CRISPR-Cas13 system targeting Ebola GP gene and used for inhibiting Ebola virus replication and application of CRISPR-Cas13 system

Abstract

The invention belongs to the field of biomedicine, and discloses a CRISPR-Cas13 system targeting an Ebola GP gene and used for inhibiting Ebola virus replication and application of the CRISPR-Cas13 system. The technical problem to be solved by the invention is how to inhibit the replication of the Ebola virus. According to the CRISPR-Cas13 system for inhibiting the replication of the Ebola virus, disclosed by the invention, the CRISPR-Cas13 system contains crRNA, and the target sequence of the crRNA is SEQ ID No.1 in a sequence table. Experimental results prove that the crRNA provided by the invention can target the genome sequence of the Ebola virus, can reduce the protein level of the virus gene, and also can degrade the targeted virus gene, so that the replication ability of the virus is inhibited. The crRNA and the CRISPR-Cas13 system composed of the crRNA can be used for treating and / or preventing diseases caused by the Ebola virus.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a CRISPR-Cas13 system that targets the Ebola GP gene to inhibit Ebola virus replication. Background Technology

[0002] Ebola virus is a virus belonging to the family Filoviridae. It causes Ebola hemorrhagic fever, a highly fatal acute hemorrhagic disease characterized by multi-organ damage. Different types of Ebola virus vary considerably in pathogenicity, with the Zaire strain being the most widespread and having a high mortality rate.

[0003] The Ebola virus genome is that of a single-stranded, negative-sense RNA virus belonging to the Filoviridae family. Its genome contains approximately 19,000 to 20,000 nucleotide base pairs. The Ebola virus genome encodes seven known proteins, including: NP (core protein), primarily used to encapsulate and protect viral RNA; GP (glycoprotein), a surface glycoprotein that allows the virus to enter and interact with host cells; VP40, a viral capsid protein involved in the assembly and release of viral particles; VP35, a multifunctional protein that plays a crucial role in viral replication and suppression of the host's immune response; VP30, which plays a role in viral replication and contributes to the synthesis of new viral RNA; VP24, involved in viral replication and suppression of the host's immune response; and L protein, a protein expressed by the L gene, which is the virus's RNA-dependent RNA polymerase and plays a key role in viral replication and transcription.

[0004] The CRISPR-Cas13 system is currently the only CRISPR system discovered capable of targeting RNA. Cas13b proteins possess two distinct catalytic activities: ① RNase activity provided by two higher eukaryotic and prokaryotic nucleotide-binding domains; ② RNase activity catalyzing the processing of pre-crRNA and the formation of mature crRNA, exhibiting unique advantages in RNA-specific recognition, RNA cleavage, and transactivation activities.

[0005] The defense mechanisms of the CRISPR-Cas13b system mainly include: ① recognition and binding of pre-crRNA: newly transcribed pre-crRNA recognizes and binds to the REC leaf of Cas13b through the 5' stem-loop structure of crRNA, forming an intermediate transition state of the pre-crRNA-Cas13b complex; ② formation of mature crRNA: the conformation of conserved residues between the Helical-1 and HEPN2 domains in the NUC region changes, thereby forming an acid-base catalytic center that catalyzes the enzymatic cleavage of pre-crRNA to form mature crRNA; ③ activation of the enzymatic activity of the crRNA-Cas13b complex: target ssRNA enters the crRNA-Cas13b complex and undergoes base pairing with crRNA, inducing a conformational change in Cas13b, thereby activating the enzymatic activity of the crRNA-Cas13b complex; ④ degradation of target RNA: under the guidance of crRNA, the HEPN domain of Cas13b catalyzes the enzymatic cleavage of target ssRNA. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to inhibit the replication of Ebola virus.

[0007] To address the aforementioned technical problems, the present invention first provides a CRISPR-Cas13 system for inhibiting Ebola virus replication, wherein the CRISPR-Cas13 system contains crRNA, and the target sequence of the crRNA is SEQ ID No.1 in the sequence listing.

[0008] The aforementioned CRISPR-Cas13 system also contains the Cas13 protein.

[0009] Specifically, the Cas13 protein is the Cas13b protein.

[0010] Furthermore, the CRISPR-Cas13 system is a reagent or kit. The CRISPR-Cas13 system may contain only the crRNA, or it may consist of the crRNA and the Cas13 protein.

