CN111534544A - Method for high-throughput screening of eukaryotic cell and virus interaction target gene - Google Patents
Method for high-throughput screening of eukaryotic cell and virus interaction target gene Download PDFInfo
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Abstract
The invention relates to a method for screening interacting target genes of eukaryotic cells and viruses in a high-throughput manner. Then the cell library is uniformly divided into two parts, cultured, and genomic DNA is collected and extracted, and the sgRNA abundance of the cell library is detected by a high-throughput sequencing method respectively, and the interaction target gene of the eukaryote and the virus is screened. The invention has the greatest advantage that the eukaryotic organism and virus interaction target gene is screened in the whole genome range under the condition of no precondition. Compared with the traditional method for researching the interaction target gene of the eukaryote and the virus, the method can screen the interaction target gene of the eukaryote and the virus to the maximum extent, and has the advantages of low cost, high efficiency and wide range.
Description
Technical Field
The invention belongs to the technical field of gene editing and high-throughput sequencing, and relates to a method for screening eukaryotic cell and virus interaction target genes in a high-throughput manner.
Background
Viruses are an important class of microorganisms, small in size, simple in structure, usually contain only one nucleic acid (DNA or RNA), and must survive in a living cell. The virus itself is very little nucleic acid, containing only a few essential genes, neither complete genes involved in metabolism, nor various replicable enzyme systems, and therefore, after leaving the host cell, the virus is still alive but is not alive and dormant. Since the virus itself does not have genes that perform the entire life activity, the virus can only propagate by virtue of the organelles and intracellular environment of living cells.
The virus is an important pathogenic microorganism and is closely related to a series of animal and plant diseases, such as influenza virus and immunodeficiency virus which infect mammals, baculovirus which infect insects, tobacco mosaic virus which infect plants, rice dwarf virus and the like. The viruses are widely existed and seriously threaten the life safety of a great number of animals and plants including human beings. In order to prevent and treat virus diseases, countless scientific researchers have intensively researched to analyze genome and crystal structures of a large number of viruses, find ways of infecting eukaryotes by a plurality of viruses, clarify a plurality of eukaryote signal paths closely related to virus infection, and lay a foundation for interaction research of eukaryotes and viruses to a certain extent. However, at present, a lot of viruses harm animals and plants, and people cannot take charge of the viruses at present.
Gene editing is an important genetic manipulation technology for researching functional genomes, and four generations of genetic manipulations including meganuclease, zinc finger nuclease, transcription factor activator like nuclease, CRISPR and the like have been developed at present. The CRISPR/Cas9 system is currently the most widely used. The CRISPR/Cas9 system is a gene editing technology derived from the bacterial acquired immune system. At present, gene editing has been successfully realized in species including human, mouse, fruit fly, Arabidopsis, rice, etc. Because the design and construction are simple, the cost is low, and the editing efficiency is high, the application range of the CRISPR/Cas9 system is not limited to single-gene editing at present, and the application of the CRISPR/Cas9 system is expanded to multi-gene editing and even whole-genome editing. CRISPR/Cas9 system-mediated whole genome editing has been achieved in human, mouse, Drosophila, rice and other species. CRISPR/Cas9 system-mediated whole genome editing has achieved important achievements in the research fields of drug target gene screening, tumorigenesis, immune response and the like.
The CRISPR/Cas9 mediated whole genome editing means is comprehensively used, and the method has important significance for solving the interaction research between eukaryotes and viruses.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for high-throughput screening of eukaryotic cell and virus interaction target genes.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for screening eukaryotic cell and virus interaction target gene with high flux comprises the following steps:
(1) constructing a eukaryotic CRISPR/Cas whole genome editing vector library delivered by a transgenic system;
(2) stably integrating the vector library constructed in the step (1) to a eukaryotic cell genome to obtain a eukaryotic CRISPR/Cas whole genome editing cell library;
(3) uniformly dividing the cell library constructed in the step (2) into two parts, wherein one part is infected by virus to be used as an experimental group, the other part is used as a control group, then simultaneously collecting two groups of cells, and respectively extracting genome DNA;
(4) and (3) taking all the genomes extracted in the step (3) as templates, designing primers to amplify the sgRNA fragments of each group of cells, performing high-throughput sequencing, counting the abundance of the sgRNA, and screening the interaction target genes of the eukaryotic cells and the viruses, wherein the screening standard of the target genes is p-value < 0.05.
