CN113684209A

CN113684209A - sgRNA capable of effectively editing pig PCBP1 gene and application thereof

Info

Publication number: CN113684209A
Application number: CN202110756558.2A
Authority: CN
Inventors: 欧阳红生; 逄大欣; 谢子聪; 齐春云; 唐小春
Original assignee: Chongqing Research Institute Of Jilin University
Current assignee: Chongqing Research Institute Of Jilin University
Priority date: 2021-07-05
Filing date: 2021-07-05
Publication date: 2021-11-23

Abstract

The invention provides a sgRNA capable of effectively knocking out a porcine PCBP1 gene, which is characterized in that: the sgRNA specifically targets the 485 th to 504 th sequence of the pig PCBP1 gene, and the nucleotide sequence of the coding gene is shown in SEQ ID NO. 1. According to the invention, the PCBP1 gene is successfully knocked out in the PK15 cell line by using the CRISPR/Cas9 technology, and the result shows that the knocked-out cell line can inhibit the proliferation of CSFV in cells to a certain extent, so that the PCBP1 knocked-out gene editing pig with the capacity of resisting CSFV is possible to prepare, and the method has important significance for screening research of swine fever virus resisting medicine targets and genetic breeding of antiviral pigs.

Description

sgRNA capable of effectively editing pig PCBP1 gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a sgRNA capable of effectively editing a porcine PCBP1 gene and a PCBP1 knockout cell line obtained by applying the sgRNA.

Background

Poly (rC) binding protein 1 is an RNA or DNA binding protein of 38kDa relative molecular weight, belonging to the family of PCBPs. Members of the PCBP family include PCBP1-4, hnRNP K five members, PCBP1 is an intron-free gene, and it has been reported that PCBP1 protein can inhibit RLR-mediated signaling pathways by degrading adaptor protein MAVS. At the same time, the protein can promote cGAS to be combined with DNA in cytoplasm in a virus infection-dependent mode, and recent literature shows that the PCBP1 protein can be combined with N of Classical Swine Fever Virus (CSFV)^proInteract to promote the replication of CSFV. Therefore, the gene locus can be used as a potential drug target for resisting CSFV and has important significance for preparing transgenic pigs resisting CSFV.

CRISPR/Cas9 is an adaptive immune defense formed during long-term evolution of bacteria and archaea, and can be used to fight invading viruses and foreign DNA. The CRISPR/Cas9 system provides immunity by integrating fragments of invading phage and plasmid DNA into the CRISPR and using the corresponding CRISPR RNAs (crRNAs) to direct degradation of homologous sequences. Later, researchers discovered that it was a more accurate and versatile gene editing tool that could be used to delete, add, activate, or repress genes of interest from other organisms, including human, mouse, pig, cow, sheep, zebrafish, bacteria, drosophila, yeast, nematode, and crop plants. Accurate editing of the genome, such as conditional gene knockout, gene knock-in, gene replacement, point mutation, and the like, can be achieved using this mechanism. The CRISPR/Cas9 technology is favored by the convenience of operation and high-efficiency gene editing capacity, and after the technology is made up for a plurality of defects of the traditional gene editing technology, the technology has wide application prospect in the field of gene editing. The invention is based on a CRISPR/Cas9 fixed-point editing platform established in the early stage, takes porcine fetal fibroblasts and PK-15 cells as research objects, and researches the influence of the porcine PCBP1 gene on the infection of the classical swine fever virus.

Disclosure of Invention

The invention aims to provide a sgRNA sequence capable of effectively knocking out a pig PCBP1 gene, and a pig PCBP1 gene knockout cell and a pig model can be prepared by adopting a CRISPR/Cas9 system based on the sgRNA.

To achieve the above object, the present invention first provides sgRNA specifically targeting the porcine PCBP1 gene, which targets the 485 th to 504 th sequences of the porcine PCBP1 gene.

