CN104762321A - Knockout vector construction method based on CRISPR/Cas9 system target knockout KHV gene and crNRA prototype thereof - Google Patents

Abstract

A method for constructing a knockout vector and its crRNA component for targeted knockout of the KHV gene based on a CRISPR/Cas9 system, and relates to a method for constructing a knockout vector and its crRNA component. It is to solve the problem of insufficient KHVD suppression in existing methods. Methods: 1. Synthesize crRNA-TK and crRNA-DP primers, treat with boiling water, and anneal to obtain two DNA double-stranded annealed products; 2. Prepare pX330-puro vector; 3. Digest pX330-puro vector and dephosphorize Reaction to recover the linearized vector; 4. Ligate the linearized vector with two annealed products to obtain the ligated product; 5. Transfer the ligated product into competent cells, extract the recombinant plasmid DNA, and name it pX330-puro TK or pX330 -puro DP recombinant vector. The invention is based on the directional knockout of key KHV genes, has high targeting accuracy, and has obvious inhibitory effect on intracellular viruses.

Description

KHV gene-targeted knockout vector construction method and crRNA components based on CRISPR/Cas9 system

technical field

The invention relates to a method for constructing a knockout vector and its crRNA original.

Background technique

Carp herpes virus disease (KHVD) is an acute infectious disease of carp and koi caused by the infection of carp herpes virus (KHV or CyHV-3). serious economic loss. The virus is an envelope-coated virus with a diameter of 170-230nm, which contains an icosahedral symmetrical nucleocapsid, belongs to the herpes family, and its genetic material belongs to double-stranded DNA. At present, the remedial measures for the outbreak of the epidemic are still mainly isolation and mass culling, and there is no effective treatment method. The study confirmed that the carp brain cell line CCB and the fin ray cell line KF-1 were KHV sensitive cells under the experimental conditions.

CRISPR: Clustered, regularly interspaced short palindromic repeats, an immune defense system for prokaryotes against foreign gene fragments—phages, plasmids, etc. The CRISPR/Cas9 system, which belongs to type III CRISPR, consists of three essential parts: crRNA, tracrRNA, and Cas9 endonuclease. In the current common px330 vector, crRNA and tracrRNA are connected to form sgRNA with stem-loop structure. The 5' end of crRNA contains a specific sequence of 20 bp, which can recognize the complementary sequence of specific DNA. In the process of performing the recognition function, the 3 (NGG) bases adjacent to the 3' of the pre-gap region of the target gene are called the PAM sequence. After intracellular transfection of px-330, the gRNA containing crRNA brings the Cas9 nuclease to the specific target site on the genome, thereby cutting the specific gene site and causing mutation, thereby realizing gene knockout. The schematic diagram of the key components of the pX330-U6-Chimenic_BB-CBh-hSpCas9 (pX330-puro) transgene donor plasmid is shown in Figure 2. The key components include the crRNA fragment and tracerRNA fragment that make up the gRNA, the Cas9 protein and the complete expression cassette of the Puro resistance gene.

At present, the CRISPR system has been widely used as an efficient DNA editing tool. It has been reported that the system can cause specific double-strand breaks in the viral genome DNA at the cellular level, thereby inhibiting the replication of the corresponding virus in the cell, such as cccDNA viruses. HBV and pre-DNA forms of EBV and HIV exist during the infection process, but there is no report on the successful inhibition of fish DNA viruses using this strategy.

Contents of the invention

The present invention aims to solve the problem of insufficient inhibition of KHVD by existing methods, and provides a method for constructing a knockout vector based on a CRISPR/Cas9 system for targeted knockout of the KHV gene and its crRNA component.

