SgRNA targeting the enhancer of locust tobr gene and application thereof in locust prevention and control

Abstract

The present application relates to the technical field of gene knockout. The present application provides sgRNA targeting Tob gene enhancer of locust and Cas9-sgRNA ribonucleoprotein complex containing the sgRNA. The present application mixes and injects the Cas9-sgRNA ribonucleoprotein complex and homologous arm repair template into locust eggs, so as to specifically knockout enhancer EH1 or EH2 of Tob gene of locust, thereby causing change of Tob gene space-time expression level, under the premise of not destroying the coding sequence of Tob protein, making the aggregation migration ability of the obtained homozygous mutant locust obviously weakened, and not affecting the basic life parameters such as survival rate and reproductive capacity. This provides a new technical approach for exploring a new type of green pest control strategy based on behavior intervention.

Description

Technical Field

[0001] This invention relates to the field of gene knockout technology, and in particular to sgRNA targeting the enhancer of the Tob gene in locusts and its application in locust control. Background Technology

[0002] Genome editing has become a cutting-edge core technology for modifying biological traits and has shown significant application potential in integrated pest management in agriculture. Locusts, as large-scale migratory pests that seriously threaten global food security, have seen their population density surge, triggering a shift from solitary to gregarious behavior. Large numbers of gregarious locusts can rapidly aggregate and migrate on a massive scale, which is key to locust outbreaks. In existing biological control technologies, RNA interference (RNAi) and gene coding region knockout remain the main methods. However, RNAi suffers from transient and incomplete silencing effects, making it difficult to achieve stable inheritance of phenotypic traits in field populations. Gene coding region knockout often results in complete loss of gene function, leading to growth and developmental defects or even lethal effects, failing to meet the need for precise and appropriate regulation of complex behaviors such as locust aggregation and migration. These technological limitations restrict the practical application of behavioral intervention strategies in locust control, especially the lack of mature methods for finely regulating locust behavior. Enhancers, as important cis-regulatory elements of gene expression, precisely regulate the expression levels of target genes spatiotemporally through epigenetic mechanisms, thereby influencing complex behavioral phenotypes. Based on epigenetic regulation strategies, compared to directly knocking out coding regions, targeting enhancers can achieve targeted downregulation of gene expression levels without disrupting protein-coding sequences, thereby creating locust strains with heritable, low-aggregation, and low-migration phenotypes for disaster control. However, an enhanced epigenetic regulation system and CRISPR site-directed knockout technology for locust behavior intervention are still lacking. Therefore, there is an urgent need to develop an epigenetic editing method that can precisely target enhancers of key behavioral genes, thereby intervening in locust aggregation and migration behavior at its source and constructing a novel green control strategy based on behavioral intervention. Summary of the Invention

[0003] The purpose of this invention is to provide sgRNA targeting the enhancer of the Tob gene in locusts and a formulation containing the sgRNA that targets and knocks out the enhancer of the Tob gene in locusts. By using a CRISPR-Cas9-mediated gene editing system, the enhancer EH1 or EH2 of the Tob gene, a key gene for aggregation and migration, is specifically knocked out, thereby achieving precise genetic regulation of locust aggregation and migration behavior.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of the Tob gene in regulating the aggregation and migration behavior of locusts, and achieves the regulation of locust aggregation and migration behavior by regulating the Tob gene through the Tob gene enhancer.

[0005] This invention provides sgRNAs targeting the Tob gene enhancer EH1 or EH2 of the locust, wherein the sgRNA targeting the Tob gene enhancer EH1 of the locust includes sgRNA1 as shown in SEQ ID NO.1 and sgRNA2 as shown in SEQ ID NO.2; and the sgRNA targeting the Tob gene enhancer EH2 of the locust includes sgRNA3 as shown in SEQ ID NO.3 and sgRNA4 as shown in SEQ ID NO.4.

[0006] The present invention provides a Cas9-sgRNA ribonucleoprotein complex targeting the Tob gene enhancer of the locust, comprising the Cas9 protein and two specific sgRNAs of the Tob gene enhancer EH1 or EH2 of the locust.

[0007] Preferably, the mass ratio of the two specific sgRNAs of the Cas9 protein and the locust Tob gene enhancer EH1 or EH2 is (1-3):1:1.

