Cucurbita fruit fly egg insertion protein LP and its application in gene editing

Abstract

The present invention discloses the cucurbit fruit fly egg entry protein LP and its application in gene editing. Its amino acid sequence is shown in SEQ ID NO: 1 in the sequence listing. The present invention demonstrates that the cucurbit fruit fly egg entry protein LP can be successfully absorbed by the ovaries of the cucurbit fruit fly and can guide the Cas9 / gRNA system into eggs and perform gene editing in offspring. This LP sequence and LP-Cas9 fusion protein have high application value in gene editing of the cucurbit fruit fly and related species.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene editing, and in particular relates to a cucurbit fruit fly egg protein LP and its application in gene editing. Background Art

[0002] The CRISPR / Cas9 system is currently the most commonly used gene editing technology. This system comprises clustered regulatory interspaced short palindromic repeats (CRISPR) and the Cas9 nuclease (CRISPR associated system 9). Guided by a specific RNA (single guide RNA, sgRNA), Cas9 can recognize specific sequences and cut target DNA. Following genomic DNA breaks, it activates the cell's DNA repair mechanism. This repair mechanism enables precise genome editing, such as conditional gene knockout, gene knock-in, gene replacement, and point mutations.

[0003] Currently, the primary method for introducing the CRISPR / Cas9 system into animals is through zygote microinjection. For example, a ribonucleoprotein complex formed by co-injecting Cas9 and sgRNA into zygotes or early embryos can be used to generate targeted mutants. Alternatively, an expression cassette containing Cas9 and sgRNA is transcribed in vitro to generate the corresponding mRNA, which is then co-injected to edit the target gene. However, zygote microinjection has significant limitations. First, this injection technique requires expensive equipment and specialized training, which limits its general application, especially for non-specialized laboratories. The injection process can easily damage the zygote and make it difficult to collect a large number of zygotes for injection. Furthermore, the timing of injection, which must occur early during cleavage, is difficult to control, especially in some ovoviviparous species that produce larvae rather than eggs. This significantly limits the application of CRISPR-Cas9 technology in various systems. Due to the limitations of zygote microinjection for the introduction of the CRISPR / Cas9 system, researchers have sought to develop alternative delivery methods.

[0004] In recent years, researchers have developed a novel CRISPR / Cas9 delivery method—receptor-mediated vertical CRISPR / Cas9 delivery for gene editing—first applied to Aedes aegypti mosquitoes. Taking advantage of the fact that Aedes mosquito eggs require receptor-mediated endocytosis to absorb yolk proteins during development, the researchers selected a peptide sequence, P2C, from Drosophila yolk proteins as a guide protein and fused it to Cas9 for expression. This fusion protein is recognized and actively absorbed by receptors on the egg surface, allowing it to successfully enter the egg and edit genes in the nucleus. This approach, also known as receptor-mediated ovarian transfer of cargo (ReMOT Control), allows Cas9 / sgRNA to be delivered into the mother and subsequently edit genes in her offspring. This ovarian delivery of germline gene editing has subsequently been successfully applied to species such as Bemisia tabaci, Tribolium castaneum, Bombyx mori, and Ixodes scapularis ticks. Compared to the classic fertilized egg injection method, this germline gene editing technique's greatest advantage is that it eliminates the reliance on microinjection, significantly reducing operational difficulty and technical labor costs. Furthermore, because insects lay large numbers of eggs, a single injection into a parent can edit a large number of offspring, improving editing efficiency. Therefore, this germline gene editing method has great potential for application in scientific research and molecular breeding.

[0005] The key to the successful implementation of this receptor-mediated ovarian-delivered gene editing technique is to identify the key sequence that guides the fusion protein into the egg. Due to differences in egg surface receptors between species, the identified lead protein sequences used vary, and different lead proteins identified within the same species also have varying abilities to guide the fusion protein into the egg. Egg entry sequences identified in other species are not suitable for ovarian-delivered gene editing in the cucurbit fruit fly. Therefore, identifying a protein sequence that can efficiently guide the fusion protein into the cucurbit fruit fly egg is of great significance for gene editing in the cucurbit fruit fly and for research on its egg developmental biology. Summary of the Invention

[0006] The purpose of the present invention is to provide the cucurbit fruit fly egg protein LP and its application in gene editing.

