A novel CRISPR RNA delivery system based on vesicular stomatitis virus and its application

Abstract

The present invention discloses a novel CRISPR RNA delivery system based on vesicular stomatitis virus and its application. The delivery system has the advantages of high delivery efficiency, strong targeting, etc. The CRISPR RNA delivery system based on vesicular stomatitis virus provided by the present invention can be used for targeted delivery of CRISPR RNA system for genetic disease treatment, bacterial and viral infection prevention and treatment, etc., and has very good clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology. Specifically, the present invention relates to a CRISPR RNA delivery system. More specifically, the present invention relates to a novel CRISPR RNA delivery system based on vesicular stomatitis virus and its application. Background Art

[0002] Clustered regularly interspaced shortpalindromic repeats (CRISPR) are a group of DNA sequences widely found in bacteria and archaea, consisting of repeat sequences and spacer sequences. Among them, the spacer sequence has homology with exogenous DNA and is a trace left by invasive nucleic acids such as bacteriophages and viruses. It can detect and destroy similar DNA in subsequent infections. The Cas sequence is located near the CRISPR sequence and encodes a protein closely related to the function of CRISPR. The CRISPR / Cas system is an acquired immune defense mechanism evolved by bacteria and archaea to resist the continuous invasion of viruses and plasmids. There are types of CRISPR / Cas systems, including type I, type II and type III. Among them, CRISPR / Cas is the most widely used as a marker gene of type II. Its mediated gene editing can be used to generate transgenic models, regulate transcription, regulate epigenetics, etc.

[0003] As a new genome editing technology, the CRISPR / Cas system can target specific genes in the genome for interference or repair. CRISPR / Cas systems from bacteria or artificial modifications have been discovered or constructed by biologists. Cas nucleases and single-stranded guide RNA (sgRNA) are the main components of the CRISPR / Cas system. The system is widely used in the discovery of new targets for disease treatment, the identification of gene functions, the establishment of animal models, and the development of gene therapy drugs. The CRISPR / Cas system has partially alleviated or completely cured certain diseases by mutating or correcting disease-related genes. However, how to effectively deliver CRISPR / Cas to target cells and target organs remains one of the important challenges faced by the use of this technology, which affects the system's stable and precise gene editing capabilities.

[0004] At present, the delivery methods of CRISPR / Cas system include: physical method, viral delivery system, non-viral delivery system. Among them, physical method is mainly used for gene editing of cells and tissues in vitro or in vitro, and its application is limited; viral delivery system mainly includes the use of adenoviral vector, lentiviral vector and retroviral vector to deliver CRISPR / Cas system, among which adenoviral vector has the following disadvantages: 1. Pre-existing immunity: adenovirus infects more people, so it is easy to have resistance in the body; 2. Long-term existence: the system delivers DNA, which will exist in terminal cells for a long time, and there will be risks such as random insertion; 3. Adeno-associated virus has problems such as small capacity; lentiviral vector and retroviral vector delivery system mainly have the risk of carcinogenicity caused by insertion, even non-integrated vectors still have the potential possibility of insertion; and although non-viral delivery system does not have the safety risk of viral vectors, its targeted receptor delivery function is still in the early research stage and has poor targeting.

[0005] The CRISPR viral delivery systems commonly used in this field have obvious disadvantages for delivering CRISPR RNA systems. Therefore, how to efficiently and specifically deliver CRISPR RNA systems into susceptible cells remains an important technical problem facing this field. Summary of the invention

[0006] In view of this, in order to overcome the above-mentioned technical defects existing in the current field, the present invention provides a novel CRISPR RNA delivery system based on vesicular stomatitis virus and its application. In addition, the delivery system provided by the present invention is the first RNA delivery system used in this field, which fills the gap in this field. The present invention has been scientifically verified by experiments and found that the CRISPR RNA delivery system based on vesicular stomatitis virus has the advantages of high delivery efficiency and strong targeting.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] A first aspect of the present invention provides a nucleic acid molecule.