[0008] The present invention also provides a formulation for targeting and knocking out the Tob gene enhancer of the locust, comprising the Cas9-sgRNA ribonucleoprotein complex and a homologous arm repair template targeting the Tob gene enhancer EH1 or EH2 of the locust.

[0009] Preferably, the sequence of the homologous arm repair template targeting the locust Tob gene enhancer EH1 is shown in SEQ ID NO.5, and the sequence of the homologous arm repair template targeting the locust Tob gene enhancer EH2 is shown in SEQ ID NO.6.

[0010] Preferably, the concentration of Cas9 protein in the formulation is 380-420 ng / μL, the concentration of sgRNA in the formulation is 130-170 ng / μL, and the concentration of homologous arm repair template in the formulation is 480-540 ng / μL.

[0011] The present invention also provides the application of the sgRNA or the preparation that targets and knocks out the enhancer of the Tob gene in locusts in locust control.

[0012] This invention also provides the application of the sgRNA or the preparation that targets and knocks out the enhancer of the Tob gene in locusts in regulating locust aggregation and migration behavior.

[0013] This invention also provides a method for controlling locusts based on intervening in their aggregation and migration behavior, comprising the following steps: (1) Inject the preparation that targets and knocks out the Tob gene enhancer EH1 or EH2 of the locust into locust eggs, and hatch and screen to obtain Tob gene enhancer knockout locust offspring; (2) The Tob gene enhancer knockout locust offspring were self-crossed to obtain homozygous strains.

[0014] Preferably, in step (1), the injection volume of the agent that targets and knocks out the enhancer of the Tob gene in each locust egg is 13-14.5 nL.

[0015] Preferably, the screening in step (1) includes: injecting a preparation that targets and knocks out the enhancer EH1 or EH2 of the Tob gene in locusts, extracting genomic DNA from the forelegs of adult locusts for amplification, and performing sequencing analysis on the PCR products.

[0016] Preferably, the primers for amplification corresponding to the formulation that targets and knocks out the Tob gene enhancer EH1 of the locust include the forward primer shown in SEQ ID NO.7 and the reverse primer shown in SEQ ID NO.8; the primers for amplification corresponding to the formulation that targets and knocks out the Tob gene enhancer EH2 of the locust include the forward primer shown in SEQ ID NO.9 and the reverse primer shown in SEQ ID NO.10.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects: This invention designs targeted sgRNAs and repair templates with homologous arms based on the two enhancers EH1 and EH2 of the Tob gene in locusts. The targeted sgRNAs are then mixed with Cas9 protein to obtain a Cas9-sgRNA-ribonucleoprotein complex, which is then mixed with the repair template with homologous arms to obtain an injection solution. This solution is then microinjected into locust eggs to selectively knock out either the Tob gene enhancer EH1 or EH2, thereby altering the spatiotemporal expression level of the Tob gene. This achieves precise genetic regulation of locust aggregation and migration behavior (e.g., without disrupting the Tob protein coding sequence) without damaging the Tob protein coding sequence. Figure 1 This knockout system exhibits high targeting specificity, low off-target effects, and the deletion of enhancers can be stably inherited by offspring. Behavioral analysis also shows that the homozygous mutant locusts have significantly reduced aggregation and migration abilities, but their basic life parameters such as survival rate and reproductive capacity are not significantly affected.