[0007] The invention discloses a cucurbit fruit fly egg protein LP, the amino acid sequence of which is shown in SEQ ID NO: 1 in the sequence listing.

[0008] The gene sequence of the cucurbit fruit fly egg protein LP is shown in the sequence listing as SEQ ID NO: 2.

[0009] Application of cucurbit fruit fly egg protein LP in gene editing.

[0010] Preferably, the LP-Cas9 / sgRNA complex is injected into Bactrocera cucurbitae eggs, which are then hatched and reared to adulthood.

[0011] The present invention has the following beneficial effects: The present invention has, for the first time, identified a leading protein sequence (LP) that enters the egg of the cucurbit fruit fly. This sequence has been shown to produce a fusion protein that is successfully absorbed by the ovaries of the cucurbit fruit fly and capable of guiding the Cas9 / gRNA system into the egg and editing the genes of the offspring. This LP sequence and LP-Cas9 fusion protein have high application value in gene editing in the cucurbit fruit fly and related species. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 To test the ability of LP to guide egg entry;

[0013] In the figure, Blank: untreated cucurbit fruit fly ovary; eGFP: cucurbit fruit fly ovary 24 hours after injection of eGFP protein; LP-eGFP: cucurbit fruit fly ovary 24 hours after injection of LP-eGFP protein; the upper half is the field of view under green fluorescence, and the lower half is the field of view under white light.

[0014] Figure 2 For the expression and purification of LP-Cas9 protein;

[0015] In the figure, M-protein molecular weight marker; lane 1-total protein of Escherichia coli Rosseta strain before IPTG induction; lane 2-total protein of Escherichia coli Rosseta strain after IPTG induction; lane 3-purified LP-Cas9 protein; the arrow indicates the target protein LP-Cas9.

[0016] Figure 3 The changes in eye phenotype of the melon fruit fly after knocking out the eye pigment gene white;

[0017] In the figure, Control is the uninjected control, and sgRNA1 and sgRNA2 are gudie RNAs designed and synthesized for different sites of the white gene. DETAILED DESCRIPTION

[0018] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0019] Example 1 Vector Construction

[0020] The amino acid sequence of the cucurbit fruit fly pentaprotein LP is shown in the sequence listing as SEQ ID NO: 1; the gene sequence of the cucurbit fruit fly pentaprotein LP is shown in the sequence listing as SEQ ID NO: 2.

[0021] LP-Cas9 expression vector construction: A DNA sequence encoding the LP protein and a ligation linker, including restriction sites, was obtained by chemical synthesis (Shanghai Sangon Biotechnology Co., Ltd.) as shown in SEQ ID NO: 5. This fragment and the pET28a-Cas9-Cys vector were double-digested with Nde I and EcoR I. The digested vector and fragment were then detected and recovered. The recovered fragment was ligated with the vector to construct the expression vector. The ligation product from the above step was transformed into competent Escherichia coli DH5α, and positive strains were screened. The cells were inoculated into LB medium containing kanamycin and cultured at 37°C, 180 rpm, for 10 hours. The expression plasmid was then extracted using a kit and transformed into E. coli Rosseta, where positive strains were screened.

[0022] The construction process of LP-eGFP expression vector is similar to the above method, except that the vector is replaced with pET30a-eGFP and the restriction enzyme cutting sites are replaced with Bgl II and EcoR I.