The present invention is based on the CRISPR/Cas9 system targeted knockout KHV gene knockout vector construction method, according to the following steps:

1. Design crRNAs based on KHV genome TK (GenBank: AB375385.1) and DP (GenBank: AY939862.1) genes, optimize and synthesize crRNA-TK primers and crRNA-DP respectively through http://crispr.mit.edu/ The primers were treated in boiling water for 15 minutes, and then annealed overnight at natural cooling to obtain two double-stranded DNA annealed products with BbsI-cut sticky ends;

2. In order to facilitate the screening of cloned cell lines, the previous work of the present invention modified the pX330 vector (pX330-U6-Chimenic_BB-CBh-hSpCas9; http://www.addgene.org/42230) capable of carrying CRISPR/Cas9, The pX330-puro vector containing the puromycin resistance gene was successfully prepared, as shown in Figure 1;

3. The pX330-puro vector was digested with BbsI, and then SAP phosphatase was used for dephosphorylation reaction to recover the linearized pX330-puro vector;

4. Use T4 ligase to connect the linearized pX330-puro vector recovered in step 3 and the two annealed products obtained in step 1, respectively, to obtain the ligated product;

5. Transfer the connection product of step 4 into competent cells. After culturing, pick a single colony for PCR verification. For a single colony that is positive for PCR verification, extract the recombinant plasmid DNA, and use BbsI enzyme to perform single enzyme on the recombinant plasmid DNA. For cross-cutting verification, the recombinant plasmid DNA that is verified as positive is named pX330-puro TK or pX330-puro DP recombinant vector.

Wherein the crRNA-TK primer sequence in step 1 is:

Upstream primer: 5'-CACCGTGCTCTTGCCCGCGAACAT-3'

Downstream primer: 5'-AAACATGTTCGCGGGCAAGAGCAC-3'

The crRNA-DP primer sequence is:

Upstream primer: 5'-CACCGCCGTGTTCCTCACGTACTCG-3'

Downstream primer: 5'-AAACCGAGTACGTGAGGAACACGGC-3'

The specific crRNA elements based on the CRISPR/Cas9 system for targeted knockout of the KHV gene are crRNA-TK and crRNA-DP. The crRNA-TK is double-stranded DNA, and its sense strand is shown in SEQ ID NO: 1 in the sequence table. The sense strand is shown as SEQ ID NO: 2 in the sequence listing; the sense strand is 5'-GTGCTCTTGCCCGCGAACAT-3', and the antisense strand is 5'-ATGTTCGCGGGCAAGAGCAC-3'.

The crRNA-DP is double-stranded DNA, its sense strand is shown in SEQ ID NO: 3 in the sequence listing, and the antisense strand is shown in SEQ ID NO: 4 in the sequence listing; the sense strand 5'-CCGTGTTCCTCACGTACTCG-3 ', antisense strand 5'-CGAGTACGTGAGGAACACGG-3'.

In the present invention, we applied the CRISPR/Cas9 system to carry out the KHV proliferation inhibition test in carp carp fin ray KF-1 cell line. In order to effectively interfere with the replication of KHV virus, we selected TK and DP related to viral replication as the targets of the CRISPR/Cas9 system. In order to improve the detection sensitivity, we designed and synthesized the taqman hydrolysis probe according to the ORF-81 gene transcribed early in the virus. The results showed that the CRISPR/Cas9 system could effectively inhibit the proliferation of KHV in carp fin ray cell lines. The schematic diagram of targeted cleavage of the TK gene is shown in Figure 3. The Cas9 endonuclease complex can be oriented to the first 5 nt of the PAM sequence (NGG) in the DNA recognition region under the guidance of gRNA (targeting double-stranded DNA reversely complementary to crRNA) position causing a double-strand break.

Beneficial effects of the present invention:

In the present invention, the pX330-puro vector with the CRISPR/Cas9 system is connected to the crRNA targeting the TK and DP genes of the KHV genome to ensure the specific destruction of its target DNA fragment. Compared with the prior art, the invention has the characteristics of safety and high efficiency, and the virus inhibition rate can reach 80%. The present invention has the following advantages: 1. High targeting accuracy and obvious intracellular virus inhibition effect; 2. Simple construction and short experiment period.

Description of drawings

Figure 1 is the pX330-puro vector plasmid map containing the puromycin resistance gene; Figure 2 is the schematic diagram of the key elements of the transgene donor plasmid; Figure 3 is the schematic diagram of the targeted cleavage of the TK gene; Figure 4 is the pX330-puro TK and pX330-puro DP cells Cloning PCR verification results; Figure 5 is the KHV virus copy Real-time PCR monitoring standard curve; Figure 6 is the virus copy number monitoring KHV proliferation; Figure 7 is the virus copy number monitoring KHV proliferation; Figure 8 is the continuous determination of virus titers in cells increase or decrease.

