation was performed by electric shock at a voltage of 220 V. Non-resistant LB liquid culture medium was added, and the culture was shaken at a constant temperature of 28°C for about 3 hours. The culture was evenly spread on LB solid culture medium containing 50 μg / ml Kan and 50 μg / ml Rif, and the culture was carried out in the dark at 28°C for 2 days until single colonies appeared.

[0022] Agrobacterium transfection: Use a pipette to remove the Agrobacterium from the plate. This is the Agrobacterium suspension used for co-culture with rice. Select a sufficient amount of callus tissue and place it in a 100ml sterile Erlenmeyer flask. Add an appropriate amount of Agrobacterium suspension and incubate at room temperature for 20 minutes, shaking occasionally. Discard the inoculum suspension and place the callus on sterile filter paper to remove excess liquid. Immediately transfer the callus to a solid co-culture medium covered with sterile filter paper and incubate in the dark at 26°C for 3 days.

[0023] Screening and culture: After 3 days of co-culture, the callus tissue needs to be cleaned. Use a 1ml blue pipette tip to sow the callus on the co-culture medium into a sterilized Erlenmeyer flask, add sterile water to rinse both sides, and rinse once with sterile water containing 500ul / L carbenicillin for the third time. After the excess water is sucked away by a pipette, the callus is transferred to sterile filter paper and the water on the callus is blown dry by the air from the clean bench. The blowing time is controlled at about 30 minutes. After the callus is dried, it is transferred to a culture medium containing hygromycin B for screening and culture. The culture conditions are 28-30℃ and dark culture; the screening time is 3-4 weeks.

[0024] Rooting of seedlings: When the differentiated seedlings grow to about 2-3cm and have obvious root systems, they can be transferred to rooting medium to allow them to grow. The rooting medium should be poured into a relatively high bottle or tube so that the rooted seedlings have enough space to grow taller. The rooting culture conditions are 28-30℃ and sterile light culture.

[0025] In some embodiments, the step of identifying positive transgenic plants further includes extracting DNA from young leaves of individual T0 and T1 generations, amplifying the sgRNA targeting site using target-specific primers, detecting the amplified products by 1% gel electrophoresis, and recovering specific, correctly sized fragments from the gel for sequencing. Positive transgenic plants confirmed by sequencing were designated osotu2-ko (osotu2-5# and osotu2-13#), confirming the successful construction of the transgenic rice line.

[0026] In some embodiments, the step of identifying positive transgenic plants further comprises:

[0027] The resistance of the overexpressing mutant osotu2-ko transgenic line to southern rice black-streaked dwarf virus was tested by artificial inoculation with SRBSDV.

[0028] In some embodiments, the induction medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, pH=5.8.

[0029] In some embodiments, the secondary culture medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, 50 mg / L hygromycin, 300 mg / mL cephalosporin, and pH=5.8.

[0030] In some embodiments, the rooting medium comprises: 1 / 2 MS 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, pH=5.8.

[0031] In some embodiments, the co-culture medium comprises: 24.1 g / L N6 medium, 2 mg / L 2,4-D, 200 μmol / L acetosyringone, and pH=5.2.

[0032] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0033] This study constructed the YL-Hu-OsOTU2 vector and introduced it into the rice variety Nipponbare using Agrobacterium-mediated transfection. Two stably inherited transgenic rice lines, named osotu2-5# and osotu2-13#, were obtained. Their resistance to southern rice black-streaked dwarf virus (SRBSDV) was also analyzed, which will help deepen our understanding of rice viruses and delve into the molecular mechanisms of interaction between rice and SRBSDV. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 :Consequences of osotu2 mutation in osotu2-ko mutant transgenic rice.

[0035] Figure 2: Disease symptoms of osotu2-ko mutant transgenic and control Nipponbare after SRBSDV infection.

[0036] Figure 3 Detection of viral load in transgenic osotu2-KO mutant and control Nipponbare rice after SRBSDV infection. Mock: healthy rice; SRBSDV: southern rice black-streaked dwarf virus (SRBSDV)-infected rice; NIP: Nipponbare rice.

[0037] The following examples are provided to better illustrate the present invention, but are not intended to limit the present invention to these examples. Non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above invention are still within the scope of protection of the present invention. DETAILED DESCRIPTION

[0038] The rice varieties used in this series of experiments are: Nipponbare

[0039] The relevant culture medium components are as follows:

[0040] Induction medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601) 24.1 g / L, 2 mg / L 2,4-D, pH = 5.8.