[0011] The application of the CRISPR-Cas13 system in the preparation of products that inhibit Ebola virus replication is also within the scope of protection of this invention.

[0012] The application of the CRISPR-Cas13 system in the preparation of products for the treatment and / or prevention of diseases caused by Ebola virus is also within the scope of protection of this invention.

[0013] The present invention also provides biological materials related to crRNA, wherein the crRNA is the crRNA described above, and the biological material is any one of B1) to B3) below: B1) Transcribe the DNA molecule containing the crRNA; B2) An expression cassette containing the DNA molecule described in B1); B3) A recombinant vector containing the DNA molecule described in B1) or a recombinant vector containing the expression cassette described in B2).

[0014] The present invention also provides a biomaterial composition containing the crRNA-associated biomaterial.

[0015] Furthermore, the biomaterial composition also contains biomaterials related to the Cas13 protein, said biomaterials being any one of C1) to C3) below: C1) A nucleic acid molecule encoding the Cas13 protein; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1) or a recombinant vector containing the expression cassette described in C2).

[0016] The application of the biomaterial composition in the preparation of products that inhibit Ebola virus replication.

[0017] The application of the biomaterial composition in the preparation of products for the treatment and / or prevention of diseases caused by Ebola virus is also within the scope of protection of this invention.

[0018] Experimental results demonstrate that the crRNA of this invention can target the Ebola virus genome sequence, reduce the protein level of viral genes, and degrade the targeted viral genes, thereby inhibiting viral replication. The crRNA of this invention and the CRISPR-Cas13 system it comprises can be used to treat and / or prevent diseases caused by Ebola virus.

[0019] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0020] Figure 1 Functional validation and screening of crRNA were performed using Cas13b stably expressed cells. GFP represents green fluorescence, BF represents bright field, and Merge represents the superposition of GFP and BF channels.

[0021] Figure 2Transfecting 293T cells with PspCas13b and crRNA plasmids significantly inhibited the expression of target genes. Figure A shows the fluorescence microscopy results, with GFP showing a green fluorescent signal, BF representing bright field, and Merge indicating the superposition of GFP and BF channels; Figure B shows the Western blot results. NT indicates no co-transfection with pC0043-PspCas13b-GP-crRNA1 plasmid.

[0022] Figure 3 The Ebola minimal genome system was used to verify that crRNA1 significantly inhibited the replication of ebolatrVLP. "-L" indicates the control group without co-transfection of pCAGGS-L plasmid, "+L" indicates co-transfection of pCAGGS-L plasmid, and "L+GP-crRNA1" indicates co-transfection of pCAGGS-L and pC0043-PspCas13b-GP-crRNA1 plasmid containing the GP-crRNA1 sequence. Detailed Implementation

[0023] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following embodiments are commercially available. All quantitative experiments in the following embodiments were performed in at least three replicates, and the results were averaged.

[0024] The following examples use Graphpad 8.0 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0025] Preparation of 293T cells stably expressing cas13b: 293T cells were obtained by stably transfecting the YOE-LV005-pspcas13b plasmid. The YOE-LV005-pspcas13b plasmid is a product of Yuanjing Biotechnology.

[0026] pC0043-PspCas13b-crRNA-backbone and pC0046-EF1a-PspCas13b-NES-HIV plasmid (hereinafter referred to as PspCas13b plasmid) are both products of Qingdao Keruisibo Biotechnology Co., Ltd.

[0027] GP-P2A-GFP: This is a recombinant vector obtained by replacing the DNA fragment between the KpnI and XhoI recognition sequences of the pcDNA3.1(+) vector with the DNA fragment shown in SEQ ID No. 4.

[0028]

[0029] The references for plasmids pCAGGS-NP, pCAGGS-VP35, pCAGGS-VP30, pCAGGS-L, p4cis-vRNA-Rluc, pCAGGS-T7, and pCAGGS-Tim1 are Hoenen, T., Watt, A., Mora, A., Feldmann, H. Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes. J. Vis. Exp.(91), e52381, doi:10.3791 / 52381 (2014).

[0030] PGL3-Basic plasmid: promega.

[0031] 293T cells were cultured in DMEM medium containing 10% serum and 1% penicillin and streptomycin antibiotics. Both DMEM medium (catalog number C11965500BT) and FBS (catalog number 10099141C) were Gibco products.

[0032] Both the single-luciferase and dual-luciferase assay kits are products of Promega (Renilla-Glo® Luciferase Assay System, catalog number E2710; dual-luciferase assay kit, catalog number E1910).

