CN111534577A - Method for high-throughput screening of essential genes and growth inhibitory genes of eukaryotes - Google Patents
Method for high-throughput screening of essential genes and growth inhibitory genes of eukaryotes Download PDFInfo
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Abstract
The invention relates to a method for screening eukaryotic essential genes and growth inhibitory genes in high flux, which comprises the steps of firstly establishing a eukaryotic CRISPR/Cas whole genome editing vector library, then transfecting eukaryotic cells with the vector library, then uniformly dividing the cells into two parts, directly collecting cells on one part and extracting genomes for later use, collecting the cells and extracting the genomes for later use after about 1 month of culture on one part, and collecting the cells and extracting the genomes for later use after about 2 months of culture on the other part. Comparing the sgRNA abundances of the three groups of cells by a PCR amplification method and a high-throughput sequencing technology, and finally analyzing essential genes and growth inhibitory genes of eukaryotes. The delivery system of the vector library is a piggyBac transposon system, does not need to package viruses, can directly transfect eukaryotic cells, greatly saves the cost, and has a great promoting effect on the research of essential genes and growth inhibitory genes of eukaryotic organisms.
Description
Technical Field
The invention belongs to the technical field of high-throughput sequencing, and relates to a method for high-throughput screening of essential genes and growth inhibitory genes of eukaryotes.
Background
As more and more organisms complete whole genome sequencing, functional genome research increasingly becomes the key field of life science research, and in the face of massive functional genes, the traditional forward genetics of researching the functional genes one by one is diligent, and the technical scheme of researching the functional genes with high throughput is imperative to develop. The methods for high-throughput research of functional genomes that have been widely used at present mainly include high-throughput RNAi technology, RNA-Seq technology, high-throughput CRISPR technology and the like, wherein the high-throughput CRISPR technology is increasingly widely used due to low price and high efficiency.
The essential gene is a gene which is necessary for an organism to complete the life cycle of the organism, is a core gene of the organism, and the deletion or mutation of the essential gene can cause the mutation or even death of the organism. The research of essential genes is important for the research of functional genomes of organisms, and has basic guiding significance for the improvement of biological germplasm and the creation of mutant materials. The growth inhibitory genes are genes having inhibitory action on the growth and reproduction of organisms, and the transcriptional expression of the genes tends to inhibit the proliferation of the organisms, wherein many of the growth inhibitory genes are secretory genes and many of the growth inhibitory genes are genes which interact with the external environment of the organisms. The research of the growth inhibitory gene is of great significance to the research of functional genome and the research of biological growth and reproduction.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for high-throughput screening of essential genes and growth inhibitory genes of eukaryotes.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for screening eukaryotic essential genes and growth inhibitory genes in high flux 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 the eukaryotic cell genome to form the eukaryotic whole genome editing cell library. Uniformly dividing the eukaryotic whole genome editing cell library into three parts, wherein one part of cells is directly collected and then extracted with genome, one part of cells is cultured for 1 month by using a complete culture medium, the other part of cells is cultured for 2 months by using the complete culture medium, and then the cells are collected and the genome is extracted;
(3) designing a forward primer and a reverse primer in a U6 promoter part and a sgRNA scaffold part of the vector library obtained in the step (1), and then respectively using the three groups of cell genomes collected in the step (2) as templates to amplify sgRNA fragments;
(4) and (4) respectively carrying out high-throughput sequencing on the three groups of sgRNA fragments amplified in the step (3), counting the abundance of the sgRNAs, and analyzing essential genes and growth inhibitory genes.
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 gene editing vector mediated by a transgenic system, and constructing to obtain 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 preferable technical proposal, the synthesized single-stranded oligonucleotide library is cloned to a gene editing vector mediated by a transgenic system to construct and obtain a gene editing vector library.
As one of preferable technical means, in the step (4), under the condition that p-value is <0.05, sgRNA-depleted genes are essential genes, and sgRNA-enriched genes are growth-inhibitory genes.
The invention has the beneficial effects that:
the invention firstly establishes a eukaryotic organism CRISPR/Cas whole genome editing vector library, then transfects eukaryotic cells with the vector library, then uniformly divides the cells into two parts, one part directly collects the cells and extracts the genome for standby, and the other part collects the cells and extracts the genome for standby after being cultured for about 2 months. Comparing the sgRNA abundances of the three groups of cells by a PCR amplification method and a high-throughput sequencing technology, and finally analyzing essential genes and growth inhibitory genes of eukaryotes.
