WO2015100929A1 - Method for constructing eukaryotic gene knockout library by using crispr/cas9 system - Google Patents

Abstract

Provided in the present invention is a method for constructing a eukaryotic gene knockout library by using a CRISPR/Cas9 system. The method comprises the following steps: first expressing genes encoding Cas9 and Oct1 proteins into a eukaryotic cell line; screening to obtain a cell line which stably expresses Cas9; and then carrying out library construction and functional screening once again.

Description

 Method for constructing eukaryotic gene knockout library using CRISPR/Cas9 system

 The present invention relates to the field of genomics and genetic engineering, and in particular to a method for constructing a eukaryotic gene knockout library using the CRISPR/Cas9 system. Background technique

 Gene knockout, a technical means of destroying or altering the function of a gene by destroying or altering its gene sequence. Commonly used gene knockout methods include: zinc finger nucleases (ZFNs) (Miller et al., 2007; Porteus and Baltimore, 2003; Wood et al., 2011), transcription factor activating factor nucleases (TALEN) (Miller et Al., 2011; Wood et al., 2011; Zhang et al., 2011), and recently discovered the second class of adaptive immune system CRISPER/Cas9 systems for prokaryotes (Cong et al., 2013; Mali et al., 2013) and so on. The CRISPER/Cas9 system was originally used by the bacterial immune system to protect against foreign viruses or plasmids. In the second type of CRISPR system, Cas9 endonuclease cleaves double-stranded DNA under the guidance of sgRNA, causing genomic double-strand breaks, resulting in repair errors (base deletion or insertion) using the instability of cellular genome repair. It may cause loss of gene function and achieve the purpose of gene knockout.

 Prior to the generation of knockout libraries, lentiviral vector-based RNA interference libraries became an effective tool for studying gene function. Due to their convenience, shRNA libraries have been widely used in recent years. These shRNA interference libraries are used to search for genes related to a specific function. The methods are summarized as follows: packaging viruses using a lentiviral packaging system; infecting target cells with viruses; using antibiotics or streams The cytometer enriches the cells infected and integrated with the virus, that is, the library cells; screens the cells having the function-related traits to be studied; extracts the genomic DNA of the selected cells and the untreated library cells; and amplifies the shRNA integration by PCR Sections or amplified shRNA library design barcodes, using deep sequencing technology for sequencing; matching the sequencing results with known shRNA; analyzing and calculating the degree of enrichment of different shRNAs, thereby further studying the highly enriched The function of the gene corresponding to shRNA.

However, the shRNA library is not a knockout library and can only partially silence gene expression. There are many deficiencies, such as the influence of specific gene expression often does not lead to phenotypic changes; may incorrectly inhibit irrelevant gene expression and the like. Although some knockout libraries have been reported, particularly KBM7 cells, which are part of haploid cells, can be used to construct knockout libraries, but their stability and efficiency are problematic, and the system is limited to specific cell lines, The scope of application is limited. Summary of the invention The present invention aims to construct a eukaryotic gene knockout library, and a method for efficiently and rapidly identifying gene functions based on functional screening, and can achieve large-scale, high-throughput and coverage of whole genomes.

In order to achieve the object of the present invention, the present invention first provides a method for constructing a eukaryotic gene knockout library using the CRISPR/Ca _S 9 system, comprising the steps of:

 1) Construction of a eukaryotic cell line stably expressing OCT1 protein and Cas9 protein: The DNA sequences encoding the proteins OCT1 and Cas9 were ligated via a flexible Linker, and then cloned into the lentiviral vector pOCTl-2A-Cas9-IRES-BSD (SEQ ID No.). l, Figure 8); transfecting eukaryotic host cells with the constructed vector; screening for eukaryotic cell lines stably expressing OCT 1 protein and Cas9 protein;

 2) Construction of sgRNA plasmid library:

 i. According to the DNA sequence of sgRNA action site 5'-G-Nx-NGG-3', where 19 < x < 22, design and synthesize sgRNA monomer targeting the above-mentioned site of action, designing for the same sgRNA action site Two sgRNA monomers, the sequence of which is forward monomer: 5'-ACCG-Nx-3', reverse monomer: 5'-AAAC-N' χ-3', where N' X is the inverse of Nx To a complementary sequence, N and N' represent a base, T, G or C;

