CRISPR-Cas9 system-based phage genome editing vector as well as editing method and application thereof

Abstract

The invention discloses a bacteriophage genome editing vector based on a CRISPR-Cas9 system as well as an editing method and application of the bacteriophage genome editing vector. According to the invention, a plasmid containing a cas9 gene and an sgRNA element is successfully constructed, a specific primer is designed for an editing site, and a pTarget plasmid is obtained; a donor DNA sequence is constructed on a vector, and a pEdit plasmid is obtained. After the bacteriophage is infected with host bacteria containing pEdit plasmids, the mutant bacteriophage is reversely screened through pTarget plasmids, and efficient and rapid gene editing, including gene deletion mutation, mononucleotide replacement mutation and insertion mutation, of the bacteriophage genome is achieved. The method is simple to operate and high in gene editing efficiency, promotes the structural analysis and functional research development of the staphylococcus aureus bacteriophage genome, provides a new strategy for the transformation of engineering bacteriophages, and has wide application prospects and market values.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene editing, and specifically relates to a phage genome editing vector based on the CRISPR-Cas9 system, and an editing method and application thereof. Background Art

[0002] Staphylococcus aureus is a common Gram-positive bacterium that is widely present in nature. As an important clinical pathogen and foodborne pathogen, Staphylococcus aureus can cause serious infections in humans and animals, including skin and soft tissue infections, pneumonia, sepsis, and arthritis. At present, antibiotics are mainly used to treat infections caused by Staphylococcus aureus in clinical practice, and some countries and regions have also begun to try phage therapy. However, the wild-type phages currently isolated from nature still have some limitations in clinical treatment, and because the functions of most open reading frames of phages are unknown, their functional genomics research has lagged behind. Therefore, it is urgent to develop a gene editing method for Staphylococcus aureus phages.

[0003] At present, the methods of phage genome editing mainly include random mutagenesis, classical homologous recombination system, phage recombination strategy of electroporated DNA, etc. However, these traditional methods are not only time-consuming and labor-intensive, but also have low editing efficiency, which greatly limits the in-depth study of the structure and function of phage genomes. In recent years, with the discovery of various types of CRISPR-Cas systems, it has been transformed into an efficient gene editing tool and has been widely used in animals, plants, bacteria and other organisms, but its application in phage genome editing is still in the exploratory stage.

[0004] The CRISPR-Cas system is an adaptive immune defense system used by bacteria and archaea to protect themselves from invasion by exogenous phages or plasmids. According to the type of Cas protein, the CRISPR-Cas system can be divided into two major categories, seven types, and 54 subtypes. Among them, the effector protein of the type II CRISPR-Cas system is Cas9, which has been widely used in the field of gene editing due to its relatively simple structure. At present, the widely used Cas9 protein is SpCas9 derived from Streptococcus thermophilus, but its protein molecular weight is large, which poses a severe challenge to the efficiency of gene editing. The type II CRISPR-Cas system promotes the degradation of these two RNAs by the host RNaseIII through base complementary pairing between tracrRNA and pre-crRNA, and then further processes them to form mature crRNA. The crRNA and Cas9 nuclease complex recognizes PAM, and after complementary pairing with the original spacer sequence base, it cuts the exogenous gene. The CRISPR-Cas system combined with the homologous recombination method can effectively exert the function of phage genome editing.

[0005] So far, the CRISPR-Cas system has been widely used to edit the genomes of other bacterial phages, such as Salmonella and Pseudomonas aeruginosa. However, there is no widely applicable editing method reported for the Staphylococcus aureus phage genome. At the same time, studies have reported that the editing effect of phage genomes in phages with larger genomes is often unstable. Therefore, by modifying the CRISPR-Cas9 system so that it can be widely used in the editing of Staphylococcus aureus phage genomes, it will provide new strategies for subsequent genetic biological function research and the modification of engineered phages. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a phage genome editing vector based on the CRISPR-Cas9 system.

[0007] The technical problem that the present invention needs to solve is to provide a pTarget plasmid.

[0008] The technical problem that the present invention also solves is to provide a CRISPR-Cas dual-plasmid system.

[0009] The technical problem that the present invention needs to solve is to provide an engineered bacterium for a phage genome editing vector.

[0010] The technical problem that the present invention needs to solve is to provide a method for constructing the phage genome editing vector.

[0011] The final technical problem to be solved by the present invention is to provide a phage genome editing method based on the CRISPR-Cas9 system.

[0012] Technical solution: In order to solve the above technical problems, the first aspect of the present invention provides a phage genome editing vector based on the CRISPR-Cas9 system, wherein the phage genome editing vector comprises a resistance screening marker, a promoter PrpsL, a Cas9 nuclease gene, an endogenous U6 promoter, an sgRNA sequence, an endogenous U6 terminator and a replicon, and the Cas9 nuclease gene is selected from the SeCas9 nuclease gene of Staphylococcus epidermidis (S. epidermidis).