[0041] Subculture medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: 15HBZ0601) 24.1 g / L, 2,4-D, 50 mg / L hygromycin, 300 mg / mL cephalosporin, pH = 5.8.

[0042] Co-culture medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601) 24.1 g / L, 2 mg / L 2,4-D, 200 μmol / L acetosyringone, pH = 5.2.

[0043] Rooting medium: 1 / 2MS (Manufacturer: Haibo Biotechnology Co., Ltd., Product No.: HB8469-6)

[0044] 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, pH = 5.8.

[0045] Example 1: Rice YL-Hu-OsOTU2 vector construction

[0046] CRISPR / Cas9 gene editing vector construction plan, using overlapping PCR to construct the sgRNA expression cassette. The PCR primer sequences are:

[0047] SEQ ID NO:4

[0048] YL-Hu-OsOTU2-F:CAGTGGTCTCATGCATCACGGCTTTCAGACAATATAA;

[0049] SEQ ID NO:5

[0050] YL-Hu-OsOTU2-R:CAGTGGTCTCAAAACTGTTAATTCCTCCACCAGAA;

[0051] The specific steps are as follows:

[0052] Primers were designed based on the OsOTU2 sequence shown in SEQ ID NO:1 on the CRISPR-GE website (http: / / skl.scau.edu.cn / ). A sgRNA fragment (target sequence binding region shown in SEQ ID NO:3) was cloned from rice. The PCR product was gel-recovered. Using the pYLsgRNA-OsU3 vector as a template, the target sequence was ligated to the U3 promoter and gRNA scaffold, respectively. The two PCR products were mixed and used as templates. Overlapping PCR was used to connect the U3 promoter, target sequence, and gRNA scaffold to form the sgRNA expression cassette. The gRNA expression cassette product was mixed with the uncut pYLCRISPR / Cas9Pubi-H plasmid and digested with BsaI at 37°C for 15 minutes. After enzyme digestion, add 1.5 μl of 10× DNA Ligase Buffer and 35 U of T4 ligase. Perform 15 cycles of enzyme digestion and ligation using a variable temperature cycling program (37°C for 5 min, 10°C for 5 min, and 20°C for 5 min). Select positive clones and sequence them to confirm the successful construction of the expression vector YL-Hu-OsOTU2. Recombinant plasmids were obtained and their quality was verified by 1% agarose gel electrophoresis.

[0053] After product recovery and purification, the sgRNA expression cassette was ligated to the YL-Hu expression vector using a cut-and-ligate method. The ligated product was heat-shocked and transformed into competent E. coli. Positive clones were selected for sequencing, and the recombinant vectors that were sequenced correctly were subsequently transformed into Agrobacterium tumefaciens GV3101.

[0054] Example 2: Rice genetic transformation

[0055] 1) Callus Induction and Subculture: Select freshly harvested mature rice seeds, remove the husks, and pour into a 50ml centrifuge tube. Add 15ml of 75% ethanol to disinfect for 1 minute. Discard the ethanol, rinse three times with sterile water, and then discard. Add 15ml of 30% sodium hypochlorite to disinfect for 20 minutes. Discard the sodium hypochlorite and rinse 5-6 times with sterile water. Use a pipette to remove excess water, transfer the seeds to induction medium, and culture in a 28°C light incubator for 3 weeks. Use pre-sterilized forceps to transfer the emerging callus to subculture medium and subculture in a 28°C light incubator for 1 week.

[0056] 2) Transformation and Culture of Agrobacterium: The plasmid containing the target vector was transformed into Agrobacterium tumefaciens GV3101 (Manufacturer: Shanghai Weidi, Catalog No.: AC1003S) using the following steps: 5 μL of plasmid was added to 100 μL of competent Agrobacterium GV3101, and the mixture was pipetted and mixed. The mixture was added to a sterile electrode cup pre-cooled at 4°C, and transformed by electroporation at 220 V. Non-resistant LB liquid medium was added, and the cells were shaken and cultured at 28°C for about 3 h. The cells were evenly spread on LB solid medium containing 50 μg / ml Kan and 50 μg / ml Rif, and cultured in the dark at 28°C for 2-3 days until single colonies appeared.

