Preparation of sgRNA and HaCaT cell models of human genome sequences at high-frequency integration sites of targeted high-risk HPV
阅读说明：本技术 靶向高危型HPV高频整合位点处人基因组序列的sgRNA和HaCaT细胞模型的制备 (Preparation of sgRNA and HaCaT cell models of human genome sequences at high-frequency integration sites of targeted high-risk HPV ) 是由 汪辉 马丁 任慈 丁文成 李晓敏 熊劲风 于 2020-04-22 设计创作，主要内容包括：本发明涉及一种靶向高危型HPV高频整合位点处人基因组序列的sgRNA和HaCaT细胞模型的制备。本发明针对HPV16在13q22和HPV18在8q24区域的整合位点分别设计特异性的sgRNA,并得到sgRNA双链DNA,将sgRNA双链引物分别与CRISPR/Cas9载体连接获得sgRNA表达载体,筛选得到sgRNA打靶质粒；运用克隆构建及PCR扩增分别合成含有高危型人乳头状瘤病毒HPV基因的线性Donor片段,与sgRNA打靶质粒共转染HaCaT细胞,获得HPV基因定点整合的HaCaT细胞模型。本发明运用CRISPR/Cas技术建立的HPV16及HPV18在人基因组高频位点整合的细胞模型,促进了HaCaT细胞的生长增殖,为高危型HPV整合在宫颈癌发病中的机制研究提供新的方法。(The invention relates to preparation of sgRNA and HaCaT cell models of human genome sequences at high-frequency integration sites of targeted high-risk HPV. Specific sgRNA is respectively designed aiming at the integration sites of HPV16 in the region of 13q22 and HPV18 in the region of 8q24, sgRNA double-stranded DNA is obtained, sgRNA double-stranded primers are respectively connected with a CRISPR/Cas9 vector to obtain a sgRNA expression vector, and a sgRNA targeting plasmid is obtained through screening; respectively synthesizing linear Donor segments containing high-risk human papilloma virus HPV genes by cloning construction and PCR amplification, and co-transfecting HaCaT cells with sgRNA targeting plasmids to obtain a HaCaT cell model integrated with HPV genes at fixed points. The invention uses the cell models of HPV16 and HPV18 integrated at the high-frequency locus of the human genome established by the CRISPR/Cas technology to promote the growth and proliferation of HaCaT cells and provide a new method for the mechanism research of the high-risk HPV integration in the onset of cervical cancer.)
1. A sgRNA targeting a human genome sequence at a high-frequency integration site of high-risk HPV (human papilloma virus), which is characterized in that: the high-risk HPV is respectively 16 type human papilloma virus, and when the high-frequency integration site of the HPV is 13q22 site, the 13q22-sgRNA sequence is as follows: TACAGCCACTCACAGCCCGC, as shown in SEQ ID No. 1;
or when the high-risk HPV is human papilloma virus type 18 and the high-frequency integration site of the HPV is 8q24 site, the sequence of 8q24-sgRNA is as follows: ACAAATAACTGTAAAGAGTA, as shown in SEQ ID No. 2.
2. A DNA sequence corresponding to the sgRNA of claim 1, characterized in that: the 8q24-sgRNA sequence corresponding DNA is:
8q24-sgRNA-DNA F：ACAAATAACTGTAAAGAGTA
R：TACTCTTTACAGTTATTTGT；
or, the 13q22-sgRNA sequence corresponding DNA is:
13q22-sgRNA-DNA F：TACAGCCACTCACAGCCCGC
R：GCGGGCTGTGAGTGGCTGTA。
3. a preparation method of a HaCaT cell model for high-risk HPV gene site-specific integration is characterized by comprising the following steps: the method comprises the following steps:
1) designing a sgRNA sequence aiming at a high-frequency integration site of high-risk HPV in a human genome and combining with base pairing rule specificity to obtain double-stranded DNA of the sgRNA sequence;
2) CAACCG is connected to the left end of a sense strand of the double-stranded DNA sequence of the sgRNA, and AAACNNNC is connected to the two ends of an antisense strand of the double-stranded DNA sequence of the sgRNA, wherein NNN represents the antisense strand sequence of the sgRNA; preparing double-stranded DNA of the sgRNA into double-stranded primers, and performing enzyme digestion connection on the double-stranded primers and a pSpCas9(BB) -2A-GFP expression vector through Bpil restriction nuclease to construct an sgRNA expression plasmid;
3) transfecting the sgRNA expression plasmids into target cells respectively, and screening effective sgRNA targeting plasmids through a T7E1 experiment;
4) the efficient sgRNA targeting plasmid and a linear Donor fragment containing high-risk Human Papilloma Virus (HPV) genes are used for co-transfecting HaCaT cells, and the HaCaT cell model for the fixed-point integration of the high-risk HPV genes is obtained.