[0013] Among them, the sequence of the phage genome editing vector is shown in SEQ ID No.1.

[0014] The second aspect of the present invention further provides a pTarget plasmid, wherein the pTarget plasmid is obtained by connecting the spacer fragment of the target gene with the phage genome editing vector, and the spacer fragment of the target gene is designed and amplified according to the PAM site requirements.

[0015] The third aspect of the present invention also provides a CRISPR-Cas dual-plasmid system, which includes the pTarget plasmid and the pEdit plasmid. The pEdit plasmid is obtained by introducing the upstream homology arm fragment and the downstream homology arm fragment of the target gene in the phage genome into the plasmid, and the upstream homology arm fragment and the downstream homology arm fragment of the target gene are obtained by designing primers and amplifying by PCR.

[0016] The fourth aspect of the present invention also provides an engineered bacterium, which contains the phage genome editing vector based on the CRISPR-Cas9 system, the pTarget plasmid or the CRISPR-Cas dual-plasmid system.

[0017] The fifth aspect of the present invention also provides a method for constructing the gene editing vector, comprising the following steps:

[0018] 1) The promoter PrpsL, Secas9, endogenous U6 promoter and sgRNA gene sequences were connected and introduced into the pUC57 plasmid to obtain the SeCas9-pUC57 plasmid. The gene fragments of the SeCas9 editing system components were amplified by PCR using the SeCas9-pUC57 plasmid as a template;

[0019] 2) Connect the gene fragments of the SeCas9 editing system components to the linear vector to obtain a phage genome editing vector based on the CRISPR-Cas9 system.

[0020] Preferably, the linear vector includes but is not limited to the linear vector after double restriction digestion of pRAB11, and the endonuclease includes but is not limited to BamH I and Kpn I.

[0021] The sixth aspect of the present invention also provides a method for constructing the pTarget plasmid, comprising the following steps:

[0022] 1) constructing the phage genome editing vector based on the CRISPR-Cas9 system;

[0023] 2) According to the PAM site requirements of SeCas9 nuclease, determine the editing site on the target gene, design the targeting sequence, and construct it into the phage genome editing plasmid of the CRISPR-Cas9 system obtained in step 1) to obtain the pTarget plasmid.

[0024] The seventh aspect of the present invention also provides a phage genome editing method based on the CRISPR-Cas9 system, comprising the following steps:

[0025] 1) constructing the phage genome editing vector based on the CRISPR-Cas9 system;

[0026] 2) According to the PAM site requirements of the SeCas9 nuclease, the editing site on the target gene is determined, the targeting sequence is designed, and constructed onto the phage genome editing plasmid of the CRISPR-Cas9 system obtained in step 1) to obtain the pTarget plasmid, which is then electroporated into Staphylococcus aureus competent cells to obtain a targeted strain;

[0027] 3) 200 bp DNA sequences upstream and downstream of the editing site were constructed onto the vector to obtain the pEdit plasmid, which was then electroporated into Staphylococcus aureus competent cells to obtain the editing strain;

[0028] 4) Infecting the edited strain obtained in step 3) with the phage to induce homologous recombination of the phage, then harvesting the phage, and further infecting the targeted strain in step 2) with the phage again, obtaining phage mutants through reverse screening, and achieving gene fragment deletion, insertion or site-directed mutation editing of the phage genome.

[0029] Wherein, the PAM site selection requirement in step 2) is 5'-NNGRRT-3', and the target gene includes the gp036 gene or the gp170 gene.

[0030] Wherein, the bacteriophage in step 4) includes but is not limited to Staphylococcus aureus philPLA-RODI phage.

[0031] The phage philPLA-RODI used in the present invention is a phage with a larger genome, with a genome size of 142348 bp, which makes it difficult to establish the editing method of the present invention. The present invention achieves the editing of phages with larger genomes by modifying the CRISPR-Cas9 system and establishing a dual plasmid system.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The phage genome CRISPR-Cas dual-plasmid editing system provided by the present invention, wherein the Cas protein is selected from Staphylococcus epidermidis, has a small molecular weight, a simple structure, and is convenient for gene editing. The present invention can achieve gene fragment deletion, insertion, and site-directed mutation editing of the phage genome through homologous recombination combined with reverse screening. The gene editing method has stable performance, high efficiency, convenience, and powerful functions. The gene editing method of the present invention has high editing efficiency, which can reach up to 100%, and the editing method of the present invention is short in time, and the required edited phage can be harvested in as fast as 3 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1This is a schematic diagram of the structure of the reverse screening plasmid pTarget in the CRISPR-Cas dual-plasmid system of the present invention.

[0034] Figure 2 A schematic diagram of the structure of the template plasmid pEdit in the CRISPR-Cas dual-plasmid system of the present invention.

[0035] Figure 3 This is a diagram showing the results of plating the second-generation phage philPLA-RODI-Δgp036 on TSB double-layer plates.