[0033] Free of EDTA protease inhibitor: Roche, catalog number 04693132001.

[0034] Cell lysis buffer: 150 mM NaCl, 50 mM Tris-HCl pH 8.0, 1 tablet / 50 ml of EDTA protease inhibitor-free buffer, 1% (v / v) NP40 lysis buffer; NP40 was purchased from Solarbio (N8030).

[0035] The HRP-labeled GFP antibody was a product of Proteintech (catalog number HRP-66002), the GP antibody was a product of Sinopharm (catalog number 40304-T46), the HRP-labeled β-actin antibody was a product of Proteintech (catalog number HRP-60008), and the HRP anti-rabbit secondary antibody was a product of Zhongshan Jinqiao (catalog number ZB-2301).

[0036] Example 1 1. crRNA sequence design The crRNA target sequence targeting the Ebola virus GP gene was designed as follows: GP-crRNA1: 5′-AATTACCCAAAGAAAGAATGATGTCCCTCTT-3′ (SEQ ID No. 1); GP-crRNA2: 5′-CACGTCAGTTGCCACTCCATTCCCTTCGAG-3′ (SEQ ID No. 2); GP-crRNA3: 5′-TGGAAGCAAGTCGATCATACAGGAAGAAAG-3′ (SEQ ID No. 3); GP-crRNA4: 5′-CTTGTATATTGTCTCATTCAGCTGGAGC-3′ (SEQ ID No. 4).

[0037] 2. Screening for target crRNA sequences capable of cleaving RNA CACC and CAAC were added to both ends of each target sequence to synthesize single-stranded DNA with the same sequence and single-stranded DNA with the reverse complementary sequence as a primer pair. The primer pair was annealed in an annealing system to obtain the annealing product. pC0043-PspCas13b-crRNA-backbone was digested with BbsI to obtain the plasmid backbone. The plasmid backbone and the annealing product were ligated to obtain the recombinant plasmid with the correct sequence, which is the crRNA plasmid. It can transcribe crRNA targeting the target sequences (GP-crRNA1~GP-crRNA5). Each recombinant plasmid is named as recombinant plasmid pC0043-PspCas13b-GP-crRNA1~pC0043-PspCas13b-GP-crRNA5.

[0038] In HEK293T six-well plates containing Cas13b stably expressed cells, 4 x 10^5 cells were seeded per well, and the medium was changed by 2 ml 1 hour before transfection. Each well was transfected with GP-P2A-GFP (0.2 μg), non-target crRNA plasmid (i.e., pC0043-PspCas13b-crRNA-backbone, NT), or target crRNA plasmid (1.0 μg), respectively. Lipofectamine 3000 was used as the transfection reagent. Thirty-six hours after transfection, the cells were photographed under a 10x Zeiss fluorescence microscope. The results showed that the cells transfected with pC0043-PspCas13b-GP-crRNA1 had the lowest fluorescence signal, indicating that the crRNA targeting GP-crRNA1 had the most significant inhibitory effect on the GP gene. Figure 1 ).

[0039] 3. Further detection of GP-crRNA1 inhibitory effect In a 6-well plate containing 293T cells, 4 x 10^5 cells were seeded per well. GP-P2A-GFP (1.0 μg), PspCas13b plasmid (1.0 μg), and pC0043-PspCas13b-GP-crRNA1 plasmid (0, 0.2, 0.5, 1.0, 1.5, and 2.0 μg) from step 2 were transfected using a lip3000 transfector. The brightness of GFP was observed using a fluorescence microscope 36 hours after transfection. After discarding the supernatant, wash twice with PBS, add 3 ml of pre-chilled PBS, and then gently scrape the cells off with a cell scraper, gently pipetting them into centrifuge tubes. Centrifuge at 1000 g for 3 min at 4°C to collect the cells. Add 150 μl of cell lysis buffer to each tube of cells, lyse on ice for 15-30 min, then centrifuge at 16000 g for 10 min at 4°C. Take 100 μl of the supernatant for SDS-PAGE electrophoresis and Western blotting, and ECL imaging. The primary antibodies used were GP antibody, HRP-labeled GFP antibody, or HRP-labeled β-actin antibody, and the secondary antibody was HRP anti-rabbit secondary antibody.

