CRISPR-Cas13a-based crRNA sequence and plasmid for targeting vibrio parahaemolyticus and application of crRNA sequence and plasmid

Abstract

The invention discloses a crRNA sequence and a plasmid for targeting vibrio parahaemolyticus based on CRISPR-Cas13a (clustered regularly interspaced short palindromic repeats) and application of the crRNA sequence and the plasmid. According to the invention, crRNA of a targeted T2RHS-Nuc gene is designed, a plasmid of tetracycline-induced expression Cas13a protein is established, the plasmid is introduced into vibrio parahaemolyticus under the assistance of escherichia coli and targets a target gene, and after mRNA of the target gene is recognized, targeted cleavage activity is generated, so that the affiliated cleavage activity of Cas13a is activated, a transcript of a bacterial strain is randomly cleaved in the bacterial body, and the expression of the Cas13a protein is promoted. When the transcript is necessary for the growth of the vibrio parahaemolyticus, the specific death of the vibrio parahaemolyticus can be caused. The crRNA and Cas13a protein expression vector and the sterilization method thereof provided by the invention do not induce vibrio parahaemolyticus to generate drug resistance, are a good alternative method for vibrio parahaemolyticus disease source control by using a traditional bactericide, and have a very good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a crRNA and CRISPR-Cas13a protein expression plasmid for targeted inhibition and killing of Vibrio parahaemolyticus based on the CRISPR-Cas13a system, as well as plasmid transformation mediated by Escherichia coli S17-1 and applications thereof. Background Art

[0002] Vibrio parahaemolyticus is a Gram-negative bacillus. As a typical halophilic pathogen in marine environments, it is one of the major pathogens of foodborne illness in coastal areas worldwide. It enters the food chain through contamination of seafood such as shellfish and fish. Its virulence factors, such as heat-stable direct hemolysin (TDH) and TDH-related hemolysin (TRH), can cause intestinal mucosal damage and inflammation in humans. Clinically, the main symptoms are acute onset, abdominal pain, vomiting, diarrhea, and watery stools. The bacterium has a strong adaptability to its environment and is halophilic and acidophilic. It can grow in culture media with a salt content of 2.5%-3.5%, but will not grow if the salt concentration is below 0.5%. It will die after being treated in ordinary vinegar for 1-3 minutes. Its optimal culture temperature is 30°C-37°C and a pH of 8.0-8.5. It will not grow below pH 6. It has low resistance to high temperatures and can be killed by treatment at 50°C for 20 minutes, 65°C for 5 minutes, or 80°C for 1 minute. If traditional prevention and control measures fail to completely inactivate the bacterium, it may multiply again during cold chain transportation, processing, and storage, leading to an increased risk of foodborne illness. Furthermore, Vibrio parahaemolyticus is prone to forming biofilms, which further enhances its resistance to disinfectants and antibiotics, making it a difficult-to-eradicate source of contamination in the food industry.

[0003] Current prevention and control technologies for Vibrio parahaemolyticus mainly include physical sterilization technologies: high-temperature sterilization (such as pasteurization) and ultraviolet irradiation are commonly used methods in the food industry, but high-temperature treatment will destroy the nutritional content and taste of seafood, and is not suitable for products such as ready-to-eat seafood and raw sashimi. Ultraviolet irradiation is limited by its penetration ability and is difficult to kill deep-seated contaminating bacteria, and the sterilization effect is unstable. Chemical prevention and control methods: Although chemical reagents such as chlorine-containing disinfectants and organic acids can inhibit bacterial growth, excessive use can easily lead to chemical residues, which does not meet the current safety requirements of "clean production" and "green food". The misuse of antibiotics (such as tetracycline and chloramphenicol) in aquaculture has led to the emergence of multidrug-resistant strains of Vibrio parahaemolyticus, exacerbating the difficulty of clinical treatment. Biological control technologies: Phage therapy and probiotic intervention are emerging approaches that work through specific lysis or competitive inhibition. However, phages have a narrow host range (targeting only specific serotypes), and long-term use may induce phage-resistant bacterial mutations. The antibacterial effect of probiotics is affected by environmental pH, salt concentration, and differences in the host's intestinal flora, resulting in insufficient stability in practical applications. Exploration of gene editing applications: Existing research on bacterial control based on the CRISPR-Cas system focuses primarily on Gram-positive bacteria (such as Listeria). A mature technical solution for the Gram-negative bacterium Vibrio parahaemolyticus has yet to be developed due to issues such as the outer membrane barrier and low CRISPR system delivery efficiency.

[0004] Given the high pathogenicity and strong environmental adaptability of Vibrio parahaemolyticus, as well as the shortcomings of existing technologies, there is an urgent need to develop novel control technologies that are highly specific, safe, efficient, and independent of host metabolic status. As an RNA-targeted gene editing tool, the CRISPR-Cas13a system, with its highly specific recognition capabilities mediated by a single crRNA, offers a new approach to addressing the challenges of Gram-negative bacterial control. Summary of the Invention

[0005] The purpose of the present invention is to provide a crRNA for targeting and killing Vibrio parahaemolyticus based on the CRISPR-Cas13a system and a plasmid expressing the Cas13a protein and its application, so as to solve the shortcomings of insufficient specificity of sterilization methods such as antibiotics, as well as the problems of low efficiency of crRNA targeting Vibrio parahaemolyticus and insufficient plasmid expressing the Cas13a protein due to the reliance on CRISPR-Cas13a technology.

[0006] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0007] In a first aspect, the present invention provides a crRNA that specifically targets the T2RHS-Nuc gene of Vibrio parahaemolyticus, including crRNA1 or crRNA2, the nucleotide sequence of the crRNA1 is shown in SEQ ID NO.1, and the nucleotide sequence of the crRNA2 is shown in SEQ ID NO.2.

[0008] crRNA1: AAAAATGAAAGCTTTGACCAAGG;

[0009] crRNA2:TGCTTTTGCAAATACAGCCGCAT.

[0010] The nucleotide sequence of the T2RHS-Nuc gene is shown in SEQ ID NO.3.

[0011] In a second aspect, the present invention also provides a bactericidal plasmid for Vibrio parahaemolyticus, which contains the above-mentioned crRNA specifically targeting Vibrio parahaemolyticus, a nucleotide sequence encoding LsCas13a protein, and a tetracycline-inducible expression element.

[0012] The LsCas13a protein is derived from the pC003 plasmid (Addgene plasmid library: 79152), and the nucleotide sequence of the LsCas13a protein is shown in SEQ ID NO.4.

[0013] The tetracycline-inducible expression element is derived from the pC008 plasmid (Addgene plasmid library: 79157), and the nucleotide sequence of the tetracycline-inducible expression element is shown in SEQ ID NO.5.

[0014] The bactericidal plasmid is based on the pBBR1 plasmid as a backbone, and the nucleotide sequence of the pBBR1 plasmid is shown in SEQ ID NO.6.

[0015] In a third aspect, the present invention also provides the use of the aforementioned bactericidal plasmid in targeted killing of Vibrio parahaemolyticus for purposes other than disease diagnosis and treatment.

[0016] In a fourth aspect, the present invention also provides a recombinant bacterium containing the above-mentioned bactericidal plasmid.

[0017] The host strain of the recombinant bacteria is Escherichia coli.

[0018] In a fifth aspect, the present invention also provides the use of the above-mentioned recombinant bacteria in the preparation of a bactericidal agent against Vibrio parahaemolyticus.

[0019] In a sixth aspect, the present invention also provides a method for targeted elimination of Vibrio parahaemolyticus based on the CRISPR-Cas13a system, wherein the above-mentioned bactericidal plasmid is heat-shock transformed into Escherichia coli competent cells; and Escherichia coli carrying the bactericidal plasmid and Vibrio parahaemolyticus are co-incubated.

[0020] The Escherichia coli is preferably Escherichia coli S17-1.

[0021] The bactericidal plasmid is a pBBR1-TetR-Cas13a-crRNA1 plasmid or a pBBR1-TetR-Cas13a-crRNA2 plasmid. The nucleotide sequence of the pBBR1-TetR-Cas13a-crRNA1 plasmid is shown in SEQ ID NO.7; the nucleotide sequence of the pBBR1-TetR-Cas13a-crRNA2 plasmid is shown in SEQ ID NO.8.

[0022] The bactericidal plasmid is constructed by the following method:

[0023] First, the crRNA synthesized by Genentech was cloned into the pC003 plasmid;

[0024] Secondly, the LsCas13a protein coding gene and crRNA from the pC003 plasmid were amplified using primers trace-F and PAM-QJ-R;

[0025] Then, the tetR inducible expression element was amplified from the pC008 plasmid using ptet-F and ptet-R primers;

[0026] At the same time, pBBR1 plasmid (sequence shown in SEQ ID NO. 6) was linearized by PCR using primers p2-F and p1-R;

[0027] Finally, the LsCas13a protein coding gene, crRNA, tetR inducible expression element, and PCR linearized pBBR1 amplified by PCR were amplified by Novozymes The Ultra One Step Cloning Kit was used to connect them and construct a bactericidal plasmid.

[0028] The present invention optimizes the crRNA design and delivery system that relies on the CRISPR-Cas13a system to achieve precise cleavage of specific gene mRNA of Vibrio parahaemolyticus and activate the accessory cleavage activity of the Cas13a protein. If the essential genes for the growth of Vibrio parahaemolyticus are cleaved, the strain is inhibited and killed. The above method provides a feasible idea for the safe control of Vibrio parahaemolyticus in seafood.

[0029] The present invention has the following beneficial effects:

[0030] 1. The present invention uses a highly sensitive CRISPR-Cas13a system to specifically target the endogenous transcripts of Vibrio parahaemolyticus, thereby achieving specific targeted sterilization. The system can target the endogenous mRNA of a specific strain through specific crRNA. After recognizing the specific mRNA, it will produce targeted cleavage activity and accessory cleavage activity, leading to the specific death and elimination of Vibrio parahaemolyticus.

[0031] 2. The present invention establishes a plasmid based on CRISPR-Cas13a for targeted elimination of Vibrio parahaemolyticus. The plasmid has the function of simultaneously expressing Cas13a protein and carrying crRNA and tetracycline-inducible expression elements. The construction is convenient and fast, and the expression of Cas13a protein can be controlled by adding tetracycline. The Cas13a protein expression vector is introduced into Vibrio parahaemolyticus by conjugation transfer, which greatly improves the transformation efficiency and effect.

[0032] 3. The present invention provides an application based on the CRISPR-Cas13a system for targeted elimination of Vibrio parahaemolyticus, which is expected to develop a new type of Vibrio parahaemolyticus bactericide. Since this method is targeted sterilization, it is not easy to produce drug resistance, which is of great significance for reducing the harm of this strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the construction process of the pBBR1-tetR-Cas13a-crRNA plasmid.

[0034] Figure 2 The antibacterial effects of the experimental examples of the present invention are shown in Table 1. Different lowercase letters represent significant differences (p<0.05). DETAILED DESCRIPTION

[0035] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0036] The present invention is further illustrated below with reference to the examples. Specific experimental steps or conditions not specified in the following examples are, unless otherwise specified, performed in accordance with conventional experimental steps or conditions described in the literature in this field. The experimental reagents and consumables described in the following examples are all from conventional biochemical reagent companies unless otherwise specified.

[0037] Unless otherwise indicated, embodiments of the present invention will employ conventional techniques in the fields of molecular biology and the like, which are within the capabilities of those skilled in the art.

[0038] Example 1: Construction of CRISPR-Cas13a-crRNA system

[0039] (1) Selection of specific crRNA sequence: In order to design a targeted CRISPR-Cas13a system, the T2RHS-Nuc gene sequence of Vibrio parahaemolyticus was first retrieved and downloaded from the NCBI database (NCBI accession number: WP_005479434). The site in the conserved region was selected as the target of the CRISPR-Cas13a system, and the top two candidate crRNA sequences with higher scores were obtained from the Cas13a crRNA online design website (https: / / cas13design.nygenome.org / ). The sequences are shown in SEQ ID NO. 1-2:

[0040] crRNA1: AAAAATGAAAGCTTTGACCAAGG (SEQ ID NO. 1);

[0041] crRNA2: TGCTTTTGCAAATACAGCCGCAT (SEQ ID NO. 2).