4. The method for preparing the HaCaT cell model for the site-specific integration of the high-risk HPV genes according to claim 3, which is characterized in that: in the step 1), the high-risk HPV is human papillomavirus type 16 or human papillomavirus type 18 respectively.
5. The method for preparing the HaCaT cell model for the site-specific integration of the high-risk HPV genes according to claim 4, which is characterized in that: when the high-risk HPV is HPV16 and the HPV gene integration site is 13q22 site, the sgRNA sequence is as follows: TACAGCCACTCACAGCCCGC, respectively;
or when the high-risk HPV is HPV18 and the HPV gene integration site is 8q24 site, the sgRNA sequence is ACAAATAACTGTAAAGAGTA.
6. The method for preparing the HaCaT cell model for the site-specific integration of the high-risk HPV genes according to claim 5, which is characterized in that: in the step 2), when the HPV gene integration site is 8q24 site, the double-stranded primer is:
8q24-sgRNA-primer F：CACCGACAAATAACTGTAAAGAGTA
R：AAACTACTCTTTACAGTTATTTGTC
or when the integration site of the HPV gene is 13q22 site, the sgRNA double-stranded DNA is as follows:
13q22-sgRNA3-primer F：CACCGTACAGCCACTCACAGCCCGC
R：AAACGCGGGCTGTGAGTGGCTGTAC。
7. the method for preparing the HaCaT cell model for the site-specific integration of the high-risk HPV genes according to claim 5, which is characterized in that: in the step 3), when the HPV gene integration site is 13q22 site, the sgRNA targeting plasmid consists of a CRISPR-Cas9 vector and double-stranded DNA of sgRNA targeting 13q22 site;
or when the HPV gene integration site is 8q24 site, the sgRNA targeting plasmid consists of a CRISPR-Cas9 vector and double-stranded DNA of sgRNA targeting 8q24 site.
8. The method for preparing the HaCaT cell model for the site-specific integration of the high-risk HPV genes according to claim 3, which is characterized in that: when the high-risk human papilloma virus HPV is HPV16, a linear Donor fragment containing the high-risk human papilloma virus HPV16 gene is formed by linearization of Donor skeleton plasmids, and the downstream of an ad element of the Donor skeleton plasmids is sequentially connected with a left arm2 gene, an HPV16 gene fragment, a tag gene and a right arm2 gene in a seamless mode; wherein the content of the first and second substances,
the left arm2 is shown in SEQ ID No.11,
the label gene is shown as SEQ ID No.13,
the right arm2 is shown in SEQ ID No.12,
the HPV16 gene segment is shown in SEQ ID No.10 and comprises URR gene of HPV16, E6 gene and E7 gene;
or when the high-risk human papilloma virus is HPV18, a linear Donor fragment containing the high-risk human papilloma virus HPV18 gene is formed by linearization of Donor skeleton plasmids, and the downstream of an ad element of the Donor skeleton plasmids is sequentially connected with a left arm1 gene, an HPV18 gene fragment, a tag gene and a right arm1 gene in a seamless mode; wherein the content of the first and second substances,
the left arm1 gene is shown as SEQ ID No.8,
The HPV18 gene segment is shown in SEQ ID No.7 and comprises URR gene of HPV18, E6 gene and E7 gene;
the right arm1 gene is shown in SEQ ID No.9,
the nucleotide sequence of the label gene is specifically shown as SEQ ID No.13 and consists of a CMV promoter, a green fluorescent protein gene, a puromycin gene and an SV40 PolyA sequence;
the Donor skeleton plasmid is pDC 515.