The delivery system of the vector library is a piggyBac transposon system, does not need to package viruses, can directly transfect eukaryotic cells, greatly saves the cost, and has a great promoting effect on the research of essential genes and growth inhibitory genes of eukaryotic organisms.
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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 shows that the number of essential genes and growth inhibitory genes of silkworms is close to the total number of genes of silkworms, and 6.1% and 5.1%, respectively.
FIG. 2 shows that the homology between the essential gene of Bombyx mori and other essential genes of eukaryotes is high, and the homology between the essential gene of eukaryotes and the essential gene of prokaryotes is low.
FIG. 3 shows the essential gene KEGG pathway analysis of silkworm, and the essential gene of silkworm is mainly enriched in the cell core functions of DNA, RNA metabolism, etc.
FIG. 4 shows the subcellular localization analysis of silkworm essential genes, which mainly enriches the nucleus and cytoplasm.
FIG. 5 shows the subcellular localization analysis of silkworm genes showing that there are more essential genes than growth inhibitory genes in silkworm genes localized to the nucleus or mitochondria; among silkworm genes localized to the cell membrane or secreted outside the cell membrane, growth inhibitory genes are more abundant than essential genes.
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).
Method for screening silkworm essential genes and growth inhibitory genes based on CRISPR/Cas high-throughput technology
1. Constructing or purchasing a bombyx mori CRISPR/Cas9 whole genome editing vector library using a piggyBac transposon system as a delivery system, wherein the library comprises all targeting sites of bombyx mori encoding protein genes, and the number of the targeting sites of most of the genes is 6. The method for constructing the vector library is an enzyme digestion connection method and an electrotransformation method.
2. And (2) transfecting a silkworm embryonic cell line BmE after mixing the whole genome editing vector library of the silkworm CRISPR/Cas9, which takes the piggyBac transposon system constructed in the step 1 as a delivery system, with a piggyBac transposase expression vector A3-helper (the nucleotide sequence is shown as SEQ ID NO.1) according to a molar ratio of 1:1, wherein the transfection method is a liposome transfection method or an electroporation transfection method and the like. 6-8 hours after transfection, complete medium is replaced and cultured for a period of time until the cell state returns to normal. The complete medium containing Zeocin was then replaced and continued for 2 months until all cells stably integrated the CRISPR/Cas9 editing system. The cell completion Medium was Grace's Insect Medium (Amersham biosciences) containing 10% Fetal Bovine Serum (FBS) and Penicillin-Streptomycin (Penicillin-Streptomycin, 20 ten thousand units/liter). The 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 editing cell library which is used for constructing a piggyBac transposon system as a delivery system, wherein the library capacity is 100 x and the library is named as BmEGCKLib.
3. Fetch 8 × 107The cells of the cell bank BmEGCKLib are evenly divided into three groups, and the number of the cells in each group is 4 × 107One part of the cells is directly collected and then genome is extracted for standby, and the other part of the cells is completely culturedThe medium was cultured for 1 month, another cell was cultured with the complete medium for 2 months, and then the cells were collected and the genome was extracted. Extraction of cell genomic DNA was carried out using a DNA miniprep kit from Meiji Biotech Ltd, according to the instructions.
4. Primers forward and reverse primers were designed for amplification of sgRNA fragments in the U6 promoter portion and sgRNA scaffold portion of the CRISPR/Cas9 whole genome editing vector library, respectively, with the piggyBac transposon system as the delivery system, the primer sequences were 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 three sets of cellular genomic DNAs collected in step 3 as templates, respectively, and all of the three sets of cellular genomes collected in step 3 were used.
6. And (4) respectively carrying out high-throughput sequencing on the three groups of sgRNA fragments amplified in the step (5), counting the abundance of the sgRNAs, and analyzing essential genes and growth inhibitory genes. Wherein, under the condition that the p-value is less than 0.05, the sgRNA consumed gene is an essential gene, and the sgRNA enriched gene is a growth suppressor gene.
7. The specific analysis results of silkworm essential genes and growth inhibitory genes are as follows:
1) the number of silkworm essential genes and growth inhibitory genes is close to the total number of silkworm genes, and is respectively 6.1% and 5.1%. As shown in fig. 1.
2) The silkworm essential gene has higher homology with other eukaryotic essential genes and has smaller homology with prokaryotic essential genes. As shown in fig. 2.