[0057] 3) Agrobacterium Transfection of Callus: Use a pipette to aspirate the infection solution and rinse the Agrobacterium off the plate to prepare the Agrobacterium suspension needed for rice transformation. Select a sufficient amount of callus (in good condition, bright yellow, round and firm, with particles approximately 3 mm in diameter) and place it in a 100 ml sterile Erlenmeyer flask. Add an appropriate amount of Agrobacterium suspension (ensure sufficient contact between the callus and the material). Incubate the callus at room temperature for 20 minutes, shaking occasionally. Discard the suspension and place the callus on sterile filter paper to remove excess solution. Immediately transfer the callus to a solid co-culture medium covered with a layer of sterile filter paper and incubate in the dark at 26°C for 3 days.

[0058] 4) Screening culture: After 3 days of co-culture, the callus tissue needs to be cleaned. Use a 1ml blue pipette tip to transfer the callus on the co-culture medium to a sterilized Erlenmeyer flask and rinse twice with sterile water. Rinse once with sterile water containing 500ul / L carbenicillin for the third time. After removing excess water with a pipette, transfer the callus to sterile filter paper and use the air from the clean bench to dry the water on the callus. The blowing time should be controlled at about 30 minutes. After the callus is dried, transfer it to the screening culture medium for screening culture. The culture conditions are 28-30℃ and dark culture. The screening time is 3-4 weeks.

[0059] 5) Rooting: When seedlings reach approximately 2-3 cm in height and have a visible root system, they can be transferred to rooting medium for further growth. The rooting medium should be placed in a relatively high bottle or tube to allow the seedlings ample room to grow upward. Optimal rooting conditions are sterile light culture at 28-30°C.

[0060] Example 3: Identification of positive transgenic plants

[0061] Young leaves from individual T0 and T1 plants were taken and DNA was extracted using the CTAB method. Target-specific primers were used to amplify the sgRNA targeting site. Amplified products were detected by 1% gel electrophoresis. Specific and correctly sized fragments were recovered from the gel and sequenced. The sequencing primers are as follows:

[0062] SEQ ID NO:6OsOTU2-F:TCACGGCTTCAGACAATATAA

[0063] SEQ ID NO:7OsOTU2-R:TGTTAATTCCTCCACCAGAA

[0064] The experimental results are as follows Figure 1 As shown, sequencing results showed that osotu2-5# lacked a base G at the target site, causing OsOTU2 to terminate prematurely at amino acid position 246, while osotu2-13# had a base T inserted at the target site, causing OsOTU2 to terminate prematurely at amino acid position 250. This demonstrated that the transgenic rice line was successfully constructed.

[0065] Example 4: Artificial inoculation of SRBSDV

[0066] (1) The osotu2-ko transgenic plants and the control Nipponbare rice materials were soaked and germinated for 2-3 days. After the seeds turned white, they were sown in 1L beakers, with about 30 seedlings per cup, and three biological replicates. The culture was carried out at 30℃, 16h light and 8h dark conditions.

[0067] (2) Newly hatched 1-2 instar non-virulent white-backed planthoppers were infected with SRBSDV for 3-5 days and then transferred to healthy rice seedlings for a 10-day circulation period to obtain white-backed planthoppers that can be used to spread SRBSDV. Virus inoculation experiments were conducted using white-backed planthoppers of the same instar carrying SRBSDV and healthy white-backed planthoppers. Three white-backed planthoppers per seedling were inoculated with virus-infected and non-virus-infected white-backed planthoppers on rice seedlings at the three- to four-leaf stage (about 15 seedlings). After feeding for 3 days, all the insects were swept out.

[0068] (3) After 30 days, the symptoms of the diseased rice were observed and the virus infection status of the rice was determined by qRT-PCR.

[0069] Example 5: Resistance analysis of rice after inoculation with SRBSDV

[0070] 30 days after transplanting rice, it was found that rice infected with SRBSDV showed mosaic symptoms. Compared with the control Nipponbare NIP, the two osotu2-ko mutant transgenic lines (osotu2-5# and osotu2-13#) showed mild mosaic symptoms, while the control NIP showed obvious mosaic symptoms, such as Figure 2 As shown. The diseased rice plants were mixed and sampled, with 3 biological replicates in each group, and the expression levels of the virus SRB-S2 and S10 genes were detected by qRT-PCR. Figure 3 As shown in the figure, the expression levels of S2 and S10 genes in the transgenic lines were significantly lower than those in the control. The above results indicate that the resistance of transgenic plants to SRBSDV infection is affected by the OsOTU2 gene, and that mutation of OsOTU2 in rice can significantly enhance the resistance of rice to SRBSD infection.