[0042] (2) Preparation of crRNA single-stranded upstream and downstream primers: Based on the specific crRNA oligonucleotide sequence as the upstream primer sequence, at the same time, the downstream primer sequence of the reverse complementary upstream primer sequence was designed. In order to enable the two crRNAs to be smoothly inserted into the BsaI restriction site of the pC003 plasmid (Addgene plasmid library: 79152), the aaac sequence was added to the 5'-end of the forward primer, and the tatc sequence was added to the 5'-end of the reverse primer (Table 1). The primers were synthesized by BGI.

[0043] (3) Annealing of crRNA oligonucleotide chains: Mix equal volumes of upstream and downstream primers of the forward and reverse crRNA oligonucleotides, swirl gently to mix, place on a PCR instrument and incubate at 95°C for 5 minutes, then slowly cool at room temperature to obtain successfully annealed crRNA DNA double-stranded fragments.

[0044] (4) Linearization of the pC003 vector: The pC003 vector was digested with the restriction endonuclease BsaI. The digestion system was as follows: 1 μg of pC003 plasmid, 1 μL of BsaI restriction endonuclease (NEB), 5 μL of 10× CutSmart Buffer (NEB), and ddH2O to 50 μL. Reaction conditions: After incubation at 37°C for 2 h, the linearized pC003 vector after BsaI digestion was separated and purified by 1% agarose gel electrophoresis.

[0045] Table 1: crRNA sequences and related primers

[0046]

[0047] (5) Ligation reaction between crRNA fragment and linear vector: According to the DNA Ligation Kit ligation kit operation method of Takara Company, the linearized pC003 vector was ligated with the crRNAN23 fragment formed after annealing to construct the specific CRISPR-Cas13a system plasmid pC003-Cas13a-crRNA1 or pC003-Cas13a-crRNA2 for the T2RHS-Nuc gene. The fragment was ligated with the plasmid. The specific ligation steps were as follows: 4 μL of plasmid and 1 μL of crRNA N23 fragment were placed in a PCR tube, 5 μL of Ligation Mix was added, and the mixture was thoroughly mixed and ligated at 16°C overnight, and then transformed into Escherichia coli Turbo competent cells (NEB).

[0048] (6) PCR verification of transformants: Use an inoculation loop to pick a single clone colony from the Turbo competent cell plate and suspend it in 50 μL of physiological saline. Take 5 μL of the suspension as a template and use trance-F and PAM-QJ-R primers (Table 1) to amplify the crRNA region to confirm whether the crRNAN23 sequence is successfully inserted into the predetermined crRNA cloning site (BsaI restriction site) of the pC003 plasmid, thereby completing the effective identification of the transformant. The PCR identification reaction system is: 5 μL bacterial culture, 2 μL crRNA1-F / crRNA2-F, 2 μL PAM-QJ-R, 25 μL 2×Easy Taq PCR Super Mix, and ddH2O to make up the reaction system to a total volume of 50 μL. The reaction conditions are: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 15 s, 35 cycles; 72℃ final extension for 5 min. The positive strains were sent to BGI for sequencing to ensure that the inserted crRNA1 or crRNA2 sequence was correct.

[0049] (7) Strain culture and plasmid extraction: After the colonies verified as correct clones by sequencing were expanded in LB liquid carrying chloramphenicol (25 ng / mL) resistance, 1 mL of fresh bacterial liquid was taken and frozen in a glycerol tube at -80°C. The remaining fresh bacterial liquid was used to extract plasmid DNA.

[0050] Example 2: Construction of Cas13a protein expression plasmid

[0051] In order to construct the elimination function plasmid based on Cas13a, we selected pBBR1 (sequence as shown in SEQ ID NO.6) as the basic backbone vector.

[0052] The key steps in this process include precisely inserting the LshCas13a gene fragment and crRNA guide sequence from Leptotrichia shahii and the TetR regulatory element from the pC008 plasmid (Addgene plasmid library: 79157) into the pBBR1 vector, using a one-step seamless ligation method to construct the plasmid. The vector plasmid pBBR1 itself has a plasmid replication origin (ori) and a chloramphenicol resistance gene marker, forming a stable and operable linear molecular framework.

[0053] First, the high-fidelity DNA polymerase Prime STAR Max was used to amplify the 6046 bp Cas13a+crRNA fragment from the plasmid pC003-Cas13a-crRNA1 or pC003-Cas13a-crRNA2 containing the spacer sequence, the flanking repeat sequences, and the LshCas13a coding region using the trace-F and PAM-QJ-R primers.

[0054] Secondly, the high-fidelity DNA polymerase Prime STAR Max was also used to amplify the TetR-inducible expression element (784 bp) from the pC008 plasmid using ptet-F and ptet-R primers ( Table 1 );

[0055] Furthermore, the high-fidelity DNA polymerase Prime STAR Max was also used to amplify the relevant fragment from the pBBR1 plasmid using the P2-F and P1-R primers ( Table 1 ) to achieve linearization of the pBBR1 plasmid (3782 bp);

[0056] The following reaction system was used for all three PCR amplifications: 25 μL 2× Prime STAR Max Premix, 1 μL Primer F (10 μM), 1 μL Primer R (10 μM), 100 ng to 1 μg template, and 50 μL ddH₂O. Reaction conditions: 98°C for 10 s, 55°C for 15 s, 72°C for 5 s / kb, 35 cycles.

[0057] Finally, perform the recombinase-mediated ligation reaction: refer to Novozymes products Ultra OneStep Cloning Kit instructions: Operate on ice and place 3.5 μL of Cas13a+crRNA fragment PCR product, 0.5 μL of TetR inducible expression element, and 1 μL of linearized pBBR1 plasmid into a PCR tube. Then add 5 μL of 2× ClonExpress Mix and add ddH2O to a total volume of 10 μL. Mix thoroughly with gentle vortexing, react at 50°C for 15 minutes, and immediately cool on ice.

[0058] Transform the recombinant product into E. coli S17-1 competent cells: Add the ligation product on ice to 100 μL of E. coli S17-1 competent cells. Place the cells on ice for 30 minutes, then heat shock them in a 42°C water bath for 90 seconds. Continue to ice bath for 2 minutes. Then, suspend the competent cells in 1 mL of LB liquid medium and culture them on a shaker at 37°C and 180 rpm for 1 hour. Spread the appropriate amount of bacterial solution onto an LB solid plate containing chloramphenicol (25 ng / mL) and culture at 37°C for 12-16 hours. E. coli S17-1 competent cells were prepared using the Sangon Super Competent Cell Preparation Kit, and the instructions were followed.

[0059] Identify transformants: Use an inoculating loop to pick several single colonies from the resistant plate and resuspend each in 50 μL of saline. 5 μL of this suspension was used as a PCR template for amplification of the fragment junction region using the ptet-F and PAM-QJ-R primers to verify the success of the plasmid construction. The PCR amplification system consisted of 5 μL of bacterial culture, 2 μL of ptet-F, 2 μL of PAM-QJ-R, 25 μL of 2× Easy Taq PCR Super Mix, and ddH2O to a total reaction volume of 50 μL. The reaction conditions were: initial denaturation at 95°C for 5 min, followed by one cycle of denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 1.5 min, 35 cycles, and a final extension at 72°C for 5 min. Positive clones were sent to BGI for sequencing to ensure the correct insertion sequence.

[0060] Cas13a protein expression plasmids, i.e., pBBR1-tetR-Cas13a-crRNA1 and pBBR1-tetR-Cas13a-crRNA2, were constructed by the above method.

[0061] A vector without crRNA (i.e., pBBR1-tetR-Cas13a plasmid) was constructed according to the above steps as a control.

[0062] Example 3: Evaluation of conjugation transfer and strain elimination effects

[0063] (1) Method: Escherichia coli S17-1 carrying pBBR1-tetR-Cas13a-crRNA plasmid or pBBR1-tetR-Cas13a plasmid and wild-type Vibrio parahaemolyticus ATCC 17802 were cultured in LB medium overnight;

[0064] Collect the cells of the above strains, remove the culture medium, and wash the collected cells twice with PBS (pH 7.0). Then, adjust the cell concentration to OD 600=1.0, Escherichia coli S17-1 carrying pBBR1-tetR-Cas13a-crRNA plasmid or pBBR1-tetR-Cas13a plasmid: wild-type Vibrio parahaemolyticus ATCC 17802 were mixed at a ratio of 1:3;

[0065] Pipette 10 μL of the bacterial mixture and drop it onto a 0.45 μm filter membrane in an LB plate, then culture it at 37°C overnight.

[0066] After gently washing the bacteria off the filter membrane with a coating rod, dilute the solution to an appropriate concentration and spread it on a double-resistance plate (chloramphenicol 25 μg / mL and ampicillin 200 μg / mL), and culture at 37°C overnight.

[0067] After visible colonies grew on the plate, the bacteria were picked with a toothpick and colony PCR was performed using ptet-F and PAM-QJ-R primers. The amplification system was as shown in Example 2.

[0068] Use fresh LB liquid medium to culture the positive strains at 37°C overnight, and then adjust the bacterial concentration to OD 600 =1.0, then, gradient dilution is performed, i.e., 10 times, 100 times, and 1000 times of dilution, and 100 μL is drawn from the diluents of different dilution multiples and applied to a common LB plate, a double-resistant plate with tetracycline (500 μg / mL) and chloramphenicol (25 μg / mL) is added, and at least 3 plates are applied to each strain. After 24 hours of overnight culture at 37°C, the colony growth on the plate is observed and the colonies are counted. Because tetracycline-inducible expression elements are added to the pBBR1-Cas13a-crRNA-tetR plasmid, only when tetracycline is present in the culture medium, its Cas13a protein will be expressed and the function of the targeted cutting T2RHS-Nuc gene transcript under crRNA1 or crRNA2 guidance is played. In this technology, its auxiliary cutting function is also activated, so as to achieve the cutting of the essential genes for the growth of Vibrio parahaemolyticus, and ultimately achieve the purpose of inhibiting strain growth.

[0069] (2) Data statistics and analysis

[0070] All the experimental data above were expressed as mean ± standard deviation (Mean ± SD). The DPS data processing system was used to perform significance analysis on the experimental data. When p < 0.05, it indicated that there was a significant difference between the treatments. GraphPad-Prism software was used to draw the graphs.

[0071] (3) Experimental results

[0072] When the pBBR1-tetR-Cas13a plasmid was introduced into Vibrio parahaemolyticus, the strain inhibition rate was 5.5±3.3% ( Figure 2 ), indicating that the expression of Cas13a protein has a certain negative effect on the growth of Vibrio parahaemolyticus; when pBBR1-tetR-Cas13a-crRNA1 and pBBR1-tetR-Cas13a-crRNA2 were introduced into Vibrio parahaemolyticus, the inhibition rates were 87.3±1.8% and 75.3±4.1%, respectively ( Figure 2 ), it can be seen that pBBR1-tetR-Cas13a-crRNA1 has better antibacterial effect.

[0073] In a word, the present invention designs two crRNAs according to T2RHS-Nuc genes, and constructs and relies on tetracycline to carry out Cas13a protein induction expression pBBR1-tetR-Cas13a-crRNA plasmid, with the assistance of Escherichia coli S17-1, successfully imports the plasmid into Vibrio parahaemolyticus bacteria, and realizes the killing effect of Vibrio parahaemolyticus, thus, this method can be used as an important prevention and control method of Vibrio parahaemolyticus in the environment. Compared with traditional methods, because the method directly targets the transcript of Vibrio parahaemolyticus, therefore, the method is not easy to cause Vibrio parahaemolyticus to develop drug resistance, and has good application potential.