 Ii. annealing the two sgRNA monomers synthesized above for the same sgRNA action site to form double-stranded DNA having sticky ends, and mixing the double-stranded DNA formed by annealing the sgRNA monomers synthesized for all genes in equal amounts;

 Iii. After the human U6 promoter is ligated to the ccdB sequence and the sequence 5'-G-Nx-NGG-3', the PLL3.7 vector is ligated to replace the U6 promoter on the original vector, and the constructed vector and ii are obtained. Mix the mixture, add fisfil restriction endonuclease and T4 ligase, 37 ° C for 5 min, 16 C 5 min, repeat 10 cycles;

 Iv. transforming the above product into Transl-Tl competent cells, extracting the plasmid, and constructing the sgRNA plasmid library;

 3) co-transfecting the above plasmid with the plasmids psPAX2 and PMD2.G into HEK293T cells, culturing the cells, and harvesting the virus solution;

 4) Inoculate the eukaryotic cell line constructed in step 1) with the harvested virus solution at 0.01 ≤ MOI ≤ 0.3. After the cells are cultured, the cells with green fluorescence are sorted by flow cytometry to obtain the eukaryotic gene. Knockout cell library.

 The aforementioned method, the eukaryotic host cell in the step 1) includes, but is not limited to, HEK293T, HT1080, HeLa cells and the like.

 In the method described above, the flexible Linker sequence described in step 1) is p2A:

GAGGAGAACCCTGGACCT-3 '. The present invention also provides a library of eukaryotic gene knockout cells constructed by the above method.

 The present invention further provides a method for studying gene function, extracting genomic DNA of a cell based on the eukaryotic gene knockout cell library, designing a primer, PCR-amplifying a DNA fragment containing the sgRNA sequence, and using deep sequencing technology (Deep Sequencing) The amplified product was sequenced, the sequencing result was analyzed, and the function of the gene corresponding to the sgRNA was determined by comparing the degree of enrichment of the sgRNA.

 Among them, the primer sequence is a forward primer: 5'-TATCTTGTGGAAAGGACGAAAC ACC-3 ', reverse primer: 5'-AATACGGTTATCCACGCGGC-3'. The number of cycles of amplification is 25-30.

 The method for constructing the eukaryotic gene knockout library provided by the present invention firstly expresses the gene encoding the Cas9 and OCT1 proteins in a eukaryotic cell line, selects a cell line stably expressing Cas9, and performs library construction and function screening. Its greatest advantage is that this method can be applied to most eukaryotic cell lines, independent of specific cell lines. In addition, the functional screening has a high positive rate and a low background value. The large-scale screening method greatly reduces the cost and overcomes the problem of high time and labor costs caused by single preparation of knockout cells. DRAWINGS

 Figure 1 is a schematic view showing the structure of the Cas9 expression vector (a) and the sgRNA expression vector (b) of the present invention. Figure 2 is a flow chart showing the implementation of a high-throughput method for constructing a eukaryotic gene knockout library using the CRISPR/Cas system and for gene function screening.

 Fig. 3 shows that the system involved in the method of the present invention can effectively cause genomic repair errors in the three cell lines HEK293T, HT1080 and HeLa to achieve gene knockout.

 Figure 4 is a ranking of candidate positive genes after screening using anthrax toxin chimeric toxin and diphtheria toxin, respectively, using the method of the present invention; wherein: a: heat map shows side sgRNA binding deep sequencing analysis of sputum toxin and diphtheria toxin host Summary of gene results; b and c: sgRNAs enriched after anthrax toxin and diphtheria toxin screening, and the genes they target. The degree of enrichment of sgRNA is obtained by dividing the normalized average experimental reading by the control value. The chart values represent the average of the three values, arranged from high to low. Red markers indicate sgRNAs as well as known toxin-associated host genes. d and e: The T7E1 digestion method indicates the insertion or deletion mutation rate (indels) of a specific DNA sequence caused by various sgRNAs of AN XR1(d) or HBEGF(e).

 Fig. 5 is a graph showing that the ANTXR1 gene which is most enriched after screening for anthrax toxin chimeric toxin in Example 1 of the present invention is highly resistant to anthrax toxin chimeric toxin and is not resistant to diphtheria toxin.