[0071] The sequences of the quantitative primers are as follows:

[0072] qSRB-S2-F:CATCGACCAAGTTCAACCCG; SEQ ID NO:8

[0073] qSRB-S2-R:AAGAAGTCTGCGGGTGAAGA; SEQ ID NO:9

[0074] qSRB-S10-F:TTGACATAGCGCCCGATCTT; SEQ ID NO:10

[0075] qSRB-S10-R:ACGCGATGTGGGGTGATTTA; SEQ ID NO:11

[0076] In summary, the experimental results show that compared with the control NIP, transgenic rice plants with the osotu2-ko mutant significantly enhance rice resistance to SRBSDV infection (including phenotypic symptoms and viral load in diseased plants). Therefore, the present invention successfully generated SRBSDV-resistant transgenic rice through gene editing, which has important implications for agricultural development and national food security, and achieved unexpected technical results.

Claims

1. Application of the rice deubiquitinase OsOTU2 gene in Southern rice blackstreaked dwarf virus (SRBSDV)-resistant crop breeding, the gene sequence of which is shown in SEQ ID NO:

1.

2. The use according to claim 1, wherein the protein sequence encoding the OsOTU2 gene is shown in SEQ ID NO:

2.

3. The use according to any one of claims 1 to 2, wherein the crop is preferably rice, corn, wheat, oats and barley; more preferably rice, and most preferably Nipponbare.

4. The use according to any one of claims 1 to 3, wherein the resistance of rice to Southern rice black streaked dwarf virus (SRBSDV) is increased by negatively regulating the expression of the OsOTU2 gene.

5. The use according to any one of claim 4, wherein the negative regulation of OsOTU2 gene expression is achieved by the Crispr / Cas9 system; the nucleotide sequence of the Crispr / Cas9 system target is shown in SEQ ID NO:

3.

6. A method for preparing transgenic rice resistant to Southern rice black streaked dwarf virus (SRBSDV), comprising the following steps: 1) Construction of rice OsOTU2 gene knockout vector; 2) Rice genetic transformation, Agrobacterium transformation and callus induction; 3) Identification of positive transgenic plants.

7. The method according to claim 6, wherein the rice OsOTU2 knockout vector construction comprises the following steps: Primers were designed based on the OsOTU2 sequence shown in SEQ ID NO: 1, and the gene sgRNA fragment was cloned from rice. The PCR product was recovered from gel; Using the pYLsgRNA-OsU3 vector as a template, the target sequence was ligated to the U3 promoter and gRNA scaffold, respectively. The two PCR products were mixed as templates, and the U3 promoter, target sequence, and gRNA scaffold were ligated together using overlapping PCR to form an sgRNA expression cassette. The gRNA expression cassette product and the uncut pYLCRISPR / Cas9Pubi-H plasmid were mixed and digested with BsaI at 37°C for 15 min. After digestion, Ligase Buffer and T4ligase were added, and the cells were subjected to 15 cycles of temperature cycling, with the cycle program being 37°C for 5 min, 10°C for 5 min, and 20°C for 5 min. Positive clones were selected and sequenced to confirm that the rice OsOTU2 gene knockout expression vector YL-Hu-OsOTU2 had been successfully constructed.

8. The method according to claim 7, wherein the primer sequences are as follows: YL-Hu-OsOTU2-F:CAGTGGTCTCATGCATCACGGCTTCAGACAATATAA; YL-Hu-OsOTU2-R:CAGTGGTCTCAAAACTGTTAATTCCTCCACCAGAA.

9. A method for improving rice resistance to southern rice black-streaked dwarf virus, comprising targeting and editing the rice deubiquitinase OsOTU2 gene using CRISPR / Cas9 technology, wherein the nucleotide sequence of the OsOTU2 gene is shown in SEQ ID NO:

1.

10. An sgRNA, wherein the target sequence binding region thereof is shown in SEQ ID NO: 3.

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