[0074] >SEQ ID NO: 3 (full length of T2RHS-Nuc gene)

[0075] ATGATTCCTCAATTTGTTATCCCTCTCACCAACTGCCTTGGTCAAAGCTTTCATTTTTCCA

[0076] GTCAGCCAATCCCAAAAGGTGAACACAAAAAGTTTGACTCAGAACAGAGTGCGAAGG

[0077] CGTTTCTAGATGACTTCGTACCGCTTAGCTCAAGCCGTGTTGAAGAGTTGTATCATCTAC

[0078] TTGGTCAGTTTCCCCCAAACGTACCGGATGAAGAACTGACACCAGAGCTTTATGCGGCT

[0079] GTATTTGCAAAAGCACTTGTGAATGGTTCTCTTTATGTCGCTAGTTTCCCTAAAACAAAG

[0080] AAAAACGCGACAATATCCAGTGAACCAACACCGGTACCTAAGCAAGTAAAAGCAAAGT

[0081] CTAAGCAGAACAAAGCACATACATCTTCTAAAACCAAGCTAAGAATTCAGCCTCTGCA

[0082] AAGCCTTTACAGACGGGCAGCGAATGTCATGAAAAAGCGGGAGATCCGGTTTTCTCTAGT

[0083] AACGGGCGAAGAAATATTAAACACTCAATGATGTAGAGCTTCCCAATGGCTTCGTTTGGTC

[0084] AAGAACGTATCGCTCATCCAAAGCTTCCCGAAATCAAGGTCTTGGTTACGGTTGGCGAC

[0085] ATGCTTTTCAATTTGAACTGAAAGAAGTCACAGATGAAAAGCACAACGTCACCTCATGG

[0086] GAATTTGTAAGTGATTCTGCTGACGAGATTGAATTTGAACCAGTTGAGCATGGTTCAACA

[0087] AGTTATCAAGTCTATGTCGGAGCAAGCTGTCATTTCCTAAATCCAAATACACGAATTGTC

[0088] ACTCTCTCTAGTGGTGATCAATATCGTTTTGAGTTGGTAGAAGACATTTGGCTATTAAAAC

[0089] AAATCCGCAATGGAATCTTTTCGACGTTCCAACTTCGTTATTCAAGAAACCACCGCCTAG

[0090] TAGAAGTTGCACATAACAAGCGACCAGTTCTTGAGTGTCAATATGATAAACAAGGGCGC

[0091] CTTGTTGAATTGCTGAACGCAAAGACAGAACAAGTATTAACAACATATATCTATGACGAG

[0092] CAAGACGATCTTGTTGGTGCGACGAATGATTTGGGTTTGACTGAGCGATATGAGTATCAA

[0093] GACCAACATTTGATAGCCAAACGTGTTCGACCAACAGGGTTTACGCATCACTTTGAATG

[0094] GTCGGGAGAAGGCTCGAGTGCTAAGTGCATTAGAAACTTTGGTGACTCAGGTATTTATG

[0095] ATTATCGATTCCATTATGAGGGTGCAAAGTCCTCTTATTCTGACTCTTTAGATAATGAGTG

[0096] GACTTTCATTCATGATGAGCAAGGTCATCTGCTCGAAAAATCGAGCCCGACAGGAAGAA

[0097] CGTGGCAATGGCACTATGATCATTTAGGGCGTAAAGAGAAAGCGGTATTCCCAGATAACT

[0098] CGACAACACAGTACCAGTACAATCAGCAAGGCCAATTAATTTCCAAACTGCACTCTTCC

[0099] GGTGCTCAAATTCAATATGGTTATGACAGCTTAGGTAAGCTTGTAAAAACGGTATCTCCT

[0100] GATGGTGATCTTGAAAAGGCCTATTACAACAGTCTTGGCCAACGAGTGTGGGATATCGAT

[0101] GCTCTAGGTTGCGTAACGGAGTATGAATACGATAAACATGGTCAAGTCGTAAAGAGAGA

[0102] GTCTGAAGATGGTAAGAAGAGCCGTTGGTGGTGGGATAAACAACAAAGATTAGTCGCTC

[0103] ATGAAGTAGATGGCACTTTACTTCGCTACAGTTATGGCGCTACCGATTTAGTTAATGGGAT

[0104] TGCGTATCCCGATGGATGTGTTGCCCAGATTTCCTATGATGATTACGGACGCCGCACATCG

[0105] ATAAGATATTTCAACGACGAAGACAAAGTCGGTTACAGTGAAGAGTACGCTTATGACGA

[0106] GTTTAGTCGTGTTGCACAGATACACACACCAGAAGGTGTCACGTCTTATCAATGGGGAG

[0107] CTTTGGCTCAACAAGAAGCGGTAATCTTCCCTGATGGCAGCCATATTTCCTACGAGTACG

[0108] ATCAACAACGTAACCTAACGAAATTAGTTCGAAGTGATGGTCTAGCGTTTGAGTTTTTGT

[0109] ACGACAGTGAAGGTTTACTATCAGGTACCGTTGGATTTGATGGGCTACACAGCCAATTCA

[0110] AATACGACTCTATGGGAAGAATCATCCGAAAAGATGTTGCAGATAGGACCGTTTTATACA

[0111] GCTATGACGATGCTGGCTTTCTTCAGCATATTAAAGCGGGAAATGGAAAAAATATTGTTG

[0112] AGAATCACTTTAACTATACGTTAGGTGGAAGACTGACTTTAGCCTCTAATCGTCATCAAA

[0113] CTCTACAGTATCAATATTCCTCATTTGGACATTTAACTAAGCGTATTCAAGGTCAATTTGA

[0114] AATCGGTGAAGAGTTTAACCGAGTCGGACAGCGTGTTTCGCAAACCTTACCTGACAAG

[0115] ACGTCACTTAACTTTTCATACGACACCAATGGTAGGCTTTCCGAAATCCGGTTTAGCGAT

[0116] GACTCATTACCAAAAATTGAGTTCCAATATGATGTTATGGGGAGGCTTTCTGTCACTGAA

[0117] ACTGAGAGTTTCAGAGAGAGCAAACTTTATGACGGTGTCGGTCGATTAGTCGAGCAGCA

[0118] ATGGTCAGGTAGAGAAAAAAAATACATATACAATGCACAGAACCGAATCTCTTCAATTTT

[0119] AGATAATACTGCAGGGGCAACTCACTATCAGTACGATACCCTCGGGCACGTTACAAAGG

[0120] TTAGTGAAGCCAGTTCGACTTCAACATTTGAATCAGATAGCTTTGGAAATCCCGCTTTGG

[0121] CTGATTCTAAGGTAATGAGTGACCGCATTGAAGCTTATGCAGGTGTTCATTACAAATACG

[0122] ACCAACAAGGTAATCAAGTTAAACGAGAAGGCGATGGCACGGTTCAAAAACGTGTGTT

[0123] TGACGCATTAAATCAACTGGTAGAAGTACATGGTGATTCGAGTATTAGTCACTATGAATAT

[0124] GATGCATTAGGCCGTAGAACAAAGAAAATTACTCAACATGGTATCACGGAGTTTCTTTGG

[0125] GAAGGTGAGCGTTTACTCGGAGAACGCACTGCCGATGGCTTTCGTTGGTACTTATATCAA

[0126] CCAGAAACCTACATCCCATTGGCTGTGTTAGAAAATGGCTCAATTTATTTGTATGAATGTG

[0127] ATCAGGTTGGTAAACCGGAACGACTAAAAGACAGTGCCGGAAATATCGTTTGGAGTGCA

[0128] AGTTATGATGTCCATGGATTTGCTTCTATAGACGTTGAAGAGGTGAGAAATCCACTTCGC

[0129] TTTCAGGGACAGTATTTCGACCAAGAAACCAACTTACATTACAACCTCGCTCGGTATTAC

[0130] GACCCTAAGCTAGGACGTTTTATCCAACAAGACCCGATATCTATTGCTGGTGGCATTAAC

[0131] CATTACCAATATGCAGTCAATCCTATCCAGTGGATTGACCCAACAGGTTTCCTTTGTGAA

[0132] GAAGGGCTAAAACGCTTACAGCAAATGCTGGCTGATTATCAGGCGCAAAACAACGTTCC

[0133] CCAAGAGGTTTGCGATCAGATTTTAGAGGCGGCAAAAGAGTCATCCGTCGGAGAAGAT

[0134] GGTGTTCGCTCGCAAGTCAAAATCCGCAAACCCAATGGTAAAAACAATATTCGATATGAA

[0135] TACGATTTAGACCATATTGATTGCAAGAAAAACGAGATTACTTTTTATCGCCATATCAATT

[0136] ATTCAGATGGTATTAAACGCAAAGTTCAATATGTTGTAGGCATAGAAGAAATTGTCCAAA

[0137] TCATCCCCTTGAATAAAAGCCAAAGTGGGAGTGTTGCGAATCCTAATGGCGGTTCTTTAG

[0138] ACAGAGATTGTGGGCTTAAGTTAAATAACGAAATAGTTGGCGGGGTACCTGCAAACCAT

[0139] GCTGGACACCATCAAATTAGTATCAATATTGCTCAGGAATATCCTGTAATGCATAGAGCTG

[0140] CCGAACTTGGTTACAACATTAACAGAGGTACAAACGGGATTGCACTTCCGACTAATATTG

[0141] AAACATCTGTCGAAACAGGTCTTCCTTTACACGTGGGTAGACATCTAAGTGCAAGGCAC

[0142] GAAGGTTCAGCTGATGCTTTAGTTCGACGAGAAATGAGAACGTTGCAAACTAAGTATGA

[0143] TCGTGGTTTTATCGATGATATGACTCTTGTAGATGAAATTGGAAAAACAGAAAGTAGAAT

[0144] TAGGAATGCTCTGGAAAAGAATGAAGTTCGATTGCAATTGGATGATCCGCACTGGAAAA

[0145] GTAGGAAGTAG

[0146] >SEQ ID NO:4(Nucleotide sequence of LsCas13a protein)