As one of the preferable technical schemes, the eukaryote is silkworm, fruit fly, human, etc., and the virus is a virus capable of infecting the eukaryote.
As one of the preferable technical proposal, the specific method of the step (1) is as follows:
(1-1) designing targeting sites, designing about 6 targeting sites for each gene, and synthesizing a single-stranded oligonucleotide library containing the targeting sites by means of a DNA chip;
(1-2) cloning the obtained single-stranded oligonucleotide library to a transgenic vector to construct a gene editing vector library.
As one of the further preferred technical schemes, according to the Cas action rule, the editing sites of all genes encoding proteins are designed at the whole genome level of eukaryotes, and the targeting sites have the following rules:
5 '-NNNNNNNNNNNNNNNNNNNNN-NGG-3', the designed sgRNA sequence is consistent with the sequence of the target site on the genome, and the following rules are provided: 5 '-G-nnnnnnnnnnnnnnnnnnnnnnnnn-3'; designing the target-targeting sites of the genes of all eukaryotic coding proteins according to the rule.
As a further preferred technical scheme, the synthetic single-stranded oligonucleotide library is cloned to a transgenic vector to construct a gene editing vector library.
As one of the preferable technical proposal, the specific method of the step (2) is as follows: stably integrating the vector library constructed in the step (1) to a eukaryotic cell genome to obtain a CRISPR/Cas whole genome editing cell library.
As one of the preferable technical proposal, the specific method of the step (3) is as follows: uniformly dividing the cell library constructed in the step (2) into two parts, wherein one part is infected by virus until the number of cells is reduced to 5%, the other part is cultured for the same time under normal conditions by using a complete culture medium, then simultaneously collecting two groups of cells, and respectively extracting genomic DNA.
As one of the preferable technical solutions, in the step (4), compared with the control group, the gene enriched or consumed by the sgRNA of the experimental group is the candidate target gene for the interaction between the eukaryotic cell and the virus.
The invention has the beneficial effects that:
the invention firstly constructs a eukaryotic organism CRISPR/Cas whole genome editing vector library, and then stably transfects a eukaryotic organism cell line with the vector library to construct a eukaryotic organism whole genome editing mutant cell library. The cell library was then evenly divided into two parts, one of which was infected with the virus and the other was incubated in virus-free medium for the same period of time. And simultaneously collecting and extracting genomic DNA of the cells of the two groups of experiments, respectively detecting the sgRNA abundance of the cells by using a high-throughput sequencing method, and screening the interaction target genes of the eukaryote and the virus. The invention has the greatest advantage that the eukaryotic organism and virus interaction target gene is screened in the whole genome range under the condition of no precondition. Compared with the traditional method for screening the eukaryotic organism and virus interaction target genes, the method provided by the invention can screen the eukaryotic organism and virus interaction target genes to the maximum extent, and is low in cost, high in efficiency and wide in range. Has great significance for screening eukaryotic antiviral target genes and researching virus-host interaction.
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In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a flow chart of the operation of the present invention;
FIG. 2 shows that a large number of silkworm BmNPV-resistant genes reported in the prior art are all in the BmNPV-resistant target gene library screened by us, wherein dark scatters are silkworm BmNPV-resistant genes reported in the prior art;
FIG. 3 is a KEGG pathway enrichment analysis.
FIG. 4 is a graph of sgRNA scores for phagosome pathway genes;
FIG. 5 is a schematic diagram of the key pathway and inhibitory pathway of BmNPV invasion.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Silkworm virosis is the most harmful to the sericulture among a plurality of silkworm diseases, and particularly silkworm Nuclear Polyhedrosis Virus (Bombyx mori Nuclear viruses NPV) often brings destructive attacks to the sericulture. The present invention will be described below by taking silkworms and silkworm nuclear polyhedrosis viruses as examples, but the present invention is not limited to silkworms and silkworm nuclear polyhedrosis viruses.
All the following specific experimental methods, which are not indicated, are carried out according to accepted experimental methods and conditions, for example, according to the instructions provided by the manufacturers of reagents and consumables, or according to the classic laboratory book "molecular cloning guidelines" (third edition, J. SammBruke et al).