Detailed ways

The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

Specific embodiment 1: This embodiment is based on the knockout vector construction method of the CRISPR/Cas9 system targeted knockout KHV gene, which is carried out according to the following steps:

1. Design crRNAs based on KHV genome TK (GenBank: AB375385.1) and DP (GenBank: AY939862.1) genes, optimize and synthesize crRNA-TK primers and crRNA-DP respectively through http://crispr.mit.edu/ The primers were treated in boiling water for 15 minutes, and then annealed overnight at natural cooling to obtain two double-stranded DNA annealed products with BbsI-cut sticky ends;

2. In order to facilitate the screening of cloned cell lines, the previous work of the present invention modified the pX330 vector (pX330-U6-Chimenic_BB-CBh-hSpCas9; http://www.addgene.org/42230) capable of carrying CRISPR/Cas9, The pX330-puro vector containing the puromycin resistance gene was successfully prepared, as shown in Figure 1, the full length of the recombined px330puro vector is 10046bp, as shown in SEQ ID NO: 21 in the sequence listing;

3. The pX330-puro vector was digested with BbsI, and then SAP phosphatase was used for dephosphorylation reaction to recover the linearized pX330-puro vector;

4. Use T4 ligase to connect the linearized pX330-puro vector recovered in step 3 and the two annealed products obtained in step 1, respectively, to obtain the ligated product;

5. Transfer the connection product of step 4 into competent cells. After culturing, pick a single colony for PCR verification. For a single colony that is positive for PCR verification, extract the recombinant plasmid DNA, and use BbsI enzyme to perform single enzyme on the recombinant plasmid DNA. For cross-cutting verification, the recombinant plasmid DNA that is verified as positive is named pX330-puro TK or pX330-puro DP recombinant vector.

Wherein the crRNA-TK primer sequence in step 1 is:

Upstream primer: 5'-CACCGTGCTCTTGCCCGCGAACAT-3'

Downstream primer: 5'-AAACATGTTCGCGGGCAAGAGCAC-3'

The crRNA-DP primer sequence is:

Upstream primer: 5'-CACCGCCGTGTTCCTCACGTACTCG-3'

Downstream primer: 5'-AAACCGAGTACGTGAGGAACACGGC-3'

The single enzyme digestion reaction system in step 3 is as follows:

Element Dosage 200ng/μL pX330-puro vector 5μL Buffer 3μL BbI 2μL ddH ₂ O 20 μL

Enzyme digestion reaction conditions: react at 37°C for 3h.

Reaction conditions for dephosphorylation in step 3: linearized pX330-puro carrier 30 μL, SAP 1 μL, Buffer 5 μL, ddH ₂ O 14 μL, reacted for 30 minutes, and stopped the action of SAP at 60°C for 15 minutes.

The connection system in step 4 is as follows:

Element Dosage Linearized pX330-puro vector 0.5μL Annealed product 7.5μL T4 DNA ligase 1μL Ligase Buffer 1μL

Ligation reaction conditions: react at 17°C for 12h.

The sequence of primers for identifying the pX330-puro TK positive plasmid in Step 5 is:

Upstream primer: 5'-CACCGTAAACTGACAGGTCGTGCAT-3'

Downstream primer: 5'-AAACATGTTCGCGGGCAAGAGCAC-3';

The primer sequence for pX330-puro DP positive plasmid identification is:

Upstream primer: 5'-CACCGTAAACTGACAGGTCGTGCAT-3'

Downstream primer: 5'-AAACCGAGTACGTGAGGAACACGGC-5';

The upstream detection primers of each positive plasmid used the human U6 promoter sequence (5'-CACCGTAAACTGACAGGTCGTGCAT-3'), and the downstream primers used the downstream primers for the synthesis of each crRNA annealing product. Detection PCR reaction conditions are as follows: pre-denaturation at 94°C for 10 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; after cycling, extend at 72°C for 10 min and store at 4°C.