The invention discovers that the sequence is adopted as a target sequence through a large amount of screening, and has higher targeting efficiency and knockout efficiency.

Aiming at the target sequence on the pig PCBP1 gene, the invention designs specific sgRNA, and further discovers that the sgRNA corresponding to the nucleotide sequence shown in SEQ ID NO.1 can realize higher targeting efficiency and PCBP1 gene knockout efficiency.

As a more preferable technical scheme, the nucleotide sequence of the sgRNA coding gene of the specific targeting pig PCBP1 gene is shown in SEQ ID No. 1.

The invention also provides a CRISPR/Cas9 gene knockout vector containing the sgRNA of the specific targeted pig PCBP1 gene.

As a more preferable technical scheme, the CRISPR/Cas9 gene knockout vector is a vector connected with the sgRNA of the specific targeted pig PCBP1 gene.

The invention also provides a construction method of the CRISPR/Cas9 gene knockout vector, which comprises the following steps: and connecting the sgRNA of the specific targeted pig PCBP1 gene with a vector to obtain the CRISPR/Cas9 gene knockout vector.

As a more preferable technical scheme, the construction method comprises the following steps: synthesizing a nucleotide sequence of a coding gene of the sgRNA as shown in SEQ ID No.1, wherein a complementary chain of the coding gene is shown in SEQ ID No.2, and then respectively annealing DNA sequences of the single-stranded sgRNAs to form 1 oligonucleotide chain of the sgRNAs targeting different loci of the unique intron of pig PCBP 1; the oligonucleotide was then ligated into PX330 plasmid vector.

The invention also provides application of the sgRNA of the specific targeted pig PCBP1 gene or the CRISPR/Cas9 gene knockout vector containing the sgRNA in pig PCBP1 gene knockout.

The invention also provides a method for preparing a PCBP1 gene knockout cell line, which comprises the following steps: introducing a CRISPR/Cas9 gene knockout vector containing sgRNA of the specific targeted pig PCBP1 gene into cells to knock out the PCBP1 gene. The CRISPR/Cas9 gene knockout vector can be introduced into cells by conventional biological methods such as transfection. The method for preparing the PCBP1 gene knockout cell can screen positive cells by adopting conventional methods such as PCR and the like after introducing the CRISPR/Cas9 gene knockout vector containing the sgRNA of the specific targeted pig PCBP1 gene into the cells.

The invention also provides a method for preparing the PCBP1 gene knockout pig model, which realizes the knockout of the PCBP1 gene of the pig by using the CRISPR/Cas9 gene knockout carrier.

The invention also provides a kit comprising the sgRNA, the kit functioning as at least one of the following one to five: firstly, specifically recognizing a porcine PCBP1 gene; knocking out a porcine PCBP1 gene; thirdly, carrying out gene editing on the PCBP1 gene of the pig; fourthly, preventing the pig from being infected by virus; and fifthly, breeding or preparing the gene editing pig.

The beneficial effects of the invention at least comprise:

on the premise of one sgRNA in a pig genome capable of specifically recognizing the pig PCBP1 gene, the PCBP1 gene is successfully knocked out in a PK15 cell line by using a CRISPR/Cas9 technology, and the result shows that the knocked-out cell line can inhibit the proliferation of CSFV in cells to a certain extent, so that the PCBP1 knocked-out gene editing pig with the inherent CSFV resistance capability is possible to prepare, and the method has important significance for screening research of swine fever virus resistance medicament targets and genetic breeding of antiviral pigs.

The pig PCBP1 gene knockout method provided by the invention has strong practicability, and provides an effective method and a foundation for the functional research and application of the pig PCBP1 gene.