[0036] Figure 4 The CRISPR-Cas dual-plasmid system of the present invention realizes genome editing in bacteriophage philPLA-RODI; and a PCR verification result diagram of the gp036 gene deletion mutation in bacteriophage philPLA-RODI.

[0037] Figure 5 The CRISPR-Cas dual-plasmid system of the present invention realizes genome editing in bacteriophage philPLA-RODI; a schematic diagram of inserting the fluorescent marker gene GFP into the bacteriophage philPLA-RODI for PCR verification.

[0038] Figure 6 The CRISPR-Cas dual-plasmid system of the present invention realizes genome editing in bacteriophage philPLA-RODI; and a PCR verification result diagram of inserting the fluorescent marker gene GFP into bacteriophage philPLA-RODI.

[0039] Figure 7 The CRISPR-Cas dual-plasmid system of the present invention realizes genome editing in bacteriophage philPLA-RODI; and a sequencing result diagram of point mutation in bacteriophage philPLA-RODI. DETAILED DESCRIPTION

[0040] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] The materials and sources used in the specific implementation of the present invention are as follows:

[0042] 1. Strains and plasmids

[0043] pRAB11 vector (Helle, L., Kull, M., Mayer, S., Marincola, G., Zelder, M.-E., Goerke, C., Wolz, C. and Bertram, R. (2011) Vectors for improved Tet repressor-dependent gradual gene induction or silencing in Staphylococcus aureus. Microbiology, 157, 3314-3323.), E. coli IM08B, (Monk, IR, Tree, JJ, Howden, BP, Stinear, TP and Foster, TJ (2015) Complete Bypass of RestrictionSystems for Major Staphylococcus aureus Lineages.mBio,6,e00308-00315.) Staphylococcus aureus RN4220 and bacteriophage philPLA-RODI (3.Gutiérrez,D.,Vandenheuvel,D.,Martínez,B.,Rodríguez,A.,Lavigne,R.,García,P.and Wommack,KE(2015)Two phages,phiIPLA-RODI and phiIPLA-C1C,lyse mono-and dual-species staphylococcal biofilms.ApplEnviron Microbiol,81,3336-3348.) were all preserved in the laboratory.

[0044] 2. Main reagents

[0045] Primestar max DNA Polymerase, DNA Marker, restriction endonuclease BamH I, Kpn I were purchased from Bao Biotechnology (Dalian) Co., Ltd.; restriction endonuclease BsaI and T4 Polynucleotide kinase (PNK) were purchased from Biotech Co., Ltd.; Clone express II one step cloning kit, ClonExpress UltraOne Step Cloning Kit, and 2×Taq Master Mix were purchased from Novozymes Biotech Co., Ltd.; T4 DNA ligase and viral genome RNA / DNA extraction kit were purchased from Takara; kanamycin (Kan), chloramphenicol (Cm), and LB medium were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; tryptone soy agar medium (TSA) and tryptone soy broth (TSB) were purchased from Qingdao Haibo Biotechnology Co., Ltd.; rapid plasmid mini-extraction kit and agarose gel DNA recovery kit were purchased from TIANGEN; other reagents were domestically produced analytical grade.

[0046] 3. Main instruments and equipment

[0047] PCR instrument, electrophoresis instrument, and Gel Doc XR+ gel imager were all purchased from Bio-RAD, USA; pipettes, electroporators, electroporation cups, high-speed desktop centrifuges, and constant temperature metal baths were all purchased from Eppendorf, Germany; IS-RDS3 constant temperature oscillating shaker was purchased from Crystal; and constant temperature incubators were purchased from Shanghai Yuejin Medical Equipment Factory.

[0048] Example 1 Construction of SeCas9-pRAB11 editing system plasmid

[0049] The composition of the SeCas9-pRAB11 editing system plasmid is as follows Figure 1 As shown, its sequence is SEQ ID No. 1. The specific construction method is as follows:

[0050] 1. Amplification of DNA sequences of SeCas9 editing system components

[0051] 1) Download the promoter PrpsL, Secas9, endogenous U6 promoter and sgRNA gene sequences from the NCBI database.

[0052] PrpsL sequence:

[0053]

[0054] SecAs9 sequence:

[0055]

[0056]

[0057] Endogenous U6 promoter sequence:

[0058]

[0059] sgRNA gene sequence:

[0060]

[0061] The above sequence was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis and named as SeCas9-pUC57 plasmid. The specific plasmid sequence is shown in SEQ ID No: 3 in the sequence table.

[0062] 2) Using SeCas9-pUC57 plasmid as a template, the gene fragments of the SeCas9 editing system components were obtained by PCR amplification.

[0063] The primer sequences used for PCR amplification are:

[0064] SeCas9-F:

[0065]

[0066] SeCas9-R:

[0067]

[0068] The reaction system is shown in Table 1:

[0069] Table 1 PCR reaction system

[0070]

[0071] The reaction program was as follows: 98°C for 3 min; 98°C for 15 s, 55°C for 15 s, 72°C for 1 min, 35 cycles; 72°C for 10 min, 4°C∞.