[0147] ATGGGAAATTTATTTGGACATAAGAGATGGTATGAAGTTCGTGATAAAAAAGATTTTAA

[0148] AATAAAACGGAAAGTAAAAGTTAAAAGGAACTATGATGGAAACAAATACATTTTAAAT

[0149] ATTAATGAAAATAATAATAAAGAAAAAATTGATAATAATAAATTTATTAGAAAATATATTA

[0150] ATTACAAAAAAAATGATAATATTCTTAAAGAATTTACAAGAAAATTTCATGCAGGAAAT

[0151] ATTCTATTTAAGTTGAAAGGTAAAGAAGGAATCATAAGAATAGAAAATAATGATGATTT

[0152] TTTGGAAACAGAAGAAGTTGTATTATATATAGAAGCATATGGAAAATCTGAGAAATTGA

[0153] AAGCATTGGGGATTACAAAAAAGAAAATAATAGATGAAGCAATTAGGCAAGGAATAAC

[0154] TAAAGATGATAAAAAAATAGAAATAAAGAGACAAGAGAATGAAGAAGAAATAGAGAT

[0155] AGATATCAGAGATGAGTATACTAATAAAACACTAAATGATTGTTCAATAATATTAAGAAT

[0156] AATAGAAAATGATGAATTAGAAACAAAAAAATCAATTTACGAAATTTTTAAAAATATTA

[0157] ACATGAGTTTATATAAGATTATAGAAAAAATAATTGAAAATGAAACAGAAAAAGTATTT

[0158] GAAAATCGATATTATGAAGAACATTTAAGAGAAAAATTATTGAAAGATGATAAAATAGA

[0159] TGTTATTTTGACTAATTTTATGGAAATAAGAGAAAAAATAAAAAGTAATTTAGAAATTC

[0160] TGGGTTTTGTAAAATTTTATCTTAATGTTGGTGGTGATAAAAAGAAATCTAAAAATAAA

[0161] AAAATGCTTGTTGAAAAATTTTAAATATAAATGTAGATTTAACAGTAGAAGATATAGC

[0162] TGATTTTGTTTATAAAAAGAGTTAGAATTTTGGAATATTACTAAAAGAATTGAAAAAAGTAA

[0163] AAAAAGTTAATAATGAATTTCTTGAAAAGAGAAATAGAACATATATAAAATCTTTAT

[0164] GTACTATTAGATAAGCACGAAAAATTTAAAATAGAAAGAGAAAATAAAAAAGATAAAAA

[0165] TTGTAAAATTTTTTTGTAGAAAAATAAAAAATAATATAGTATAAAAAAAAAATAGAAAAA

[0166] ATTTTAGCAGAATTTAAAAATAGATGAATTAAAAAAAATTAGAAAAAAGAACTAAAAA

[0167] AAGGAAATTGTGATACAGAAATTTTTGGAATATTTAAAAAACATTATAAAAGTTAATTTT

[0168] GATTCGAAAAAATTTTCAAAGAAATCTGATGAAGAAAAAGAACTATATAAAATCATATA

[0169] TCGATATTGAAAGGAAGAATTGAAAAAATATTGGTAATGAAAAAAAAGTCAGATTA

[0170] AAAAAATGGAAAAAATAGAAATAGAAAAAATTTTAAATGAAAGTATATTATCTGAAA

[0171] AAATTTAAAAAGAGTTAAGCAATATACGTTAGAGCATATTATGTATTTAGGGAAATTAA

[0172] GACATAATGATATAGATATGAACAGTAAATACTGATGATTTTTCTAGACTTCACGCTA

[0173] AAGAGGAATTGGATTTAGAATTGATTACTTTTTTTGCTTCTACCAATATGGAATTAAATA

[0174] AGATTTTTTCTAGAGAAAAATAAATAATGAGAAAATATAGATTTCTTTGGAGGAGATC

[0175] GAGAAAAAAATTACGTACTTGATAAAAAAAATTTTAAATTCAAAAAATAAAAAATAATAAG

[0176] AGACTTAGATTTTATAGATAATAAAAAATAACATTACAAATAATTTTATTAGGAAATTTAC

[0177] AAAGATAGGAACAAATGAAAGAAACAGGATATTACATGCGATTAGCAAGGAAGAGA

[0178] TTTACAAGGAACGCAAGATGACTATAATAAAGTCATAAATATAATTCAAAATTTAAAAA

[0179] TATCAGATGAAGAAGTGTCAAAAGCATTAAATTTAGATGTAGTATTTAAAGATAAAAAAA

[0180] AATATAATTACTAAAAATAAATGATATTAAAAATTCAGAAGAAAAATAATAATGATATTAAAT

[0181] ATTTACCATCCTTTTCTAAAGTACTTCCTGAAATATTAAATTTTATATGGAATAATCCTAA

[0182] GAATGAACCATTTGATACTATAGAAAACAGAAAAATAGTATTAAATGCTTTAATATATGT

[0183] GAATAAAGAATTGTATAAGAAATTAATTTTAGAAGATGATTTAGAGAAAATGAAGTA

[0184] AGAATATTTTTTACAAGAATTAAAGAAACTTTAGGAAACATTGATGAAATAGATGAA

[0185] AATATTATAGAAAATTATTATAAAAATGCTCAGATTTCTGCTTCCAAAAGGTAATAATAAA

[0186] GCTATTAAAAAAATACAGAAAAAAGTAATAGAATGTTATATTGGATATTTGAGAAAAAA

[0187] TTATGAGGAACTTTTTGATTTTTCAGATTTTAAAATGAATATTCAAGAAATTAAAGAAAC

[0188] AAATAAAGACATTAATGATAATAAAACTTACGAAAGAATAACTGTAAAGACTAGTGAT

[0189] AAAACTATAGTAATAAATGATGATTTTGAATATATAATTTCAATATTGCACTGTTAAATA

[0190] GTAATGCGGTTATAAATAAAATTAGAAATAGATTTTTTGCAACATCAGTTTGGTTAAATA

[0191] CTTCAGAATATCAAAATTATATAGATATTTAGATGAAATAATGCAATTAAATACTTTGAG

[0192] AAAATGAATGTATAACTGAAAATTGGAATCTTAAATTTAGAAGAATTTATTCAAAAAAATGA

[0193] AAGAGATAGAAAAAAGACTTTGATGATTTTAAAATACAAACTAAAAAGAAATTTTTAA

[0194] TAATTATTATGAGGATATAAAAAATAATATATTAACTGAATTTAAAGATGATATTAATGGT

[0195] TGTGATGTTTTAGAGAAAAATTAGAAAAATTGTAATTTTTGATGATGAAACTAAATT

[0196] TGAGATAGATAAAAAATCGAATATATTACAAGATGAACAAAGTAAGTTATCAAACATAA

[0197] ATAAAAAGGATTGAAAAAAAGGTTGATCAGTATAATAAAAGATAAAGATCAGGAAAT

[0198] AAAAGGAAAATATTGTGTAGATAATATTTAATTCTGATTTTTAAAAAAAATACAAAAA

[0199] AGAAATAGATAATTTAATAGAAGATATGGAATCAGAGAATGAAAATAAATTTCAAGAAA

[0200] TATATTATCCTAAAAAAGGAATGAATTATATATTTATAAAAAGAATTTTTTTTAAA

[0201] TATTGGAAATCCAAATTTTGATAAAATTTATGGATTAATTTCAAATGATATTAAATGGCT

[0202] GATGCAAAATTTTTTATTTAATATTGATGGTAAAAATTTAGAAAAAAATAAAATATCTGAA

[0203] ATTGATGCAATATTGAAAAAATCTAAATGATAAACTAAATGGATATTCTAAAAAACAA

[0204] AGAGAAGTATATAAAAAATTGAAAGAAAATGATGACTTTTTTGCAAAAATATACAA

[0205] AATAAAAAACTATAAATCATTTGAAAAAGATTATAATAGAGTTTCTGAATACAAAAAAAAT

[0206] TAGAGATTTGGTAGAATTTAATTATTGAATAAATAGAAAGTTATTTGATAGATAAAT

[0207] TGGAAACTTGCTATTCAAATGGCTAGATTTGAAAGAGATATGCACTATATTGTAAATGG

[0208] GCTAAGAGAATTAGGAATTATTAAGTTAAGTGGCTACAATACTGGAATAAGTAGAGCTT

[0209] ATCCTAAGCGTAATGGATCTGATGGCTTTTATACAACAACAGCATACTATAAATTTTTTG

[0210] ATGAAGAATCTTATAAAAAATTTGAGAAAATATGTTATGGATTTGGAATTGATTTGTCTG

[0211] AAAATTCTGAAATTAATAAGCCGGAAAATGAAAGTATTAGAAACTATATTTCACATTTC

[0212] TATATTGTAAGAAATCCGTTTGCTGATTACAGTATTGCAGAACAAATTGATAGAGTTTCT

[0213] AATTTATTATCATATAGTACACGTTATAATAATTCAACTTATGCAAGTGTATTTGAAGTAT

[0214] TTAAAAAAGATGTGAATTTAGATTATGATGAATTAAAGAAAAAATTTAAACTTATCGGT

[0215] AATAATGATATACTTGAAAGATTAATGAAACCTAAAAAAGTTTCTGTTTTAGAATTAGA

[0216] AAGTTATAATTCTGATTATATTAAAAACTTGATAATAGAACTTTTAACAAAAATAGAAAA

[0217] TACGAATGATACGTTATAA

[0218] >SEQ ID NO:5 (Nucleotide sequence of tetracycline-inducible expression element)

[0219] ATGATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGA

[0220] GGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCA

[0221] GCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTTAGCCATTG

[0222] AGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGA

[0223] TTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGAG

[0224] CAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCA

[0225] ATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGC

[0226] TGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAA

[0227] GAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCG

[0228] AATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATAT

[0229] GCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCTTAA

[0230] >SEQ ID NO:6 (pBBR1 plasmid)

[0231] CTCGGGCCGTCTCTTGGGCTTGATCGGCCTTCTTGCGCATCTCACGCGCTCCTGCGGC

[0232] GGCCTGTAGGGCAGGCTCATACCCCTGCCGAACCGCTTTTGTCAGCCGGTCGGCCACG

[0233] GCTTCCGGCGTCTCAACGCGCTTTGAGATTCCCAGCTTTTCGGCCAATCCCTGCGGTG

[0234] CATAGGCGCGTGGCTCGACCGCTTGCGGGCTGATGGTGACGTGGCCCACTGGTGGCC

[0235] GCTCCAGGGCCTCGTAGAACGCCTGAATGCGCGTGTGACGTGCCTTGCTGCCCTCGAT

[0236] GCCCCGTTGCAGCCCTAGATCGGCCACAGCGGCCGCAAACGTGGTCTGGTCGCGGGT

[0237] CATCTGCGCTTTGTTGCCGATGAACTCCTTGGCCGACAGCCTGCCGTCCTGCGTCAGC

[0238] GGCACCACGAACGCGGTCATGTGCGGGCTGGTTTCGTCACGGTGGATGCTGGCCGTC

[0239] ACGATGCGATCCGCCCCGTACTTGTCCGCCAGCCACTTGTGCGCCTTCTCGAAGAACG

[0240] CCGCCTGCTGTTCTTGGCTGGCCGACTTCCACCATTCCGGGCTGGCCGTCATGACGTA

[0241] CTCGACCGCCAACACAGCGTCCTTGCGCCGCTTCTCTGGCAGCAACTCGCGCAGTCG

[0242] GCCCATCGCTTCATCGGTGCTGCTGGCCGCCCAGTGCTCGTTCTCTGGCGTCCTGCTG

[0243] GCGTCAGCGTTGGGCGTCTCGCGCTCGCGGTAGGCGTGCTTGAGACTGGCCGCCACG

[0244] TTGCCCATTTTCGCCAGCTTCTTGCATCGCATGATCGCGTATGCCGCCATGCCTGCCCCT

[0245] CCCTTTTGGTGTCCAACCGGCTCGACGGGGGCAGCGCAAGGCGGTGCCTCCGGCGGG

[0246] CCACTCAATGCTTGAGTATACTCACTAGACTTTGCTTCGCAAAGTCGTGACCGCCTACG

[0247] GCGGCTGCGGCGCCCTACGGGCTTGCTCTCCGGGCTTCGCCCTGCGCGGTCGCTGCGC

[0248] TCCCTTGCCAGCCCGTGGATATGTGGACGATGGCCGCGAGCGGCCACCGGCTGGCTCG

[0249] CTTCGCTCGGCCCGTGGACAACCCTGCTGGACAAGCTGATGGACAGGCTGCGCCTGC

[0250] CCACGAGCTTGACCACAGGGATTGCCCACCGGCTACCCAGCCTTCGACCACATACCCA

[0251] CCGGCTCCAACTGCGCGGCCTGCGGCCTTGCCCCATCAATTTTTTTAATTTTCTCTGGG

[0252] GAAAAGCCTCCGGCCTGCGGCCTGCGCGCTTCGCTTGCCGGTTGGACACCAAGTGGA

[0253] AGGCGGGTCAAGGCTCGCGCAGCGACCGCGCAGCGGCTTGGCCTTGACGCGCCTGGA

[0254] ACGACCCAAGCCTATGCGAGTGGGGGCAGTCGAAGGCGAAGCCCGCCCGCCTGCCCC

[0255] CCGAGCCTCACGGCGGCGAGTGCGGGGGTTCCAAGGGGGCAGCGCCACCTTGGGCA

[0256] AGGCCGAAGGCCGCGCAGTCGATCAACAAGCCCCGGAGGGGCCACTTTTTGCCGGAG

[0257] GGGGAGCCGCGCCGAAGGCGTGGGGGAACCCCGCAGGGGTGCCCTTCTTTGGGCAC

[0258] CAAAGAACTAGATATAGGGCGAAATGCGAAAGACTTAAAAATCAACAACTTAAAAAA

[0259] GGGGGGTACGCAACAGCTCATTGCGGCACCCCCCGCAATAGCTCATTGCGTAGGTTAA

[0260] AGAAAATCTGTAATTGACTGCCACTTTTACGCAACGCATAATTGTTGTCGCGCTGCCGA

[0261] AAAGTTGCAGCTGATTGCGCATGGTGCCGCAACCGTGCGGCACCCTACCGCATGGAGA

[0262] TAAGCATGGCCACGCAGTCCAGAGAAATCGGCATTCAAGCCAAGAACAAGCCCGGTC

[0263] ACTGGGTGCAAACGGAACGCAAAGCGCATGAGGCGTGGGCCGGGCTTATTGCGAGGA

[0264] AACCCACGGCGGCAATGCTGCTGCATCACCTCGTGGCGCAGATGGGCCACCAGAACG

[0265] CCGTGGTGGTCAGCCAGAAGACACTTTCCAAGCTCATCGGACGTTCTTTGCGGACGGT

[0266] CCAATACGCAGTCAAGGACTTGGTGGCCGAGCGCTGGATCTCCGTCGTGAAGCTCAA

[0267] CGGCCCCGGCACCGTGTCGGCCTACGTGGTCAATGACCGCGTGGCGTGGGGCCAGCC

[0268] CCGCGACCAGTTGCGCCTGTCGGTGTTCAGTGCCGCCGTGGTGGTTGATCACGACGAC

[0269] CAGGACGAATCGCTGTTGGGGCATGGCGACCTGCGCCGCATCCCGACCCTGTATCCGG

[0270] GCGAGCAGCAACTACCGACCGGCCCCGGCGAGGAGCCGCCCAGCCAGCCCGGCATTC

[0271] CGGGCATGGAACCAGACCTGCCAGCCTTGACCGAAACGGAGGAATGGGAACGGCGC

[0272] GGGCAGCAGCGCCTGCCGATGCCCGATGAGCCGTGTTTTCTGGACGATGGCGAGCCG

[0273] TTGGAGCCGCCGACACGGGTCACGCTGCCGCGCCGGTAGCACTTGGGTTGCGCAGCA

[0274] ACCCGTAAGTGCGCTGTTCCAGACTATCGGCTGTAGCCGCCTCGCCGCCCTATACCTTG

[0275] TCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGA

[0276] AGCCGGCGGCACCTCGCTAACGGATTCACCGTTTTTATCAGGCTCTGGGAGGCAGAAT

[0277] AAATGATCATATCGTCAATTATTACCTCCACGGGGAGAGCCTGAGCAAACTGGCCTCA

[0278] GGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAACCAG

[0279] CAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCGAATT

[0280] TGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCACCAGGC

[0281] GTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGC

[0282] AGTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAAC

[0283] AAAATATTAACGCTTACAATTTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGG

[0284] CGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAA

[0285] GGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGC

[0286] CAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCACCGCGGTGGC

[0287] GGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGA

[0288] TACCGTCGACCTCGAGGGGGGGCCCGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTT

[0289] AATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCT

[0290] CACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAA

[0291] TGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAA

[0292] ACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGC

[0293] GTATTGGGCGCATGCATAAAAACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAG

[0294] CCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTT

[0295] GCGTATAATATTTGCCCATGGACGCACACCGTGGAAACGGATGAAGGCACGAACCCAG

[0296] TTGACATAAGCCTGTTCGGTTCGTAACAGAAGCCACTGGAGCACCTCAAAAACACCAT

[0297] CATACACTAAATCAGTAAGTTGGCAGCATCACCCGACGCACTTTGCGCCGAATAAATA

[0298] CCTGTGACGGAAGATCACTTCGCAGAATAAATAAATCCTGGTGTCCCTGTTGATACCGG

[0299] GAAGCCCTGGGCCAACTTTTGGCGAAAATGAGACGTTGATCGGCACGTAAGAGGTTC

[0300] CAACTTTCACCATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATT

[0301] TTCAGGAGCTAARGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGAT

[0302] ATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTAC

[0303] CTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATA

[0304] AGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCG

[0305] GAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTT

[0306] GTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCA

[0307] CGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAA

[0308] ACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCT

[0309] GGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCC

[0310] GTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGA

[0311] TTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTA

[0312] CAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTATTGGT

[0313] GCCCTTAAACGCCTGGTTGCTACGGCGGAGTTGTTCGGTAAATTGTCACAACGCCGCC

[0314] AGGTGGCACTTTTCGGGGAAATGTGCGCGCCCGCGTTCCTGCTGGCGCTGGGCCTGTT

[0315] TCTGGCGCTGGACTTCCCGCTGTTCCGTCAGCAGCTTTTCGCCCACGGCCTTGATGATC

[0316] GCGGCGGCCTTGGCCTGCATATCCCGATTCAACGGCCCCAGGGCGTCCAGAACGGGCT

[0317] TCAGGCGCTCCCGAAGGT

[0318] >SEQ ID NO:7 (pBBR1-TetR-Cas13a-crRNA1 plasmid)

