A gene editing injection for intervening locust swarm behavior and a pest control method

Abstract

This invention provides a gene-editing injection solution and a method for controlling locust swarming behavior, belonging to the field of insect gene-editing technology. The gene-editing injection solution of this invention comprises the following components: sgRNA, Cas9 protein, and modified donor DNA in a concentration ratio of 1:2:1~4; the nucleotide sequence of the sgRNA is shown in SEQ ID NO.1; the nucleotide sequence of the modified donor DNA is shown in SEQ ID NO.2. This invention uses a CRISPR-Cas9-mediated gene-editing system to edit functionally encoded genes in locusts. henna By inserting nucleic acid sequences into specific promoter regions, changes in the protein expression of functional genes can be achieved without altering the protein-coding sequence.

Description

Technical Field

[0001] This invention belongs to the field of insect gene editing technology, and in particular relates to a gene editing injection solution for intervening in locust swarming behavior and a method for pest control. Background Technology

[0002] The CRISPR-Cas9 system represents a significant technological breakthrough in life sciences, enabling targeted DNA double-strand breaks through RNA guidance. In gene knock-in technology, CRISPR-Cas9-mediated DNA double breaks can achieve precise insertion of exogenous sequences via homology-directed repair. However, the efficiency of this insertion system is limited by factors such as donor DNA design. Especially in non-model organisms with large genomes and multiple repetitive sequences, this system-mediated precise sequence knock-in still faces technical bottlenecks such as low efficiency, off-target effects, and unclear genetic background.

[0003] In the field of insect gene editing, CRISPR-Cas9 technology has been successfully applied to species such as fruit flies, but it mainly targets the knockout of protein-coding sequences or the insertion of oligonucleotide chains. For species with large genomes and multiple repetitive sequences, such as locusts, the current technology has the following prominent problems: (1) lack of precise editing strategies for functional elements in non-coding regions; (2) optimization of effective gene knock-in systems in the context of genomes rich in repetitive sequences; (3) lack of effective schemes for optimizing donor DNA design and delivery.

[0004] Therefore, developing a site-directed nucleic acid sequence insertion system suitable for locusts, especially editing technology for functional elements such as promoters, is of great scientific significance and application value for promoting locust functional genomics research and developing new pest control strategies. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a gene-editing injection for intervening in locust swarming behavior and a method for pest control. This invention uses a CRISPR-Cas9-mediated gene-editing system to edit functionally encoded genes in locusts. henna This technology allows for the insertion of nucleic acid sequences into specific promoter regions without altering the protein-coding sequence, thereby achieving changes in the protein expression of functional genes. This verifies the feasibility of inserting nucleic acid sequences into important functional regions of the locust genome. The system can edit and insert nucleic acid sequences of a certain length, modifying the sequences of functional elements in the locust genome, and providing a new technical approach for locust gene function research and pest control.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a gene-editing injection for intervening in locust swarming behavior, comprising the following components: sgRNA, Cas9 protein, and modified donor DNA; The concentration ratio of sgRNA, Cas9 protein and modified donor DNA is 1:2:2; The nucleotide sequence of the sgRNA is shown in SEQ ID NO.1; The nucleotide sequence of the modified donor DNA is shown in SEQ ID NO.2.

[0007] Preferably, the sgRNA targets henna The promoter base sequence of the gene is the PAM region of NGG.

[0008] Preferably, the henna The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0009] Preferably, the concentration of the sgRNA is 150 ng / μL, the concentration of the Cas9 protein is 300 ng / μL, and the concentration of the modified donor DNA is 300 ng / μL.

[0010] This invention also provides a locust control method, comprising the following steps: The gene-editing injection solution was microinjected into locust eggs, and the resulting samples were screened to obtain the gene-editing properties of the locusts. henna The sequence of the PAM region, a functional element of the gene promoter, is inserted into the individual to control locusts by intervening in their swarming behavior.

[0011] Preferably, the injection volume of the gene-editing injection solution is 13-15 nL per locust egg.

[0012] Compared with the prior art, the present invention has the following beneficial effects: 1. Knock-in of nucleic acid sequences into functional element regions of large genomes with multiple repetitive sequences This invention utilizes an in vitro luciferase reporter system to analyze the core regions of functional elements such as promoters. Screening for sgRNA sites within the core promoter region requires a PAM region with an NGG base sequence, and the seed and non-seed regions of the sgRNA must show no off-target sites when aligned with the entire locust genome. Donor double-stranded DNA is optimized for insertion efficiency using C6-PEG10. The final result is a homozygous gene-edited strain, which can be identified through genomic fragment analysis, enabling the creation of a nucleic acid sequence platform for specific functional elements.

[0013] 2. Gene expression regulation is achieved without modifying the protein-coding sequence.