Example (b):
the silkworm embryonic cell line (The Bombyx mori electrophoretic cell line, BmE) used in this example was a cell line commonly used in biological experiments (PMID: 17570024).
Bombyx mori nuclear polyhedrosis virus (BmNPV) contains green fluorescent protein (EGFP) as a commonly used biological material (PMID: 29875158).
The specific working flow is shown in figure 1.
1. The bombyx mori embryonic cell line BmE whole genome editing cell library.
1) Constructing or purchasing a bombyx mori CRISPR/Cas9 whole genome editing vector library mediated by a piggyBac transposon system, wherein 6 targeting sites are designed for most genes, and 6 gene editing vectors are constructed; in total, 94000 gene editing vectors were designed and constructed.
2) Transfecting the silkworm embryo cell line BmE by the whole genome editing mutant library of the silkworms constructed in the step 1) and a piggyBac transposase expression vector A3-helper (the nucleotide sequence of the mutant is shown as SEQ ID NO.1) according to a molar ratio of 1:1, wherein the transfection method comprises but is not limited to a liposome transfection method, an electroporation transfection method and the like. Then, a complete culture medium containing Zeocin is used for screening for 2 months to construct a bombyx mori BmE cell whole genome editing cell library. The cell completion medium was Grace insect medium (Grace's institute medium, semer feishal) containing fetal bovine serum (FBS, semer feishal) and Penicillin-Streptomycin (Penicillin-Streptomycin, 20 ten thousand units/liter) at a volume concentration of 10%. The culture conditions were 27 ℃ and Zeocin was purchased from Silmer Feishel corporation at a working concentration of 200. mu.g/ml.
2. Preparation of Bombyx mori nuclear polyhedrosis virus (BmNPV)
1) Propagation of BmNPV virus. BmE cells were infected with Bombyx mori nuclear polyhedrosis virus (BmNPV), and after 48 hours, the medium was collected, and BmNPV virus (PMID:29875158) was collected.
2) Calculation of the multiplicity of infection of the BmNPV Virus
Infecting BmE cells by the BmNPV collected in the step 1) by using a gradient dilution method, and calculating the virus infection complex number.
3. Uniformly dividing the bombyx mori BmE cell whole genome editing cell library constructed in the step 1 into two parts, repeatedly infecting one part of the bombyx mori BmNPV cell whole genome editing cell library with low multiplicity of infection (MOI ═ 1) for 4 times at intervals of one day, then sorting cells which do not emit green fluorescence by using a flow cytometer, culturing the other part of the bombyx mori BmNPV cell whole genome editing cell library with a complete culture medium for the same time, and then simultaneously collecting two groups of cells for later use.
4. And (4) respectively extracting genomic DNA from the two groups of cells collected in the step (3) for later use.
5. Designing a pair of primers for amplifying the sgRNA fragment according to the bombyx mori CRISPR/Cas9 whole genome editing vector library mediated by the piggyBac transposon system constructed in the step 1.
The forward primer is > gD-F, 5-NNNNNNNNNNNNTAAATCACGCTTTCAATA, N represents a base A, T, G or C, and is shown as SEQ ID NO. 2;
the reverse primer is > gD-R, 5-NNNNNNNNNNNNCGACTCGGTGCCACTTT, and N represents a base A, T, G or C, as shown in SEQ ID NO. 3.
6. And (3) taking the two groups of genomes extracted in the step 4 as templates, amplifying the sgRNA fragment by using the primer pair gD-F/gD-R described in the step 5, and then performing high-throughput sequencing, wherein all the genomes obtained in the step 4 are used for PCR amplification.
7. And (3) counting the abundances of two groups of sgRNAs through analyzing the high-throughput sequencing data in the step (6), and analyzing and screening the bombyx mori anti-BmNPV target gene, wherein compared with a control group, the gene enriched or consumed by the sgRNAs of the experimental group is a candidate target gene for interaction of the bombyx mori cells and BmNPV.
8. Screening result of bombyx mori BmNPV (BmNPV) resistant target gene
1) A large number of silkworm BmNPV-resistant genes reported in the literature are in the BmNPV-resistant target gene bank screened by us. See fig. 2.