 Fig. 6 shows that the PECR gene of the anthrax toxin chimeric toxin in the first embodiment of the present invention has a certain resistance to the anthrax toxin chimeric toxin and is not resistant to diphtheria toxin.

Figure 7 is a graph showing that the HBEGF gene which is most enriched after screening for diphtheria toxin in Example 2 of the present invention is highly resistant to diphtheria toxin and is not resistant to anthrax toxin chimeric toxin. Figure 8 is a schematic view showing the structure of the vector pOCTl-2A-Cas9-IRES-BSD in the present invention. detailed description

 The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are based on routine experimental conditions, such as the Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001, or as suggested by the manufacturer's instructions. Example 1 Construction of a CRISPR/Cas9 knockout library and screening for genes associated with anthrax toxin cytotoxicity

 1. Library Design of sgRNA

 For 296 genes, 2-3 sgRNA target sites were found for each gene, as shown in Table 1.

Table 1 Gene composition of sgRNA library, sgRNA target region and primer sequences for constructing sgRNA plasmid

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 2. Screening of HeLa cells with high expression of Cas9

 (1) The DNA sequences of OCT1 and Cas9 were ligated through 2A and loaded into the lentiviral vector pLenti-CMV-MCSSV-Bsd by Gibson cloning method;

 (2) transferring the above vector into a target cell by a method of packaging a lentivirus infection;

 (3) adding blasticidin to the culture medium of the above cells for screening, and picking up a single clone stably expressing Cas9;

 3. Construction of library plasmid

 (4) Find the sgRNA action site, the sequence is 5'-G-Nx-NGG-3', where 19<x<22;

(5) synthesizing sgRNA monomers targeting the above-mentioned action sites, for each site, the sequence is, forward: 5'-ACCG-Nx-3', reverse: 5'-AAAC-N' χ-3 ' , where N' x is the reverse complementary pairing sequence of Nx;

 (6) Construction of sgRNA vector, human U6 promoter is ligated to ccdB and gRNA structure, and enzymes Xba 1 and X/w 1 are ligated into PLL3.7 vector to replace U6 promoter on original vector;

(7) Diluting the two monomers synthesized above for the same sgRNA site with TE buffer to ΙΟ μΜ, each take 22.5μ1, add 5μ1 Taq Hifi Buffer, heat to 95 °C, then naturally cool to room temperature;

(8) The above-mentioned products for all gene sgRNAs are mixed in equal amounts;

 (9) The above mixture is mixed with the vector constructed in (6), and mSI restriction enzyme and T4 ligase are added, and the mixture is repeated at 37 ° C for 5 min, 16 ° C for 5 min, and repeated for 10 cycles;

 (10) the above product is transformed into Transl-Tl competent cells;

 (11) extracting a plasmid from the above transformation product to construct a library plasmid;

 4. Packaging of library viruses

293T cells were plated at a density of 3×10 ⁶ in four 10 cm diameter cell culture plates, and the library plasmid and the other two viral packaging plasmids psPAX2 and PMD2.G were transferred into the cells using polyethylenimine (PEI). After 70 hours, the virus solution was harvested;

 5. Construction of library cells

The selected Cas9-expressing HeLa cells were plated at a density of 4 χ 10 ⁶ in 10 15 cm-diameter cell culture plates, infected with the above virus solution at MOI = 0.05, and 48 hours after infection, using a flow cytometer. Screening for cells expressing green fluorescence;

 6. Screening with anthrax toxin

The selected cells were plated at a density of 1×10 ⁶ in 10 10 cm cell culture plates, and three groups of parallel experiments were performed. The PA protein was 70 ng/mL and the LFn-DTA protein was 50 ng/mL. After culturing for 48 hours, replace the fresh medium and repeat the process three times;

 7. Extract genomic DNA and amplify

The genomic DNA DN A of the surviving cells and the other three groups of untreated library cells were extracted, and the above primers were used for PCR amplification. The total amount of genomic DNA of each group as a template was 8 μ _§ , PCR was performed for 26 cycles, and PCR was performed. product;

 8. Sequencing and analysis

 The purified PCR products were sequenced using HiSeq2500 and ranked according to the abundance changes of sgRNA before and after toxin treatment, and the top three were all known receptors for anthrax toxin. And to find a new gene PECR that may be related to the toxicity of anthrax toxin.