9. The method for preparing the HaCaT cell model for the site-specific integration of the high-risk HPV genes according to claim 3, which is characterized in that: in the step 4), during transfection, the linear Donor fragment and the sgRNA targeting plasmid are wrapped by a transfection reagent PEI, the ratio of the total mass of the linear Donor fragment and the sgRNA targeting plasmid to the transfection reagent is 1:3, and the mass ratio of the linear Donor fragment to the sgRNA targeting plasmid is 1: 1.
10. A HaCaT cell model of claim 3, wherein: the cells are HaCaT cells.
Technical Field
The invention relates to the technical field of medical biology, in particular to preparation of sgRNA and HaCaT cell models of human genome sequences at high-frequency integration sites of targeted high-risk HPV.
Background
Cervical cancer is the third largest malignancy in women worldwide, with a high incidence of the first gynecological malignancy. Infection with Human Papilloma Virus (HPV) has been identified as a causative agent of cervical cancer, most of which are caused by persistent infection with high-risk HPV, of which HPV16 and HPV18 are the two most important types. In the early stages of HPV infection, the viral genome replicates as extrachromosomal episomal DNA as the host genome replicates. As the precancerous lesion progresses, the integration of the oncogene of the virus with the host genome gradually occurs, and most lesions show the integration of the viral gene at the stage of progressing to cervical cancer, and the integration is closely related to the progression of the precancerous lesion to cancer and is taken as a marker of the disease progression.
The existing studies on HPV have mostly focused on the role of E6 and E7 oncogenes, ignoring the important role of HPV integration. According to TCGA data, in the cervical cancer positive to HPV16, 76% of tumors find the integration of HPV viral genes, and in the cervical cancer tissues positive to HPV18, 100% of tumors have the integration of HPV viral genes. The research shows that the in vitro transfection of HPV can promote the immortalization of normal epithelial cells, but the malignant transformation of the cells still needs the participation of other gene abnormal events, such as the mutation of Ras gene or the deletion of DCC gene, the genetic detection of the immortalized cells caused by the HPV transfection shows that a large number of unstable events, such as diploid, polyploid, chromosome deletion, translocation and the like, are generated in the cell genome. The chromosome aneuploidy and mutation of cells after HPV gene transfection indicate that the occurrence of the cervical cancer has important participation of cell instability events besides the role of E6/E7.
HPV integration is an important cause of induced genomic instability. A large body of research data confirms that: integration of HPV into the human genome does not occur randomly, but there are multiple high frequency integration sites. Most of the sites are distributed near fragile sites, oncogenes or cancer suppressor genes of the genome, and the integration not only destroys the structure and function of key genes, but also causes a large amount of amplification, deletion, rearrangement, translocation and the like of the genome near the integration sites, resulting in strong instability of the genome. Research shows that HPV genes integrated near MYC in HeLa cells are amplified by nearly 30 copies, including the URR region of the HPV genes, which is rich in an epithelial-specific enhancer element, and the continuous amplification of the HPV genes induces strong genomic instability, causes multiple fragmentation and repair of genomic DNA at the integration site, leads to massive amplification of the MYC genes, and promotes cell canceration. It can be seen that integration of HPV into the host genome is a critical event in the development of major cervical diseases.
However, the E6 and E7 genes are only one of the expression products of HPV infection and integration host cells, and the inevitable relation between E6/E7 gene expression and cell canceration is not yet proved by the phenomena, the molecular abnormal events of cancer cell genes are all final products of cells after long-term clonal evolution, and the biological process that HPV integration induces genome instability and further promotes cell canceration cannot be known. Because of the characteristics of the HPV virus, the complete HPV virus with biological activity cannot be cultured in vitro, and the HPV gene transfected cell model cannot reveal the real pathogenic mechanism of the HPV. Therefore, we need to establish a new cell model to reduce the biological process of HPV integration pathogenesis, and provide a new method and theoretical basis for the research of HPV integration carcinogenesis.