Verify effect of the present invention by following experiment:

Experiment 1: Transient transfection of pX330-puro TK and pX330-puro DP inhibits the proliferation of KHV

A. Count KF-1 cells, passage in DMEM nutrient solution containing 10% FBS, glutamine, double antibody (100U/ml penicillin; 100μg/ml streptomycin) in 24-well culture plate at 1.0×10 ⁴ /well, 25 Cultivate for 24 hours at ℃, 5% CO ₂ to make the confluence of adherent cells reach 75%, discard the culture medium, wash the monolayer cells 3 times with serum-free and antibiotic-free DMEM, add 450 μL serum-free and antibiotic-free DMEM for later use.

B. Inoculation Add DMEM containing 4.5×10 ² TCID50KHV to each well, and after 2 hours at 25°C, replace the culture medium with 3% FBS, glutamine, double antibody (100U/ml penicillin; 100ug/ml streptomycin ) in DMEM at 25° C., 5% CO ₂ .

C. 0.5 days after exposure (0.5dpi), the prepared 2 μg pX330-puro TK and pX330-puro DP mixed plasmids were transfected into the fin ray cell line with LTX regent (Invitrogen). The proliferation of KHV in transfected cells was measured by fluorescent quantitative Real-time PCR and half the cell infection amount of the virus.

The primers for Real-time PCR detection of KHV virus ORF81 gene based on taqman hydrolysis probe detection principle are as follows:

5'-AGAGGTCTATGCGCGACTAT-3'

5'-FAM-AGACACTGAGAGCGTCATCGGTCA-BHQ1-3'

5'-CACATCTTGCCGGTGTACTT-3'

Experiment 2: KF-1 cells stably expressing pX330-puro TK and pX330-puro DP inhibit KHV proliferation

A. The pX330-puro TK and pX330-puro DP vectors were linearized with ApaLI endonuclease, and recovered by cold ethanol precipitation for later use.

B. Count KF-1 cells, subculture in 6-well plate at 2×10 ⁵ /well in DMEM medium containing 10% FBS and no antibiotics, and culture at 25°C 5% CO ₂ for 48 hours, so that the cell confluency rate is greater than 80%. Wash the cells twice with PBS without Ca ²⁺ and Mg ²⁺ ions, and then add 1.5 mL of serum-free optiMEM to each well.

C. Using LTX regent (Invitrogen), 2 μg of the prepared linearized vector per well was used to transfect the KF-1 fin ray cell line.

D. One day after transfection, screen with puromycin (Sigma aldrich) containing 500ng/ml, and replace the culture medium every 2-3 days. After 2 weeks, clones were picked by cloning ring and identified by PCR. The identification primers are as follows:

TK clone: Upstream primer: 5'-GATTCCTTCATATTTGCATATAC-3'

Downstream primer: 5'-ATGTTCGCGGGCAAGAGCAC-3'

DP clone: Upstream primer: 5'-GATTCCTTCATATTTGCATATAC-3'

Downstream primer: 5'-CGAGTACGTGAGGAACACGG-3'

Detection PCR reaction conditions are as follows: pre-denaturation at 94°C for 10 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; after cycling, extend at 72°C for 10 min and store at 4°C. Then electrophoresis was performed on 1% agarose gel containing EB, and the test results showed that the cloned cells were all positive, and the results are shown in FIG. 4 .

E. Count the cloned KF-1 cells and spread them evenly on a 96-well plate. When the confluence of the cells reaches 50%, add DMEM containing 100TCID50KHV to each well, and after 2 hours at 25°C, replace the culture medium with 3% FBS, glutamine, double antibody (100U/ml penicillin; 100μg/ml streptomycin) in DMEM at 25° C., 5% CO ₂ . The culture medium was changed every 3 days.

F, 7 days after inoculation, DNA extraction kit (Tiangen) was used to extract the supernatant and cell DNA respectively, and the aforementioned taqman hydrolysis probe Real-time PCR was used to detect the viral genome copy number. 400nM primers and 80nM taqman probes were added to each reaction, and three replicates were set up for each sample. The results are shown in Figure 5-7; , the T-ORF81 standard carrier was serially diluted, and the corresponding curve was obtained by using the taqman probe hydrolysis method. Figure 5 is used for the absolute quantification of the virus copy number. Virus copy number monitoring KHV proliferation is shown in Figure 6. Through the Real-time PCR detection method, using intracellular DNA as a template, the virus content at 7dpi is 1/3 of that in the control cells, and the difference is obvious, indicating that the transient vector introduced into the cells Effectively inhibits virus proliferation in infected cells. Virus copy number monitoring KHV proliferation is shown in Figure 7. Through the Real-time PCR detection method, using the cell supernatant and intracellular DNA as templates, the virus copy numbers inside and outside the cells at 7dpi were significantly inhibited by the stably expressed Cas9 and specific gRNA .