Drawings

FIG. 1 is a schematic diagram of the design of sgRNA targeting pig PCBP1 gene;

FIG. 2 is a sequencing peak diagram of the cleavage efficiency of 3 sgRNAs targeting the porcine PCPB1 gene;

FIG. 3 shows the detection results of PCBP1 knockout positive clone immunoblot;

FIG. 4 is a proliferation profile of CSFV in wild type PK-15 cells and PCBP1 knock-out PK-15 cell clones.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

According to the invention, the nucleotide sequence of the pig PCBP1 gene is downloaded from a database, based on the analysis of the pig PCBP1 gene sequence, the screening of the target site of the pig PCBP1 gene and the design and screening of sgRNA aiming at the target site are carried out according to the design principle of identifying the target site by CRISPR/Cas9, and the sgRNA-95 with the highest knockout efficiency is finally obtained by screening, as shown in SEQ ID NO.1, and the CRISPR/Cas9 knockout vector containing the sgRNA is constructed, so that the high-efficiency knockout of the pig PCBP1 gene is realized.

In the following examples, the effects of 3 sgrnas designed according to the present invention for different target sites are described as examples.

Example 1 design and Synthesis of sgRNA sequences

3 sgRNA sequences of targeted pig PCBP1 sites are designed and synthesized, specifically, sequences from 402 th site to 421 th site of sgRNA-91 targeted pig gene, sequences from 485 th site to 504 th site of sgRNA-95 targeted pig PCBP1 gene, and sequences from 371 th site to 390 th site of sgRNA-97 targeted pig PCBP1 gene. Annealing the DNA sequences of 6 single-stranded sgRNAs to form 3 oligonucleotide chains of the sgRNAs targeting different loci of the unique intron of pig PCBP 1; the oligonucleotide was then ligated into PX330 plasmid vector.

The sequences of the 3 sgrnas and the sequences of their action sites are, respectively, the coding gene sequences of sgRNA-91, sgRNA-95, and sgRNA-97 and the sequences of their action sites are as follows:

sgRNA-91 sequence: 5-GCGCGGATCAACATCTCGGA-3;

sequence of sgRNA-91 site of action: 5-TCCGAGATGTTGATCCGCGC-3;

sgRNA-95 sequence: 5-CGCTATGATCATCGACAAGC-3;

sequence of sgRNA-95 site of action: 5-GCTTGTCGATGATCATAGCG-3;

sgRNA-97 sequence: 5-GTCGGTTAAGAGGATCCGCG-3;

sequence of sgRNA-97 site of action: 5-CGCGGATCCTCTTAACCGAC-3.

The targeting pattern of 3 different sgrnas against PCBP1 gene is shown in fig. 1, the cyan rectangle box represents the only exon of PCBP1 gene on which three sgrnas were designed, the relative positions are shown in fig. 1, and F and R represent the positions of the sequencing primers relative to the target site.

Example 2 evaluation and screening of high-potency sgRNAs

After the expression vectors of 3 sgRNAs are further sequenced, the large extraction of plasmids and the ethanol precipitation of the plasmids are carried out, the expression vectors of PX330 of three sgRNAs with certain concentrations after purification are introduced into fetal fibroblasts of pigs by an electroporation transfection mode, the genome of each group of cells is extracted 72 hours after transfection, then a PCR reaction is carried out by using specific primers for detecting mutation efficiency, the obtained PCR product is sent to sequencing on the one hand, the cleavage efficiency of each sgRNA is preliminarily evaluated by analyzing a sequencing peak diagram, and simultaneously the residual PCR product is used for connecting pLB vectors or T7E1 analysis to accurately evaluate the cleavage efficiency of each sgRNA to a target gene.

The sequencing peak map of 3 different sgRNA cleavage efficiencies is shown in fig. 2, a red arrow indicates a cleavage site of Cas9 protein, and as theory, after sequencing by using an upstream primer F, a peak overlapping condition is observed before and after the cleavage site, the sgRNA-91 cleavage efficiency is the lowest and close to 0, the sgRNA-97 cleavage efficiency is higher, and the sgRNA-95 cleavage efficiency is the highest.