3) The silkworm essential gene KEGG pathway analysis shows that the silkworm essential gene is mainly enriched on cell core functions such as DNA and RNA metabolism. As shown in fig. 3.
4) The subcellular localization analysis of the silkworm essential genes shows that the silkworm essential genes mainly enrich cell nucleuses and cytoplasm. As shown in fig. 4.
5) The sub-cellular positioning analysis of the silkworm gene shows that the necessary gene in the silkworm gene positioned to the nucleus or the mitochondrion is more than the growth inhibitory gene; among silkworm genes localized to the cell membrane or secreted outside the cell membrane, growth inhibitory genes are more abundant than essential genes. As shown in 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> a method for screening eukaryotic essential genes and growth inhibitory genes in high throughput
<130>2020
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aaatcaactt gtgttatagt cacggatttg ccgtccaacg tgttcctcaa aaagttgaag 60
accaacaagt ttacggacac tattaattat ttgattttgc cccacttcat tttgtgggat 120
cacaattttg ttatattttt aaacaaagct tggcactggc cgtcgtttta caacgtcgtg 180
actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 240
gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 300
atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 360
gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 420
acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 480
gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 540
aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 600
taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 660
gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 720
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
ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 1140
ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 1200
acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 1260
taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 1320
tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 1380
cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 1440
ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 1500
gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 1560
gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 1620
aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 1680
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
ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 2040
gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 2100
cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 2160
tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 2220
cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 2280
ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 2340
gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 2400
tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 2460
ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 2520
gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg 2580
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 (3)
1. A method for screening eukaryotic essential genes and growth inhibitory genes in high flux 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 the eukaryotic cell genome to form the eukaryotic whole genome editing cell library. Uniformly dividing the eukaryotic whole genome editing cell library into three parts, wherein one part of cells is directly collected and then extracted with genome, one part of cells is cultured for 1 month by using a complete culture medium, the other part of cells is cultured for 2 months by using the complete culture medium, and then the cells are collected and the genome is extracted;
(3) designing a forward primer and a reverse primer in a U6 promoter part and a sgRNA scaffold part of the vector library obtained in the step (1), and then respectively using the three groups of cell genomes collected in the step (2) as templates to amplify sgRNA fragments;
(4) and (4) respectively carrying out high-throughput sequencing on the three groups of sgRNA fragments amplified in the step (3), counting the abundance of the sgRNAs, and analyzing essential genes and growth inhibitory genes.
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 gene editing vector mediated by a transgenic system, and constructing to obtain a gene editing vector library.
3. The method according to claim 1, wherein in step (4), under the condition that p-value is <0.05, sgRNA-depleted genes are essential genes, and sgRNA-enriched genes are growth-inhibitory genes.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160251648A1 (en) * | 2013-10-28 | 2016-09-01 | The Broad Institute Inc. | Functional genomics using crispr-cas systems, compositions, methods, screens and applications thereof |
| US20180340176A1 (en) * | 2015-11-09 | 2018-11-29 | Ifom Fondazione Istituto Firc Di Oncologia Molecolare | Crispr-cas sgrna library |
| US20200216837A1 (en) * | 2016-08-17 | 2020-07-09 | Katholieke Universiteit Leuven | Drug-target identification by rapid selection of drug resistance mutations |
-
2020
- 2020-05-07 CN CN202010379323.1A patent/CN111534577A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160251648A1 (en) * | 2013-10-28 | 2016-09-01 | The Broad Institute Inc. | Functional genomics using crispr-cas systems, compositions, methods, screens and applications thereof |
| US20180340176A1 (en) * | 2015-11-09 | 2018-11-29 | Ifom Fondazione Istituto Firc Di Oncologia Molecolare | Crispr-cas sgrna library |
| US20200216837A1 (en) * | 2016-08-17 | 2020-07-09 | Katholieke Universiteit Leuven | Drug-target identification by rapid selection of drug resistance mutations |
Non-Patent Citations (4)
| Title |
|---|
| WANG T等: "Identification and characterization of essential genes in the human genome", 《SCIENCE》 * |
| ZHOU YX等: "High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells", 《NATURE》 * |
| 常珈菘: "家蚕全基因组编辑细胞库的构建及其应用", 《中国优秀博硕士学位论文全文数据库(博士)农业科技辑(月刊)》 * |
| 王干诚等: "基于CRISPR/Cas9***高通量筛选研究功能基因", 《遗传》 * |
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Application publication date: 20200814 |