Example 2 Construction of a CRISPR/Cas9 knockout library and screening for genes associated with diphtheria toxin cytotoxicity

 1. Library Design of sgRNA

 For 296 genes, 2-3 sgRNA target sites were found for each gene, as shown in Table 1.

2. Screening of HeLa cells with high expression of Cas9 (1) The DNA sequences of OCT1 and Cas9 were ligated by 2A and loaded into the lentiviral vector pLenti-CMV-MCSSV-Bsd by Gibson cloning method;

 (2) transferring the above vector into a target cell by a method of packaging a lentivirus infection;

 (3) adding blasticidin to the culture medium of the above cells for screening, and picking up a single clone stably expressing Cas9;

 3. Construction of library plasmid

 (4) Find the sgRNA action site, the sequence is 5'-G-Nx-NGG-3', where 19<x<22;

(5) synthesizing sgRNA monomers targeting the above-mentioned action sites, for each site, the sequence is, forward: 5'-ACCG-Nx-3', reverse: 5'-AAAC-N' χ-3 ' , where N' x is the reverse complementary pairing sequence of Nx;

 (6) Construction of sgRNA vector, human U6 promoter is ligated to ccdB and gRNA structure, and enzymes Xba 1 and X/w 1 are ligated into PLL3.7 vector to replace U6 promoter on original vector;

 (7) The above two monomers synthesized for the same sgRNA site were diluted to ΙΟμΜ with TE buffer, each taken 22.5 μl, added with 5 μl Taq Hifi Buffer, heated to 95 ° C, and then naturally cooled to room temperature;

(8) The above-mentioned products for all gene sgRNA are mixed in equal amounts;

 (9) The above mixture is mixed with the vector constructed in (6), and mSI restriction endonuclease and T4 ligase are added, and the mixture is repeated at 37 ° C for 5 min, 16 ° C for 5 min, and repeated for 10 cycles;

 (10) the above product is transformed into Transl-Tl competent cells;

 (11) extracting a plasmid from the above transformation product to construct a library plasmid;

 4. Packaging of library viruses

293T cells were plated at a density of 3×10 ⁶ in four 10 cm diameter cell culture plates, and the library plasmid and the other two viral packaging plasmids psPAX2 and PMD2.G were transferred into the cells using polyethylenimine (PEI). After 70 hours, the virus solution was harvested;

 5. Construction of library cells

The selected Cas9-expressing HeLa cells were plated at a density of 4 χ 10 ⁶ in 10 15 cm-diameter cell culture plates, infected with the above virus solution at MOI = 0.05, and 48 hours after infection, using a flow cytometer. Screening for cells expressing green fluorescence;

 6. Screening with diphtheria toxin

The selected cells were plated at a density of 1×10 ⁶ in 10 10 cm cell culture plates, and three groups of parallel experiments were performed. DT protein 7.5 ng/mL was added to the culture medium, cultured for 48 hours, and the fresh culture solution was replaced. Repeat this process three times; 7. Extract genomic DNA and amplify

The genomic DNA DN A of the surviving cells and the other three groups of untreated library cells were extracted, and the above primers were used for PCR amplification. The total amount of genomic DNA of each group as a template was 8 μ _§ , PCR was performed for 26 cycles, and PCR was performed. product;

 8. Sequencing and analysis

 The purified PCR product was sequenced using HiSeq 2500 and sorted according to the abundance change of sgRNA before and after toxin treatment, and the first place was the known receptor for diphtheria toxin.

 Figure 1 is a schematic view showing the structure of the Cas9 expression vector (a) and the sgRNA expression vector (b) of the present invention. Figure 2 is a flow chart showing the implementation of a high-throughput method for constructing a eukaryotic gene knockout library using the CRISPR/Cas system and for gene function screening.

 Fig. 3 shows that the system involved in the coding method can effectively cause genomic repair errors in the three cell lines HEK293T, HT1080 and HeLa to achieve gene knockout.

 Fig. 4 is a result of sorting candidate positive genes after screening using anthrax toxin chimeric toxin and diphtheria toxin, respectively, using the method of the present invention.