By comparing and evaluating the cleavage efficiency for each sgRNA, it can be seen from the sequencing peak diagram of fig. 2 that sgRNA-95 can effectively knock out a gene, and the remaining two cannot effectively knock out a gene, so that the subsequent examples were selected.

The RNA sequence of the sgRNA related by the invention is as follows:

5-CGCUAUGAUCAUCGACAAGC-3(SEQ ID NO.3)。

the complement of the RNA sequence of the sgRNA is:

5-GCUUGUCGAUGAUCAUAGCG-3(SEQ ID NO.4)。

wherein, the DNA sequence of the sgRNA related by the invention is as follows:

5-CGCTATGATCATCGACAAGC-3(SEQ ID NO.1)。

the complement of the DNA sequence of the sgRNA is:

5-GCTTGTCGATGATCATAGCG-3(SEQ ID NO.2)。

example 3

(1) The PX330 plasmid was electrotransfected.

Recovering a PK15 cell line, regulating the cell state to a transfectable state by using a serum starvation method, digesting the cells by using 0.5% of pancreatin, washing the cells for 2-3 times by using DPBS, removing the supernatant, adding opti-MEM electrotransfection buffer solution, adding 30 mu g of PX330-sgRNA95 plasmid, gently mixing the mixture by using a pipettor, gently transferring the mixture into a 0.2cm electric shock cup, and placing the electric shock cup on an electroporation apparatus for electric shock operation. After the completion of the electric shock, the electric shock cup was allowed to stand at room temperature for 3 minutes, and then the mixed solution in the electrode cup was transferred to a cell culture dish. Finally, the cell culture dish is placed in a carbon dioxide incubator at 37 ℃ for culture. After 12 hours of incubation, the medium was changed.

(2) Selection and identification of PCBP1 knockout cell clones.

After electrotransfection for 72h, the PK15 cell line is paved in a 100mm cell culture dish by a limiting dilution method, and the cell culture solution is replaced once in 2-3 days. After the cell clone grows up after 9-10 days, picking the clone into a 24-hole cell culture plate and then culturing. After 2-3 days, when the cells in the 24-well plate grow to a certain confluency, carrying out passage on the cells, simultaneously taking out partial cloned cells, cracking the cells by NP40 cracking buffer solution, and further verifying the knockout event by a PCR (polymerase chain reaction) and sequencing method.

Example 4 Western Blot to detect gene expression in PCBP1 knockout positive clones.

Recovering PCBP1 knockout positive clone from liquid nitrogen, expanding to 100mm culture dish, collecting one third cell precipitate when the confluence reaches 90%, adding 200 μ L RIPA cell lysate (containing PMSF and protease inhibitor mixture), incubating on ice for 10min, centrifuging, and taking supernatant to measure protein concentration by BCA. SDS-PAGE samples were prepared by adding 40. mu.g of protein to each sample.

The final immunoblot is shown in fig. 3, wherein #1503 and #2703 are two monoclonals with different genotypes picked after sgRNA-97 knockout, and #4004 is one monoclonal picked after sgRNA-95 knockout, and as shown in the figure, the protein water level of the knockout clone is reduced relative to that of the wild WT, wherein the PCBP1 gene in the clone #4004 is close to complete knockout.

Example 5 RT-qPCR the copy number of CSFV in positive clones at different time points was determined.

Recovering positive clone of PCBP1 knockout from liquid nitrogen, expanding to 100mm culture dish, and adding 1 × 10 per well⁵Individual cells were passaged to 6-well plates, 3 biological replicates were set at each time point, with the addition of negative controls. After passaging to 6-well plates, the cells were attached to the plates for 6 hours, and CSFV hemotoxins that had passed through a 0.22 μm filter were inoculated at an MOI of 0.01, incubated in an incubator for 2 hours, and then cultured by adding normal culture medium, and the cells were collected at time points of 12, 24, 36, and 48 hours, respectively.

