CN111549063A - Method for high-throughput screening of cell response environment extreme temperature target gene - Google Patents
Method for high-throughput screening of cell response environment extreme temperature target gene Download PDFInfo
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Abstract
The invention relates to a method for screening a cell response environment extreme temperature target gene in a high-throughput manner. Then uniformly dividing the cell library into several parts, wherein one part is cultured by using a complete culture medium under normal culture conditions and used as a control group; several other aliquots were cultured with complete medium under extreme temperature conditions until the cell number was only about 10% as experimental groups. And respectively extracting genomes from the collected cells of the experimental group and the control group, analyzing the abundance of the sgRNA by using a high-throughput sequencing method, and screening the cells to respond to the extreme temperature target genes. The method has the greatest advantages of simplicity, high efficiency and capability of screening functional genomes at the whole genome level.
Description
Technical Field
The invention belongs to the technical field of gene editing high-throughput sequencing, and relates to a method for high-throughput screening of a cell response environment extreme temperature target gene.
Background
The temperature is an important external factor influencing the life activities of organisms, and the constant temperature animal adapts to the environmental temperature through the thermoregulation center; the variable-temperature animals are more sensitive to the environmental temperature, and research reports prove that extreme temperature has great influence on the activities of agricultural pests and aggravates the harm to agricultural production (such as pollinating insects, bees, affected by the extreme temperature, the number of bees is reduced, the life state is deteriorated, the pollination ability is weakened, and the yield of crops is reduced); the plants are fixed and cannot move, the temperature of the opposite ends can only be passively accepted, and the extreme high temperature in early spring, summer and the like can cause great harm to the agricultural and forestry crops, so that the yield of the agricultural and forestry crops is reduced. As global warming becomes more severe, extreme temperatures cover the world, and have a significant impact on life activities. At present, the global warming trend is further aggravated, more serious and frequent extreme temperatures are brought, the influence of the extreme temperatures on organisms is researched, target genes of cells influenced by the extreme temperatures are screened, and the method is very important for reducing the damage of the extreme temperatures to the organisms.
The traditional technical means for researching functional genes is forward genetics, solves a large number of biological problems, and is time-consuming and labor-consuming. CRISPR/Cas 9-mediated whole genome screening is a brand new technical means for researching functional genomes, can research a large number of gene functions in a whole genome range with high flux, short period and low cost, has good effect on screening specific functional genes, is widely applied to model organisms such as people, mice, drosophila and the like at present, and achieves important research results in the aspects of screening drug target genes, antiviral target genes, immunity-related genes and the like. The screening method shows excellent effect in screening specific functional genomes.
Disclosure of Invention
In view of the above, the present invention provides a method for high-throughput screening of a cell-responsive environment extreme temperature target gene.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for screening a cell response environment extreme temperature target gene in a high-throughput manner comprises the following specific steps:
(1) constructing a 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 cell genome to obtain a CRISPR/Cas whole genome editing cell library;
(3) uniformly dividing the cell library constructed in the step (2) into a plurality of parts, wherein one part is used as a control group, the rest parts are used as experimental groups, simultaneously collecting cells of the control group and cells of the experimental groups, and respectively extracting cell genome DNA;
(4) and (4) taking all the genomes extracted in the step (3) as templates, designing primers to amplify the sgRNA fragments of each group of cells, respectively building a library, then executing high-throughput sequencing, counting the abundance of the sgRNAs, and screening the extreme temperature target genes of the cell response environment, wherein the target gene screening standard p is less than 0.05.
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 one of the further optimized technical proposal, the synthesized single-stranded oligonucleotide library is cloned to a pB-CRISPR vector by using a bypass PCR technology and an enzyme digestion connection technology, and the gene editing vector library pB-CRISPR-library is constructed.
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) into a cell genome to obtain a CRISPR/Cas whole genome editing cell library, wherein the library capacity is larger than 100 x.
As one of the preferable technical schemes, in the step (2), the cell is a eukaryotic cell or a prokaryotic cell.
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 a plurality of parts, wherein one part is used as a control group and is cultured by using a complete culture medium under the condition of the optimal cell culture temperature; and (3) respectively culturing the rest of the cells as experimental groups by using complete culture media at an extreme temperature which is higher or lower than the optimal cell culture temperature, obviously influencing the growth state of the cells until the number of the cells is only about 10%, simultaneously collecting cells of a control group and cells of the experimental groups, and respectively extracting genomic DNA of the cells.
As one of the preferable technical solutions, the specific method in step (4) is that, compared with the control group, the gene enriched or consumed by the sgRNA in the experimental group is the candidate target gene of the cell responding to the environmental extreme temperature stimulation.
The invention has the beneficial effects that:
the invention first constructs or purchases a CRISPR/Cas whole genome editing vector cell library. Then uniformly dividing the cell library into several parts, wherein one part is cultured by using a complete culture medium under normal culture conditions and used as a control group; several other aliquots were cultured at extreme temperatures with complete medium until the cell number was only about 10% as experimental groups. And respectively extracting genomes from the collected cells of the experimental group and the control group, analyzing the abundance of the sgRNA by using a high-throughput sequencing method, and screening the extreme temperature target genes of the cell response environment. The method has the greatest advantages of simplicity, high efficiency and capability of screening functional genomes at the whole genome level.