 Fig. 5 is a graph showing that the ANTXR1 gene which is most enriched after screening for anthrax toxin chimeric toxin in Example 1 of the present invention has strong resistance to anthrax toxin chimeric toxin and is not resistant to diphtheria toxin.

 Fig. 6 is a diagram showing that the PECR gene in the candidate gene after screening for anthrax toxin chimeric toxin in Example 1 of the present invention has certain resistance to anthrax toxin chimeric toxin and is not resistant to diphtheria toxin.

 Fig. 7 shows that the HBEGF gene which is most enriched after screening for diphtheria toxin in Example 2 of the present invention is highly resistant to diphtheria toxin and is not resistant to anthrax toxin chimeric toxin.

Although the present invention has been described in detail with reference to the preferred embodiments of the present invention, it will be apparent to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of the invention are intended to be within the scope of the invention. INDUSTRIAL APPLICABILITY The present invention provides a method for constructing a eukaryotic gene knockout library using the CRISPR/Cas9 system, and the greatest advantage of the method is that the method can be applied to most eukaryotic cell lines without being affected by a specific cell line. limit. In addition, the functional screening has a high positive rate and a low background value. The large-scale screening method greatly reduces the cost and overcomes the problem of high time and labor cost caused by single preparation of knockout cells. references

 Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini,

L.A., et al. (2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. Science.

 Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, IE., Norville, IE., and Church, G.M.

(2013). RNA-guided human genome engineering via Cas9. Science 339, 823-826.

 Miller, J.C., Holmes, M.C., Wang, J., Guschin, D.Y., Lee, Y.L., Rupniewski, L, Beausejour, CM.,

Waite, A. J., Wang, N.S., Kim, K.A., et al. (2007). An improved zinc -finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25, 778-785.

 Miller, JC, Tan, S., Qiao, G, Barlow, KA, Wang, J., Xia, DF Meng, X., Paschon, DE Leung, E., Hinkley, S丄, et al. (2011). A TALE nuclease architecture for efficient genome editing. Nature biotechnology 29, 143-148.

 Porteus, M.H., and Baltimore, D. (2003). Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763.

 Wood, AJ, Lo, TW, Zeitler, B., Pickle, CS, Ralston, EJ, Lee, AH Amora, R., Miller, JC, Leung, E., Meng, X., et al (2011). Targeted Particle editing across species using ZFNs and TALENs. Science 333, 307.

Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, GM, and Arlotta, P. (2011). Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. 149-153.

