CN107435051B - Cell line gene knockout method for rapidly obtaining large fragment deletion through CRISPR/Cas9 system - Google Patents
Cell line gene knockout method for rapidly obtaining large fragment deletion through CRISPR/Cas9 system Download PDFInfo
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Abstract
The invention relates to a method for quickly knocking out a cell line gene with large fragment deletion by a CRISPR/Cas9 system, belonging to the field of genetic engineering and genetic modification. The invention modifies a pX458 vector to enable the vector to carry DsRed2 and ECFP, then designs a plurality of specific sgRNA sites aiming at a target gene, connects the sgRNA sites into the modified vector, sorts cell groups by using a flow cytometer after transfecting a cell line, can very quickly obtain single cells with edited genomes, then amplifies a single cell DNA sequence by using PCR, and can select the single cells with large fragment deletion from the single cells by gel electrophoresis. According to the invention, the CRISPR/Cas9 system, the flow cytometer single cell sorting and the fluorescent protein screening on the expression vector are combined, so that the positive monoclonal with large fragment deletion can be obtained in a short time, and the gene knockout working efficiency of a cell line is greatly improved.
Description
Technical Field
The invention relates to a method for quickly knocking out a cell line gene with large fragment deletion by a CRISPR/Cas9 system, belonging to the field of genetic engineering and genetic modification.
Background
The CRISPR-Cas9 system is one of the most widely studied and utilized genome editing techniques in recent years, in contrast to traditional genome editing methods: compared with Zinc Finger Nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR-Cas9 system has the following advantages: the structure is simple, and the modification and the operation are very easy; the genome editing efficiency is high, and gene knockout individuals are easy to obtain; in the practical use process, species limitation does not exist, so that the method is widely applied to gene knockout model making of animals, plants, cell lines and the like.
The traditional cell line gene knockout needs to obtain positive monoclonal cells after transfection through multiple rounds of resistance screening and dilution, which is time-consuming and labor-consuming and has high false positive rate. After the CRISPR/Cas9 system cuts genomic DNA, deletion or insertion of a few bases is formed in most cases, and the minor change cannot be detected by traditional gel electrophoresis; and others, such as: the direct sequencing method, the PCR enzyme digestion detection method, the T7E1 enzyme digestion detection method and the high-resolution dissolution curve detection method can detect whether the targeted site generates base change or not, but specific number of base deletion or insertion cannot be obtained; the PAGE electrophoresis detection method and the fluorescence PCR method can accurately obtain the detailed information of the base change, but the PAGE electrophoresis detection method is complex to operate, and the fluorescence PCR method needs expensive instruments and equipment, so that the PAGE electrophoresis detection method and the fluorescence PCR method are not suitable for high-throughput screening and large-scale popularization.
Disclosure of Invention
The invention aims to provide a cell line gene knockout method for rapidly obtaining large-fragment deletion through a CRISPR/Cas9 system, the detection method is simple and easy to implement, and large-fragment DNA deletion can be obtained by simultaneously carrying out multi-site targeting on a target gene.
In order to achieve the purpose, the invention adopts the technical scheme that:
a cell line gene knockout method for rapidly obtaining large fragment deletion through a CRISPR/Cas9 system comprises the following steps:
1) selecting a target gene to be knocked out, and using online sgRNA design software to obtain at least two sgRNA sequences, wherein the interval between the sgRNA sequences is 50-100 bp;
2) adding enzyme cutting sites into the sgRNA sequence obtained in the step 1) and synthesizing single-stranded primers, annealing the paired primers to obtain corresponding double-stranded DNA fragments with sticky ends, respectively connecting the double-stranded DNA fragments into CRISPR/Cas9 system expression vectors with different fluorescent reporter genes, correspondingly connecting one double-stranded DNA fragment with one CRISPR/Cas9 system expression vector with the fluorescent reporter genes, equivalently mixing the constructed expression vectors, and transferring the mixed expression vectors into cells;
3) after the cells are cultured, a flow cytometer is used for positive screening, and monoclonal cells containing all fluorescent reporter gene signals are separated;
4) carrying out amplification culture on the obtained monoclonal cells, taking the cells after 10-15 days, and obtaining genome DNA by a direct lysis method;
5) designing a PCR detection primer containing a target site, carrying out PCR amplification by taking the genomic DNA obtained in the step 4) as a template, carrying out gel electrophoresis detection on an amplification product, and selecting a large fragment-deleted homozygote cell line.