In recent years, the emergence of CRISPR (regularly clustered interspaced short palindromic repeats) technology brings great convenience for the research of molecular biology by vast researchers. Under the recognition of the targeted sgRNA, the Cas9 nuclease protein can cut a DNA double strand at a specific locus of a genome, once the DNA double strand is broken, a cell can spontaneously start a non-homologous end repair mechanism to connect broken ends, and because the repair mode has low fidelity, the base mismatching, insertion, deletion and the like of the broken locus are easily caused, the normal function of the gene is weakened or even lost, so that the purpose of gene knockout is achieved. On the other hand, if a homology repair template containing HPV DNA is provided while the sgRNA acts, the cell initiates a homologous end repair mechanism to introduce the HPV DNA into the fracture site, so that the site-directed knock-in of the HPV gene is realized, and the sufficient technical support is provided for establishing a cell model of HPV site-directed integration.
However, there are still difficulties in successfully establishing a cell model for site-directed integration of HPV genes. First, cells have a preference in repairing DNA breaks, and in general, cells will prefer non-homologous end repair mechanisms over homologous end repair mechanisms, greatly limiting the efficiency of gene knock-in. Furthermore, in vitro knock-in still has many uncontrollable factors. The feasibility of gene knock-in is greatly reduced along with the length of a knock-in fragment, and most of current researches realize single-gene disease related gene point mutation or small fragment tags insertion of special sites by a CRISPR (clustered regularly interspaced short palindromic repeats) and Donor combined method, such as gene mutation for correcting sickle-shaped cell anemia, and knock-in of an EGFP (enhanced green fluorescent protein) gene at an AAVS1 site, wherein the length of the knock-in gene is mostly not more than 1000 bp. Moreover, in most of the in vitro cell experiments at present, viruses are taken as vectors for gene knock-in, so that the probability of random integration of target genes is increased, and more biological safety problems are brought.
At present, cases of establishing a gene knock-in cell model by using the CRISPR technology are still rare, and most researches are still in continuous technical exploration and method improvement. Researches show that the Donor Donor in a linear double-chain or linear single-chain form is more beneficial to knock-in than the Donor Donor in a ring form, and researches also prove that the increase of the lengths of the homologous arms at the two ends of the Donor Donor within a certain range can also improve the efficiency of gene knock-in, and the homologous arm with the length of more than 1000bp can reduce the efficiency of gene knock-in. More researchers found that many small molecule compounds, such as cell cycle synchronization drugs and DNA ligase inhibitors, could improve the possibility of gene knock-in.
Until now, no mature HPV gene integrated cell model exists, which is largely due to the technical difficulty of in vitro large fragment gene site-specific knock-in, so that a safe and effective gene knock-in system needs to be established to provide technical support for the HPV gene site-specific integrated cell model.
Disclosure of Invention
According to big data analysis, the HPV gene segment with the highest integration frequency is about 1600bp, which contains E6 and E7 genes called oncogenes and Upstream Regulatory Region (URR) for positively regulating the expression of E6 and E7 genes, and exceeds the conventional knock-in gene length, and considering the specific experimental operation steps, a label gene, such as fluorescent protein gene and drug resistance gene, is usually attached to the end of the integration gene to facilitate the screening and enrichment of cells with positive integration, which undoubtedly greatly increases the difficulty of HPV gene knock-in the technical aspect. The invention provides preparation of sgRNA and HaCaT cell models of human genome sequences at high-frequency integration sites of targeted high-risk HPV, which comprises the steps of analyzing and obtaining two sites with the highest integration frequency of 13q22 and 8q24 from big data of HPV integration hot spots, screening sgRNA targeting plasmids of the targeted high-frequency integration sites, and respectively constructing and synthesizing linear Donor segments containing HPV16 and HPV18 genes. Meanwhile, HaCaT cells from normal epithelial cells are selected as host cells, and the non-tumor cell lines do not contain HPV and are more in line with the pathogenic characteristics of HPV. Through co-transfection of sgRNA targeting plasmids targeting specific sites and linear Donor fragments containing HPV genes in epithelial cells HaCaT, an in-vitro cell model integrating the HPV genes at the specific sites of a cell genome is established by means of a homologous recombination repair mechanism of cells to DNA broken ends, knocking-in of the HPV genes at the specific sites of the genome is verified through a specific PCR experiment and a first-generation sequencing method, and finally, the cell model integrating the HPV at fixed points is established.