[0018] This invention, based on an epigenetic regulation strategy, utilizes a CRISPR-Cas9-mediated gene editing system to target any enhancer region of the Tob gene, a key gene for aggregation and migration behavior in locusts, and knock out a fragment, thereby eliminating its regulatory effect on the Tob gene and reducing the locusts' aggregation and migration abilities. This achieves precise genetic manipulation from non-coding regulatory elements to behavioral phenotypes. Unlike traditional coding region knockouts that result in complete loss of protein function, this invention achieves fine-tuning of gene expression levels and precise alteration of behavioral phenotypes without destroying the Tob protein coding sequence. This technology directly intervenes in the aggregation and migration behavior of locusts through targeted enhancer epigenetic regulation. It can not only be used to verify the knockout of any enhancer or other cis-regulatory elements in the locust genome, but can also be extended to the functional study of epigenetic regulatory elements in other non-model insects, providing a new technical approach for exploring novel green pest control strategies based on behavioral intervention. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the principle of homology repair in gene editing. Figure 2 This diagram shows the location of sgRNAs on the genome and the sequences of four sgRNAs. Figure 3 The results are the predictions for sgRNA off-target effects. Figure 4 For sgRNA editing efficiency detection ( Figure 4 In this context, A represents the editing efficiency of sgRNA1 and sgRNA2 used in the construction of the EH1 knockout mutant, and B represents the editing efficiency of sgRNA3 and sgRNA4 used in the construction of the EH2 knockout mutant. Figure 5 This is a schematic diagram of hybridization and interspecific self-pollination; Figure 6 Schematic diagram of sequencing and electrophoresis of EH1 and EH2 homozygous knockout mutants ( Figure 6 In this diagram, A represents the sequencing results of the homozygous knockout mutant, and B represents the electrophoresis image. Figure 7 Molecular-level detection results for EH1 and EH2 knockout homozygous lines (Figure) Figure 7 In this diagram, A represents the quantitative detection of mature RNA and precursor RNA, B represents the protein detection result, and C represents the protein grayscale scan result. Figure 8 Results of mutant behavior detection ( Figure 8 In this text, A represents the change in the order parameter of the aggregation and migration behavior of the EH1 knockout mutant, and B represents the change in the order parameter of the aggregation and migration behavior of the EH2 knockout mutant. Detailed Implementation

[0020] This invention provides the application of the Tob gene in regulating the aggregation and migration behavior of locusts.

[0021] In this invention, the Tob gene is regulated by the Tob gene enhancer, thereby controlling the aggregation and migration behavior of locusts.

[0022] This invention provides sgRNAs targeting the Tob gene enhancer EH1 or EH2 in locusts. The sgRNAs targeting the Tob gene enhancer EH1 in locusts include sgRNA1 and sgRNA2. The nucleotide sequence of sgRNA1 is shown in SEQ ID NO.1, specifically TTGCCGCATTCCTGCCCGC; the nucleotide sequence of sgRNA2 is shown in SEQ ID NO.2, specifically GCCGACAGATTCTACCTGC. The sgRNAs targeting the Tob gene enhancer EH2 in locusts include sgRNA3 and sgRNA4. The nucleotide sequence of sgRNA3 is shown in SEQ ID NO.3, specifically AGGTGAAACCAGCCAGCACAGG; the nucleotide sequence of sgRNA4 is shown in SEQ ID NO.4, specifically CGCTTCAGCCCCGCGTATAAGG.

[0023] The present invention provides a Cas9-sgRNA ribonucleoprotein complex targeting the Tob gene enhancer of the locust, comprising the Cas9 protein and two specific sgRNAs of the Tob gene enhancer EH1 or EH2 of the locust.

[0024] In this invention, the preferred mass ratio of the two specific sgRNAs of the Cas9 protein and the locust Tob gene enhancer EH1 or EH2 is (1-3):1:1, further preferably (1.5-2.5):1:1, and even more preferably 2:1:1.

[0025] The present invention also provides a formulation for targeting and knocking out the Tob gene enhancer of the locust, comprising the Cas9-sgRNA ribonucleoprotein complex and a homologous arm repair template targeting the Tob gene enhancer EH1 or EH2 of the locust.

[0026]

[0027] In this invention, the concentration of Cas9 protein in the formulation is preferably 380-420 ng / μL, more preferably 390-410 ng / μL, and even more preferably 400 ng / μL; the concentration of sgRNA in the formulation is preferably 130-170 ng / μL, more preferably 140-160 ng / μL, and even more preferably 150 ng / μL; the concentration of homologous arm repair template in the formulation is preferably 480-540 ng / μL, more preferably 490-520 ng / μL, and even more preferably 500 ng / μL.

[0028] The present invention also provides the application of the sgRNA or the preparation that targets and knocks out the enhancer of the Tob gene in locusts in locust control.

[0029] This invention also provides the application of the sgRNA or the preparation that targets and knocks out the enhancer of the Tob gene in locusts in regulating locust aggregation and migration behavior.

[0030] This invention also provides a method for controlling locusts based on intervening in their aggregation and migration behavior, comprising the following steps: (1) The preparation that targets and knocks out the Tob gene enhancer of the locust is injected into locust eggs, and the locusts with the Tob gene enhancer knocked out are hatched and screened to obtain Tob gene enhancer knockout locust offspring; (2) The Tob gene enhancer knockout locust offspring were self-crossed to obtain homozygous strains.