Adding 15 ug of RNA template, inverting by two-step method to obtain cDNA as qPCR template, and setting qPCR program as follows: pre-denaturation at 95 ℃ for 15 min; performing 40 cycles at 95 ℃ for 10s in the following three steps; 59 ℃,20 s; 72 ℃ for 30 s; melt Curve.

The copy number of CSFV in clone is shown in figure 4, and clone #4004 is selected to carry out a CSFV infection experiment at a cell level, compared with wild WT, the virus copy number in clone #4004 is obviously lower than WT at different time points of CSFV infection, which indicates that the PCBP1 gene knockout can obviously inhibit the replication of CSFV in a target cell.

The porcine PCBP1 gene is knocked out by using a CRISPR/Cas9 knocking-out system. Through the specific selection of the pig PCBP1 gene target site and the specific design of the sgRNA aiming at the target site, the sgRNA capable of efficiently targeting the pig PCBP1 gene is obtained, the targeting efficiency of the CRISPR/Cas9 knockout vector is obviously improved, and further the knockout efficiency of the pig PCBP1 gene is obviously improved. Experiments prove that the CRISPR/Cas9 knockout vector provided by the invention can knock out the PCBP1 gene of the pig with high efficiency, and the construction efficiency of PCBP1 gene knockout cells and PCBP1 gene knockout pigs is obviously improved.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> Chongqing institute of Jilin university

<120> sgRNA capable of effectively editing porcine PCBP1 gene and application thereof

<130> 2021

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 1

cgctatgatc atcgacaagc 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 2

gcttgtcgat gatcatagcg 20

<210> 3

<211> 20

<212> RNA

<213> Artificial sequence (artificial sequence)

<400> 3

cgcuaugauc aucgacaagc 20

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

gcuugucgau gaucauagcg 20

Claims

1. An sgRNA that can effectively knock out pig PCBP1 gene, its characterized in that: the sgRNA specifically targets the sequence from position 485 to 504 of the porcine PCBP1 gene.

2. The sgRNA of claim 1, which is effective in knocking out the PCBP1 gene of a pig, wherein: the nucleotide sequence of the coding gene is shown in SEQ ID NO. 1.

3. A CRISPR/Cas9 gene knockout vector, characterized in that: the CRISPR/Cas9 gene knockout vector is a vector connected with sgRNA of the specific targeted pig PCBP1 gene.

4. The method of constructing a knock-out vector of claim 3, wherein: synthesizing a nucleotide sequence of a coding gene of the sgRNA as shown in SEQ ID No.1, wherein a complementary chain of the coding gene is shown in SEQ ID No.2, and then respectively annealing DNA sequences of the single-stranded sgRNAs to form 1 oligonucleotide chain of the sgRNAs targeting different loci of the unique intron of pig PCBP 1; the oligonucleotide was then ligated into PX330 plasmid vector.

5. The application of the sgRNA capable of effectively knocking out porcine PCBP1 gene or the CRISPR/Cas9 gene knockout vector containing the sgRNA in porcine PCBP1 gene knockout of claim 1.

6. A method of making a PCBP1 gene knockout cell line, comprising: introducing a CRISPR/Cas9 gene knockout vector containing sgRNA of the specific targeted pig PCBP1 gene into cells to knock out the PCBP1 gene.

7. A method for preparing a PCBP1 gene knockout pig model is characterized in that: the CRISPR/Cas9 gene knockout vector is used for realizing the knockout of the porcine PCBP1 gene.

8. A kit, characterized in that: comprises the sgRNA, and the function of the kit is at least one of the following one to five:

firstly, specifically recognizing a porcine PCBP1 gene;

knocking out a porcine PCBP1 gene;

thirdly, carrying out gene editing on the PCBP1 gene of the pig;

fourthly, preventing the pig from being infected by virus;

and fifthly, breeding or preparing the gene editing pig.