In order to achieve the above purpose, the present invention designs a sgRNA targeting a human genome sequence at a high-frequency integration site of high-risk HPV (human papilloma virus), wherein the high-risk HPV is human papilloma virus type 16, and when the high-frequency integration site of HPV is 13q22, the 13q22-sgRNA sequence is: TACAGCCACTCACAGCCCGC, as shown in SEQ ID No. 1;
or when the high-risk HPV is human papilloma virus type 18 (HPV18) and the high-frequency integration site of the HPV is 8q24 site, the sequence of 8q24-sgRNA is ACAAATAACTGTAAAGAGTA, and is shown as SEQ ID No. 2.
The invention also provides a DNA sequence corresponding to the sgRNA, wherein the DNA corresponding to the 8q24-sgRNA sequence is as follows:
the invention also provides a preparation method of the HaCaT cell model for the fixed-point integration of the high-risk HPV genes, which comprises the following steps:
1) aiming at a high-frequency integration site of high-risk HPV (human papilloma virus) in a human genome and combining with a base pairing rule, specifically designing a sgRNA sequence and obtaining double-stranded DNA of the sgRNA sequence (the sgRNA sequence contains a 20bp high-specificity gene sequence which can be combined with a target sequence of 8q24 site or 13q22 site through base pairing to form a compound respectively, and the compound guides a nuclease Cas9 protein to cut the double-stranded DNA at a target site and introduces DNA double-strand break);
2) CAACCG is connected to the left end of a sense strand of the double-stranded DNA sequence of the sgRNA, and AAACNNNC is connected to the two ends of an antisense strand of the double-stranded DNA sequence of the sgRNA, wherein NNN represents the antisense strand sequence of the sgRNA; preparing double-stranded DNA of the sgRNA into double-stranded primers, and performing enzyme digestion connection on the double-stranded primers and a pSpCas9(BB) -2A-GFP expression vector through Bpil restriction nuclease to construct an sgRNA expression plasmid;
3) transfecting target cells with the sgRNA expression plasmids respectively, and screening effective sgRNA targeting plasmids through a T7E1 experiment (the sgRNA targeting plasmids can specifically and efficiently recognize and target-cut specific gene sequences at 8q24 or 13q22 sites);
4) the efficient sgRNA targeting plasmid and a linear Donor fragment containing high-risk Human Papilloma Virus (HPV) genes are used for co-transfecting HaCaT cells, and the HaCaT cell model for the fixed-point integration of the high-risk HPV genes is obtained.
Further, in the step 1), the high-risk type HPV is human papillomavirus type 16 (HPV16) or human papillomavirus type 18 (HPV18), respectively.
Still further, when the high-risk HPV is HPV16 and the HPV gene integration site is 13q22 site, the sgRNA sequence is: TACAGCCACTCACAGCCCGC are provided.
Or when the high-risk HPV is HPV18 and the HPV gene integration site is 8q24 site, the sgRNA sequence is ACAAATAACTGTAAAGAGTA.
Still further, in the step 2), when the integration site of the HPV gene is 8q24 site, the double-stranded primer is:
still further, in the step 3), when the HPV gene integration site is 13q22 site, the sgRNA targeting plasmid consists of a CRISPR-Cas9 vector and double-stranded DNA of sgRNA targeting 13q22 site (capable of specifically and efficiently recognizing and targeting cutting a specific gene sequence at 13q22 site);
or when the HPV gene integration site is 8q24 site, the sgRNA targeting plasmid consists of CRISPR-Cas9 vector and double-stranded DNA of sgRNA targeting 8q24 site (capable of specifically and efficiently recognizing and targeting cutting a specific gene sequence at 8q24 site).