Drawings
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 the enrichment analysis of the target gene of the BmE cells of silkworms under the extremely low temperature (4 ℃) condition by the KEGG pathway;
FIG. 3 shows the enrichment analysis of the target gene of the BmE cells of silkworms under the extreme high temperature (30 ℃) condition by the KEGG pathway;
FIG. 4 shows the subcellular localization ratio of the bombyx mori BmE cell response extreme temperature gene. (Mit, mitochondria; ERM, endoplasmic reticulum membrane; GOLM, Golgi membrane; PM, cytoplasmic membrane).
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
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).
A method for screening a bombyx mori embryonic cell line BmE response environment extreme temperature target gene by using a CRISPR/Cas9 high-throughput technology (figure 1):
1. constructing or purchasing a bombyx mori CRISPR/Cas9 whole genome knockout vector library taking a piggyBac transposon system as a delivery system, wherein the library comprises all targeting sites of bombyx mori encoding protein genes, the number of the targeting sites of most of the genes is 6, and the total number of sgRNA vectors is 94000. The method for constructing the vector library is an enzyme digestion connection method and an electrotransformation method.
2. The bombyx mori CRISPR/Cas9 whole genome knockout vector library which takes the piggyBac transposon system constructed in the step 1 as a delivery system and a piggyBac transposase expression vector A3-helper (the nucleotide sequence is shown as SEQ ID NO.1) are mixed according to a molar ratio of 1: 1, transfecting the silkworm embryonic cell line BmE after mixing, wherein the transfection method is a liposome transfection method or an electroporation transfection method and the like. 6-8 hours after transfection, the complete culture medium is replaced and cultured for a period of time under normal culture conditions until the cell state returns to normal. The complete Medium containing Zeocin was then replaced and screening continued for 2 months until all cells stably integrated the CRISPR/Cas9 knock-out system, the cell Medium was Grace Insect Medium (Grace's insert Medium) containing Fetal Bovine Serum (FBS) and Penicillin-Streptomycin (penicilin-Streptomycin, 20 ten thousand units/liter, sequomie) at a concentration of 10% by volume. The normal culture conditions were 27 ℃ and the working concentration of Zeocin was 200. mu.g/ml. Constructing a silkworm embryonic cell line CRISPR/Cas9 whole genome knockout cell library which takes the piggyBac transposon system as a delivery system, wherein the library capacity is more than 100 x, and the library is named as BmEGCKLib.
3. Take 1.2 × 108The cells of the cell bank BmEGCKLib are evenly divided into three groups, and the number of the cells in each group is 4 × 107One of the cells was a control group, cultured in complete medium under normal culture conditions (27 ℃); the other two groups were experimental groups and cultured with complete medium at 4 ℃ and 30 ℃ respectively until the experimental group cells survived only about 10%. Meanwhile, cells of a control group and an experimental group are collected, and genome DNA of the cells is extracted for later use.
1) The normal culture condition of the silkworm embryonic cell line BmE is 27 degrees, the complete culture Medium is Grace insect culture Medium (Sieve's fly et. Co.) containing 10% by volume of fetal bovine serum (FBS, Sieve fly et. Co.) and the culture Medium is changed every other day.
2) Extraction of cell genomic DNA was carried out using a DNA miniprep kit from Meiji Biotech Ltd, according to the instructions.
4. Designing a forward primer and a reverse primer for amplifying a sgRNA fragment in a U6 promoter part and a sgRNA scaffold part of a CRISPR/Cas9 whole genome knockout vector library which is used as a delivery system in a piggyBac transposon system respectively, wherein the primer sequences are as follows:
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.
5. The sgRNA fragments were amplified using the primer pairs designed in step 4 using the genomic DNA of the experimental and control cells collected in step 3 as templates, respectively, and the two cellular genomes collected in step 3 were used in their entirety.
6. And (4) respectively carrying out high-throughput sequencing on the sgRNA fragments of the experimental group and the control group amplified in the step (5), counting the abundance of the sgRNA, and screening the response environment extreme temperature target gene of the silkworm embryonic cell line BmE.
7. The result of the bombyx mori embryonic cell line BmE response extreme temperature target gene screening is as follows (under the condition that p-value is less than 0.05):
1) KEGG passage enrichment analysis silkworm BmE cells target gene enrichment condition under extreme low temperature (4 ℃). The sgRNA-depleted genes in the figure indicate that these pathways are critical for the survival of bombyx mori BmE cells at extremely low temperatures, including steroid biosynthesis, etc. (the stability of biofilms under extremely low temperature conditions is very important). The sgRNA-enriched genes in the figure indicate that these pathways are detrimental to the survival of bombyx mori BmE cells under extreme low temperature conditions. Many of the pathways overlap with essential genes of bombyx mori BmE cells, and mainly related to RNA and protein, but do not have related DNA pathways in the essential genes. Indicating that an appropriate reduction in the level of cellular activity (RNA and protein metabolism) can help the cell resist extreme low temperatures. See fig. 2.