[0031] In this invention, the injection volume of the agent that targets and knocks out the enhancer of the Tob gene in each locust egg is preferably 13-14.5 nL, more preferably 13.5-14 nL, and even more preferably 13.8 nL.

[0032] In this invention, the locust eggs are preferably eggs laid within 2 hours.

[0033] In this invention, the preferred temperature for hatching the locust eggs is 28°C, and the preferred hatching time is 14 days.

[0034] In this invention, the screening includes: injecting a preparation that targets and knocks out the enhancer EH1 or EH2 of the Tob gene in locusts, extracting genomic DNA from the forelegs of adult locusts and amplifying it, and then sequencing and analyzing the PCR products.

[0035] In this invention, the primers for amplification corresponding to the formulation that targets and knocks out the enhancer EH1 of the Tob gene in locusts include the forward primer shown in SEQ ID NO.7 and the reverse primer shown in SEQ ID NO.8, wherein the specific sequence shown in SEQ ID NO.7 is ggcctgtttctattttggttgg; and the specific sequence shown in SEQ ID NO.8 is cggctgcaattgtcaaCGTT.

[0036] In this invention, the primers for amplification corresponding to the formulation that targets and knocks out the enhancer EH2 of the Tob gene in locusts include the forward primer shown in SEQ ID NO.9 and the reverse primer shown in SEQ ID NO.10, wherein the specific sequence shown in SEQ ID NO.9 is gcacatatatccatgcccga; and the specific sequence shown in SEQ ID NO.10 is aaatgccgggatggttccttt.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] (a) Obtaining sgRNA and homologous arm repair templates

[0040] The two enhancers EH1 and EH2 of the Tob gene from the migratory locust were used as target genes. The sequence of the Tob gene is shown in SEQ ID NO. 11; the sequence of EH1 is shown in SEQ ID NO. 11. As shown in NO.12, the specific sequence is TGCAGTGGCGGTCTATTTCCGGCGGCCGCCCCTCGGGCAATCAATATCGGTGGCGAGCGCCGTGGCGTGCTGTGCCACTGGCGTGGAGTGGGAGTGCGCCGCGCCAGCCGCAGCCACGCCGCTTGCGCAAGTGCCGACCGCGCTTGCGTCACCGCCGGCGGTCACGTGACCGCCGCCCCTCCAGCTGCGTGACTATCTTTGG TGGCCGCCACGCCGCACCCCAACCTTGCACAAGGCACGTTGCCCGCCACGGGCCGCGCCCCGCGCCCCCGCCCCTAGCCTCACCCCTCCCTCACTCACTCTCTCTCGCTCTCTCTCTCTCTCTCGTGTTCTGGCCTCGCAATCCGCCCTGCTCCCAACCCTTCCAACTCGCTACAGTAGGGTCGACGCTACAGCTGTGC; the sequence of EH2 is as SEQ. ID As shown in NO.13, the specific sequence is: CAGCAGCCCTTCCGACTTTCCCTCATTCATCGCCGCCTCCGCTGCCCCTGTCCGTCCTCTCGCGCCTGCGCCCCCTCCGTTCTGCGAAGCGCGGGGCGCGACCGTAGTATCGGTTCCGCGAAAGGCTCCGCCAGGCAGAAATACCCGCGTAAACAGCCTGCGGCAGCGGCAGGTTTCGCTGCGCCCG GCCGGGCCCGGCGCTGCGGCGGTGACGTCAGAGGCGCGATCAATACGACTGACCTACGTCACAGTGGCACGCGCAGCGCGGCGCAGGGCCACCCCAGCCGCTATTGTCGGGGAGGGGGAGGTGGGGGGGAAACCACCCTGGCGCGTCAGAAATAGCGCGCCAGCGGCGGCGAGCGCGCTTACCACAGTCAGTCGAGCCGAGTC.

[0041] sgRNA design and off-target prediction were performed using CasOT software. The selected sgRNA sequences are as follows: Figure 2 As shown, the predicted off-target efficiency is as follows: Figure 3 As shown.