2) The enrichment analysis of KEGG pathway shows that the bombyx mori BmNPV-resistant genes screened by the method are mainly distributed in the reported pathways such as Phagsome, Notch signalling pathway, Wnt signalling pathway and the like, and meanwhile, new pathways such as MAPK signalling-flow and Dorso-ventral axis formation are also discovered. See fig. 3.
3) sgRNA analysis showed that the Phagsome pathway key gene v-ATPase gene was highly enriched for sgRNA. See fig. 4.
4) Analyzing a key interaction pathway between the bombyx mori BmE cell line and BmNPV. The left picture shows sgRNA enrichment of numerous genes of the Phagsome pathway, which indicates that the Phagsome pathway is a key pathway for BmNPV infection and is also a target pathway for BmNPV resistance of silkworms. The right panel shows sgRNA consumption of the Wnt signaling pathway key gene, indicating that Wnt signaling pathway is a BmNPV infection inhibition pathway. See fig. 5.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Sequence listing
<110> university of southwest
<120> method for high-throughput screening of eukaryotic cell and virus interaction target gene
<130>2020
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actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 240
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atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 360
gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 420
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gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 540
aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 600
taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 660
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tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 780
ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 840
taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 900
gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 960
aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 1020
gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 1080
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aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 1740
actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 1800
gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 1860
atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 1920
atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 1980
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cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 2160
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cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 2280
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ttgccacaga ggactattag aggtaagaat aaacattgtt ggtcaacttc aaagtccacg 3780
aggcgtagcc gagtctctgc actgaacatt gtcagatctc aaagaggtcc gacgcgtatg 3840
tgccgcaata tatatgaccc acttttatgc ttcaaactat tttttactga tgagataatt 3900
tcggaaattg taaaatggac aaatgctgag atatcattga aacgtcggga atctatgaca 3960
ggtgctacat ttcgtgacac gaatgaagat gaaatctatg ctttctttgg tattctggta 4020
atgacagcag tgagaaaaga taaccacatg tccacagatg acctctttga tcgatctttg 4080
tcaatggtgt acgtctctgt aatgagtcgt gatcgttttg attttttgat acgatgtctt 4140
agaatggatg acaaaagtat acggcccaca cttcgagaaa acgatgtatt tactcctgtt 4200
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ggtgaatact acgtgaagga gttatcaaag cctgtgcacg gtagttgtcg taatattacg 4500
tgtgacaatt ggttcacctc aatccctttg gcaaaaaact tactacaaga accgtataag 4560
ttaaccattg tgggaaccgt gcgatcaaac aaacgcgaga taccggaagt actgaaaaac 4620
agtcgctcca ggccagtggg aacatcgatg ttttgttttg acggacccct tactctcgtc 4680
tcatataaac cgaagccagc taagatggta tacttattat catcttgtga tgaggatgct 4740
tctatcaacg aaagtaccgg taaaccgcaa atggttatgt attataatca aactaaaggc 4800
ggagtggaca cgctagacca aatgtgttct gtgatgacct gcagtaggaa gacgaatagg 4860
tggcctatgg cattattgta cggaatgata aacattgcct gcataaattc ttttattata 4920
tacagccata atgtcagtag caagggagaa aaggttcaaa gtcgcaaaaa atttatgaga 4980
aacctttaca tgagcctgac gtcatcgttt atgcgtaagc gtttagaagc tcctactttg 5040
aagagatatt tgcgcgataa tatctctaat attttgccaa atgaagtgcc tggtacatca 5100
gatgacagta ctgaagagcc agtaatgaaa aaacgtactt actgtactta ctgcccctct 5160
aaaataaggc gaaaggcaaa tgcatcgtgc aaaaaatgca aaaaagttat ttgtcgagag 5220