[0009] Further, the nucleic acid molecule sequentially comprises nucleic acid sequences encoding the following operably linked elements: sgRNA, N protein, P protein, M protein, target molecule, L protein;

[0010] Preferably, the target molecules include Cas proteins, CRISPR single base editors, CRISPR lead editors, antimicrobial peptides, and antibodies;

[0011] More preferably, the Cas protein includes Cas9, Cas12, Cas13, Cas3, Cas4, Cas1, Cas2, Cas10, Cas5, Cas6, Cas7, Cas8, Cas7-11;

[0012] More preferably, the CRISPR single base editor comprises CBE and ABE;

[0013] Most preferably, the Cas protein is Cas9, Cas12, or Cas13;

[0014] Most preferably, the Cas protein is CasRx in Cas13;

[0015] More preferably, the antimicrobial peptides include microbial antimicrobial peptides, animal antimicrobial peptides, human antimicrobial peptides, and plant antimicrobial peptides;

[0016] Most preferably, the nucleic acid sequence of the nucleic acid molecule is as shown in SEQ ID NO:1.

[0017] In a specific embodiment of the present invention, the specific profile of the nucleic acid comprising the target molecule and sgRNA is VSV-NT-pre-sgRNA-CasRx.

[0018] The second aspect of the present invention provides a recombinant vesicular stomatitis virus vector expressing G protein on its surface.

[0019] Further, the viral vector comprises the nucleic acid molecule described in the first aspect of the present invention;

[0020] Preferably, the viral vector is composed of the following operably linked elements: N protein, P protein, M protein, L protein, viral genome;

[0021] More preferably, the viral vector further comprises a G protein.

[0022] The vesicular stomatitis virus (VSV) described in the present invention belongs to the genus Vesiculovirus of the family Rhabdoviridae, and is an enveloped single-stranded negative-strand RNA (ssRNA) virus. The length of the VSV genome is about 11 kb. From the 3' end to the 5' end, there are 5 non-overlapping genes N, P, M, G, and L, which encode 5 different major proteins, namely, nucleoprotein (N), phosphate (P), matrix (M), sugar (G), and RNA polymerase (L). There is an untranslated leader sequence at the 3' end of the N gene, a non-translated region at the 5' end, and spacer sequences between the genes. The leader RNA is the earliest viral transcript in the infected cell.

[0023] In a specific embodiment of the present invention, the recombinant vesicular stomatitis virus vector expressing G protein on the surface is a replication-deficient recombinant vesicular stomatitis virus vector (rVSVΔG-sgRNA-Cas-G). This replication-deficient recombinant virus vector still has the ability to infect target cells, but does not replicate, and thus does not directly cause damage to target cells. The recombinant vesicular stomatitis virus vector expressing G protein on the surface described in the present invention includes replication-deficient and replicative recombinant vesicular stomatitis virus vectors expressing G protein on the surface, therefore, both replication-deficient and replicative recombinant vesicular stomatitis virus vectors expressing G protein on the surface are within the protection scope of the present invention.

[0024] In a specific embodiment of the present invention, the sgRNA (single guide RNA) is a guide RNA, thereby guiding the Cas protein to perform site-specific cutting of nucleic acids.

[0025] The third aspect of the present invention provides a recombinant host cell.

[0026] Further, the recombinant host cell comprises the nucleic acid molecule described in the first aspect of the present invention, and / or the viral vector described in the second aspect of the present invention;

[0027] Preferably, the host cell includes eukaryotic cells and prokaryotic cells;

[0028] More preferably, the host cell is a eukaryotic cell;

[0029] Most preferably, the eukaryotic cells include mammalian cells, plant cells, yeast cells, fungal cells;

[0030] Most preferably, the eukaryotic cell is a mammalian cell.

[0031] In a specific embodiment of the present invention, the recombinant host cell is mainly used to construct a recombinant vesicular stomatitis virus vector (rVSVΔG-sgRNA-Cas-target glycoprotein) expressing a target glycoprotein on its surface.

[0032] The fourth aspect of the present invention provides a recombinant vesicular stomatitis virus vector expressing a target glycoprotein on its surface.

[0033] Further, the viral vector comprises the nucleic acid molecule described in the first aspect of the present invention;

[0034] Preferably, the viral vector is composed of the following operably linked elements: N protein, P protein, M protein, L protein, viral genome;

[0035] Preferably, the target glycoprotein includes Spike protein and influenza HA protein.