[0072] Prepare 1% agarose gel for gel electrophoresis. After electrophoresis at 110 V for 40 min, cut the target band and use an agarose gel DNA recovery kit to recover fragment A. The specific operation steps are carried out according to the kit instructions.

[0073] 2. Vector digestion

[0074] The pRAB11 plasmid was extracted according to the instructions of the rapid plasmid extraction kit and double-digested with BamH I and Kpn I. The enzyme digestion reaction system is shown in Table 2. 20 systems were prepared and the reaction system was placed at 37°C for enzyme digestion for 3.5 hours.

[0075] Table 2 Enzyme digestion reaction system

[0076]

[0077] Prepare 1% agarose gel for gel electrophoresis. After 40 min of electrophoresis at 110 V, cut the target band and use an agarose gel DNA recovery kit to recover the double-enzyme digestion product B. The specific operation steps are carried out according to the kit instructions.

[0078] 3. Connection conversion

[0079] The above fragments A and B were connected using Clone express II one step cloning kit. The in vitro connection reaction system is shown in Table 3:

[0080] Table 3 Ligation reaction system

[0081]

[0082]

[0083] The ligation reaction conditions were 37°C for 30 min, and then the ligation product was transformed into E. coli IM08B competent cells, spread on LB solid culture medium plates containing 50 μg / mL kanamycin, and inverted in a 37°C incubator for overnight culture. The next day, a single colony was picked for colony PCR verification, and PCR-positive clones were selected. After the plasmid was extracted, it was sent to the company for sequencing, and the plasmid with correct sequencing was named SeCas9-pRAB11 editing plasmid.

[0084] Example 2pTarget plasmid construction

[0085] 1. The experiment takes the gp036 gene deletion mutation as an example. According to the PAM site requirements, the 30 bp upstream is selected as the spacer. The spacer primer sequence is:

[0086] gp036-SeCas9-F:5'-caccgtcaaatgttaaagttattagaagaagcag-3'

[0087] gp036-SeCas9-R: 5'-taacctgcttcttctaataactttaacatttgac-3'.

[0088] 2. The two completely complementary single-stranded oligonucleotides are phosphorylated, annealed, and connected to obtain a connection product. The specific reaction system and reaction conditions are shown in Tables 4 and 5:

[0089] 1) Phosphorylation:

[0090] Table 4 Phosphorylation reaction system

[0091]

[0092] The above reaction system was incubated at 37° C. for 1 h, and the obtained product was a phosphorylation reaction product.

[0093] 2) Annealing:

[0094] Add 2.5 μL 1M NaCl to the above phosphorylation reaction product, and the PCR reaction program is 95℃ for 1 min, 80℃ for 3 min, 70℃ for 3 min, 60℃ for 3 min, 50℃ for 3 min, 40℃ for 3 min, 30℃ for 3 min, 25℃ for 3 min, 4℃∞; dilute the obtained annealing product 20 times to obtain the required spacer fragment.

[0095] 3) Connection:

[0096] Table 5 Ligation reaction system

[0097]

[0098]

[0099] The reaction program was as follows: 37°C for 2 min; 16°C for 5 min, 25 cycles; 50°C for 5 min, 80°C for 5 min, and 10°C infinity.

[0100] 3. Transform the ligation product into competent E. coli IM08B, spread it on LB solid medium plate with 50μg / mL kanamycin, and invert it in a 37℃ incubator for overnight culture. The next day, pick a single colony for colony PCR verification, select PCR positive clones, extract plasmids and send them to the company for sequencing, and name the plasmid with correct sequencing results as pTarget-gp036 plasmid.

[0101] The pTarget plasmid is a shuttle plasmid that can replicate and propagate in E. coli and Staphylococcus aureus; the plasmid is kanamycin-resistant in E. coli, with a screening concentration of 50 μg / mL, and chloramphenicol-resistant in Staphylococcus aureus, with a screening concentration of 15 μg / mL. The plasmid expresses SeCas9 nuclease in Staphylococcus aureus, and when used in conjunction with the pEdit plasmid, the genome of the phage philPLA-RODI can be edited to achieve gene deletion, insertion, and site-directed mutagenesis.

[0102] Example 3 pEdit plasmid construction

[0103] The composition of pEdit plasmid is as follows Figure 2The sequence is SEQ ID No: 2. The specific construction method is as follows:

[0104] 1. Using the phage philPLA-RODI genome as a template, select 200bp upstream and downstream of the editing site, and design primers to amplify the homology arm fragments up and down. The experiment takes the gp036 gene deletion mutation of the phage philPLA-RODI as an example.

[0105] gp036-up-F:

[0106]

[0107] gp036-up-R:

[0108]

[0109] gp036-down-F:

[0110]

[0111] gp036-down-R:

[0112]

[0113] The PCR reaction system and procedure are as follows:

[0114] Table 6 PCR reaction system

[0115]

[0116]

[0117] The reaction program was as follows: 98°C for 3 min; 98°C for 15 s, 55°C for 15 s, 72°C for 7 s, 35 cycles; 72°C for 10 min, 4°C∞.