[0319] GCTCTAGTTAGCCTAATCGCATAATTATTTATTATAGTATAATTCTTATTTTTTTTCAACCT

[0320] AAAAATTTAAAACATCTCCAAAAATTTTCGTTTCAGAACAACCAAGCAACCATATTCA

[0321] AAAAACAATAAAAAATGAGCAAGAATTGAAATTTTATTCTCACTCAGAAGTTATTTTTA

[0322] TTAAATATCACTTTTCGATATTGGGGTGGTCTATATCAATTTAAAAGACAGAATAGATAA

[0323] TTCTTTAGAGTTTTAGTCCCCTTCGATATTGGGGTGGTCTATATCCCTTGGTCAAAGCTT

[0324] TCATTTTTGTTTTAGTCCCCTTCGATATTGGGGTGGTCTATATCCCATCCTAATTTCTTGC

[0325] TGATGAGATATTTATTTCTAATTTTTCTATTTTGTCTTTATTTTCAATACTTTCAATCCTATT

[0326] TTTCTCTTTATTAATAATATAGAACCACCCTATACTATTATACCATATTTTTTGATTTTTCA

[0327] AAATTCCAATATTTTGTTTTGTGAAATTTTTTCTCCCATTGTCACTTCTCCTGCAAGTAC

[0328] CTTCATTTTTGAAACTGATCTTCTGTCAGGATAATGGAACGGATTGATGAATTTTCTGG

[0329] AGCGAGCATTGATAACTGTTTTTCTGCCAGTTCGATTTTTTCTTTTGTTTTCGACCTCAT

[0330] TATATATACCGATTTTTGAAGCTGATAATATCCCTTTTCTATCAATTTTTTCCTAAAAGTC

[0331] CTATATTCAAATCTCTCAACATCTGTCTGCATAGGAAAATCATACATAAGCAGACCAAA

[0332] ATACTCAATACTCATAGTCCATCACGCTCAATGTCGGAATTATCACTTCTTCATCTTTTAC

[0333] AAAATAATTTCGTATACTATCCAAATAATAGTCTACCGCTTGGAAAAAATCATATTTCTTA

[0334] TTGTTAAATAATACCTTCTGCTGTGCTACAAGAAGTATTTTTTGCCTTATTTCCTTACTTA

[0335] ATTTCACTTCATTCAAAATATCCTTGTACATATAAACAAGATAATCCACCATAGGACGAA

[0336] AAACCTCTATTATATCATCAGAAAAATTATAGGCATTAAACTGTGACTTATGATGTAATC

[0337] CTAAACTTGGATGAAATCCTTTTGCTACAATCTTTGATGATATTATAGCTCTTAAAATCAT

[0338] ATATCCATAATTAAGTGCAGAATTCACTCCATCTTCATCAAATCTTTTAAAACTATTACTA

[0339] TACAATTCCTGAAAATATATCCTTGAAGCTATTGCTTCCTGATGTTCTGCACTCGCATCA

[0340] TCTTTTTTCAAGTTTTCCTTATATGTTTTCAGTCTTTCAATGGAAATATCACTTTTTTCAA

[0341] GATACTCTAACAATGCTCTTTGATTTTCAATCTTATTCTCCACTATCCTGCTCCACAATTT

[0342] TTCCTTTTTCTCTTTTTCCCACTCAATCTGCTCATTTATTCGTAAAGTCACTTGAAAATG

[0343] ATTAAATAATCCCAGCGAATGAATTTCAGGCTGATGTTTCTCGTTGCAAATAATAATCGG

[0344] AATGTTATTTTCCACCAGCCTCAACTGCAAAATCGCACTAATCTTACAATAGCAGTTTT

[0345] CAATAACTATCGCAGATATATCATTCAAAGAAATCTTATTTTTCTCATCATTATTGTCTTC

[0346] ATCAACCATTAAAGCTGATTATTCGATATTGACAAATCATCAGCCCTTGTTATGTGAATT

[0347] ATATTGGGCATTTTAATCATACTCCTTATAAATTTCATTCTTATAACGTATCATTCGTATTT

[0348] TCTATTTTTGTTAAGTTCTATTATCAAGTTTTTAATATAATCAGAATTATAACTTTCTAA

[0349] TTCTAAACAGAAACTTTTTTAGGTTTCATTAATCTTTCAAGTATATCATTATTACCGATA

[0350] AGTTTAAATTTTTTCTTTAATTCATCATAATCTAATTCACATCTTTTTTAAATACTTCAA

[0351] ATACACTTGCATAAGTTGAATTATTATAACGTGTACTATATGATAATAAATTAGAAACTCT

[0352] ATCAATTTGTTCTGCAATACTGTAATCAGCAAACGGATTTCTTACAATATAGAAATGTGA

[0353] AATATAGTTTCTAATACTTTCATTTTCCGGCTTATTAATTTCAGAATTTTCAGACAAATCA

[0354] ATTCCAAATCCATAACATATTTTCTCAAATTTTTTATAAGATTCTTCATCAAAAATTTAT

[0355] AGTATGCTGTTGTTGTATAAGCCATCAGATCCATTACGCTTAGGATAAGCTCTACTTA

[0356] TTCCAGTATTGTAGCCACTTAACTTAATAATTCCTAATTCTCTTAGCCCATTTACAATATA

[0357] GTGCATATCTCTTTCAAATCTAGCCATTTGAATAGCAAGTTTCCAATTTATATCTATCAAA

[0358] TAACTTTCTATTTTATTCAAATAATTAAATTCTACCAAATCTCTAATTTTTTTGTATTCAG

[0359] AAACTCTATTATAATCTTTTTCAAATGATTTATAGTTTTTATTTTGTATATTTTTTGCAAAA

[0360] AAGTCATCATTTTCTTTCAATTTTTTTATATACTTCTCTTTGTATTCTTTAGAATATCCATT

[0361] TAGTTTATCATTTAGATTTTTCAATATTGCATCAATTTCAGATATTTTATTTTTTCTAATATT

[0362] TTTACCATCAATATTAAATAAAAATTTTGCATCAGCCATTTTAATATCATTTGAAATTAAT

[0363] CCATAAATTTTATCAAAATTTGGATTTCCAATATTTAAAAATAAATTCTTTTTATAAATATA

[0364] TAATTCATTCTTACGTTCTTTAGGATAATATATTTCTTGAAATTTATTTTCATTCTCTGATT

[0365] CCATATCTTCTATTAAATTATCTATTTCTTTTTTGTATTTTTTTAAAAAATCAGAATTAAAT

[0366] ATTATTCTACACAATATTTTACTCTTTATTTCCTGATCTTTATCTTTTATATACTGATCAAC

[0367] CTTTTTTTTCAAATCCTTTTTATTTATGTTTGATAACTTTCTTTGTTCATCTTGTAATATAT

[0368] TCGATTTTTTATCTATCTCAAATTTAGTTTCATCATCAAAAATTACAATTTTTTCTAATTTT

[0369] TTCTCTAAAACATCACAACCATTAATATCATCTTTAAATTCAGTTAATATATTATTTTTTAT

[0370] ATCCTCATAATAATTATTAAAAATTTCTTTTTTAGTTTGTATTTTAAAATCATCAAAGTCT

[0371] TTTTCTATCTCTTTCATTTTTTGAATAAATTCTTTCTAAATTAAGATTCCAATTTTCAGTTAT

[0372] ACATTCATTTCTCAAAGTATTTAATTGCATTATTTCATCTAAAATATCTATAATATTTTGAT

[0373] ATTCTGAAGTATTTAACCAAACTGATGTTGCAAAAAATCTATTTCTAATTTTATTTATAA

[0374] CCGCATTACTATTTAACAGTGCAAATATTGAAATTATATATTCAAAATCATCATTTATTAC

[0375] TATAGTTTTATCACTAGTCTTTACAGTTATTCTTTCGTAAGTTTTATTATCATTAATGTCTT

[0376] TTATTTGTTTCTTAATTTCTTGAATATTCATTTTAAAATCTGAAAAATCAAAAAGTTCCT

[0377] CATAATTTTTTCTCAAATATCCAATATAACATTCTATTACTTTTTTCTGATATTTTTTAATA

[0378] GCTTTATTATTACCTTTTGAAGCAGAAATCTGAGCATTTTTATAATAATTTTCTATAATATT

[0379] TTCATCTATTTCATCAATGTTTCCTAAAGTTTTCTTTAATTCTTGTAAAAATATATTCTTAC

[0380] TTTCATTTTCTTCTAAATCATCTTCTAAAATTAATTTCTTATACAATTCTTTATTCACATAT

[0381] ATTAAAGCATTTAATACTATTTTTTCTGTTTCTATAGTATCAAATGGTTCATTCTTAGGATT

[0382] ATTCCTATATAAATTTAATATTTCAGGAAGTACTTTAGAAAAGGATGGTAAATATTTAATA

[0383] TCATTATTATTTTCTTCTGAAATTTTAATATCATTTATTTTAGTAATTATATTTTTTTTATCTT

[0384] TAAATACTACATCTAAATTTAATGCTTTTGACACTTCTTCATCTGATATTTTTAAATTTTG

[0385] AATTATATTTATGACTTTATTATAGTCATCTTGCGTTCCTTGTAAATCTCTTTCCTTGCTAA

[0386] TCGCATGTAATATCCTGTTTCTTTCATTTGTTCCTATCTTTGTAAATTTCCTAATAAAATTA

[0387] TTTGTAATGTTATTTTTATTATCTATAAAATCTAAGTCTCTTATTATTTTTATTTTTGAATTT

[0388] AAAATTTTTTTATCAAGTACGTAATTTTTTTCTCGATCTCCTCCAAAGAAATCTATATTTT

[0389] CATCATTATTTATATTTTCTCTAGAAAAAATCTTATTTAATTCCATATTGGTAGAAGCAAA

[0390] AAAAGTAATCAATTCTAAATCCAATTCCTCTTTAGCGTGAAGTCTAGAAAAATCATCAG

[0391] TATTTACTGTTGTCATATCTATATCATTATGTCTTAATTTCCCTAAATACATAATATGCTCTA

[0392] ACGTATATTGCTTAACTCTTTTTAAAATTTTTTCAGATAATATACTTTCATTTAAAAATTTTT

[0393] TCTATTTCTATTTTTTCCATTTTCTTTAATCTGACTTTTTGTTCATTTACCAATATTTTTTC

[0394] AATTCTTCCTTTCAAATATCGATATATGATTTTATATAGTTCTTTTTCTTCATCAGATTTCT

[0395] TTGAAAATTTTTTCGAATCAAAATTAACTTTATAATGTTTTTTAAATATTCCAAAAATTTC

[0396] TGTATCACAATTTCCTTTTTTTTAGTCTTTTTTCTAATTTTTTATTAATTCATCTATTTTAAA

[0397] TTCTGCTAAAATTTTTTCTATTTTTTCTTTTATACTATTATTTTTTATATTTTCTACAAAAA

[0398] ATTTTACAATTTTATCTTTTTTATTTTCTCTTTCTATTTTAAATTTTTCGTGCTTATCTAATA

[0399] GTACATAAGATTTTATATATGTTCTATTTCTTCTCTTTTCAAGAAATTCATTATTAACTTTT

[0400] TTTACTTTTTCAATTCTTTTAGTAATATTCCAAAATTCTAACTCTTTTATAACAAAATCAG

[0401] CTATATCTTCTACTGTTAAATCTACATTTATATTTAAAATTTTTTCAACAAGCATTTTTTTA

[0402] TTTTTAGATTTCTTTTTATCACCACCAACATTAAGATAAAATTTTACAACCCAGAATT

[0403] TCTAAATTACTTTTTATTTTTTCTCTTATTTCCATAAAATTAGTCAAAATAACATCTATTTT

[0404] ATCATCTTTCAATAATTTTTCTCTTAAATGTTCTTCATAATATCGATTTTCAAATACTTTTT

[0405] CTGTTTCATTTTCAATTATTTTTTCTATAATTCTTATATAAACTCATGTTAATATTTTTAAAA

[0406] ATTTCGTAAATTGATTTTTTTGTTTCTAATTCATCATTTCTATTATTCTTAATATTATTGAA

[0407] CAATCATTTAGTGTTTTATTAGTATACTCATCTCTGATATCTATCTCTATTTCTTCTTCATT

[0408] CTCTTGTCTCTTTTATTTCTATTTTTTTATCATCTTTAGTTATTCCTTGCCTAATTGCTTCAT

[0409] CTATTATTTTCTTTTTTGTAATCCCCAATGCTTTCAATTTCTCAGATTTTCCATATGCTTCT

[0410] ATATATAATACAACTTCTTCTGTTTCCAAAAAATCATCATTATTTTCTATTCTTATGATTCC

[0411] TTCTTTACCTTCAACTTAAATAGAATATTTCCTGCATGAAATTTTCTTGTAAATTCTTTA

[0412] AGAATATTATCATTTTTTTTGTAATTAATATATTTTCTAATAAATTTATTATTATCAATTTTT

[0413] TCTTTATTATTATTTTCATTAATATTTAAAAATGTATTTGTTTCCATCATAGTTCCTTTTAACT

[0414] TTTACTTTCCGTTTTATTTTAAAATCTTTTTTATCACGAACTTCATACCATCTCTTATGTC

[0415] CAAATAAATTTCCCATTCCAATCTCCTCGTTTCTACTTTAATCTAATAAAATATTTTTAAA

[0416] TTAAATCAATTTTACATCTTTCTAATCAAAAATACAATTTTCCATTTTTAGTATACCACAT

[0417] CAATATTAAATCTCAAAAAAATAAGGAGCCGTCAAACATAGAGATCCTTTCTCCTCTTT

[0418] AGATCTTTTGAATTCTTTTCTCTATCACTGATAGGGAGTGGTAAAATAACTCTATCAACG

[0419] ATAGAGTGTCAACAAAAATTAGGAATTAATGATGTCTAGATTAGATAAAAGTAAAGTGA

[0420] TTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAA

[0421] ACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAG

[0422] CGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTG

[0423] CCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGAT

[0424] GTGCTTTACTAAGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGA

[0425] AAAACAGTATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCAC

[0426] TAGAGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGG

[0427] AAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTAT

[0428] GCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCT

[0429] TCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGT

[0430] GGGTCTTAAGCAAGTTGACATAAGCCTGTTCGGTTCGTAACAGAAGCCACTGGAGCAC

[0431] CTCAAAAACACCATCATACACTAAATCAGTAAGTTGGCAGCATCACCCGACGCACTTT

[0432] GCGCCGAATAAATACCTGTGACGGAAGATCACTTCGCAGAATAAATAAATCCTGGTGT

[0433] CCCTGTTGATACCGGGAAGCCCTGGGCCAACTTTTGGCGAAAATGAGACGTTGATCGG

[0434] CACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTACCGGGCGTATTTTT

[0435] TGAGTTATCGAGATTTTCAGGAGCTAARGAAGCTAAAATGGAGAAAAAAATCACTGGA

[0436] TATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCA

[0437] GTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGAC

[0438] CGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGA

[0439] TGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGAT

[0440] AGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTG

[0441] GAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGT

[0442] GTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCT

[0443] CAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAA

[0444] CTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGA

[0445] TGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATG

[0446] CTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAG

[0447] GCAGTTATTGGTGCCCTTAAACGCCTGGTTGCTACGGCGGAGTTGTTCGGTAAATTGTC

[0448] ACAACGCCGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGCCCGCGTTCCTGCTGGC

[0449] GCTGGGCCTGTTTCTGGCGCTGGACTTCCCGCTGTTCCGTCAGCAGCTTTTCGCCCAC

[0450] GGCCTTGATGATCGCGGCGGCCTTGGCCTGCATATCCCGATTCAACGGCCCCAGGGCG

[0451] TCCAGAACGGGCTTCAGGCGCTCCCGAAGGTCTCGGGCCGTCTCTTGGGCTTGATCGG

[0452] CCTTCTTGCGCATCTCACGCGCTCCTGCGGCGGCCTGTAGGGCAGGCTCATACCCCTG

[0453] CCGAACCGCTTTTGTCAGCCGGTCGGCCACGGCTTCCGGCGTCTCAACGCGCTTTGAG

[0454] ATTCCCAGCTTTTCGGCCAATCCCTGCGGTGCATAGGCGCGTGGCTCGACCGCTTGCG

[0455] GGCTGATGGTGACGTGGCCCACTGGTGGCCGCTCCAGGGCCTCGTAGAACGCCTGAA

[0456] TGCGCGTGTGACGTGCCTTGCTGCCCTCGATGCCCCGTTGCAGCCCTAGATCGGCCAC

[0457] AGCGGCCGCAAACGTGGTCTGGTCGCGGGTCATCTGCGCTTTGTTGCCGATGAACTCC

[0458] TTGGCCGACAGCCTGCCGTCCTGCGTCAGCGGCACCACGAACGCGGTCATGTGCGGG

[0459] CTGGTTTCGTCACGGTGGATGCTGGCCGTCACGATGCGATCCGCCCCGTACTTGTCCG

[0460] CCAGCCACTTGTGCGCCTTCTCGAAGAACGCCGCCTGCTGTTCTTGGCTGGCCGACTT

[0461] CCACCATTCCGGGCTGGCCGTCATGACGTACTCGACCGCCAACACAGCGTCCTTGCGC

[0462] CGCTTCTCTGGCAGCAACTCGCGCAGTCGGCCCATCGCTTCATCGGTGCTGCTGGCCG

[0463] CCCAGTGCTCGTTCTCTGGCGTCCTGCTGGCGTCAGCGTTGGGCGTCTCGCGCTCGCG

[0464] GTAGGCGTGCTTGAGACTGGCCGCCACGTTGCCCATTTTCGCCAGCTTCTTGCATCGCA

[0465] TGATCGCGTATGCCGCCATGCCTGCCCCTCCCTTTTGGTGTCCAACCGGCTCGACGGG

[0466] GGCAGCGCAAGGCGGTGCCTCCGGCGGGCCACTCAATGCTTGAGTATACTCACTAGAC

[0467] TTTGCTTCGCAAAGTCGTGACCGCCTACGGCGGCTGCGGCGCCCTACGGGCTTGCTCT

[0468] CCGGGCTTCGCCCTGCGCGGTCGCTGCGCTCCCTTGCCAGCCCGTGGATATGTGGACG

[0469] ATGGCCGCGAGCGGCCACCGGCTGGCTCGCTTCGCTCGGCCCGTGGACAACCCTGCT

[0470] GGACAAGCTGATGGACAGGCTGCGCCTGCCCACGAGCTTGACCACAGGGATTGCCCA

[0471] CCGGCTACCCAGCCTTCGACCACATACCCACCGGCTCCAACTGCGCGGCCTGCGGCCT

[0472] TGCCCCATCAATTTTTTTAATTTTCTCTGGGGAAAAGCCTCCGGCCTGCGGCCTGCGCG

[0473] CTTCGCTTGCCGGTTGGACACCAAGTGGAAGGCGGGTCAAGGCTCGCGCAGCGACCG

[0474] CGCAGCGGCTTGGCCTTGACGCGCCTGGAACGACCCAAGCCTATGCGAGTGGGGGCA

[0475] GTCGAAGGCGAAGCCCGCCCGCCTGCCCCCCGAGCCTCACGGCGGCGAGTGCGGGG

[0476] GTTCCAAGGGGGCAGCGCCACCTTGGGCAAGGCCGAAGGCCGCGCAGTCGATCAAC

[0477] AAGCCCCGGAGGGGCCACTTTTTGCCGGAGGGGGAGCCGCGCCGAAGGCGTGGGGG

[0478] AACCCCGCAGGGGTGCCCTTCTTTGGGCACCAAAGAACTAGATATAGGGCGAAATGCG

[0479] AAAGACTTAAAAATCAACAACTTAAAAAAGGGGGGTACGCAACAGCTCATTGCGGCA

[0480] CCCCCCGCAATAGCTCATTGCGTAGGTTAAAGAAAATCTGTAATTGACTGCCACTTTTA

[0481] CGCAACGCATAATTGTTGTCGCGCTGCCGAAAAGTTGCAGCTGATTGCGCATGGTGCC

[0482] GCAACCGTGCGGCACCCTACCGCATGGAGATAAGCATGGCCACGCAGTCCAGAGAAA

[0483] TCGGCATTCAAGCCAAGAACAAGCCCGGTCACTGGGTGCAAACGGAACGCAAAGCG

[0484] CATGAGGCGTGGGCCGGGCTTATTGCGAGGAAACCCACGGCGGCAATGCTGCTGCATC

[0485] ACCTCGTGGCGCAGATGGGCCACCAGAACGCCGTGGTGGTCAGCCAGAAGACACTTT

[0486] CCAAGCTCATCGGACGTTCTTTGCGGACGGTCCAATACGCAGTCAAGGACTTGGTGGC

[0487] CGAGCGCTGGATCTCCGTCGTGAAGCTCAACGGCCCCGGCACCGTGTCGGCCTACGT

[0488] GGTCAATGACCGCGTGGCGTGGGGCCAGCCCCGCGACCAGTTGCGCCTGTCGGTGTT

[0489] CAGTGCCGCCGTGGTGGTTGATCACGACGACCAGGACGAATCGCTGTTGGGGCATGG

[0490] CGACCTGCGCCGCATCCCGACCCTGTATCCGGGCGAGCAGCAACTACCGACCGGCCCC

[0491] GGCGAGGAGCCGCCCAGCCAGCCCGGCATTCCGGGCATGGAACCAGACCTGCCAGCC

[0492] TTGACCGAAACGGAGGAATGGGAACGGCGCGGGCAGCAGCGCCTGCCGATGCCCGAT

[0493] GAGCCGTGTTTTCTGGACGATGGCGAGCCGTTGGAGCCGCCGACACGGGTCACGCTG

[0494] CCGCGCCGGTAGCACTTGGGTTGCGCAGCAACCCGTAAGTGCGCTGTTCCAGACTATC

[0495] GGCTGTAG

[0496] >SEQ ID NO:8 (pBBR1-TetR-Cas13a-crRNA2 plasmid)