[0036] Further, the recombinant vesicular stomatitis virus vector expressing the target glycoprotein on the surface is constructed on the basis of the recombinant vesicular stomatitis virus vector expressing the G protein on the surface (rVSVΔG-sgRNA-Cas-G) described in the second aspect of the present invention, that is, the host cell expressing the target glycoprotein is infected with rVSVΔG-sgRNA-Cas-G to obtain the replication-deficient recombinant vesicular stomatitis virus expressing the target glycoprotein on the surface (rVSVΔG-sgRNA-Cas-target glycoprotein). The recombinant vesicular stomatitis virus vector expressing the target glycoprotein on the surface described in the present invention includes the replication-deficient type and the replicative type, therefore, the replication-deficient type and the replicative type of the recombinant vesicular stomatitis virus vector expressing the target glycoprotein on the surface are both within the protection scope of the present invention.

[0037] Furthermore, the recombinant vesicular stomatitis virus vector expressing Spike protein on the surface of the present invention can infect all cells with ACE2 receptors on the surface, and has the advantages of rapid onset of action and high titer. The delivered RNA is easily degraded in the cell, reducing the risk of random insertion and having higher safety.

[0038] A fifth aspect of the present invention provides a CRISPR RNA delivery system.

[0039] Furthermore, the delivery system comprises the viral vector described in the fourth aspect of the present invention.

[0040] Furthermore, the delivery system may further comprise other components.

[0041] The sixth aspect of the present invention provides a method for constructing the viral vector described in the second aspect of the present invention.

[0042] Further, the method comprises transfecting the nucleic acid molecule and vesicular stomatitis virus G protein plasmid described in the first aspect of the present invention into a host cell;

[0043] Preferably, the method comprises transfecting N protein plasmid, P protein plasmid, L protein plasmid and T7 RNA polymerase plasmid into host cells simultaneously;

[0044] More preferably, the host cell includes eukaryotic cells and prokaryotic cells;

[0045] Most preferably, the host cell is a eukaryotic cell;

[0046] Most preferably, the eukaryotic cells include mammalian cells, plant cells, yeast cells, fungal cells;

[0047] Most preferably, the eukaryotic cell is a mammalian cell.

[0048] In a specific embodiment of the present invention, the host cell is a BHK-21 cell. First, the BHK-21 cells are plated in a 6-well plate and cultured with a DMEM medium containing 10% fetal bovine serum. When the cells grow to about 90%, the vesicular stomatitis virus packaging plasmids N, P, VSVG, L, VSVΔG-sgRNA-Cas (the nucleic acid molecules described in the first aspect of the present invention) and T7 RNA polymerase are transfected with Lipofectamine™ 3000 Transfection Reagent to construct a recombinant vesicular stomatitis virus vector expressing G protein on the surface.

[0049] The seventh aspect of the present invention provides a method for constructing the viral vector described in the fourth aspect of the present invention.

[0050] Furthermore, the method comprises transfecting the target glycoprotein plasmid into a host cell, and then infecting the host cell with the viral vector described in the second aspect of the present invention when syncytia appear in the host cell;

[0051] Preferably, the target glycoprotein includes Spike protein and influenza HA protein;

[0052] Preferably, the host cell includes eukaryotic cells and prokaryotic cells;

[0053] More preferably, the host cell is a eukaryotic cell;

[0054] Most preferably, the eukaryotic cells include mammalian cells, plant cells, yeast cells, fungal cells;

[0055] Most preferably, the eukaryotic cell is a mammalian cell.

[0056] In a specific embodiment of the present invention, the host cell is a Vero cell. The Vero cells are first plated in a 6-well plate and cultured with DMEM medium containing 10% fetal bovine serum. When the cells grow to about 90%, the target glycoprotein particles are transfected with Lipofectamine™3000 Transfection Reagent. When syncytia appear in the cells, the cells are infected with rVSVΔG-sgRNA-Cas-G virus to obtain a recombinant vesicular stomatitis virus vector expressing the target glycoprotein on the surface.

[0057] An eighth aspect of the present invention provides a kit for targeted delivery of a CRISPR RNA system.

[0058] Furthermore, the kit comprises the viral vector described in the fourth aspect of the present invention.

[0059] The ninth aspect of the present invention provides a pharmaceutical composition for treating and / or preventing genetic diseases, bacterial infectious diseases, and viral infectious diseases.