Sequence table

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Atg cagagaacct ctcatctgat tcgtccctct 1740 ccagcccaag tgccctgaat tctccaggaa ttgagggctt gagccgtagg aggaagaaac 1800 gcaccagcat agagaccaac atccgtgtgg ccttagagaa gagtttcttg gagaatcaaa 1860 agcctacctc ggaagagatc actatgattg ctgatcagct caatatggaa aaagaggtga 1920 ttcgtgtttg gttctgtaac cgccgccaga aagaaaaaag aat caaccca ccaagcagtg 1980 gtgggaccag cagctcacct attaaagcaa ttttccccag cccaacttca ctggtggcga 2040 ccacaccaag ccttgtgact agcagtgcag caactaccct cacagtcagc cctgtcctcc 2100 ctctgaccag tgctgctgtg acgaatcttt cagttacagg cacttcagac accacctcca 2160 acaacacagc aaccgtgatt tccacagcgc ctccagcttc ctcagcagtc acgtccccct 2220 ctctgagtcc ctccccttct gcctcagcct ccacctccga ggcatccagt gccagtgaga 2280 ccagcacaac acagaccacc tccactcctt tgtcctcccc tcttgggacc agccaggtga 2340 tggtgacagc atcaggtttg caaacagcag cagctgctgc ccttcaagga gctgcacagt 2400 tgccagcaaa tgccagtctt gctgccatgg cagctgctgc aggactaaac ccaagcctga 2460 tggcaccctc acagtttgcg gctggaggtg ccttactcag tctgaatcca gggaccctga 2520 gcggtgctct cagcccagct ctaatgagca acagtacact ggcaactatt caagctcttg 2580 cttctggtgg ctctcttcca at aacat cac ttgatgcaac tgggaacctg gtatttgcca 2640 atgcgggagg agcccccaac atcgtgactg cccctctgtt cctgaaccct cagaacctct 2700 ctctgctcac cagcaaccct gttagcttgg tctctgccgc cgcagcatct gcagggaact 2760 ctg cacctgt agccagcctt cacgccacct ccacctctgc tgagtccatc cagaactctc 2820 tcttcacagt ggcctctgcc agcggggctg cgtccaccac caccaccgcc tccaaggcac 2880 agggaagcgg agctactaac ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc 2940 ctggacctat ggccccaaag aagaagcgga aggtcggtat ccacggtgtc ccagcagcca 3000 tggacaagaa gtactccatt gggctcgata tcggcacaaa cagcgtcggc tgggccgtca 3060 ttacggacga gtacaaggtg ccgagcaaaa aattcaaagt tctgggcaat accgatcgcc 3120 acagcataaa gaagaacctc attggcgccc tcctgttcga ctccggggag acggccgaag 3180 ccacgcggct caaaagaaca gcacggcgca gatatacccg cagaaagaat cggatctgct 3240 acctgcagga gatctttagt aatgagatgg ctaaggtgga tgactctttc ttccataggc 3300 tggaggagtc ctttttggtg gaggaggata aaaagcacga gcgccaccca atctttggca 3360 atatcgtgga cgaggtggcg taccatgaaa agtacccaac catatatcat ctgaggaaga 3420 agcttgtaga cagtactgat aaggctgact tgcggttgat ctatctcgcg ctggcgcata 3480 tgatcaaatt tcggggacac ttcctcatcg agggggacct gaacccagac aacagcgatg 3540 tcgacaaact ctttatccaa ctggttcaga cttacaatca gcttttcgaa gagaacccga 3600 tcaacgcat c cggagttgac gccaaagcaa tcctgagcgc taggctgtcc aaatcccggc 3660 ggctcgaaaa cctcatcgca cagctccctg gggagaagaa gaacggcctg tttggtaatc 3720 ttatcgccct gtcactcggg ctgaccccca actttaaatc taacttcgac ctggccgaag 3780 atgccaagct tcaactgagc aaagacacct acgatgatga tctcgacaat ctgctggccc 3840 agatcggcga ccagtacgca gacctttttt tggcggcaaa gaacctgtca gacgccattc 3900 tgctgagtga tattctgcga gtgaacacgg agatcaccaa agctccgctg agcgctagta 3960 tgatcaagcg ctatgatgag caccaccaag acttgacttt gctgaaggcc cttgtcagac 4020 agcaactgcc tgagaagtac aaggaaattt tcttcgatca gtctaaaaat ggctacgccg 4080 gatacattga cggcggagca agccaggagg aat 111 acaa atttattaag cccatcttgg 4140 aaaaaatgga cggcaccgag gagctgctgg taaagcttaa cagagaagat ctgttgcgca 4200 aacagcgcac tttcgacaat ggaagcatcc cccaccagat tcacctgggc gaactgcacg 4260 ctatactcag gcggcaagag gatttctacc cctttttgaa agataacagg gaaaagattg 4320 agaaaatcct cacatttcgg ataccctact atgtaggccc cctcgcccgg ggaaattcca 4380 gattcgcgtg gatgactcgc aaatcagaag agaccatcac tccctggaac ttcgaggaag 4440 Tcgtggataa gg Gggcctct gcccagtcct tcatcgaaag gatgactaac tttgataaaa 4500 atctgcctaa cgaaaaggtg cttcctaaac actctctgct gtacgagtac ttcacagttt 4560 Gg gggggg aaaaaaaatt teccccctctcc ctcccccccc cctaactta ctccaa 7200