In addition, according to the Reed-Muench method, the virus titer was counted to measure the proliferation of KHV in transfected cells and control cells, and the inhibitory effect of antiviral elements on the process of KHV infection and proliferation was analyzed. The results are shown in Figure 8 and Table 1. In Figure 8, -○-indicates control, -■-indicates TK, -▲-indicates DP, and -▼-indicates TK&DP. Figure 8 is the continuous measurement of the increase and decrease of virus titer in the cells. By continuously counting the virus titer of the cell supernatant at 7-11dpi, the results show that: compared with the control, the cell supernatant of DP and TK&DP clones can no longer produce cytopathic changes at 10dpi Effect, the virus in the supernatant of the TK clone was also maintained at a low level.

Table 1 Determination of the increase or decrease of half the cell infection amount of virus in cells TCID50

the TCID ₅₀ (8dpi) TCID ₅₀ (9dpi) control 10 ^3.86 10 ^5.18 CRISPR/Cas9 10 ^2.52 10 ^3.44 and control ratio 1/22 1/55

After the control cells were infected with KHV, the virus could effectively proliferate in the cells, showing a higher infection titer.

Claims (5)

1. The knockout vector construction method based on CRISPR/Cas9 system targeted knockout KHV gene, is characterized in that the method is carried out according to the following steps: 1. Design crRNAs based on the TK and DP genes of the KHV genome, synthesize crRNA-TK primers and crRNA-DP primers respectively, treat them in boiling water for 15 minutes, then cool down naturally and anneal overnight to obtain two DNA double strands with BbsI enzyme-cut sticky ends annealed product; 2. The pX330 vector capable of carrying CRISPR/Cas9 was transformed, and the pX330-puro vector containing the puromycin resistance gene was successfully prepared; 3. The pX330-puro vector was digested with BbsI, and then SAP phosphatase was used for dephosphorylation reaction to recover the linearized pX330-puro vector; 4. Use T4 ligase to connect the linearized pX330-puro vector recovered in step 3 and the two annealed products obtained in step 1, respectively, to obtain the ligated product; 5. Transfer the connection product of step 4 into competent cells. After culturing, pick a single colony for PCR verification. For a single colony that is positive for PCR verification, extract the recombinant plasmid DNA, and use BbsI enzyme to perform single enzyme on the recombinant plasmid DNA. For cross-cutting verification, the recombinant plasmid DNA that is verified as positive is named pX330-puro TK or pX330-puro DP recombinant vector. 2. The knockout vector construction method based on CRISPR/Cas9 system targeted knockout KHV gene according to claim 1, characterized in that the single enzyme digestion reaction system in step 3 is as follows: Enzyme digestion reaction conditions: react at 37°C for 3h. 3. The method for constructing a knockout vector for targeted knockout of KHV gene based on CRISPR/Cas9 system according to claim 1, characterized in that the dephosphorylation reaction conditions in step 3: 30 μL of linearized pX330-puro vector, 1 μL of SAP, Buffer 5 μL, ddH ₂ O 14 μL, after reacting for 30 minutes, stop the action of SAP at 60°C for 15 minutes. 4. The knockout vector construction method based on CRISPR/Cas9 system targeted knockout KHV gene according to claim 1, characterized in that the connection system in step 4 is as follows: Ligation reaction conditions: react at 17°C for 12h. 5. The crRNA element based on the CRISPR/Cas9 system targeted knockout KHV gene is characterized in that the crRNA element is crRNA-TK and crRNA-DP, and the crRNA-TK is double-stranded DNA, and its sense strand is as SEQID in the sequence table NO: 1, the antisense strand is shown in SEQ ID NO: 2 in the sequence listing; the crRNA-DP is double-stranded DNA, and its sense strand is shown in SEQ ID NO: 3 in the sequence listing, the antisense The chain is shown as SEQ ID NO: 4 in the Sequence Listing.