[0060] Furthermore, the pharmaceutical composition comprises the nucleic acid molecule described in the first aspect of the present invention, and / or the viral vector described in the fourth aspect of the present invention, and / or the CRISPR RNA delivery system described in the fifth aspect of the present invention.

[0061] The tenth aspect of the present invention provides the application of any of the following aspects:

[0062] (1) Use of the nucleic acid molecule described in the first aspect of the present invention in constructing a CRISPR RNA delivery system;

[0063] (2) Use of the nucleic acid molecule described in the first aspect of the present invention in constructing a recombinant vesicular stomatitis virus vector expressing a target glycoprotein on its surface;

[0064] (3) Use of the viral vector described in the second aspect of the present invention in constructing a recombinant vesicular stomatitis virus vector expressing a target glycoprotein on its surface;

[0065] (4) Use of the viral vector described in the second aspect of the present invention in constructing a recombinant host cell;

[0066] (5) The recombinant host cell described in the third aspect of the present invention is used to construct a recombinant vesicular stomatitis virus vector expressing a target glycoprotein on its surface;

[0067] (6) Use of the viral vector described in the fourth aspect of the present invention in the preparation of a pharmaceutical composition for treating and / or preventing genetic diseases, bacterial infectious diseases, and viral infectious diseases;

[0068] (7) Use of the CRISPR RNA delivery system described in the fifth aspect of the present invention in the preparation of a pharmaceutical composition for treating and / or preventing genetic diseases, bacterial infectious diseases, and viral infectious diseases;

[0069] (8) Use of the kit described in the eighth aspect of the present invention in a targeted delivery system of CRISPR RNA;

[0070] Preferably, the viral infectious disease comprises a SARS-CoV-2 infectious disease.

[0071] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0072] (1) The CRISPR RNA delivery system based on vesicular stomatitis virus provided by the present invention can deliver any RNA sequence. In addition, the protein on the surface of the recombinant VSV virus vector in the system can be changed according to actual needs to target different cells, which has the advantages of high delivery efficiency and strong targeting.

[0073] (2) The CRISPR RNA delivery system based on vesicular stomatitis virus provided by the present invention has very good clinical application prospects and can be used for targeted delivery of CRISPR RNA systems for genetic disease treatment, bacterial and viral infection prevention and treatment, etc.;

[0074] (3) In the past, viral vectors commonly used in CRISPR viral delivery systems were all used to deliver DNA. Currently, there is no delivery system suitable for targeting CRISPR RNA. The present invention provides a CRISPR RNA delivery system for the first time, filling the gap in this field.

[0075] (4) The present invention unexpectedly discovered through comparative experiments that recombinant vesicular stomatitis virus (VSV) vector delivery can express Cas protein faster than conventional adeno-associated virus (AAV) vector delivery, that is, the effect of CRISPR delivery using the VSV vector described in the present invention is significantly better than that using conventional AAV vectors, that is, the recombinant VSV vector described in the present invention has achieved unexpected technical effects in delivering CRISPR. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0077] Figure 1 A schematic diagram showing the genome structure of the recombinant VSV viral vector VSVΔG-sgRNA-Cas;

[0078] Figure 2 A fluorescence result graph showing the infection of BHK-21 cells with the recombinant VSV viral vector rVSVΔG-sgRNA-Cas-G having VSVG protein on its surface constructed by the present invention;

[0079] Figure 3 An experimental flow chart showing the construction of the present invention to obtain a recombinant VSV viral vector rVSVΔG-sgRNA-Cas-G with VSVG protein on the surface and a recombinant VSV viral vector rVSVΔG-sgRNA-Cas-S with S protein on the surface;

[0080] Figure 4 A fluorescence result graph showing the infection of COS7-ACE2 cells by the recombinant VSV viral vector rVSVΔG-sgRNA-Cas-S with S protein on the surface constructed by the present invention;

[0081] Figure 5 A protein immunoblot image showing proteins extracted from COS7-ACE2 and 293T-ACE2 cells infected with the recombinant VSV virus vector rVSVΔG-sgRNA-Cas-S with S protein on the surface constructed by the present invention;