[0023] Example 2 Protein Expression

[0024] In this example, the proteins expressed in large quantities are secreted LP-eGFP and LP-Cas9. Positive strains were grown in LB medium at 37°C and 180 rpm. 3 ml of the bacterial suspension was transferred to 300 ml of LB medium at a 1:100 ratio and cultured at 37°C and 180 rpm with shaking for 3 hours. IPTG was then added to a final concentration of 0.5 mM and induced for another 4 hours. The culture was then centrifuged at 6000 rpm for 10 minutes at 4°C, and the supernatant and precipitate were collected separately. LP-eGFP protein was expressed in the supernatant and displayed a green color. The supernatant was retained and the precipitate was discarded. LP-Cas9 protein was expressed in the precipitate, so the precipitate was suspended in 20 ml of 1× PBS and added with 1% Tween by volume. The precipitate was disrupted by sonication in an ice bath at 35% power, 3 seconds on, 3 seconds off, for 45 minutes. The supernatant and precipitate were then centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant and precipitate were collected separately. The collected precipitate was suspended and mixed with 20 ml of Buffer A (5 mM EDTA, 50 mM Tris-HCl; pH 8.0), and centrifuged at 4°C, 10,000 rpm for 20 min; the supernatant was discarded, and the precipitate was washed and mixed with 20 ml of Buffer B (5 mM EDTA, 50 mM Tris-HCl, 2 M urea; pH 8.0), and centrifuged at 4°C, 10,000 rpm for 20 min (repeat Buffer B once); the supernatant was discarded, and 20 ml of denaturing solution (0.1 M Tris-HCl, 10 mM Dissolve the precipitate with DTT, 8M urea; pH 8.0), shake on a shaker at 37°C, 220 rpm, for 1 hour; centrifuge at 4°C, 10,000 rpm, for 10 minutes, collect the supernatant and precipitate, dialyze the supernatant (the dialysate is 50 times the volume of the supernatant), dialyze at 4°C for more than 16 hours, change the dialysate the next day, and dialyze for another day. Finally, centrifuge at 4°C, 10,000 rpm, for 10 minutes. Keep the supernatant containing the target protein and store it at -80°C until use. Figure 2 ).

[0025] Example 3 Female Injection

[0026] Select newly emerged (less than 6 hours) female melon flies and inject LP-eGFP protein (1 μL, 0.5 μg) or an equal amount of control eGFP protein into the female's chest using a syringe and microinjection needle. 24 hours after injection, dissect the female flies and remove the ovaries. Rinse the ovaries repeatedly with PBS buffer and then press them into slides. Observe and photograph under a fluorescence microscope ( Figure 1 ).

[0027] like Figure 1 As shown, 24 hours after the injection of LP-eGFP protein, the ovaries of the melon fruit fly appeared green under fluorescence, proving that LP has the ability to guide into the eggs.

[0028] Example 4 Gene Editing

[0029] The purified LP-Cas9 protein was mixed with a guide RNA (gRNA) designed and synthesized for the eye pigment gene white of the melon fruit fly to form an RNP complex and injected into the fertilized eggs of the melon fruit fly. The primer sequence of the gRNA is shown in SEQ ID NO: 3 or SEQ ID NO: 4 in the sequence listing. TM sgRNA was synthesized using the In Vitro Transcription and Screening System (Takara, Dalian) kit.

[0030] First, collect the melon fruit fly eggs. The egg collection method is: put the slices of mature pumpkin into the culture dish, cover the mouth of the dish with plastic wrap and poke small holes in the plastic wrap to facilitate the melon fruit fly to lay eggs. Continue to observe the egg-laying situation and continue to collect newly laid eggs (within 30 minutes). Use an injection device to inject the LP-Cas9 / sgRNA complex (LP-Cas9 protein concentration 200ng / μl, sgRNA concentration 150ng / μl) into the eggs, and the injection volume is 0.05μl. The injected eggs are placed in an environment of 20℃ and 60% relative humidity to hatch and continue to be raised to adults. Observe the changes in its eye color to determine whether the gene editing is successful ( Figure 3 ).

[0031] like Figure 3 As shown, cucurbit flies with mutated eye color were observed in the hatched eggs, confirming the gene editing activity of the LP-Cas9 protein.

[0032] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A melon fruit fly egg protein LP, characterized in that Its amino acid sequence is shown in SEQ ID NO: 1 in the sequence listing.

2. The egg protein LP of the melon fly according to claim 1, characterized in that The gene sequence of the cucurbit fruit fly egg protein LP is shown in the sequence listing as SEQ ID NO:

2.

3. Application of cucurbit fruit fly egg entry protein LP in gene editing.

4. The use of the melon fly egg protein LP in gene editing according to claim 3, characterized in that The LP-Cas9 / sgRNA complex was injected into the melon fruit fly eggs, which hatched and continued to be raised to adults.

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