[0118] Prepare 1% agarose gel for gel electrophoresis. After electrophoresis at 110V for 40 minutes, cut the target band and use an agarose gel DNA recovery kit to recover the up and down fragments. The specific operation steps are carried out according to the kit instructions.

[0119] 2. The up and down fragments were recombined and spliced ​​together with the pRAB11 double-digested (BamH I and Kpn I) linear vector in Example 1 in vitro using ClonExpress Ultra One Step Cloning Kit to obtain the pEdit plasmid. The in vitro recombination reaction system is shown in Table 7:

[0120] Table 7 Ligation reaction system

[0121]

[0122] The ligation reaction conditions were 50°C for 15 min, and then the ligation product was transformed into E. coli IM08B competent cells, spread on LB solid culture medium plates containing 50 μg / mL kanamycin, and inverted in a 37°C incubator for overnight culture. The next day, a single colony was picked for colony PCR verification, and PCR-positive clones were selected. After the plasmid was extracted, it was sent to the company for sequencing, and the plasmid with correct sequencing was named pEdit-gp036 plasmid.

[0123] The pEdit plasmid is a shuttle plasmid that can replicate and propagate in Escherichia coli and Staphylococcus aureus; the plasmid is kanamycin-resistant in Escherichia coli, with a screening concentration of 50 μg / mL, and is chloramphenicol-resistant in Staphylococcus aureus, with a screening concentration of 15 μg / mL. The plasmid provides a homologous recombination template for phage philPLA-RODI in Staphylococcus aureus, and can be used in conjunction with the pTarget plasmid to edit the genome of phage philPLA-RODI to achieve gene deletion, insertion, and site-directed mutagenesis.

[0124] Example 4 CRISPR-Cas dual plasmid system achieves efficient gene deletion on bacteriophage philPLA-RODI

[0125] The CRISPR-Cas dual plasmid system composed of pTarget and pEdit plasmids can achieve efficient deletion of different genes in phage philPLA-RODI. The experiment took the gp036 gene as an example and performed gene deletion on phage philPLA-RODI. Figure 4 PCR verification results of gp036 gene deletion on bacteriophage philPLA-RODI.

[0126] 1. The pTarget-gp036 plasmid described in Example 2 and the pEdit-gp036 plasmid described in Example 3 were electroporated into Staphylococcus aureus RN4220 competent cells, and the successfully electroporated strains were named pTarget-gp036-RN4220 and pEdit-gp036-RN4220, respectively. The specific operations were as follows: two RN4220 competent cells (80 μL each) were placed on ice for 5 min, and then taken out and placed at room temperature for 5 min; 10 μL of the above two plasmids were added to each, gently mixed, and then transferred to an electric shock cup, and the voltage of the electric transformer was adjusted to 2100 V for electroporation; immediately after the electroporation was completed, 800 μL of TSB medium was added, mixed evenly, and then transferred to a 1.5 mL Eppendorf tube, and cultured at 37°C for 2-3 h; 5000 rpm Centrifuge for 3 minutes, discard 900 μL of supernatant, resuspend the remaining 100 μL of precipitate and spread it on a TSA solid medium plate containing 15 μg / mL chloramphenicol, invert and incubate overnight at 37°C incubator. Only bacteria with successful electroporation of plasmids can grow on the plate. The next day, pick a single colony and place it on 4 mL of liquid TSB (cm + ) medium and cultured overnight at 37°C in a shaking incubator.

[0127] 2. Bacteriophage gp036 gene deletion editing

[0128] 1) Step 1: One round of editing (this process can be completed in 1 day)

[0129] The pEdit-gp036-RN4220 bacterial suspension was expanded at a dilution of 1:100 in 3 mL TSB (cm + ) medium, add 30 μL of overnight cultured pEdit-gp036-RN4220 bacterial solution, and culture at 37°C 180 rpm in a shaking incubator for about 2.5 h. At this time, add 10 μL of phage philPLA-RODI stock solution (titer 3.7×10 9 PFU / mL), and cultured on a shaker at 37°C and 180 rpm for about 5 hours; 1 mL of the lysate was aspirated, and centrifuged at 4°C and 10000 rpm for 10 minutes. The supernatant was transferred with a syringe to pass through a 0.22 μm filter membrane. The resulting lysate was named RODI-Δgp036 first-generation phage.

[0130] 2) Step 2: Second round of Target (this process can be completed in 1 day)

[0131] The pTaregt-gp036-RN4220 bacterial suspension was expanded at a dilution of 1:100 in 3 mL TSB (cm +) medium, add 30 μL of overnight cultured pTaregt-gp036-RN4220 bacterial solution, and culture at 37°C 180 rpm in a shaking incubator for about 2.5 h. At this time, add 100 μL of RODI-Δgp036 first generation phage stock solution (titer 2×10 3 PFU / mL), and cultured on a shaker at 37°C and 180 rpm for about 5 hours; 1 mL of the lysate was aspirated, and centrifuged at 4°C and 10000 rpm for 10 minutes. The supernatant was transferred with a syringe to pass through a 0.22 μm filter membrane. The resulting lysate was named RODI-Δgp036 second-generation phage.