[0042] sgRNA synthesis was performed according to the GeneArt™ Precision gRNA Synthesis Kit (Invitrogen) instructions. First, PCR amplification was performed to obtain a DNA template containing a tracrRNA fragment and the T7 promoter. The mixture consisted of: 12.5 μL Phusion™ High-Fidelity PCR Master Mix, 1 μL Tracr Fragment + T7 Primer Mix, 1 μL 0.3 μM TargetF / R oligonucleotide mix, and 10.5 μL Nuclease-free water. After in vitro transcription, DNA was removed by incubation with DNase I (Thermo Scientific) at 37°C for 15 minutes. The sgRNA was purified using the GeneJET RNA Cleanup and Concentration Micro Kit (Thermo Scientific). The purified product was dissolved in RNase-free water, its concentration was determined using Narodrop One, and diluted to 1500 ng / μL. The solution was then stored at -80°C for later use.

[0043] The sgRNAs targeting the Tob gene enhancer EH1 of the locust are sgRNA1 and sgRNA2, and the sgRNAs targeting the Tob gene enhancer EH2 of the locust are sgRNA3 and sgRNA4. The sequence of sgRNA1 is shown in SEQ ID NO.1, specifically TTGCCGCATTCCTGCCCGC; the sequence of sgRNA2 is shown in SEQ ID NO.2, specifically GCCGACAGATTCTACCTGCCGG; the sequence of sgRNA3 is shown in SEQ ID NO.3, specifically AGGTGAAACCAGCCAGCACAGG; and the sequence of sgRNA4 is shown in SEQ ID NO.4, specifically CGCTTCAGCCCCGCGTATAAGG.

[0044] Efficiency assay of sgRNA: Each sgRNA was mixed with Cas9 protein and incubated at 37°C for 15 min to prepare the Cas9-sgRNA ribonucleoprotein (RNP) complex. The final concentration of sgRNA was 150 ng / μL, and the final concentration of Cas9 protein was 400 ng / μL. The liquid was injected into freshly laid locust eggs within 2 hours by microinjection, with 13.8 nL per egg. The eggs were incubated at 30°C for 72 h and then sampled. The eggs were added to 45 μL of 50 mmol / L NaOH solution and lysed by heating at 95°C for 30 min using a PCR instrument. After lysis, 5 μL of 1 mol / L Tris-HCl solution (pH 8.0) was added. The resulting product was the genomic DNA template.

[0045] PCR amplification was performed on DNA within 230-300 bp surrounding the sgRNA. The amplified DNA sequence of sgRNA1 was sgRNA1-check, as shown in SEQ ID NO.14, specifically: ggcctgtttctattttggttgggtaatggttgtgagctattatgtggtctgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaAAAAAAAAAAAAAAAGAAAGAAAAGAAAAAAAGAGGTAGAATCTACCGTACAATTGGTGAAGACATACGCCTCTGACAACATAATGAAGCACCGTCGCCCGCTCGGAGGCCCACCTGCTACCGTCGCCGCATGCGCTACGGGGCGGCGTTGCCGCATTCCTGCCCGCCGGCGCCTGCAGTGGCGGTCTATTTC; the amplified DNA sequence of sgRNA2 was sgRNA2-check, as shown in SEQ ID NO. As shown in NO.15, the specific sequence is TCGTGTTCTGGCCTCGCAATCCGCCCTGCTCCCAACCCTTCCAACTCGCTACAGTAGGGTCGACGCTACAGCTGTGCGCCATTCTGCCGACAGATTCTACCTGCCGGGAGACTAATTAACAGCAACCAAAGACGCCCAATTTCCTTTAGCATTGGAAAAAGCTTAGAAACGTCAGTAATTCATTCAAGTTCATAACATCTGAAATTAACGAGataataataatgggtactaattgatgcgctgcagtggagtagccgagcgtttcaaggcgccttgatgca; the DNA sequence amplified by sgRNA3 is sgRNA3-check, as shown in SEQ ID NO.As shown in 16, the specific sequence is GATTAACCGCCGGTTTCATCGCTGAAAACTGACCAGCGAACGTTTTGGAGTATCGCCGGAGAGGGATCAGAAAGCGAGACGGCGGTGGAGAGGTGAAACCAGCCAGCACAGGTGGCCGGGCGGCCGGCAGAGGTCAGCAGCCCTTCCGACTTTCCCC CTCATTCATCGCCGCCTCCGCTGCCCCTGTCCGTCCTCTCGCGCCTGCGCCCCCTCCGTTCTGCGAAGCGCGGGGCGCGACCGTAGTATCGGTTCCGCGAAAGGCTCCGCCAGGCAGAAATACCCGCGTAAACAG; the DNA sequence amplified by sgRNA4 is sgRNA4-check, and the sequence is such as SEQ ID As shown in NO.17, the specific sequence is ACAGTCAGTCGAGCCGAGTCCTAGGCCGTGCCGGTCTCAGACCGAAATACATCGCGCACCCACAGGTCAGCGAGCTGTCACGCTTCAGCCCCGCGTATAAGGACAGCAGAAT TATCCTCAAATGTCAGTGCTTCCAACGTTGCTATCTACCGGGACAATAAAGTGGCTCGTAATACTGCAGTATCGGTTTTCTTCtgccggccgcggtggccgaggggactgaggcgcttcagtca. .