[0014] This invention, through quantitative detection, detected significant [significant effects] at both the RNA and protein levels. henna Reduced gene expression levels enable indirect regulation of specific gene expression through genomic functional elements before transcription. Attached Figure Description

[0015] Picture 1 This is a schematic diagram illustrating the principles of microinjection and gene editing; Picture 2 This is a schematic diagram of target region sequence insertion; Picture 3 It involves extracting homozygous brain tissue for testing. henna Expression at the levels of gene RNA and protein. Detailed Implementation

[0016] This invention provides a gene-editing injection for intervening in locust swarming behavior, comprising the following components: sgRNA, Cas9 protein, and modified donor DNA; The concentration ratio of sgRNA, Cas9 protein and modified donor DNA is 1:2:2; The nucleotide sequence of the sgRNA is shown in SEQ ID NO.1, specifically CGCGCTCTCATATATATAACTGG; The nucleotide sequence of the modified donor DNA is shown in SEQ ID NO.2: ATTTAGTGTATGCTGATTTTGTTTCCATAGATAATTAAAACCTTTTCTGATCGATCGTGGTTCAAATATACTCTTTGCTCGCAACATCCATTTCCGTTTACGCCGCATGGCATGTATGTGTGGATGAGAAGTTGAAAAGAAGATAAAGTGTAATA.

[0017] In this invention, the sgRNA targets henna The promoter base sequence of the gene is the PAM region of NGG; henna

[0018] This invention also provides a locust control method, comprising the following steps: The gene-editing injection solution was microinjected into locust eggs, and the resulting samples were screened to obtain the gene-editing properties of the locusts. henna The sequence of the PAM region, a functional element of the gene promoter, is inserted into the individual to control locusts by intervening in their swarming behavior.

[0019] In this invention, the injection volume of the gene editing injection solution is 13~15 nL / locust egg, preferably 13.5~14.5 nL / locust egg, and more preferably 13.8 nL / locust egg.

[0020] 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.

[0021] Example 1

[0022] This invention provides a donor double-stranded DNA optimized with 5' C6-PEG10, a system mixed with Cas9 protein and target region gRNA, which is then microinjected into locust eggs to achieve intracellular delivery of the drug. henna Sequential insertion of specific regions of promoter functional elements (e.g.) Picture 1 (As shown). Specifically: 1. Target site selection Select protein-coding genes with clearly defined functional elements. henna (SEQ ID NO.3) The core element portion of the promoter region was identified using an in vitro luciferase reporter system. sgRNA sites were screened within the core promoter region. sgRNAs required the NGG base sequence in the PAM region to function, and no off-target sites were found in the seed and non-seed regions of the sgRNA upon alignment with the entire locust genome. Based on this principle, CASOT 1.0 software was used to predict sgRNA sites in the upstream region, selecting the sgRNA target site with the fewest off-target sites for subsequent operations.

[0023] The sgRNA sequence was determined to be CGCGCTCTCATATATATAACTGG (SEQ ID NO.1).

[0024] 2. Donor DNA Synthesis

[0025] Based on the designed sgRNA target site, the cleavage site was determined, and 50 bp upstream and downstream were selected as homologous arms to design primers with homologous arms. The forward primer sequence with homologous arms is AGTGGAAGAGGGGGAGGGGGGCGTGCGTGCAGGCGCGCTCTCATATATATATTTAGTGTATGCTGAT (SEQ ID NO.4); the reverse primer sequence is CCACCTGCTCTGGCCCCGCGCTGTCTACGGCGGCGGCCGCTCACCCAGTTTATTACACTTTATCTTCTTTTCAAC (SEQ ID NO.5).

[0026] Double-stranded DNA donors were used, and the 5' ends were modified with C6-PEG10. Primers with homologous arms required modification for amplification, using the C6-PEG10 modification type. Primer synthesis and modification were performed by Shanghai Zhanbiao Biotechnology Co., Ltd.

[0027] The donor DNA sequence was selected as a single copy of the locust's own sequence (155 bp), as follows: ATTTAGTGTATGCTGATTTTGTTTCCATAGATAATTAAAACCTTTTCTGATCGATCGTGGTTCAAATATACTCTTTGCTCGCAACATCCATTTCCGTTTACGCCGCATGGCATGTATGTGTGGATGAGAAGTTGAAAAGAAGATAAAGTGTAATA (SEQ ID NO.2).

[0028] 3. Knock-in system microinjection

[0029] The injection mixture contained Cas9 protein (ThermoFisher), sgRNA, and modified donor DNA. All components were thoroughly mixed and incubated at room temperature for 10 min, then placed on ice until injection. Locust eggs produced within 2 hours of injection were pre-separated, cleaned with water, and fixed onto 1% agarose gel plates. Each egg was injected with 13.8 nL of the mixture, the injection site being the area from the egg well to the end. Injected eggs were cleaned every 24 h, placed on 1% agarose medium to maintain humidity, and incubated at 30°C until hatching.

[0030] Three different concentrations were selected to test the optimal experimental concentration of the donor. The ratios of sgRNA, Cas9 protein, and donor concentration were 1:2:1, 1:2:2, and 1:2:4 for microinjection into locust eggs. Individuals injected with nuclease-free water served as controls. After hatching, DNA was extracted from each individual, and the genomic DNA at the target sites was analyzed.