Still further, when the high-risk human papilloma virus is HPV16, a linear Donor fragment containing a high-risk human papilloma virus HPV16 gene (according to a targeting site of sgRNA in a 13q22 region, 500bp gene sequences upstream and downstream of a CRISPR-Cas9 cleavage site are selected as left arm2 and right arm2 of a Donor plasmid respectively, and are connected with an HPV16 gene fragment and a tag gene by a seamless cloning method to construct a Donor plasmid containing an HPV16 gene) is linearized from the Donor backbone plasmid, and a left arm2 gene (shown as SEQ ID No. 11), an HPV16 gene fragment, a tag gene (SEQ ID No.13) and a right arm2 gene (shown as SEQ ID No. 12) are seamlessly connected downstream of an ad element of the Donor backbone plasmid; wherein the content of the first and second substances,
the HPV16 gene fragment is shown in SEQ ID No.10, and comprises URR gene (1 … … 832bp), E6(833 … … 1309bp) and E7 gene (1310 … … 1606bp) of HPV 16;
or, when the high-risk human papilloma virus is HPV18, linear Donor fragments containing high-risk human papilloma virus HPV18 gene (left arm1, right arm1, left arm2 and right arm2 are homologous arms, 500bp gene sequences upstream and downstream of CRISPR-Cas9 cleavage site are selected as left arm1 and right arm1 of Donor plasmid according to the targeting site of 8q24 region sgRNA, and are connected with HPV18 gene fragment and tag gene by seamless cloning method to construct Donor plasmid containing HPV18 gene, which is formed by linearization of Donor backbone plasmid, and left arm1 gene (shown in SEQ ID No. 8), HPV18 gene fragment, tag gene (shown in SEQ ID No.13) and right arm1 gene (shown in SEQ ID No. 9) are connected in sequence at the downstream of the ad element of Donor backbone, wherein,
the HPV18 gene fragment is shown in SEQ ID No.7 and comprises URR gene (1 … … 828bp), E6(829 … … 1305bp) and E7 gene (1306 … … 1623bp) of HPV 18;
the nucleotide sequence of the tag gene is specifically shown as SEQ ID No.7, and consists of CMV promoter (1 … … 347bp), green fluorescent protein (EGFP) gene (351 … … 956bp), puromycin gene (PURO) (1059 … … 1658bp) and SV40 PolyA (1687 … … 1846bp) sequences, so that the puromycin gene and the green fluorescent protein gene can be stably expressed in cells with positive integration, and the cells show resistance to puromycin drugs and green fluorescence;
the Donor skeleton plasmid is pDC515 (the linear Donor fragment is formed by amplifying a specific PCR primer, and the linear Donor fragment and the targeting plasmid transfect cells together);
further, in the step 4), during transfection, the linear Donor fragment and the sgRNA targeting plasmid are wrapped by a transfection reagent (PEI), the ratio of the total mass of the linear Donor fragment and the sgRNA targeting plasmid to the transfection reagent is 1:3(μ g: μ g), and the mass ratio of the linear Donor fragment to the sgRNA targeting plasmid is 1: 1.
The invention also provides the HaCaT cell model, and the cell is a HaCaT cell.
The invention has the beneficial effects that:
according to the invention, a series of targeting sgRNA sequences are designed according to gene sequences of 13q22 and 8q24 regions, after in vitro activity detection, sgRNAs with high-efficiency cutting activity are screened out, and paired and constructed to a CRISPR/Cas9 vector to obtain 13q22 and 8q24 site targeting plasmids which have good gene editing activity in cells.
According to the target gene position of 8q24 site targeting plasmid and HPV18 gene sequence, DNA fragments of
The invention uses 8q24 locus targeting plasmid and HPV18 linear Donor fragment to transfect cell together, under the condition that the homologous arm sequence at two ends of Donor is only 500bp, the 3.5kb length exogenous DNA containing HPV18 gene is knocked into 8q24 locus of cell genome, then single clone cell screening experiment is carried out, finally the single clone cell knocked-in at 8q24 locus of HPV18 gene is obtained, and HACaT cell model of HPV18 gene fixed point integration is successfully constructed. The invention uses 13q22 locus targeting plasmid and HPV16 linear Donor fragment to transfect cell together, successfully knocks the 3.5kb exogenous DNA containing HPV16 gene into 13q22 locus of cell genome, then carries out single clone cell screening experiment, finally obtains the single clone cell knocked in 13q22 locus of HPV16 gene, successfully constructs the HaCaT cell model of HPV16 gene fixed point integration.