[0082] Figure 6 Shows the protein immunoblot image obtained by extracting proteins from 293T cells infected with adeno-associated virus vector AAV9-CasRx. DETAILED DESCRIPTION

[0083] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and are not to be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0084] Example 1 Construction of recombinant VSV virus vector VSVΔG-sgRNA-Cas

[0085] 1. Experimental Materials

[0086] (1) Plasmids: N, P, VSVG, L, T7 RNA polymerase, and Spike plasmids were from Addgene;

[0087] (2) Cells: BHK-21 (Baby Hamster Kidney-21 hamster kidney cells), Vero (African green monkey kidney cells), were obtained from ATCC, and COS7-ACE2 (African green monkey kidney cells that highly express ACE2 receptors) were purchased from Beijing Weitongda Biotechnology Co., Ltd.;

[0088] (3) Reagents: Lipofectamine™ 3000 Transfection Reagent (Invitrogen L3000015); Opti-MEM (Invitrogen); DMEM (Invitrogen 10313021); PBS (Gibco C20012500BT); fetal bovine serum (Gibco); Anti-HA tag antibody (abcam ab9110); Hoechst 33258 staining solution (Biyuntian C1018); Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed SecondaryAntibody (Invitrogen A31572); 4% paraformaldehyde fixative (Biyuntian P0099).

[0089] 2. Construction of recombinant VSV virus vector VSVΔG-sgRNA-Cas

[0090] Plasmid: rVSV-ΔGFP-SARS-Δ19aa

[0091] pXR001_EF1a-CasRx-2A-EGFP (Miaoling plasmid)

[0092] EGFP_pUC57 (GenScript)

[0093] Reagent: PrimeSTAR® HS DNA Polymerase with GC Buffer (Takara R044B)

[0094] In-Fusion® Snap Assembly Master Mix (Takara 638948)

[0095] NotI (NEB R3189S)

[0096] MluI (NEB R3198V)

[0097] E.coli HST08 Premium Competent Cells (Takara 9128)

[0098] The recombinant VSV genome containing the CasRx gene was constructed based on the two plasmids rVSV-ΔGFP-SARS-Δ19aa and pXR001_EF1a-CasRx-2A-EGFP using a seamless connection method. The rVSV-ΔGFP-SARS-Δ19aa plasmid was double-digested with NotI and MluI overnight, electrophoresed, and gel recovered to obtain the vector fragment. The plasmid pXR001_EF1a-CasRx-2A-EGFP was used as a template for PCR to obtain the CasRx gene fragment. The two fragments were seamlessly connected at a ratio of vector: CasRx fragment = 1:3 at 50°C for 15 minutes using In-Fusion® Snap Assembly Master Mix (Takara). The product was transformed into Escherichia coli HST08 competent cells, coated with LB plates containing AMP antibiotics, cultured at 37°C overnight, single clones were picked, and sequencing was performed to obtain a recombinant VSV genome VSV-CasRx plasmid containing the CasRx gene (sequence as SEQ ID NO: 1), which is the recombinant VSV virus vector VSVΔG-CasRx.

[0099] The VSV-CasRx-GFP plasmid was constructed based on the VSV-CasRx plasmid (the GFP part can be replaced with any sgRNA by restriction ligation or seamless ligation, and the vector, vector1, vector2, and vector3 parts in the following steps have been marked in the plasmid sequence). The constructed VSV-CasRx-GFP plasmid is shown in SEQ ID NO: 2:

[0100] (1) Using EGFP_pUC57 plasmid as template, PCR was performed to obtain the EGFP fragment, and using VSV-CasRx plasmid as template, PCR was performed to obtain the vector fragment, and the two fragments were connected by seamless connection to obtain a plasmid with the sequence vector1;

[0101] (2) using the vector1 plasmid as a template to generate the vector1 fragment by PCR, using the VSV-CasRx plasmid as a template to generate the vector2 fragment by PCR, and connecting the two fragments by seamless connection to obtain a plasmid with a sequence of vector1_2;

[0102] (3) Using the vector1_2 plasmid as a template, PCR was performed to obtain the vector1_2 fragment, and using the VSV-CasRx plasmid as a template, PCR was performed to obtain the vector3 fragment, and the two fragments were connected by seamless connection to obtain the final VSV-CasRx-GFP plasmid;

[0103] (4) The GFP gene portion of the VSV-CasRx-GFP plasmid was double-digested with KpnI and SpeI, and ligated to replace it with any sgRNA, such as VSV-NT-pre-sgRNA-CasRx. The sequence of the VSV-NT-pre-sgRNA-CasRx is shown in SEQ ID NO: 3.