[0046] The four sgRNA detection primers were designed as follows: sgRNA1-check-F:ggcctgtttctattttggttggg (as shown in SEQ ID NO.18) sgRNA1-check-R: GAAATAGACCGCCACTGCA (as shown in SEQ ID NO.19) sgRNA2-check-F:TCGTGTTCTGGCCTCGCAAT (as shown in SEQ ID NO.20) sgRNA2-check-R:tgcatcaaggcgccttgaaa (as shown in SEQ ID NO.21) sgRNA3-check-F:GATTAACCGCCGGTTTCATC (as shown in SEQ ID NO.22) sgRNA3-check-R:CTGTTTACGCGGGTATTTC (as shown in SEQ ID NO.23) sgRNA4-check-F: ACAGTCAGTCGAGCCGAGTCCTA (as shown in SEQ ID NO.24) sgRNA4-check-R:tgactgaagcgcctcagt (as shown in SEQ ID NO.25) PCR amplification was performed using 2×PhantaMax Master Mix (Vazyme) reagent, with the following composition: 25 μL 2×PhantaMax Master Mix; 20 μL ddH2O; 1 μL template; 2 μL check-F forward primer; 2 μL check-R reverse primer.

[0047] The PCR procedure is as follows:

[0048] PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for Fast NGS sequencing and point mutation analysis. Based on the analysis report provided by Beijing Qingke Biotechnology Co., Ltd., the editing efficiencies of the four sgRNAs were calculated as follows: Figure 4 As shown, all are above 70%, and all can be used for subsequent knockout experiments.

[0049]

[0050] After the company returns the sequence and synthesizes the plasmid, new primers will be designed to amplify the fused homologous arm fragments. The primer sequences are as follows: dsDNA1-F:tgttgactgctttaccttta (as shown in SEQ ID NO.26) dsDNA1-R:tcaaCGTTATTAATACTaat (as shown in SEQ ID NO.27) dsDNA2-F: GAGGTAACTCGTCTATAGCA (as shown in SEQ ID NO.28) dsDNA2-R:attCCGACGACCGGTCATTT (as shown in SEQ ID NO.29) PCR amplification was performed using 2×PhantaMax Master Mix (Vazyme) reagent, with the following composition: 25 μL 2×PhantaMax Master Mix; 20 μL ddH2O; 1 μL template; 2 μL forward primer; 2 μL reverse primer.

[0051] The PCR procedure is as follows:

[0052] After amplification, the gel was purified using the Promega Wizard SV Gel and PCR Clean-Up System kit according to the instructions. The concentration was measured using Nanodrop, and the gel was stored at -20°C for later use.

[0053] (II) Preparation of injection solution for targeting and knocking out the Tob gene enhancer in locusts

[0054] The specific sgRNA1, sgRNA2 and Cas9 protein obtained above were mixed in a ratio of 1:1:2 to prepare the Cas9-sgRNA ribonucleoprotein (RNP) complex. Then, the homologous arm repair template dsDNA1 obtained above was mixed with the Cas9-sgRNA RNP complex and reacted in a PCR instrument at 37°C for 15 min to allow the Cas9 protein to fully bind with the gRNA, thus obtaining the injection solution for targeting and knocking out the enhancer EH1 of the Tob gene in locusts.