[0132] The second generation phage of RODI-Δgp036 (titer of 4×10 6 PFU / mL) was serially diluted with TSB medium to 10 -6 , and select appropriate gradients for plating on TSB double-layer plates. The specific plating operation is as follows: TSA and TSB are mixed evenly in a volume ratio of 1:2 to obtain TSB semi-solid medium; 50 μL of overnight cultured pTaregt-gp036-RN4220 bacterial solution and 10 μL 10 -3 Gradient RODI-Δgp036 second-generation phage, then add 4 mL of TSB semi-solid medium cooled to about 45°C, mix well, and pour along the edge of the plate on TSA (cm + ) solid culture medium plate; after it solidifies, invert it and place it in a 37℃ incubator for overnight culture. A single plaque will appear on the double-layer plate after overnight culture. Figure 3 The results of plating the second-generation phage of RODI-Δgp036 on TSB double-layer plates.

[0133] 3) Step 3: Three rounds of Target (this process can be completed in 1 day)

[0134] Use 12-well plates to expand the bacterial culture at a dilution of 1:100: in 3 mL TSB (cm + ) culture medium, add 30 μL of overnight cultured pTaregt-gp036-RN4220 bacterial solution, and culture at 37°C 180 rpm shaking for about 2.5 hours. At this time, pick out 10 single plaques plated in the second round of Target process and culture them at 37°C 180 rpm shaking for about 5 hours; aspirate 1 mL of lysate, centrifuge at 4°C 10000 rpm for 10 minutes, transfer the supernatant with a syringe to pass it through a 0.22 μm filter membrane, and the obtained lysate is named RODI-Δgp036 third-generation phage.

[0135] 3. Use the viral genomic RNA / DNA extraction kit to extract the RODI-Δgp036 third-generation phage genomic DNA, and use it as a PCR template to amplify it using the gp036 gene external primers (out) and internal primers (in). The PCR product was subjected to gel electrophoresis on a 1% agarose gel. The size of the bands in the gel run result graph can be used to determine whether the gp036 gene has been successfully deleted. Figure 4 Further, the PCR product of the successful deletion of the gp036 gene was sent to the company for sequencing to confirm the successful deletion of the gp036 gene. The specific PCR verification reaction system is shown in Table 8:

[0136] Table 8 PCR verification reaction system

[0137]

[0138]

[0139] The reaction program was as follows: 95°C for 3 min; 95°C for 15 s, 55°C for 15 s, 72°C for 10 s, 30 cycles; 72°C for 10 min, 4°C∞.

[0140] The PCR primer sequences for gp036 gene deletion are:

[0141] gp036-in-F:5'-agtaatgaccatgtaacacgaga-3'

[0142] gp036-in-R:5'-acactccgttaataagcttacct-3'

[0143] gp036-out-F:5'-agggtgaaagcttatgttcgtg-3'

[0144] gp036-out-R:5'-tgcgttcatttaataccctcct-3'.

[0145] from Figure 4 From the PCR verification results of the philPLA-RODI gp036 gene deletion mutation, it can be seen that when the 10 randomly selected plaques were verified with internal primers, no bands were produced; while when verified with external primers, the edited phage was smaller than the wild-type phage band and the size was correct; further, the sequencing results also showed that the gp036 gene was successfully deleted, and the editing efficiency was 100%.

[0146] Example 5 CRISPR-Cas dual plasmid system achieves efficient gene insertion on bacteriophage philPLA-RODI

[0147] The CRISPR-Cas dual plasmid system composed of pTarget and pEdit plasmids can achieve efficient gene insertion in phage philPLA-RODI. The experiment took the gp170 gene as an example, and inserted the fluorescent marker gene GFP after the philPLA-RODI gp170 gene. The specific GFP sequence is shown in SEQ ID No: 4 in the sequence list.

[0148] 1. The pTarget-gp170 plasmid was constructed according to the method described in Example 2. At the end of the gp170 gene, 30 bp upstream was selected as a spacer according to the PAM site requirement and constructed on the pTarget plasmid. The spacer primer sequence is:

[0149] gp170-SeCas9-F:5'-cacctgaataattttggaagtcagattacaacgg-3'

[0150] gp170-SeCas9-R:5'-taacccgttgtaatctgacttccaaaattattca-3'

[0151] 2. The pEdit-gp170 plasmid was constructed according to the method described in Example 3. The homology arm fragment up of the plasmid contained the spacer of the gp170 mentioned above, and a point mutation was performed at a certain spacer base site near PAM so that the amino acid encoded at this position underwent a synonymous mutation. The primer sequences for amplifying the homology arms up and down and GFP were:

[0152] gp170-up-F:

[0153]

[0154] gp170-up-R:

[0155]

[0156] gp170-GFP-F:

[0157]

[0158] gp170-GFP-R:

[0159]

[0160] gp170-down-F:

[0161]

[0162] gp170-down-R:

[0163]

[0164] 3. The pTarget-gp170 plasmid and pEdit-gp170 plasmid were electroporated into Staphylococcus aureus RN4220 competent cells, and the successfully electroporated strains were named pTarget-gp170-RN4220 and pEdit-gp170-RN4220, respectively. According to the phage genome editing method in Example 4, one round of Edit, two rounds of Target, and three rounds of Target were performed to collect the third generation of RODI-gp170-GFP phage.