[0104] In addition, nuclear localization sequences can be added to both ends of the Cas protein of the plasmid, and the GFP segment can be replaced with any sgRNA by enzyme cutting or seamless connection. The nuclear localization sequence is a conventional nuclear localization sequence in the art.

[0105] 3. Experimental results

[0106] The genome structure of the constructed recombinant VSV virus vector VSVΔG-sgRNA-Cas is shown in Figure 1 The vector VSVΔG-sgRNA-Cas (3'-5') is composed of CRISPR RNA (sgRNA), N protein, P protein, M protein, Cas protein, and L protein, and the Cas protein is CasRx in Cas13.

[0107] Example 2 Construction of replication-defective recombinant VSV virus rVSVΔG-sgRNA-Cas-G with VSVG protein on the packaging surface

[0108] 1. Construction of rVSVΔG-sgRNA-Cas-G virus

[0109] BHK-21 cells were plated in a 6-well plate and cultured with DMEM medium containing 10% fetal bovine serum. When the cells grew to about 90%, VSV (vesicular stomatitis virus) packaging plasmids N, P, VSVG, L, VSVΔG-sgRNA-Cas and T7 RNA polymerase were transfected with Lipofectamine™ 3000 Transfection Reagent, wherein the transfection system required for each well of the 6-well plate is shown in Table 1;

[0110] Slowly drip the liquid in tube B into tube A, mix well, let stand at room temperature for 15 minutes, then add to BHK-21 cells and shake well. After 6 hours, change the medium to DMEM medium containing 5% fetal bovine serum, collect the virus after 40-46 hours, filter the cell culture supernatant with a 0.45 micron filter membrane, and store it in a -80℃ refrigerator after aliquoting.

[0111] Table 1 Transfection system

[0112]

[0113] 2. Amplification of rVSVΔG-sgRNA-Cas-G virus

[0114] BHK-21 cells were plated in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum. When the cells grew to about 90%, 2 μg of VSVG plasmid was transfected with Lipofectamine™ 3000 Transfection Reagent. After 6 hours, the culture medium was changed to DMEM medium containing 5% fetal bovine serum. When syncytia containing 3-4 cells appeared in the cells, the cells were infected with rVSVΔG-sgRNA-Cas-G virus (infection multiplicity MOI = 0.1). After 1 hour, the cells were cultured in new DMEM medium containing 5% fetal bovine serum. After about 36 hours, the virus was collected, the cell culture supernatant was filtered through a 0.45 μm filter membrane, and stored in a -80℃ refrigerator after aliquoting.

[0115] 3. rVSVΔG-sgRNA-Cas-G virus titer determination

[0116] Vero cells were plated in a 24-well plate and cultured in DMEM medium containing 10% fetal bovine serum. When the cells grew to 80%-90%, the virus was diluted 10 times with serum-free DMEM and added to the cells. Each concentration gradient was repeated for 3 wells. After 1 hour, the culture medium was changed to DMEM medium containing 5% fetal bovine serum. After 24 hours, the cells were fixed, permeabilized, blocked and immunostained. The HA tag is connected to the Cas protein, so anti-HA antibodies can be used to mark the Cas protein expressed after the virus-infected cells, and the virus titer can be determined by observation and counting under a fluorescence microscope. The calculation formula is as follows:

[0117] Titer (TU / mL) = (X+Y×10)×1000 / 2 / volume of virus solution added to well X

[0118] When the concentration of the added virus is from high to low, the number of cells in the last two wells of the concentration gradient where fluorescent cells can be seen are recorded as X and Y, respectively, and TU (Transducing units) is the transduction unit.

[0119] 4. Experimental results

[0120] The results showed that the titer of the replication-deficient recombinant VSV virus rVSVΔG-sgRNA-Cas-G with VSVG protein on the surface packaged by plasmid could reach 4×10 6 TU / mL, can infect a variety of cells and express the CRISPR system in cells (see Figure 2 ), Figure 2 Infection of BHK-21 cells is used as an example.