[0055] The specific sgRNA3 and sgRNA4 obtained above were mixed with Cas9 protein in a ratio of 1:1:2 to prepare the Cas9-sgRNA ribonucleoprotein (RNP) complex. Then, the homologous arm repair template dsDNA2 obtained above was mixed with the Cas9-sgRNA RNP complex and reacted in a PCR instrument at 37°C for 15 min to allow the Cas9 protein to fully bind with the gRNA, thus obtaining the injection solution for targeting and knocking out the enhancer EH2 of the locust Tob gene.

[0056] The injection solution for targeting and knocking out the Tob gene enhancer in locusts contains Cas9 protein at a concentration of 400 ng / μL, two sgRNAs at a concentration of 150 ng / μL each, and a homologous arm repair template at a concentration of 500 ng / μL.

[0057] Example 2 (one)

[0059] The two injection solutions obtained in Example 1, which targeted the knockout of the Tob gene enhancer in locusts, were injected into locust eggs within 2 hours of being laid using microinjection. The eggs were cultured into adult locusts and then sequenced. The number of injected eggs, the number of hatched eggs, the number of adult locusts, and the number of mutants were recorded during the process, as shown in Table 1.

[0060] Table 1. Statistical results of mutants

[0061] Mutant identification: After the eggs hatch and develop into adult insects, a small amount of foreleg tissue was added to 45 μL of 50 mmol / L NaOH solution and lysed using a PCR instrument at 95℃ for 30 min. After lysing, 5 μL of 1 mol / L Tris-HCl solution (pH 8.0) was added. The resulting product was the genomic DNA template.

[0062] Mutant detection primers were designed upstream and downstream of EH1 and EH2, as follows: EH1-checkF:ggcctgtttctattttggttgg (as shown in SEQ ID NO.7) EH1-checkR:cggctgcaattgtcaaCGTT (as shown in SEQ ID NO. 8) EH2-checkF:gcacatatatccatgcccga (as shown in SEQ ID NO. 9) EH2-checkR:aaatgccgggatggttccttt (as shown in SEQ ID NO.10) PCR amplification was performed using 2×PhantaMax Master Mix (Vazyme) reagent, with the following system: 25 μL 2×PhantaMax Master Mix; 20 μL ddH2O; 1 μL template; 2 μL forward primer; 2 μL reverse primer.

[0063] PCR amplification procedure:

[0064] PCR products were sent to Tianyi Huiyuan Company for sequencing and analysis. Chimeric G0 generations with two enhancers knocked out at specific sites were obtained through screening. Figure 5 The mating and propagation process shown resulted in a homozygous line in the G2 generation, followed by further interspecific self-pollination to expand the population. Sequencing results and electrophoresis diagrams of the knockout mutant are shown below. Figure 6 As shown.

[0065] Genomic DNA was extracted from the forelegs of EH1 knockout homozygous adults using the alkaline lysis method, and from the forelegs of EH2 knockout homozygous adults using the same method. After PCR using mutant identification primers, the products were sent to Tianyi Huiyuan Company for sequencing and analysis. The sequencing results of the knockout mutant and the electrophoresis diagram of the deleted fragments are shown below. Figure 6 As shown. (two)

[0067] Brain tissue was collected from locusts on the third day of the fourth instar of the two homozygous strains mentioned above. RNA and protein were extracted using the Trizol method, and cDNA was obtained by reverse transcription using the Tiangen reverse transcription kit. qPCR and Western blot detection were then performed.

[0068] The primers for quantifying mature RNA are: Mature-F: AGCAACTACATCAAGCAGCG (as shown in SEQ ID NO.30) Mature-R: TACAGCTCGTGGTAGGGGAA (as shown in SEQ ID NO.31) Quantifying precursor RNA includes primers inside introns and primers across introns and adjacent exons.

[0069] The primers inside the introns are: Pre-F1: GCCATGTATTGCCGCATGTT (as shown in SEQ ID NO.32) Pre-R1: GGGACGTTAGGTGAAGCACA (as shown in SEQ ID NO.33) The primers spanning introns and adjacent exons are: Pre-F2: GGCGAAGAGGAAGGCTGTTC (as shown in SEQ ID NO.34) Pre-R2: TGCCGAGCACCTCGGATAATA (as shown in SEQ ID NO.35) qPCR and western blot results are as follows Figure 7 As shown, both mutant homozygous lines exhibited a significant decrease in Tob expression levels at both RNA and protein levels.