[0165] 4. Use the viral genomic RNA / DNA extraction kit to extract the RODI-gp170-GFP third-generation phage genomic DNA, and use it as a PCR template to amplify it with the gp170 gene external primer (out) and verification primer (verify). The PCR product was subjected to gel electrophoresis on a 1% agarose gel. The size of the band in the gel run result graph can be used to determine whether the GFP gene has been successfully inserted. Figure 5 , 6 Further, the PCR product successfully inserted with the GFP gene was sent to the company for sequencing to confirm the successful insertion of the GFP gene.

[0166] The PCR verification primer sequences for gp170 insertion into the GFP gene are:

[0167] gp170-verify-F1:5'-acggattgtattagtggtggtt-3'

[0168] gp170-GFP-R1: 5'-ccttctggcattgctgattt-3'

[0169] gp170-GFP-F2: 5'-ccaattggtgatggtccagt-3'

[0170] gp170-verify-R2:5'-tggtctcctctgtacgttctaaca-3'

[0171] gp170-out-F:5'-ccggaagtattctagcaatcg-3'

[0172] gp170-out-R: 5'-aaaagtaaagacctacagccatacc-3'.

[0173] Figure 6The PCR verification results of inserting the fluorescent marker gene GFP into the bacteriophage philPLA-RODI. Figure 6 The results showed that when 12 randomly selected phage plaques were verified using external primers, the edited phage had a larger band than the wild-type phage and the size was correct; when verified using verification primers, the edited phage with the GFP gene successfully inserted was able to run out of the band; further, sequencing results also showed that the GFP gene was successfully inserted, with an editing efficiency of 75%.

[0174] Example 6 CRISPR-Cas dual plasmid system achieves efficient site-directed mutagenesis on bacteriophage philPLA-RODI

[0175] The CRISPR-Cas dual plasmid system consisting of pTarget and pEdit plasmids can achieve efficient site-directed mutagenesis in phage philPLA-RODI. The gp106 gene is taken as an example, and the specific steps are as follows:

[0176] 1. The pTarget-gp106 plasmid was constructed according to the method described in Example 2. The upstream 30 bp of the gp106 gene was selected as a spacer according to the PAM site requirements and constructed on the pTarget plasmid. The spacer primer sequence is:

[0177] gp106-SeCas9-F: 5'-cacccaaaaggtgctcctagtggagaagctacgg-3'

[0178] gp106-SeCas9-R: 5'-taacccgtagcttctccactaggagcaccttttg-3'

[0179] 2. The pEdit-gp106 plasmid was constructed according to the method described in Example 3. The homology arm fragment up of the plasmid contained the spacer of gp106, and the first base near PAM was mutated from G to T. The primer sequences for amplifying the homology arms up and down were:

[0180] gp106-up-F:

[0181] 5'-actagttttttatttggatccaggagaaaaatagatggcatttaactac-3'

[0182] gp106-up-R:

[0183] 5'-aatcctgacgtagcttctccactaggagca-3'

[0184] gp106-down-F:

[0185] 5'-ggagaagctacgtcaggatttgtaagatggataaaagaa-3'

[0186] gp106-down-R:

[0187] 5'-ctcagatctgttaacggtaccctgagttgtagattcagtgagttcctt-3'

[0188] 3. The pTarget-gp106 plasmid and pEdit-gp106 plasmid were electroporated into Staphylococcus aureus RN4220 competent cells, and the successfully electroporated strains were named pTarget-gp106-RN4220 and pEdit-gp106-RN4220, respectively. After one round of Edit, two rounds of Target, and three rounds of Target according to the phage genome editing method in Example 4, the RODI-gp106-point third-generation phage was collected.

[0189] 4. Use the viral genome RNA / DNA extraction kit to extract the RODI-gp106-point third-generation phage genomic DNA, and use it as a PCR template. Amplify it with the verification primers (verify) of the gp106 gene, and send the PCR product directly to the company for sequencing.

[0190] The PCR primer sequences for gp106 single nucleotide substitution mutations are:

[0191] gp106-verify-F:5'-cgcctcttactgaaacacagaa-3'

[0192] gp106-verify-R:5'-tgagttgtagattcagtgagttcct-3'.