[0121] Example 3 Construction of a replication-deficient recombinant VSV virus rVSVΔG-sgRNA-Cas-S with Spike protein packaged on its surface

[0122] The present invention constructs a replication-deficient recombinant VSV virus vector with VSVG or Spike protein on the surface to achieve the delivery of CRISPR system RNA. The overall construction process is divided into two parts. First, the replication-deficient recombinant VSV virus rVSVΔG-sgRNA-Cas-G with VSVG protein on the surface is constructed, and then the Vero cells expressing Spike protein are infected with rVSVΔG-sgRNA-Cas-G to obtain the replication-deficient recombinant VSV virus rVSVΔG-sgRNA-Cas-S with Spike protein on the surface. See the specific experimental flow chart Figure 3 .

[0123] 1. Experimental methods

[0124] Vero cells were plated in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum. When the cells grew to about 90%, 2 μg of Spike plasmid was transfected with Lipofectamine™ 3000 Transfection Reagent. After 6 hours, the culture medium was changed to DMEM medium containing 5% fetal bovine serum. When syncytia appeared in the cells, the cells were infected with rVSVΔG-sgRNA-Cas-G virus (2.5×10 4 -1×10 5 TU / mL), washed three times with PBS containing 1% fetal bovine serum after 1 hour to remove the residual rVSVΔG-sgRNA-Cas-G, and cultured in a new DMEM medium containing 5% fetal bovine serum. After about 24 hours, the virus was collected, and the cell culture supernatant was filtered with a 0.45 micron filter membrane, and stored in a -80°C refrigerator after aliquoting. If necessary, the virus can be packaged in large quantities and concentrated. rVSVΔG-sgRNA-Cas-S can infect cells with ACE2 receptors on the surface, and its titer determination method is the same as in Example 2.

[0125] 2. Experimental results

[0126] The results showed that the titer of the replication-deficient recombinant VSV virus rVSVΔG-sgRNA-Cas-S packaged with Spike protein on the surface could reach 1×10 6 TU / mL, can infect cells expressing ACE2 and express the CRISPR system in cells (see Figure 4 ), Figure 4 Infection of COS7-ACE2 cells is taken as an example.

[0127] Comparative Example 1 Comparison of the effects of CRISPR delivery by replication-deficient recombinant VSV virus vector and adeno-associated virus vector

[0128] 1. Experimental Methods

[0129] This comparative example verifies the effect of using the vesicular stomatitis virus (VSV) vector of the present invention and the conventional adeno-associated virus (AAV) vector to deliver CRISPR. The specific comparative experimental methods are as follows:

[0130] COS7-ACE2 and 293T-ACE2 cells were plated in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum. When the cells grew to about 90%, rVSVΔG-sgRNA-Cas-S was used to infect COS7-ACE2 and 293T-ACE2 cells, and the virus titer was 3×10 6 TU / mL, 1 mL was added to each well, and the medium was replaced with DMEM medium containing 5% fetal bovine serum after 1 hour. The medium was discarded 24 hours after virus infection, and the cells were washed once with PBS. The cell pellets were collected to extract protein, and the protein was quantified. Western blotting experiments were performed to detect the expression of Cas protein.

[0131] 293T cells were plated in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum. When the cells grew to about 90%, 293T cells were infected with AAV9-Cas at an MOI of 3×10 5 The virus was diluted to 8.4 × 10 11 vg / mL, 1 mL was added to each well, and the medium was replaced with DMEM medium containing 10% fetal bovine serum after 12 hours. The medium was discarded 72 hours and 96 hours after virus infection, and the cells were washed once with PBS. The cell pellets were collected to extract protein, and the protein was quantified. Western blotting experiments were performed to detect the expression of Cas protein.

[0132] 2. Experimental Results

[0133] The results showed that rVSVΔG-sgRNA-Cas-S infected COS7-ACE2 and 293T-ACE2 cells had a large amount of Cas protein expression after 24 hours (see Figure 5 ), while no Cas protein expression was detected in 293T cells infected with AAV9-Cas for 72 hours and 96 hours (see Figure 6 ), it can be seen that the recombinant vesicular stomatitis virus (VSV) vector delivery can express more Cas protein faster than the conventional adeno-associated virus (AAV) vector delivery, that is, the effect of CRISPR delivery using the VSV vector described in the present invention is significantly better than the conventional AAV vector, that is, the recombinant VSV vector described in the present invention has achieved unexpected technical effects in delivering CRISPR.