cataatattg atatgtgcca aagttgtttc tgactgacta ataagtataa tttgtttcta 5280
ttatgtataa gttaagctaa ttacttattt tataatacaa catgactgtt tttaaagtac 5340
aaaataagtt tatttttgta aaagagagaa tgtttaaaag ttttgttact ttatagaaga 5400
aattttgagt ttttgttttt ttttaataaa taaataaaca taaataaatt gtttgttgaa 5460
tttattatta gtatgtaagt gtaaatataa taaaacttaa tatctattca aattaataaa 5520
taaacctcga tatacagacc gataaaacac atgcgtcaat tttacgcatg attatcttta 5580
acgtacgtca caatatgatt atctttctag ggttaaataa tagtttctaa tttttttatt 5640
attcagcctg ctgtcgtgaa taccgtatat ctcaacgctg tctgtgagat tgtcgtattc 5700
tagccttttt agtttttcgc tcatcgactt gatattgtcc gacacatttt cgtcgatttg 5760
cgttttgatc aaagacttga gcagagacac gttaatcaac tgttcaaatt gatccatatt 5820
aacgatatca acccgatgcg tatatggtgc gtaaaatata ttttttaacc ctcttatact 5880
ttgcactctg cgttaatacg cgttcgtgta cagacgtaat catgttttct tttttggata 5940
aaactcctac tgagtttgac ctcatattag accctcacaa gttgcaaaac gtggcatttt 6000
ttaccaatga agaatttaaa gttattttaa aaaatttcat cacagattta aagaagaacc 6060
aaaaattaaa ttatttcaac agtttaatcg accagttaat caacgtgtac acagacgcgt 6120
cggcaaaaaa cacgcagccc gacgtgttgg ctaaaattat t 6161
<210>2
<211>30
<212>DNA
<213>Artificial
<220>
<221>misc_feature
<222>(1)..(12)
<223>n is a, c, g, or t
<400>2
nnnnnnnnnn nntaaatcac gctttcaata 30
<210>3
<211>29
<212>DNA
<213>Artificial
<220>
<221>misc_feature
<222>(1)..(12)
<223>n is a, c, g, or t
<400>3
nnnnnnnnnn nncgactcgg tgccacttt 29
Claims (6)
1. A method for screening eukaryotic cell and virus interaction target genes at high flux is characterized by comprising the following specific steps:
(1) constructing a eukaryotic CRISPR/Cas whole genome editing vector library delivered by a transgenic system;
(2) stably integrating the vector library constructed in the step (1) to a eukaryotic cell genome to obtain a eukaryotic CRISPR/Cas whole genome editing cell library;
(3) uniformly dividing the cell library constructed in the step (2) into two parts, wherein one part is infected by virus to be used as an experimental group, the other part is used as a control group, then simultaneously collecting two groups of cells, and respectively extracting genome DNA;
(4) and (3) taking all the genomes extracted in the step (3) as templates, designing primers to amplify the sgRNA fragments of each group of cells, performing high-throughput sequencing, counting the abundance of the sgRNA, and screening the interaction target genes of the eukaryotic cells and the viruses, wherein the screening standard of the target genes is p-value < 0.05.
2. The method of claim 1, wherein the eukaryote is silkworm, fruit fly, human, and the virus is a virus capable of infecting the eukaryote.
3. The method according to claim 1, wherein the specific method of step (1) is:
(1-1) designing targeting sites, designing about 6 targeting sites for each gene, and synthesizing a single-stranded oligonucleotide library containing the targeting sites by means of a DNA chip;
(1-2) cloning the obtained single-stranded oligonucleotide library to a transgenic vector to construct a gene editing vector library.
4. The method according to claim 1, wherein the specific method of step (2) is: stably integrating the vector library constructed in the step (1) to a eukaryotic cell genome to obtain a CRISPR/Cas whole genome editing cell library.
5. The method according to claim 1, wherein the specific method of step (3) is: uniformly dividing the cell library constructed in the step (2) into two parts, wherein one part is infected by the virus until the number of the cells is reduced to 5%, the other part is cultured for the same time by using a culture medium without the virus, then simultaneously collecting two groups of cells, and respectively extracting genome DNA.
6. The method according to claim 1, wherein in the step (4), the sgRNA enriched or depleted genes in the experimental group are candidate target genes for the interaction between the eukaryotic cells and the viruses compared with the control group.
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Cited By (1)
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| CN116478262A (en) * | 2022-09-28 | 2023-07-25 | 西南大学 | Application of RACK1 in resisting silkworm nuclear polyhedrosis virus BmNPV |
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Application publication date: 20200814 |