The method for quickly knocking out the cell line gene with large fragment deletion by the CRISPR/Cas9 system comprises the following steps:
1) the EGFP fluorescent reporter gene in pX458 was replaced by DsRed2 and ECFP, which were designated as: pX458-DsRed2 and pX 458-ECFP; selecting a target gene to be knocked out, and using online sgRNA design software to obtain two sgRNA sequences, wherein the interval between the two sgRNA sequences is 50-100 bp;
2) adding enzyme cutting sites to the two sgRNA sequences obtained in the step 1) and synthesizing 4 single-stranded primers, annealing the paired primers to obtain two double-stranded DNA fragments with sticky ends, and respectively connecting the double-stranded DNA fragments into vectors pX458-DsRed2 and pX458-ECFP to obtain expression vectors pX458-DsRed2-sgRNA1 and pX458-ECFP-sgRNA 2;
3) transferring expression vectors of pX458-DsRed2-sgRNA1 and pX458-ECFP-sgRNA2 into cells by means of lipofection or electrotransformation, and performing double-positive single cell sorting on DsRed2 and ECFP fluorescent reporter genes by using a flow cytometer after culturing;
4) carrying out amplification culture on the obtained monoclonal cells, taking the cells after 10-15 days, and obtaining genome DNA by a direct lysis method;
5) designing a PCR detection primer containing two target sites, carrying out PCR amplification by taking the genomic DNA obtained in the step 4) as a template, carrying out gel electrophoresis detection on an amplification product, and selecting a large fragment-deleted homozygote cell line.
When the target gene is Gfi1b in step 1), the two sgRNA sequences are respectively: 5'-AGTGACAAGCGCTAGTCCTTTGG-3', 5 '-TTACCACCAGCCCCGGGCACAGG 3'. The 4 single-stranded primers in the step 3) are respectively as follows:
M-Gfi1b-IVT-1：5‘-CACCGAGTGACAAGCGCTAGTCCTT-3’；
M-Gfi1b-IVT-2：5‘-AAACAAGGACTAGCGCTTGTCACTC-3’；
M-Gfi1b-IVT-3：5‘-CACCGTTACCACCAGCCCCGGGCAC-3’；
M-Gfi1b-IVT-4：5‘-AAACGTGCCCGGGGCTGGTGGTAAC-3’。
when the target gene is Pparg in step 1), the two sgRNA sequences are respectively: 5'-GTATACCTAACAAGATACTATGG-3', 5'-GTGAAGCTGTGCGTCATTTCAGG-3' are provided. The 4 single-stranded primers in the step 2) are respectively as follows:
M-Pparg-IVT-1：5‘-CACCGTATACCTAACAAGATACTA-3’；
M-Pparg-IVT-2：5‘-AAACTAGTATCTTGTTAGGTATAC-3’；
M-Pparg-IVT-3：5‘-CACCGTGAAGCTGTGCGTCATTTC-3’；
M-Pparg-IVT-4：5‘-AAACGAAATGACGCACAGCTTCAC-3’。
when the target gene is DSG4 in step 1), the two sgRNA sequences are: 5'-CTTAGCCGTAAGGATTGCCGAGG-3', 5'-GTGGTTGTCATCGCAATCACAGG-3' are provided. The 4 single-stranded primers in the step 2) are respectively as follows:
M-DSG4-IVT-1：5‘-CACCGCTTAGCCGTAAGGATTGCCG-3’；
M-DSG4-IVT-2：5‘-AAACCGGCAATCCTTACGGCTAAG C-3’；
M-DSG4-IVT-3：5‘-CACCGTGGTTGTCATCGCAATCAC-3’；
M-DSG4-IVT-4：5‘-AAACGTGATTGCGATGACAACCAC-3’。
in designing the primer in step 2), if the 5' starting base of the upstream primer is not G, one additional base G is added.
Culturing for 40-80h in the step 3), and then carrying out single cell sorting by using a flow cytometer. After the transfected cells are cultured for 40-80h, the cells are sorted by a flow cytometer, and the cells can be guaranteed to have higher survival rate and gene knockout efficiency by sorting in the time period.