[0134] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A recombinant vesicular stomatitis virus vector expressing G protein on its surface, characterized in that: The recombinant vesicular stomatitis virus vector is composed of the following operably connected elements: sgRNA, N protein, P protein, M protein, Cas protein, L protein, and viral genome; The sgRNA, N protein, P protein, M protein, Cas protein, and L protein in the recombinant vesicular stomatitis virus vector are connected in sequence; The nucleic acid sequence of the nucleic acid molecule formed by connecting the sgRNA, N protein, P protein, M protein, Cas protein, and L protein is shown in SEQ ID NO: 1; The recombinant vesicular stomatitis virus vector is prepared by the following method: the nucleic acid molecule composed of the sgRNA, N protein, P protein, M protein, Cas protein and L protein and the vesicular stomatitis virus G protein plasmid are simultaneously transfected into the host cell.

2. The recombinant vesicular stomatitis virus vector according to claim 1, characterized in that The recombinant vesicular stomatitis virus vector is a replication-deficient vesicular stomatitis virus vector expressing G protein on its surface.

3. A recombinant host cell, characterized in that The recombinant host cell comprises the recombinant vesicular stomatitis virus vector according to claim 1 or 2.

4. The recombinant host cell according to claim 3, characterized in that The host cell is a mammalian cell.

5. A recombinant vesicular stomatitis virus vector expressing a target glycoprotein on its surface, characterized in that: The recombinant vesicular stomatitis virus vector is a recombinant vesicular stomatitis virus vector according to claim 1 or 2 that expresses Spike protein or influenza HA protein on its surface, and the target glycoprotein is Spike protein or influenza HA protein; The recombinant vesicular stomatitis virus vector is prepared by the following method: after the target glycoprotein plasmid is transfected into a host cell, when syncytia appear in the host cell, the host cell is infected with the recombinant vesicular stomatitis virus vector according to claim 1 or 2.

6. A CRISPR RNA delivery system, characterized in that The delivery system comprises the recombinant vesicular stomatitis virus vector of claim 1 or 2 or the recombinant vesicular stomatitis virus vector of claim 5.

7. A method for constructing a recombinant vesicular stomatitis virus vector according to claim 1 or 2, characterized in that: The method comprises simultaneously transfecting a nucleic acid molecule composed of the sgRNA, N protein, P protein, M protein, Cas protein, and L protein and a vesicular stomatitis virus G protein plasmid into a host cell.

8. The method according to claim 7, characterized in that The host cell is a mammalian cell.

9. A method for constructing a recombinant vesicular stomatitis virus vector according to claim 5, characterized in that: The method comprises transfecting the target glycoprotein plasmid into a host cell, and then infecting the host cell with the recombinant vesicular stomatitis virus vector of claim 1 or 2 when syncytia appears in the host cell; The target glycoprotein is Spike protein or influenza HA protein.

10. The method according to claim 9, characterized in that The host cell is a mammalian cell.

11. A kit for targeted delivery of a CRISPR RNA system, characterized in that: The kit comprises the recombinant vesicular stomatitis virus vector according to claim 5.

12. The application of any one of the following aspects, characterized in that: The applications include: (1) Use of the recombinant vesicular stomatitis virus vector according to claim 1 or 2 in constructing a CRISPR RNA delivery system; (2) Use of the recombinant vesicular stomatitis virus vector according to claim 1 or 2 in constructing a recombinant vesicular stomatitis virus vector expressing a target glycoprotein Spike protein or influenza HA protein on its surface; (3) Use of the recombinant vesicular stomatitis virus vector according to claim 1 or 2 in constructing a recombinant host cell; (4) Use of the recombinant host cell according to claim 3 or 4 in constructing a recombinant vesicular stomatitis virus vector expressing the target glycoprotein Spike protein or influenza HA protein on its surface; (5) Use of the kit of claim 11 in a system for targeted delivery of CRISPR RNA to non-diagnostic and non-therapeutic destinations.

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