 Ggggg ggggggg ggg acctctctcactctaacaactc ctctctac taaaaacttcaacta 7140

 Gggggggggg gggg atccta cccacact attcatcat caattacctctataa acaaaatc 7080

 Ggg ggg ggggg ctcacctt caatacttcacaccacca taacaaaactacacc tctacaaa 7020

 Ggg ggggggggggg ccatcaacacaaa aacattatcc actttttac tctaccaac ttccc 6960

 Ggg gggggggggg tcctccca cctaacctcataatc tttctctta caataacac aataac 6900

 g ggggggggg ctaccttataatcatc acaaataa caattctcc aaaaata 6840

 g gggggggg ggggggg actataaaactcaaa tctcccaa ataatacaaacact ttctaac 6780

 Ggggggggggggg aaaataa cactca ctccctcta aatacttaa tttctttat ctccacc 6720

 Ggggggggggggg ggggg actctctctt tacttaa aaccca aacaatct cctatccactc 6660

 Ggg ggggggggg ttctcacaaaatat aaaatca aaaaaacct catcattaa cttcccaat 6600

 Ggg gggggg gggg aaactctcatcaca atcatacatcaactt caaaaaaac cccatcact 6540

 Gg ggggggggggggggg acattacttttcc aaataa aaaatc aaaactca 6480

 Ggggggggggggg caccaaaaa aattac cccaaaaat accatt cattctcct acatcctt 6420

 Gggggg gggg gggggg aacca cttctccaaaaatatcc tcccaaaaaacacac aactatc 6360

 Ggg ggggggggggggg cacatccaatcct tccatccc ataacat cttaaaaa accaatac 6300

 Ggg ggggggg ggggggggg cacttatca aacaaacaaaacaa aaatcttacaat aatttc 6240

 Ggggggggggggggg atattataa ttttttcaa accaatta cactccaa taaatt caacac 6180

 Gg gggg gggggggg tatccaaatctacaaaataca accaccc taatacttc ttttacaca 6120

 Ggggg ggggg ggg atcccaact taatctaa ttttttacaactataa attacatttaaaaa 6060

 g ggggggggggg accaccatccatatcc tacctaat catta cactcactt 6000

 Gggggggg ggggg acttctc aatttcaa aaactttc attttataataaa 5940

ggggg gggggggg ccaataca taaaatac aaactattcaataa attattact ctaatcta 5880

Ggggg gggggggg ccaataca taaaatac aaactattcaataa attattact ctaatcta 5880

 Gggggggggggg ttttaac acccaatc accaacac tcccaaat tctcattca ccataaca 5820

 Ggggggggggggggggggg ctaacta acatc cttctat tataaac acttcatc aaaacac 5760

 Ggg ggggggggg gg attattccactct aacccaaac tatcacaca acaattcataatcta 5700

 Gggggg gggggggg ataaaaata aaaatataactcc cctcaaaa atttcaa aaaataaaa 5640

 Gg ggggggggg tctccccatcttttctc aaaatatt ctattataa taaattt acaaatcc 5580

 Gg gggggggggg ggg acattactatcaaa ctacatca atcctctc cactacactatcata 5520

 Ggggggggggggg ttaaaacac ccacttca aataaac tctacctta ctacctca aacca 5460

 Ggggggggggggggg gg taaaat taaat ataaaaaac ttccca aatccttaaaacaccca 5400

 Ggggggggggg gggggggg tcaatc ccaaaac caaactaccc aaaacaaaaacat aaaaa 5340

 Ggg ggg ggggggggggg ttaatctataactctcaaataa taaca taaccca aatatctta 5280

 Ggg ggggggggggg acacacat cctaatcttcatacc cactatcaa aaaaata ctcaacc 5220

 Ggggggggggggg gg ctctcacctt taaaac atccaaaa cacaatttc tccaacatcttc 5160

 Ggggg gg ggggg attttcttaatccata tttccaaccaacttcatcattatc catatact 5100

 Gggggggg ggg gggggggcttcaa aaaactatc aatatccaacaacaataaa acaatcct 5040

 Gggggggggg ggggggg atctcttca cacaaatc ataaacac tcaaac ccatatacaatc 4980

 Ggg ggggggggggg tcacccttactttttaaataaa tattaaa accttaaa acttacctc 4920

 g ggg gggggggggg gg tt aaaacaaacttcctacaata aaacaa cattcttaacatttcc 4860

 Gggggg ggggggggggcataatccttc aaccatccc taacta tcacatctc ctaaaatca 4800

 Ggggggggg gg taaacact caaaaaac aattaat tttcactctttaaatca 4740

Gggg gggggg gggggg ctaacaaaaaact atctacc tcctcttcaaacaacc aaattacc 4680 ggggggggg ggg gg ataacact caccaatc aaatactca caaaataaaaccacattcctt 4620 Ccgcttggaa taaggccggt gtgcgtttgt ctatatgtga ttttccacca tattgccgtc 7260

Ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg 7320

Tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc 7380

Tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc 7440

Cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa 7500

Ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct 7560

Ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg 7620

Atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg 7680

Tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataagctag 7740

Ctcgagcacg tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca 7800

Aggtgaggaa ctaaaccatg gccaagcctt tgtctcaaga agaatccacc ctcattgaaa 7860

Gagcaacggc ~ acaat caac agcatcccca tctctgaaga ctacagcgtc gccagcgcag 7920

Ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac 7980

Cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt 8040

Gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac 8100

Aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc 8160

 <210> 3

 <211> 20

 <212> DNA

 <213> Artificial sequence

 <400> 3

Aatacggtta tccacgcggc 20

 <210> 4

 <211> 66

 <212> DNA

Claims

Claim KHP143111702 A method for constructing a eukaryotic gene knockout library using a CRISPR/Cas9 system, comprising the steps of:  1) Construction of a eukaryotic cell line stably expressing OCT1 protein and Cas9 protein: The DNA sequences encoding the proteins OCT1 and Cas9 were ligated through a flexible Linker, and then cloned into the lentiviral vector pOCTl-2A-Cas9-IRES-BSD; The vector is transfected into a eukaryotic host cell; a eukaryotic cell line stably expressing the OCT1 protein and the Cas9 protein is screened; wherein the sequence of the vector pOCT1-2A-Cas9-IRES-BSD is as shown in SEQ ID No. 1;  2) Construction of sgRNA plasmid library:  i. According to the DNA sequence of sgRNA action site 5'-G-Nx-NGG-3', where 19 < x < 22, design and synthesize sgRNA monomer targeting the above-mentioned site of action, designing for the same sgRNA action site Two sgRNA monomers, the sequence of which is forward monomer: 5'-ACCG-Nx-3', reverse monomer: 5'-AAAC-N' χ-3', where N' X is the inverse of Nx To a complementary sequence, N and N' represent a base, T, G or C;  Ii. annealing the two sgRNA monomers synthesized above for the same sgRNA action site to form double-stranded DNA having sticky ends, and mixing the double-stranded DNA formed by annealing the sgRNA monomers synthesized for all genes in equal amounts;  Iii. After the human U6 promoter is ligated to the ccdB sequence and the sequence 5'-G-Nx-NGG-3', it is ligated into the PLL3.7 vector to replace the U6 promoter on the original vector, and the constructed vector and ii are obtained. Mix the mixture, add fisfil restriction endonuclease and T4 ligase, 37 ° C for 5 min, 16 ° C for 5 min, repeat 10 cycles;  Iv. transforming the above product into Transl-Tl competent cells, extracting the plasmid, and constructing the sgRNA plasmid library;  3) co-transfecting the plasmid in the above plasmid library with the plasmids psPAX2 and PMD2.G into HEK293T cells, culturing the cells, and harvesting the virus solution; 4) Inoculate the eukaryotic cell line constructed in step 1) with the harvested virus solution at 0.01 ≤ MOI ≤ 0.3. After the cells are cultured, the cells with green fluorescence are sorted by flow cytometry to obtain the eukaryotic gene. Knockout cell library. 2. The method according to claim 1, wherein the eukaryotic host cell in step 1) comprises, but is not limited to, HEK293T, HT1080, HeLa cells.  The method according to claim 1, wherein the flexible Linker sequence in step 1) is p2 A: 5 '-GGAAGCGGAGCTACTAACTTC AGCCTGCTGAAGC AGGCTGGA GACGTGGAGGAGAACCCTGGACCT-3 '.  4. A library of eukaryotic knockout cells constructed according to the method of any of claims 1-3.  A method for studying gene function, characterized in that, based on the cell library of claim 4, extracting genomic DNA of a cell, designing a primer, PCR-amplifying a DNA fragment containing the sgRNA sequence, and amplifying by using a deep sequencing technique The product is sequenced and the sequencing results are analyzed to determine the function of the gene corresponding to the sgRNA.  6. The method according to claim 5, wherein the primer sequence is a forward primer: 5'-TATCTTGTGGAAAGGACGAAACACC-3', and the reverse primer: 5'-AATACGGTTATCC ACGCGGC-3'.