[0070] After expanding the population, 40 locusts were collected on the third day of the fourth instar for collective behavior monitoring. Behavioral videos were recorded, and order parameters were used to assess the direction of locust movement. The behavioral results are as follows: Figure 8 As shown, the locusts' aggregation and migration behavior has changed significantly.

[0071] In summary, this invention designs and obtains sgRNA targeting the Tob gene enhancer in locusts and a formulation containing this sgRNA that targets and knocks out the Tob gene enhancer in locusts. Using a CRISPR-Cas9-mediated gene editing system, fragments of two enhancers (EH1 and EH2) of the Tob gene, a key gene for aggregation and migration, are deleted in vivo through microinjection into locust eggs. This causes changes in the spatiotemporal expression level of the Tob gene, achieving precise genetic regulation of locust aggregation and migration behavior without damaging the Tob protein coding sequence.

[0072] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of the Tob gene in regulating the aggregation and migration behavior of locusts, characterized by, The aggregation and migration behavior of locusts can be regulated by controlling the Tob gene through the Tob gene enhancer.

2. An sgRNA targeting the enhancer EH1 or EH2 of the Tob gene in locusts, characterized in that, The sgRNAs targeting the Tob gene enhancer EH1 of the locust include sgRNA1 as shown in SEQ ID NO.1 and sgRNA2 as shown in SEQ ID NO.2; the sgRNAs targeting the Tob gene enhancer EH2 of the locust include sgRNA3 as shown in SEQ ID NO.3 and sgRNA4 as shown in SEQ ID NO.

4.

3. A Cas9-sgRNA ribonucleoprotein complex targeting the enhancer of the Tob gene in locusts, characterized in that, Includes the Cas9 protein and two specific sgRNAs of the locust Tob gene enhancer EH1 or EH2 as described in claim 2; The mass ratio of the two specific sgRNAs of the Cas9 protein and the locust Tob gene enhancer EH1 or EH2 described in claim 2 is (1-3):1:

1.

4. A formulation that targets and knocks out the enhancer of the Tob gene in locusts, characterized in that, Contains the Cas9-sgRNA ribonucleoprotein complex of claim 3 and a homologous arm repair template targeting the Tob gene enhancer EH1 or EH2 of the locust; The sequence of the homologous arm repair template targeting the Tob gene enhancer EH1 of the locust is shown in SEQ ID NO.5, and the sequence of the homologous arm repair template targeting the Tob gene enhancer EH2 of the locust is shown in SEQ ID NO.

6.

5. The formulation according to claim 4, characterized in that, The concentration of Cas9 protein in the formulation is 380-420 ng / μL, the concentration of sgRNA in the formulation is 130-170 ng / μL, and the concentration of homologous arm repair template in the formulation is 480-540 ng / μL.

6. The application of the sgRNA of claim 2 or the preparation that targets and knocks out the enhancer of the Tob gene in locusts according to claim 4 in the control of locusts.

7. The application of the sgRNA of claim 2 or the preparation that targets and knocks out the enhancer of the Tob gene in locusts according to claim 4 in regulating the aggregation and migration behavior of locusts.

8. A method for controlling locusts based on intervening in their aggregation and migration behavior, characterized in that, Includes the following steps: (1) Inject the preparation of the target knockout locust Tob gene enhancer EH1 or EH2 as described in claim 4 or 5 into locust eggs, hatch and screen to obtain Tob gene enhancer knockout locust offspring; (2) The Tob gene enhancer knockout locust progeny were self-crossed to obtain homozygous strains; Step (1) The injection volume of the agent that targets and knocks out the enhancer of the Tob gene in each locust egg is 13-14.5 nL.

9. The locust control method according to claim 8, characterized in that, The screening in step (1) includes: injecting a preparation that targets and knocks out the enhancer EH1 or EH2 of the Tob gene in locusts, extracting genomic DNA from the forelegs of adult locusts for amplification, and sequencing the PCR products.

10. The locust control method according to claim 9, characterized in that, The primers for amplification corresponding to the formulation that targets and knocks out the Tob gene enhancer EH1 in locusts include the forward primer shown in SEQ ID NO.7 and the reverse primer shown in SEQ ID NO.8; the primers for amplification corresponding to the formulation that targets and knocks out the Tob gene enhancer EH2 in locusts include the forward primer shown in SEQ ID NO.9 and the reverse primer shown in SEQ ID NO.10.

Images