2) KEGG passage enrichment analysis of silkworm BmE cell target gene enrichment under extreme high temperature (30 ℃). The sgRNA-depleted genes in the figure indicate that these pathways are critical for the survival of bombyx mori BmE cells at extreme high temperatures, and that these pathways comprise fatty acid synthesis and metabolism pathways, indicating that maintaining cell membrane stability is critical under extreme high temperature conditions. The sgRNA-enriched genes in the figure indicate that these pathways are detrimental to the survival of bombyx mori BmE cells under extreme high temperature conditions. See fig. 3.
3) Under the temperature condition (including extreme high temperature and extreme low temperature), sgRNA of genes positioned in a biological membrane system is obviously consumed, which shows that under the extreme temperature, the stability of the biological membrane system is important for BmE cells of silkworms; the sgRNA of the gene located in mitochondria is obviously consumed, which shows that the metabolic level is reduced under the condition of extreme temperature, and the survival of the bombyx mori BmE cells is facilitated. See fig. 4.
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.
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cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca 2640
gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact 2700
ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 2760
acagctatga catgattacg aattcgaatt cccatccccc tagaatccca aaacaaactg 2820
gttattgtgg taggtcattt gtttggcaga aagaaaactc gagaaatttc tctggccgtt 2880
attcgttatt ctctcttttc tttttgggtc tctccctctc tgcactaatg ctctctcact 2940
ctgtcacaca gtaaacggca tactgctctc gttggttcga gagagcgcgc ctcgaatgtt 3000
cgcgaaaaga gcgccggagt ataaatagag gcgcttcgtc tacggagcga caattcaatt 3060
caaacaagca aagtgaacac gtcgctaagc gaaagctaag caaataaaca agcgcagctg 3120
aacaagctaa acaatctgca gtaaagtgca agttaaagtg aatcaattaa aagtaaccag 3180
caaccaagta aatcaactgc aactactgaa atctgccaag aagtaattat tgaatacaag 3240
aagagaactc tgggggatcc ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt 3300
ttgaactata acgaccgcgt gagtcaaaat gacgcatgat tatcttttac gtgactttta 3360
agatttaact catacgataa ttatattgtt atttcatgtt ctacttacgt gataacttat 3420
tatatatata ttttcttgtt atagatatcg tgactaatat ataataaaat gggtagttct 3480
ttagacgatg agcatatcct ctctgctctt ctgcaaagcg atgacgagct tgttggtgag 3540
gattctgaca gtgaaatatc agatcacgta agtgaagatg acgtccagag cgatacagaa 3600
gaagcgttta tagatgaggt acatgaagtg cagccaacgt caagcggtag tgaaatatta 3660
gacgaacaaa atgttattga acaaccaggt tcttcattgg cttctaacag aatcttgacc 3720
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
agaaaaatat gggatctctt tatccatcag tgcatacaaa attacactcc aggggctcat 4260
ttgaccatag atgaacagtt acttggtttt agaggacggt gtccgtttag gatgtatatc 4320
ccaaacaagc caagtaagta tggaataaaa atcctcatga tgtgtgacag tggtacgaag 4380
tatatgataa atggaatgcc ttatttggga agaggaacac agaccaacgg agtaccactc 4440
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 a cell response environment extreme temperature target gene in a high-throughput manner is characterized by comprising the following specific steps:
(1) constructing a 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 cell genome to obtain a CRISPR/Cas whole genome editing cell library;
(3) uniformly dividing the cell library constructed in the step (2) into a plurality of parts, wherein one part is used as a control group, the rest parts are used as experimental groups, simultaneously collecting cells of the control group and cells of the experimental groups, and respectively extracting cell genome DNA;
(4) and (4) taking all the genomes extracted in the step (3) as templates, designing primers to amplify the sgRNA fragments of each group of cells, respectively building a library, then executing high-throughput sequencing, counting the abundance of the sgRNAs, and screening the extreme temperature target genes of the cell response environment, wherein the target gene screening standard p is less than 0.05.
2. 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.
3. The method according to claim 1, wherein the specific method of step (2) is: and (2) stably integrating the vector library constructed in the step (1) on a cell genome to obtain a CRISPR/Cas whole genome editing cell library.
4. The method according to claim 1, wherein in step (2), the cell is a eukaryotic cell or a prokaryotic cell.
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 a plurality of parts, wherein one part is used as a control group and is cultured by using a complete culture medium under the condition of the optimal cell culture temperature; and (3) respectively culturing the rest of the cells as experimental groups by using complete culture media at an extreme temperature which is higher or lower than the optimal cell culture temperature, obviously influencing the growth state of the cells until the number of the cells is only about 10%, simultaneously collecting cells of a control group and cells of the experimental groups, and respectively extracting genomic DNA of the cells.
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 of the cells responding to the environmental extreme temperature stimulation compared with the control group.
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Application publication date: 20200818 |