[0193] Figure 7 The sequencing results for the successful realization of point mutations in phage-philPLA-RODI. Site-directed mutagenesis was performed in the spacer of the selected phage philPLA-RODI gp106 gene. The site-directed mutagenesis can only efficiently replace single nucleotides at 1-8 bases close to PAM in the 30-base spacer sequence, and can perform any replacement except replacement between bases A and T, and the editing efficiency can reach 100%.

[0194] CRISPR-Cas9 system edited plasmid sequence (SEQ ID No: 1)

[0195]

[0196]

[0197]

[0198]

[0199] Homologous recombination pEdit plasmid sequence (SEQ ID No: 2)

[0200]

[0201]

[0202]

[0203]

[0204] SeCas9-pUC57 plasmid sequence (SEQ ID No: 3)

[0205]

[0206]

[0207]

[0208] GFP sequence (SEQ ID No:4)

[0209]

Claims

1. A bacteriophage genome editing vector based on the CRISPR-Cas9 system, characterized in that: The phage genome editing vector includes a resistance screening marker, a promoter P rPv , a Cas9 nuclease gene, an endogenous U6 promoter, an sgRNA sequence, an endogenous U6 terminator, and a replicon, wherein the Cas9 nuclease gene is selected from Staphylococcus epidermidis ( S. epidermidis )'s SeCas9 nuclease gene.

2. The phage genome editing vector based on the CRISPR-Cas9 system according to claim 1, characterized in that: The sequence of the phage genome editing vector is shown in SEQ ID No.

1.

3. A pTarget plasmid, characterized in that: The pTarget plasmid is obtained by connecting the spacer fragment of the target gene with the phage genome editing vector according to claim 1 or 2, and the spacer fragment of the target gene is designed and amplified according to the PAM site requirements.

4. A CRISPR-Cas dual-plasmid system, characterized in that: The CRISPR-Cas dual-plasmid system includes the pTarget plasmid described in claim 4, and also includes a pEdit plasmid, wherein the pEdit plasmid is obtained by introducing the upstream homology arm fragment and the downstream homology arm fragment of the target gene in the phage genome into the plasmid, and the upstream homology arm fragment and the downstream homology arm fragment of the target gene are obtained by designing primers and amplifying by PCR, respectively.

5. An engineered bacterium, characterized in that: The engineered bacteria contains the phage genome editing vector based on the CRISPR-Cas9 system according to claim 1 or 2, the pTarget plasmid according to claim 3, or the CRISPR-Cas dual-plasmid system according to claim 4.

6. The method for constructing a gene editing vector according to claim 1 or 2, characterized in that: The following steps are involved: 1) The promoter P rPv , Secas9 , endogenous U6 promoter and sgRNA gene sequences were connected and introduced into pUC57 plasmid to obtain SeCas9-pUC57 plasmid. SeCas9-pUC57 plasmid was used as a template to obtain the gene fragment of SeCas9 editing system components by PCR amplification; 2) Connect the gene fragments of the SeCas9 editing system components to the linear vector to obtain a phage genome editing vector based on the CRISPR-Cas9 system.

7. The method for constructing the pTarget plasmid according to claim 3, characterized in that: The following steps are involved: 1) constructing a phage genome editing vector based on the CRISPR-Cas9 system as described in claim 1 or 2; 2) According to the PAM site requirements of SeCas9 nuclease, determine the editing site on the target gene, design the targeting sequence, and construct it into the phage genome editing plasmid of the CRISPR-Cas9 system obtained in step 1) to obtain the pTarget plasmid.

8. A phage genome editing method based on the CRISPR-Cas9 system, characterized in that: The following steps are involved: 1) constructing a phage genome editing vector based on the CRISPR-Cas9 system as described in claim 1 or 2; 2) According to the PAM site requirements of SeCas9 nuclease, determine the editing site on the target gene, design the targeting sequence, and construct it into the phage genome editing plasmid of the CRISPR-Cas9 system obtained in step 1) to obtain the pTarget plasmid, which is then electroporated into Staphylococcus aureus competent cells to obtain the targeted strain; 3) 200 bp DNA sequences upstream and downstream of the editing site were constructed into the vector to obtain the pEdit plasmid, which was then electroporated into Staphylococcus aureus competent cells to obtain the editing strain; 4) Infecting the edited strain obtained in step 3) with the phage to induce homologous recombination of the phage, then harvesting the phage, and further infecting the targeted strain in step 2) with the phage again, obtaining the phage mutant strain through reverse screening, and achieving gene fragment deletion, insertion or site-directed mutation editing of the phage genome.

9. The phage genome editing method based on the CRISPR-Cas9 system according to claim 8, characterized in that: In step 2), the PAM site selection requirement is 5'-NNGRRT-3', and the target gene includes gp036 Gene or gp170 Gene.

10. The phage genome editing method based on the CRISPR-Cas9 system according to claim 8, characterized in that: The bacteriophage in step 4) includes Staphylococcus aureus philPLA-RODI phage.

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