The invention researches the influence of HPV on normal epithelial cells by using an HPV fixed-point knock-in method, and makes up the defects of the existing HPV research method. The fixed-point knock-in of two different high-risk HPV at different high-frequency integration sites of the genome provides a model basis for exploring the incidence relation between high-risk HPV integration and cervical cancer. The invention takes the URR gene of HPV as an important research object for the first time, is expected to reveal another key gene for HPV pathogenicity, and provides a new discovery for the molecular mechanism of HPV integrated carcinogenesis. The invention constructs the HPV gene site-directed integration cell model by taking the HPV negative normal epithelial cells as cell carriers, and the cell model can more approximately simulate the biological process from integration of HPV in the normal epithelial cells to final cell malignancy, thereby providing a research platform for exploring the role of HPV gene site-directed integration in normal epithelial cell malignancy transformation and the pathogenesis of HPV integration carcinogenesis.
In a third aspect, the invention claims the use of the cell model in any of:
evaluating the influence of the substance to be tested (such as specific compound, extract, small molecule, polypeptide, protein, gene, therapeutic cell, etc.) on the disease process such as tumor growth by the cell model;
targeting a test agent (e.g., a particular compound, extract, small molecule, polypeptide, protein, gene, therapeutic cell, etc.) to the cell model to evaluate the biological safety and efficacy of the test agent; establishing a new cell or animal model by or with the help of said cell model.
Experiments prove that the growth and proliferation capacity of the HaCaT cell can be promoted by using a normal human epithelial cell HaCaT (the cell is HPV negative) and knocking the HPV18 gene fragment into the 8q24 site by adopting the CRISPR/Cas9 technology, and meanwhile, the growth and proliferation capacity of the HaCaT cell can be promoted by knocking the HPV16 gene fragment into the 13q22 site by adopting the CRISPR/Cas9 technology. The method for knocking HPV gene segments into genome high-frequency integration sites by using the CRISPR/Cas9 technology provided by the invention is expected to promote HPV site-specific integration mediated cell malignant transformation, and further reveals the necessary relationship between HPV site-specific integration and cervical cancer onset.
Drawings
FIG. 1 is a schematic diagram of HPV gene site-directed knock-in by CRISPR/Cas9 technology;
FIG. 2 shows a screening process of positive monoclonal cells;
fig. 3 is a validation of the cleavage efficiency of 3 sgrnas at position 8q 24;
fig. 4 is a validation of the cleavage efficiency of 3 sgrnas at position 13q 22;
FIG. 5 is a drawing of a Donor plasmid containing HPV18 gene;
FIG. 6 is a drawing of a Donor plasmid containing HPV16 gene;
FIG. 7 shows the results of PCR validation of HPV knock-in at 8q24 locus by agarose gel electrophoresis;
FIG. 8 is a representative fluorescence result of HPV knock-in positive monoclonal at 8q24 locus;
FIG. 9 shows the result of cloning a HaCaT cell into which HPV18 gene at 8q24 site was knocked in at the fixed point;
FIG. 10 shows the results of PCR validation of HPV knock-in at position 13q22 on different monoclonals by agarose gel electrophoresis;
FIG. 11 is a representative fluorescence result of HPV knock-in positive monoclonal at position 13q 22;
FIG. 12 shows the result of cloning of HaCaT cells by site-directed knock-in of HPV16 gene at position 13q 22.
Detailed Description
The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.
In this example, the commercial and pharmaceutical sources are as follows:
sgRNA expression vector pSpCas9(BB) -2A-GFP was purchased from Addgene (cat # PX 458);
the sequence of sgRNA was designed by on-line design software (http:// www.rgenome.net/cas-designer /), and the primers used were designed by Primer3Plus on-line Primer design tool (http:// Primer3Plus. com/cgi-bin/dev/Primer3Plus. cgi), and Primer synthesis and sequencing were performed by Wuhan engine Biotechnology, Inc., unless otherwise specified;
construction and sequencing of the Donor plasmid are completed by Shanghai and Yuanbiol Co., Ltd;
4. construction used Bpil restriction enzyme (cat. FD1014) and T4 DNA ligase (cat. EL0011) from Thermo Scientific, USA;
t7 Endonuclease I was purchased from Biolabs (cat # 0041708);
x-treme GENE HP DNA Transfectin Reagent (cat # 06366236001) available from Roche;
PCR amplification related reagents were purchased from Vazyme of Nanjing (Cat: P131-AA);
8. a plasmid extraction kit (D6926-01) and a DNA extraction kit (D3396-02);
PCR product recovery kit (D6492-01) and DNA gel cutting recovery kit (D2500-01) were purchased from Omega.