The formula of the cracking solution used in the direct cracking method in the step 4) is as follows: 100mmol/L KCl, 20mmol/L Tris-HCl pH 9.0, 0.3% Triton X-100, 1.0mg/mL protease K. Experiments show that when the lysis solution with the concentration of 0.3 percent of Triton X-100 and 1.0mg/mL of protease K is adopted, better cell lysis effect can be obtained. After the single cells are obtained by sorting, the kit is not needed to extract the genome DNA, but the genome DNA can be directly obtained by a cracking method, so that the DNA extraction cost is greatly reduced, and the subsequent verification experiment efficiency is improved.
The method for quickly knocking out the large-fragment deletion cell line gene through the CRISPR/Cas9 system is characterized in that: the direct cracking method in the step 4) comprises the following specific operations: incubate at 55 ℃ for 15min, then incubate at 95 ℃ for 10 min. The invention does not need to use a kit to extract genome DNA, and only needs 25min for processing cells, thereby greatly saving the economic cost and the time cost.
And 5) comparing the amplification product to be detected with a wild type amplification product, and selecting a homozygote cell line with two chromosomes both suffering from large fragment base deletion.
The invention modifies all-in-one CRISPR/Cas9 system carrier to make it carry different fluorescence reporter genes (DsRed2 and ECFP), then designs 2-3 specific sgRNA sites aiming at target targeting genes, connects them into modified carrier respectively, extracts plasmid DNA and transfects cell line, uses flow cytometry to sort cell group with two fluorescence simultaneously, can obtain unicell with edited genome very fast, after expanding and culturing candidate unicell, obtains its genome DNA by direct cracking method, then uses PCR to amplify DNA sequence containing targeting site, selects unicell with large fragment deletion from it by simple gel electrophoresis.
According to the invention, the CRISPR-Cas9 system, the flow cytometer single cell sorting and the fluorescent protein screening on the expression vector are combined, so that the positive monoclonal with large fragment deletion can be obtained in a short time, and the gene knockout working efficiency of a cell line is greatly improved. According to the invention, by utilizing the sorting function of the flow cytometer and simultaneously combining and utilizing the fluorescent report protein on the expression vector, not only can the monoclonal be obtained in a short time, but also the positive monoclonal can be obtained with high pertinence, so that the CRISPR-Cas9 system, the sorting of the single cell of the flow cytometer and the screening of the fluorescent report protein on the expression vector are combined, the most positive monoclonal can be obtained in a short time, and the gene knockout working efficiency of a cell line is greatly improved.
Compared with the traditional cell line gene knockout method, the method has the following advantages:
1. the CRISPR-Cas9 system is used for gene knockout of a cell line, and the editing efficiency is very high;
2. the invention uses DsRed2 and ECFP as fluorescence reporter genes, and has the advantages that the excitation wavelengths of the two fluorescent proteins are far apart, and the two fluorescent proteins cannot interfere with each other in the later single cell sorting process;
3. the invention uses the flow cytometer to perform single cell sorting on DsRed2 and ECFP fluorescent reporter gene double-positive cell populations, can ensure that two or more targeted sgRNAs simultaneously enter single cells, reduces the false positive rate, and further improves the probability of obtaining large-fragment missing single cells;
4. after the single cells are obtained by sorting, the kit is not needed to extract the genome DNA, but the genome DNA can be directly obtained by a cracking method, so that the DNA extraction cost is greatly reduced, and the subsequent verification experiment efficiency is improved;
5. the invention can screen and obtain large-fragment-missing single-cell strains through simple PCR and gel electrophoresis, does not need complicated enzyme digestion verification and other processes, does not need complicated and expensive instruments, and is suitable for large-scale popularization in laboratories with basic molecular biology equipment;
6. the whole cell line gene knockout work expressed by the invention can be finished within one month, so that the time cost is greatly saved;
7. the method of the invention has wide application range and is not limited by specific cell lines and genes.