[0031] Experimental results are shown in Table 1.

[0032] Table 1. Injection effects of different buffer systems

[0033] As shown in Table 1, when the ratio of gRNA:Cas9 protein:donor is 1:2:2, that is, Cas9 protein at a concentration of 300 ng / μL, sgRNA at a concentration of 150 ng / μL, and modified donor DNA at a concentration of 300 ng / μL, the hatching rate reaches 39.76%, and individuals with accurate insertion appear, with a proportion of 4.82%, which is the optimal ratio.

[0034] 4. Editing strain testing

[0035] Genomic DNA was crudely extracted from individual locusts using an alkaline lysis method. Each locust was labeled, and the tarsal ends were cut. 27 μL of 50 mM NaOH was added, and the mixture was incubated at 90 °C for 30 min. Then, 3 μL of 1 M pH 8.0 Tris-HCl was added for neutralization. PCR verification was performed using KOD FX (TOYOBO) DNA polymerase. The PCR reaction system is as follows: Table 2 PCR reaction system

[0036] The PCR primers used for detection were designed approximately 100 bp upstream and downstream of the insertion site. The primers used for detection are as follows: RH_F: CGTAATTGGGAATCGCCAG (SEQ ID NO.6), RH_R: GTTGTGTAGTCGCTGCGCCT (SEQ ID NO. 7).

[0037] Perform PCR using the prepared PCR system according to the following procedure: Initial denaturation at 94℃ for 2 min; denaturation annealing at 98℃ for 10 s, 58℃ for 30 s, extension at 68℃ for 1 min / kb, 35 cycles; hold at 4℃.

[0038] 5 μL of the reaction product was subjected to 2% agarose gel electrophoresis. If there were bands that were clearly different in size from the wild type, they were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0039] If a clear band cannot be amplified using primers on the genome, the reaction product from the previous step is amplified again using primers from the insert sequence and primers on the genome; this is called nested PCR. If a clear band appears, sequencing is then performed.

[0040] Chimeras G0 from individuals with correctly matched genotypes are crossed. The resulting G1 generation is then subjected to PCR and sequencing to identify the knock-in of the mutant. Homozygous individuals are selected for further breeding to obtain homozygous mutant strains. Obtaining the genome of the homozygous edited strain allows for accurate sequencing of the inserted sequence (e.g., [missing information]) in the target region. Picture 2 (As shown).

[0041] Experimental results are shown in Table 3.

[0042] Table 3. Detection of homozygous mutant strains

[0043] As shown in Table 3, the survival rate of the strain was 54.10%; genotyping of the adults revealed 2 individuals carrying the insert1 fragment, accounting for 6.06%.

[0044] 5. Gene expression level detection

[0045] Trizol extracted RNA and protein from tissues and detected henna expression levels at the transcriptional and translational levels.

[0046] RNA level detection included: 1) reverse transcription of extracted RNA using SuperScript IV Reverse Transcriptase (Invitrogen); 2) detection of relative mRNA expression using the LightCycler 480 SYBR Green I Master (Roche) kit, with rp49 gene used as an internal control for correction.

[0047] Experimental results: such as Picture 3 As shown. Significant [effects] were detected at both RNA and protein levels. henna Reduced gene expression levels enable indirect regulation of specific gene expression through genomic functional elements before transcription.

[0048] As can be seen from the above embodiments, the present invention uses a CRISPR-Cas9-mediated gene editing system to edit functionally encoded genes in locusts. henna By inserting nucleic acid sequences into specific promoter regions, changes in the protein expression of functional genes can be achieved without altering the protein-coding sequence.

[0049] 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. A gene-editing injection for intervening in locust swarming behavior, characterized in that, It includes the following components: sgRNA, Cas9 protein, and modified donor DNA; The concentration ratio of sgRNA, Cas9 protein and modified donor DNA is 1:2:2; The nucleotide sequence of the sgRNA is shown in SEQ ID NO.1; The nucleotide sequence of the modified donor DNA is shown in SEQ ID NO.

2.

2. The gene editing injection solution according to claim 1, characterized in that, The sgRNA targeting henna The promoter base sequence of the gene is the PAM region of NGG.

3. The gene editing injection solution according to claim 2, characterized in that, The henna The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

4. The gene editing injection solution according to claim 1, characterized in that, The concentration of the sgRNA is 150 ng / μL, the concentration of the Cas9 protein is 300 ng / μL, and the concentration of the modified donor DNA is 300 ng / μL.

5. A method for locust control, characterized in that, Includes the following steps: The gene-editing injection solution described in any one of claims 1 to 4 was microinjected into locust eggs, and the resulting samples were screened to obtain the gene-editing solution from locusts. henna Sequence insertion into the PAM region of the gene promoter functional element in individuals can control locusts by intervening in their swarming behavior.

6. The locust control method according to claim 5, characterized in that, The injection volume of the gene-editing injection solution is 13-15 nL per locust egg.

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