[0497] GCTCTAGTTAGCCTAATCGCATAATTATTTATTATAGTATAATTCTTATTTTTTTTCAACCT

[0498] AAAAATTTAAAACATCTCCAAAAATTTTCGTTTCAGAACAACCAAGCAACCATATTCA

[0499] AAAAACAATAAAAAATGAGCAAGAATTGAAATTTTATTCTCACTCAGAAGTTATTTTTA

[0500] TTAAATATCACTTTTCGATATTGGGGTGGTCTATATCAATTTAAAAGACAGAATAGATAA

[0501] TTCTTTAGAGTTTTAGTCCCCTTCGATATTGGGGTGGTCTATATCATGCGGCTGTATTTG

[0502] CAAAAGCAGTTTTAGTCCCCTTCGATATTGGGGTGGTCTATATCCCATCCTAATTTCTTG

[0503] CTGATGAGATATTTATTTCTAATTTTTCTATTTTGTCTTTATTTTCAATACTTTCAATCCTA

[0504] TTTTTCTCTTTATTAATAATATAGAACCACCCTATACTATTATACCATATTTTTTGATTTTTC

[0505] AAAATTCCAATATTTTGTTTTGTGAAATTTTTTCTCCCATTGTCACTTCTCCTGCAAGTA

[0506] CCTTCATTTTTGAAACTGATCTTCTGTCAGGATAATGGAACGGATTGATGAATTTTCTG

[0507] GAGCGAGCATTGATAACTGTTTTTCTGCCAGTTCGATTTTTTCTTTTGTTTTCGACCTCA

[0508] TTATATATACCGATTTTTGAAGCTGATAATATCCCTTTTTCTATCAATTTTTTCCTAAAAGT

[0509] CCTATATTCAAATCTCTCAACATCTGTCTGCATAGGAAAATCATACATAAGCAGACCAA

[0510] AATACTCAATACTCATAGTCCATCAGCTCAATGTCGGAATTATCACTTCTTCATCTTTT

[0511] ACAAAATAATTTCGTATACTATCCAAATAATAGTCTACCGCTTGGAAAAAATCATATTTC

[0512] TTATTGTTAAATAATACCTTCTGCTGTGCTACAAGAAGTATTTTTTGCCTTATTTCCTTAC

[0513] TTAATTTCACTTCATTCAAAATATCCTTGTACATATAAACAAGATAATCCACCATAGGAC

[0514] GAAAAACCTCTATTATATCATCAGAAAAATTATAGGCATTAAACTGTGACTTATGATGTA

[0515] ATCCTAAACTTGGATGAAATCCTTTTGCTACAATCTTTGATGATATTATAGCTCTTAAAAT

[0516] CATATATCCATAATTAAGTGCAGAATTCACTCCATCTTCATCAAATCTTTTAAAACTATTA

[0517] CTATACAATTCCTGAAAATATATCCTTGAAGCTATTGCTTCCTGATGTTCTGCACTCGCA

[0518] TCATCTTTTTTCAAGTTTTCCTTATATGTTTTCAGTCTTTCAATGGAAATATCACTTTTTT

[0519] CAAGATACTCTAACAATGCTCTTTGATTTTCAATCTTATTCTCCACTATCCTGCTCCACA

[0520] ATTTTTCCTTTTTCTCTTTTTCCCACTCAATCTGCTCATTTATTCGTAAAGTCACTTGAA

[0521] AATGATTAAATAATCCCAGCGAATGAATTTCAGGCTGATGTTTCTCGTTGCAAATAATAA

[0522] TCGGAATGTTATTTTCCACCAGCCTCAACTGCAAAATCGCACTAATCTTACAATAGCAG

[0523] TTTTCAATAACTATCGCAGATATATCATTCAAAGAAATCTTATTTTTCTCATCATTATTGT

[0524] CTTCATCAACCATTATAAGCTGATTATTCGATATTGACAAATCATCAGCCCTTGTTATGTG

[0525] AATTATATTGGGCATTTTAATCATACTCCTTATAAATTTCATTCTTATAACGTATCATTCGT

[0526] ATTTTCTATTTTTGTTAAAAGTTCTATTATCAAGTTTTTAATATAATCAGAATTATAACTTT

[0527] CTAATTCTAAAACAGAAACTTTTTTAGGTTTCATTAATCTTTCAAGTATATCATTATTACC

[0528] GATAAGTTTAAATTTTTTCTTTAATTCATCATAATCTAAATTCACATCTTTTTTAAATACTT

[0529] CAAATACACTTGCATAAGTTGAATTATTATAACGTGTACTATATGATAATAAATTAGAAA

[0530] CTCTATCAATTTGTTCTGCAATACTGTAATCAGCAAACGGATTTCTTACAATATAGAAAT

[0531] GTGAAATATAGTTTCTAATACTTTCATTTTCCGGCTTATTAATTTCAGAATTTTCAGACA

[0532] AATCAATTCCAAATCCATAACATATTTTCTCAAATTTTTTATAAGATTCTTCATCAAAAAA

[0533] TTTATAGTATGCTGTTGTTGTATAAAAGCCATCAGATCCATTACGCTTAGGATAAGCTCT

[0534] ACTTATTCCAGTATTGTAGCCACTTAACTTAATAATTCCTAATTCTCTTAGCCCATTTACA

[0535] ATATAGTGCATATCTCTTTCAAATCTAGCCATTTGAATAGCAAGTTTCCAATTTATATCTA

[0536] TCAAATAACTTTCTATTTTATTCAAATAATTAAATTCTACCAAATCTCTAATTTTTTTGTAT

[0537] TCAGAAACTCTATTATAATCTTTTTCAAATGATTTATAGTTTTTATTTTGTATATTTTTTGC

[0538] AAAAAAGTCATCATTTTCTTTCAATTTTTTTATATACTTCTCTTTGTATTCTTTAGAATATC

[0539] CATTTAGTTTATCATTTAGATTTTTCAATATTGCATCAATTTCAGATATTTTATTTTTTCTA

[0540] ATATTTTTACCATCAATATTAAATAAAAATTTTGCATCAGCCATTTTAATATCATTTGAAA

[0541] TTAATCCATAAATTTTATCAAAATTTGGATTTCCAATATTTAAAAAATAAATTCTTTTTATA

[0542] AATATATAATTCATTCTTACGTTCTTTAGGATAATATATTTCTTGAAATTTATTTTCATTCT

[0543] CTGATTCCATATCTTCTATTAAATTATCTATTTCTTTTTTGTATTTTTTTAAAAAATCAGAA

[0544] TTAAATATTATTCTACACAATATTTACTCTTTATTTCCTGATCTTTATCTTTTATATACTGA

[0545] TCAACCTTTTTTTTCAAATCCTTTTTTATTATTGTTTGATAACTTTCTTTGTTCATCTTGTAA

[0546] TATATTCGATTTTTTATCTATCAAATTTAGTTTCATCATCAAAATTACAATTTTTTTCTA

[0547] ATTTTTTCTCTAAAACATCACAACCATTAATATCATCTTTAAATTCAGTTAATATATTATTTT

[0548] TTTATATCCTCATAATAATTATTAAAAATTCTTTTTTAGTTTGTATTTTAAAAATCATCAAA

[0549] GTCTTTTTCTATCTCTTTCATTTTTTGAATAAATTCTTCTAAAATTAAGATTCCAATTTTCA

[0550] GTTATACATTCATTTCTCAAAGTATTTAATTGCATTATTTCATCTAAAATATCTATAATATT

[0551] TTGATATTCTGAAGTATTTAACCAAACTGATGTTGCAAAAATCTATTTCTAATTTTATTT

[0552] ATAACCGCACTATTATTAAGTGCAATATTGAAATTTATTATTCAAAATCHELP

[0553] TTACTATAGTTTTATCACTAGTCTTTACAGTTATTCTTTCGTAAGTTTTATTATCATTAATG

[0554] TCTTTTATTTGTTTCTTAATTTCTCTGAATTCATTTTAAAATCTGAAAAATCAAAAAGTT

[0555] CCTCATAATTTTCTCAAATCCAATAACATTCTATTACTTTTCTGATATTTTT

[0556] ATAGCTTTATTATTACCTTTTGAAGCAGAAATCTGAGCATTTTTTAATATTTTCTATAAT

[0557] ATTTTCATCTATTTCATCAATGTTTCCTAAAGTTTTCTTATTCTTGTAAAAATATATTCT

[0558] TACTTTCATTTTCTTCTAAATCATCTTCTAAAATTAATTTCTTACAATTCTTTATTCACA

[0559] TATATTAAAGCATTTAATACTATTTTTTCTGTTTCTATAGTATCAAATGGTTCATTCTTAGG

[0560] ATTATTCCTTATTTATTTATTTCTATTATTATTTATTATTATTGTATTATTATT

[0561] ATATCATTTATTTTCTTCTGAATTTATTATTATTATTTTAGTAATTATTTTTTTTAT

[0562] CTTTAAATACTACATCTAAATTATATGCTTTTGACACTTCTTCATCTGATATTTTTAAATT

[0563] TTGAATTATTTATGACTTTATTAGTCATCTTGCGTTCCTTGTAAATCTCTTTCCTTGC

[0564] TAATCGCATGTAATATCCTGTTTCTTTCATTTGTTCCTATCTTTGTAAATTTCCTAATAA

[0565] ATTATTTGTAATGTTATTTTTATTATCTATAAAATCTAAGTCTCTTATTTTTTTTTTTTGA

[0566] ATTTAAAATTTTTTTATCAAGTAATTTTTTTCTCGATCTCCTCCAAAGAAATCTATA

[0567] TTTTCATCATTATTTATTTTCTCTAGAAAAAATCTTATTTAATTCCATATTGGTAGAAGC

[0568] AAAAAAGAATCAATTCTAAATCCAATTCCTCTTTAGCGTGAAGTCTAGAAAAATCAT

[0569] CAGTATTTACTGTTGTCATATCTATATATCATTATGTCTTAATTTCCCTAAAATACATAATATGC

[0570] TCTAACGTATATTGCTTAACTCTTTTTAAAATTTTCAGATAATACTTTCATTTAAAA

[0571] TTTTTTCTATTTCTATTTTCCATTTTCTTTAATCTGACTTTGTTCATTTACCAATTT

[0572] TTTCAATTCTTCCTTTCAAATATCGATATGATTTATAGTTCTTTTTCTTCATCAGAT

[0573] TTCTTTGAAAATTTTTTCGAATCAAAATTAACTTATTAATGTTTTTTAAAATTCCAAAAA

[0574] TTTCTGTATCACAATTTCCTTTTTTTAGTTTCTTTTTCTAATTTTTTTTATTAATTCATCTATTT

[0575] TAAATTCTGCTAAAATTTTTTCTATTTTTTTCTTTTATACTATTATTTTTTATATTTTCTACAA

[0576] AAAATTTTACAATTTTATTCTTTTTTATTTTCTCTTTCTATTTTAAATTTTTCGTGCTTATCT

[0577] AATAGTACATAAGATTTTATATATGTTCTATTTCTTTCTCTTTTCAAGAAATTCATTATTAAC

[0578] TTTTTTTACTTTTTCAATTCTTTTAGTAATATTCCAAAATTCTAACTCTTTTATAACAAAA

[0579] TCAGCTATATCTTCTACTGTTAAATCTACATTTATATTTAAAATTTTTTCAACAAGCATTT

[0580] TTTTATTTTTAGATTTCTTTTTATCACCCACCAACATTAAGATAAAATTTTACAAAACCCA

[0581] GAATTTCTAAATTACTTTTTATTTTTTTCTCTTATTTCCATAAAATTAGTCAAAATAACATC

[0582] TATTTTATCATCTTTCAATAATTTTTCTCTTAAATGTTCTCATAATATCGATTTTCAAATA

[0583] CTTTTTCTTGTTTCATTTTCAATTATTTTTTCTATAATCTTTATAAACTCATGTTAATATTTT

[0584] TAAAAATTTCGTAAATTGATTTTTTTGTTTCTAATTCATCATTTTCTATTATTCTTAATATT

[0585] ATTGAACAATCATTTAGTGTTTTATTAGTATACTCATCTCTGATATCTATCTCTATTTCTTC

[0586] TTCATTCTCTTGTCTCTTATTTCTATTTTTTTATCATCTTTAGTTATTCCTTGCCTAATTG

[0587] CTTCATCTATTATTTTCTTTTTTGTAATCCCCAATGCTTTCAATTTCTCAGATTTTCCATAT

[0588] GCTTCTATATATAATACAACTTCTTCTGTTTCCAAAAAATCATCATTATTTTCTATTCTTAT

[0589] GATTCCTTCTTTACCTTTCAACTTAAATAGAATATTTCCTGCATGAAATTTTCTTGTAAAT

[0590] TCTTTAAGAATATTATCATTTTTTTTGTAATTAATATATTTTCTAATAAATTTATTATTATCA

[0591] ATTTTTTCTTTATTATTATTTTCATTAATATTTAAAAATGTATTTGTTTCCATCATAGTTCCTT

[0592] TTAACTTTTACTTTCCGTTTTATTTTAAAATCTTTTTTATCACGAACTTCATACCATCTCT

[0593] TATGTCCAAAATAAAATTTCCCATTCCAATCTCCTCGTTTCTACTTTAATCTAATAAAATATT

[0594] TTTAAATTAAATCAATTTTACATCTTTCTAATCAAAAATACAATTTTCCATTTTTAGTATA

[0595] CCACATCAATATTAAATCTCAAAATAAGGAGCCGTCAAACATAGAGATCCTTTCTC

[0596] CTCTTTAGATCTTTTGAATTCTTTTCTCTATCACTGATAGGGAGTGGTAAAATAACTCTA

[0597] TCAACGATAGAGTGTCAACAAAAATTAGGAATTAATGATGTCTAGATTAGATAAAAGTA

[0598] AAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAAC

[0599] CCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAA

[0600] AATAAGCGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCA

[0601] CTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTT

[0602] TTAGATGTGCTTTACTAAGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCT

[0603] ACAGAAAAACAGTATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTT

[0604] TTTCACTAGAGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGC

[0605] GTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACT

[0606] GATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGA

[0607] GCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAAT

[0608] GTGAAAGTGGGTCTTAAGCAAGTTGACATAAGCCTGTTCGGTTCGTAACAGAAGCCAC

[0609] TGGAGCACCTCAAAAACACCATCATACACTAAATCAGTAAGTTGGCAGCATCACCCGA

[0610] CGCACTTTGCGCCGAATAAATACCTGTGACGGAAGATCACTTCGCAGAATAAATAAAT

[0611] CCTGGTGTCCCTGTTGATACCGGGAAGCCCTGGGCCAACTTTTGGCGAAAATGAGACG

[0612] TTGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTACCGGG

[0613] CGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAARGAAGCTAAAATGGAGAAAAAA

[0614] ATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCA

[0615] TTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTT

[0616] TTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTG

[0617] CCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGT

[0618] GATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTT

[0619] CATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAA

[0620] GATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATAT

[0621] GTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCA

[0622] ATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGAC

[0623] AAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGT

[0624] CGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAA

[0625] TTTTTTTAAGGCAGTTATTGGTGCCCTTAAACGCCTGGTTGCTACGGCGGAGTTGTTCG

[0626] GTAAATTGTCACAACGCCGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGCCCGCGTT

[0627] CCTGCTGGCGCTGGGCCTGTTTCTGGCGCTGGACTTCCCGCTGTTCCGTCAGCAGCTT

[0628] TTCGCCCACGGCCTTGATGATCGCGGCGGCCTTGGCCTGCATATCCCGATTCAACGGCC

[0629] CCAGGGCGTCCAGAACGGGCTTCAGGCGCTCCCGAAGGTCTCGGGCCGTCTCTTGGG

[0630] CTTGATCGGCCTTCTTGCGCATCTCACGCGCTCCTGCGGCGGCCTGTAGGGCAGGCTC

[0631] ATACCCCTGCCGAACCGCTTTTGTCAGCCGGTCGGCCACGGCTTCCGGCGTCTCAACG

[0632] CGCTTTGAGATTCCCAGCTTTTCGGCCAATCCCTGCGGTGCATAGGCGCGTGGCTCGA

[0633] CCGCTTGCGGGCTGATGGTGACGTGGCCCACTGGTGGCCGCTCCAGGGCCTCGTAGA

[0634] ACGCCTGAATGCGCGTGTGACGTGCCTTGCTGCCCTCGATGCCCCGTTGCAGCCCTAG

[0635] ATCGGCCACAGCGGCCGCAAACGTGGTCTGGTCGCGGGTCATCTGCGCTTTGTTGCCG

[0636] ATGAACTCCTTGGCCGACAGCCTGCCGTCCTGCGTCAGCGGCACCACGAACGCGGTC

[0637] ATGTGCGGGCTGGTTTCGTCACGGTGGATGCTGGCCGTCACGATGCGATCCGCCCCGT

[0638] ACTTGTCCGCCAGCCACTTGTGCGCCTTCTCGAAGAACGCCGCCTGCTGTTCTTGGCT

[0639] GGCCGACTTCCACCATTCCGGGCTGGCCGTCATGACGTACTCGACCGCCAACACAGCG

[0640] TCCTTGCGCCGCTTCTCTGGCAGCAACTCGCGCAGTCGGCCCATCGCTTCATCGGTGC

[0641] TGCTGGCCGCCCAGTGCTCGTTCTCTGGCGTCCTGCTGGCGTCAGCGTTGGGCGTCTC

[0642] GCGCTCGCGGTAGGCGTGCTTGAGACTGGCCGCCACGTTGCCCATTTTCGCCAGCTTC

[0643] TTGCATCGCATGATCGCGTATGCCGCCATGCCTGCCCCTCCCTTTTGGTGTCCAACCGG

[0644] CTCGACGGGGGCAGCGCAAGGCGGTGCCTCCGGCGGGCCACTCAATGCTTGAGTATA

[0645] CTCACTAGACTTTGCTTCGCAAAGTCGTGACCGCCTACGGCGGCTGCGGCGCCCTACG

[0646] GGCTTGCTCTCCGGGCTTCGCCCTGCGCGGTCGCTGCGCTCCCTTGCCAGCCCGTGGA

[0647] TATGTGGACGATGGCCGCGAGCGGCCACCGGCTGGCTCGCTTCGCTCGGCCCGTGGAC

[0648] AACCCTGCTGGACAAGCTGATGGACAGGCTGCGCCTGCCCACGAGCTTGACCACAGG

[0649] GATTGCCCACCGGCTACCCAGCCTTCGACCACATACCCACCGGCTCCAACTGCGCGGC

[0650] CTGCGGCCTTGCCCCATCAATTTTTTTAATTTTCTCTGGGGAAAAGCCTCCGGCCTGCG

[0651] GCCTGCGCGCTTCGCTTGCCGGTTGGACACCAAGTGGAAGGCGGGTCAAGGCTCGCG

[0652] CAGCGACCGCGCAGCGGCTTGGCCTTGACGCGCCTGGAACGACCCAAGCCTATGCGA

[0653] GTGGGGGCAGTCGAAGGCGAAGCCCGCCCGCCTGCCCCCCGAGCCTCACGGCGGCG

[0654] AGTGCGGGGGTTCCAAGGGGGCAGCGCCACCTTGGGCAAGGCCGAAGGCCGCGCAG

[0655] TCGATCAACAAGCCCCGGAGGGGCCACTTTTTGCCGGAGGGGGAGCCGCGCCGAAGG

[0656] CGTGGGGGAACCCCGCAGGGGTGCCCTTCTTTGGGCACCAAAGAACTAGATATAGGG

[0657] CGAAATGCGAAAGACTTAAAAATCAACAACTTAAAAAAGGGGGGTACGCAACAGCTC

[0658] ATTGCGGCACCCCCCGCAATAGCTCATTGCGTAGGTTAAAGAAAATCTGTAATTGACTG

[0659] CCACTTTTACGCAACGCATAATTGTTGTCGCGCTGCCGAAAAGTTGCAGCTGATTGCG

[0660] CATGGTGCCGCAACCGTGCGGCACCCTACCGCATGGAGATAAGCATGGCCACGCAGTC

[0661] CAGAGAAATCGGCATTCAAGCCAAGAACAAGCCCGGTCACTGGGTGCAAACGGAAC

[0662] GCAAAGCGCATGAGGCGTGGGCCGGGCTTATTGCGAGGAAACCCACGGCGGCAATGC

[0663] TGCTGCATCACCTCGTGGCGCAGATGGGCCACCAGAACGCCGTGGTGGTCAGCCAGA

[0664] AGACACTTTCCAAGCTCATCGGACGTTCTTTGCGGACGGTCCAATACGCAGTCAAGGA

[0665] CTTGGTGGCCGAGCGCTGGATCTCCGTCGTGAAGCTCAACGGCCCCGGCACCGTGTC

[0666] GGCCTACGTGGTCAATGACCGCGTGGCGTGGGGCCAGCCCCGCGACCAGTTGCGCCT

[0667] GTCGGTGTTCAGTGCCGCCGTGGTGGTTGATCACGACGACCAGGACGAATCGCTGTTG

[0668] GGGCATGGCGACCTGCGCCGCATCCCGACCCTGTATCCGGGCGAGCAGCAACTACCG

[0669] ACCGGCCCCGGCGAGGAGCCGCCCAGCCAGCCCGGCATTCCGGGCATGGAACCAGAC

[0670] CTGCCAGCCTTGACCGAAACGGAGGAATGGGAACGGCGCGGGCAGCAGCGCCTGCC

[0671] GATGCCCGATGAGCCGTGTTTTCTGGACGATGGCGAGCCGTTGGAGCCGCCGACACG

[0672] GGTCACGCTGCCGCGCCGGTAGCACTTGGGTTGCGCAGCAACCCGTAAGTGCGCTGT

[0673] TCCAGACTATCGGCTGTAG

[0674] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as such modifications are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A crRNA specifically targeting Vibrio parahaemolyticus, characterized in that The crRNA targets and recognizes the T2RHS-Nuc gene of Vibrio parahaemolyticus, including crRNA1 or crRNA2, the nucleotide sequence of the crRNA1 is shown in SEQ ID NO.1, and the nucleotide sequence of the crRNA2 is shown in SEQ ID NO.

2.

2. The crRNA specifically targeting Vibrio parahaemolyticus according to claim 1, characterized in that The nucleotide sequence of the T2RHS-Nuc gene is shown in SEQ ID NO.

3.

3. A bactericidal plasmid for Vibrio parahaemolyticus, characterized in that: Contains the crRNA specifically targeting Vibrio parahaemolyticus according to claim 1, a nucleotide sequence encoding LsCas13a protein, and a tetracycline-inducible expression element.

4. The bactericidal plasmid according to claim 3, characterized in that The nucleotide sequence of the LsCas13a protein is shown in SEQ ID NO.4; the nucleotide sequence of the tetracycline-inducible expression element is shown in SEQ ID NO.

5.

5. The bactericidal plasmid according to claim 3 or 4, characterized in that The bactericidal plasmid is based on the pBBR1 plasmid as a backbone, and the nucleotide sequence of the pBBR1 plasmid is shown in SEQ ID NO.

6.

6. Use of the bactericidal plasmid according to claim 3 in targeted killing of Vibrio parahaemolyticus for purposes other than disease diagnosis and treatment.

7. A recombinant bacterium containing the bactericidal plasmid according to claim 3.

8. The recombinant bacterium according to claim 7, characterized in that The host strain of the recombinant bacteria is Escherichia coli.

9. Use of the recombinant bacteria according to claim 7 or 8 in the preparation of a bactericidal agent against Vibrio parahaemolyticus.

10. A method for targeted elimination of Vibrio parahaemolyticus based on the CRISPR-Cas13a system, characterized in that: The bactericidal plasmid according to claim 3 is heat-shock transformed into Escherichia coli competent cells; and the Escherichia coli carrying the bactericidal plasmid and Vibrio parahaemolyticus are co-incubated.

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