Drawings
FIG. 1 is a schematic diagram of pX458-DsRed2 and pX458-ECFP vectors in example 1;
fig. 2 is a graph of sequencing peaks of sgRNA expression vectors;
FIG. 3 is a schematic diagram of single cell sorting of DsRed2 and ECFP double positive cells by flow cytometry;
FIG. 4 is a diagram showing the result of detection by gel electrophoresis of Gfi1b gene knockout positive monoclonal antibody in cell line RAW 264.7;
FIG. 5 is a diagram showing the result of Pparg gene knockout positive monoclonal gel electrophoresis detection in cell line RAW 264.7;
FIG. 6 is a diagram showing the result of gel electrophoresis detection of a positive single clone of DSG4 gene knockout in cell line HaCat.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
Example 1
And (3) transforming an all-in-one CRISPR/Cas9 system vector to obtain expression vectors with DsRed2 and ECFP respectively.
(1) The mother vector pX458 was purchased from addge (ID: 48138), and after the amplification culture, the vector DNA was linearized using the restriction enzyme EcoRI (NEB); the method comprises the following specific steps: sequentially adding 1 mu g of pX458 vector DNA into a 1.5ml centrifuge tube; 3 μ l 10 XNEB Buffer 2.1; mu.l EcoRI (NEB) was finally supplemented with water to a total volume of 30. mu.l and incubated at 37 ℃ for 2 hours. After completion of the cleavage, the cleavage product was purified using QIAquick PCR Purification Kit and recovered to 30. mu.l of ddH 2O.
(2) DNA sequences of DsRed2 (shown as SEQ ID NO. 25) and ECFP (shown as SEQ ID NO. 26) were synthesized by submitting companies (Shanghai Bailey Biotechnology Co., Ltd.), an EcoRI cleavage site and a T2A sequence were added to the 5' end of the sequence, and cloned into a pUC57 vector; then, the two DNA sequences are respectively cut by enzyme, and the specific steps are as follows: sequentially adding 1 mu g of carrier DNA into a 1.5ml centrifuge tube respectively; 3 μ l 10 XNEB Buffer 2.1; mu.l EcoRI (NEB) was finally supplemented with water to a total volume of 30. mu.l and incubated at 37 ℃ for 2 hours. After completion of the digestion, the vector and the target fragment were separated by gel electrophoresis, and after the gel containing the target fragment was removed, the digestion product was purified using QIAquick gel Purification Kit and recovered into 30. mu. lddH 2O.
(3) And (3) adding 0.5 mu l of the double-stranded DNA fragment with sticky ends obtained in the step (2) and 2 mu l of the vector DNA with the same sticky ends obtained in the step (1), adding 0.5 mu l of T4DNA ligase (NEB M0202S) and 1 mu l of 10 xT 4ligation buffer (NEB), reacting for 1 hour, transforming Escherichia coli DH5 α, coating an ampicillin resistant plate, placing in a 37 ℃ incubator for overnight culture, selecting single colonies in the next day, and carrying out sequencing verification to finally obtain DsRed2 and ECFP expression vectors which are respectively named as pX458-DsRed2 and pX458-ECFP, wherein the schematic diagram of the vectors is shown in FIG. 1.
Example 2
A method for quickly knocking out a large-fragment deleted RAW264.7 cell line Gfi1b gene by using a CRISPR/Cas9 system.
(1) Determining the specific target sites of Gfi1b (Gene ID:1276578) to be knocked out, namely sgRNA1, sgRNA 2: the mouse Gfi1b gene DNA sequence (Transcript ID: ENSMUST00000028156.7) was found in the mouse genome database ensemble (http:// asia. ensemble. org), and then 2 specific sites were determined to be selected within the target sites intron1-2 and exon2(exon ID: ENSMUSE00001307648) of the mouse Gfi1b gene as target sequences for sgRNAs using the online design software CRISPOR (http:// CRISPOR. for. net/CRISPOR. cgi): sgRNA1(SEQ ID No. 1): 5'-AGTGACAAGCGCTAGTCCTTTGG-3', sgRNA2(SEQ ID NO. 2): 5'-TTACCACCAGCCCCGGGCACAGG-3' are provided.