[0040] The results are as follows Figure 2 As shown, transfection with the pC0043-PspCas13b-GP-crRNA1 plasmid can inhibit the expression of GP, and the inhibitory effect has a dose-response effect; the more crRNA plasmid used, the better the inhibitory effect.

[0041] 4. The inhibitory effect of GP-crRNA1 on viral replication was detected using the Ebola minimal genome system. The modified Ebola minimal genome system used in this experiment can be used to study the replication and infection of the Ebola virus in a biotechnology level 2 laboratory.

[0042] On day 1, 293T cells were seeded into 6-well plates at 4 x 10^5 cells per well. On day 2 (24 hours later), each well was transfected with plasmids pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng), p4cis-vRNA-Rluc (250 ng), and pCAGGS-T7 (250 ng). On day 3, the cell culture supernatant was replaced with medium containing 5% FBS, and the resulting cell culture supernatant was the P0 cell supernatant. On day 4, 293T cells (virus-targeting cells, abbreviated as P1 cells) were seeded into 6-well plates at 4 x 10^5 cells per well. On day 5, the plasmids pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), and pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng), p4cis-vRNA-Rluc (250 ng), and pCAGGS-T7 (250 ng) were transfected into each well. Add pCAGGS-L (1000 ng) and pCAGGS-Tim1 (250 ng) to P1 generation cells; on day 6, discard the supernatant, then add the P0 cell supernatant obtained on day 3, and infect for 24 hours; on day 7, replace the supernatant with medium containing 5% FBS, and continue culturing for 72 hours, then collect the supernatant, which is the P1 cell supernatant, and continue to passage according to the above steps or freeze and store at -80 degrees Celsius.

[0043] In 6-well plates containing 293T cells, each well was seeded with 4 x 10^5 cells. On day 2 (24 hours later), each well was transfected with plasmids pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng), p4cis-vRNA-Rluc (250 ng), and pCAGGS-T7 (250 ng); as well as 1.0 μg of PspCas13b plasmid and 1.0 μg of pC0043-PspCas13b-GP-crRNA1, and 0.5 μg of PGL3-Basic plasmid as internal control. On day 3, the cell culture supernatant was replaced with medium containing 5% FBS. After 72 hours of medium replacement, the proliferation of P0 generation virus was detected. The activity of dual-luciferase was detected using the promega Dual-Luciferase Reporter Assay System (E1960). After cell lysis, 30 μl of the supernatant was collected by centrifugation, and 50 μl of dual-luciferase substrate was added to each cell for detection to determine the effect of GP-crRNA1 on viral replication.

[0044] The results showed that crRNA targeting GP-crRNA1 could significantly inhibit the production of P0 virus. Figure 3 ).

[0045] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A CRISPR-Cas13 system for inhibiting Ebola virus replication, characterized in that: The CRISPR-Cas13 system contains crRNA, and the target sequence of the crRNA is SEQ ID No. 1 in the sequence listing.

2. The CRISPR-Cas13 system according to claim 1, characterized in that: The CRISPR-Cas13 system also contains the Cas13 protein.

3. The CRISPR-Cas13 system according to claim 2, characterized in that: The Cas13 protein is the Cas13b protein.

4. The use of the CRISPR-Cas13 system according to any one of claims 1-3 in the preparation of products that inhibit Ebola virus replication.

5. The use of the CRISPR-Cas13 system according to any one of claims 1-3 in the preparation of products for the treatment and / or prevention of diseases caused by Ebola virus.

6. Biomaterials related to crRNA, characterized in that: The crRNA is the crRNA of claim 1, and the biological material is any one of B1) to B3) below: B1) Transcribe the DNA molecule containing the crRNA; B2) An expression cassette containing the DNA molecule described in B1); B3) A recombinant vector containing the DNA molecule described in B1), or a recombinant vector containing the expression cassette described in B2).

7. A biomaterial composition, characterized in that: The biomaterial composition contains the biomaterial of claim 6.

8. The biomaterial composition according to claim 7, characterized in that: The biomaterial composition further comprises a biomaterial associated with the Cas13 protein, said biomaterial being any one of C1) to C3) below: C1) A nucleic acid molecule encoding the Cas13 protein; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1) or a recombinant vector containing the expression cassette described in C2).

9. The use of the biomaterial composition of claim 7 or 8 in the preparation of products that inhibit Ebola virus replication.

10. The use of the biomaterial composition of claim 7 or 8 in the preparation of products for the treatment and / or prevention of diseases caused by Ebola virus.

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