(2) Designing a primer: designing 4 primers (Shanghai Bailey biotechnology, Inc.) with 2 pairs according to the sgRNA target sequence in the step (1), and adding BbsI enzyme cutting site at the 5' end of the primer sequence:
M-Gfi1b-IVT-1(SEQ ID NO.3)：5‘-CACCGAGTGACAAGCGCTAGTCCTT-3’；
M-Gfi1b-IVT-2(SEQ ID NO.4)：5‘-AAACAAGGACTAGCGCTTGTCACT C-3’；
M-Gfi1b-IVT-3(SEQ ID NO.5)：5‘-CACCGTTACCACCAGCCCCGGGCAC-3’；
M-Gfi1b-IVT-4(SEQ ID NO.6)：5‘-AAACGTGCCCGGGGCTGGTGGTAAC-3’。
because the sgRNA expression vector uses the U6 promoter, if there is a base G at the start site of gene transcription, the expression amount of the gene will be significantly increased, so in the primer design process, if the 5 'start base of the upstream primer is not G, an additional base G needs to be added to ensure that the gene maintains a high expression amount, in this case, a base C needs to be added to the 3' end of the corresponding downstream primer.
(3) Obtaining double-stranded DNA fragments with sticky ends through primer annealing and pairing: the 4 primers synthesized in step (2) were used for annealing in a combination of M-Gfi1b-IVT-1+ M-Gfi1b-IVT-2 and M-Gfi1b-IVT-3+ M-Gfi1b-IVT-4Fire pairing to obtain double-stranded DNA fragments with sticky ends, and the specific procedure is as follows: firstly, respectively phosphorylating two pairs of synthesized primers, wherein the phosphorylation reaction system is that primers M-Gfi1b-IVT-1+ M-Gfi1b-IVT-2 and M-Gfi1b-IVT-3+ M-Gfi1b-IVT-4 are respectively added with 1 mul (100 mul), then 1 mul 10 XT 4Ligation buffer (NEB) is added, then 0.5 mul l T4 polynuceotide Kinase (NEB M0201S) is added, and finally 6.5 mul H is added2O to the total volume of 10 mu l, fully and uniformly mixing after preparing a reaction system, and incubating for 30min at 37 ℃; taking out the reaction product, transferring the reaction product to PCR for denaturation and annealing, and carrying out the following reaction procedures: 95 ℃ for 5 min; 95-25 ℃ at-5 ℃/min.
(4) The vector DNA (pX458-DsRed2 and pX458-ECFP) was linearized with the restriction endonuclease BbsI and then purified and recovered to obtain vector DNA fragments with sticky ends, as follows: mu.g of pX458-DsRed2 vector DNA (or pX458-ECFP) was sequentially added to a 1.5ml centrifuge tube; 3 μ l 10 XNEB Buffer 2.1; mu.l of BbsI (NEB) was finally replenished with water to a total volume of 30. mu.l and incubated at 37 ℃ for 2 hours. After completion of the digestion, the digestion product was purified using QIAquick PCR purification kit and recovered to 30. mu.l of ddH2And (4) in O.
(5) And (3) adding 0.5 mu l of the double-stranded DNA fragment with sticky ends obtained in the step (3) and 2 mu l of the vector DNA with the same sticky ends obtained in the step (4), adding 0.5 mu l of T4DNA ligase (NEB M0202S) and 1 mu l of 10 XT 4ligation Buffer (NEB), transforming the Escherichia coli DH5 α after 1-hour reaction, coating ampicillin resistant plates, placing the Escherichia coli DH5 α in a 37 ℃ incubator for overnight culture, selecting single colonies for sequencing verification in the next day, and finally obtaining the sequencing peaks of expression vectors pX 458-465-b-1 and pX 458-Gfi-b-2 respectively expressing DsRed2 and EC865FP, wherein the sequencing peaks of the expression vectors of the sgRNA are shown in a sequence chart of pX458-Gfi 1-Gfi and pX 458-b-Gfi, and the sequencing peaks of the expression vectors are shown in a diagram of a 387 458-635-b-1-3876-2.
(6) The constructed expression vectors are mixed in equal mass, and then a mouse macrophage system RAW264.7 is transfected by a liposome-mediated transfection mode. The detailed steps of cell culture and transfection are as follows:
culture and passage of RAW264.7 cells: cells were purchased from ATCC cell banks and taken back to the laboratory for CO2Culturing in an incubator, after the cells are completely attached to the wall, replacing the DPBS with a liquid to wash out dead cells and cell metabolites, adding a fresh culture solution (DMEM/GLUCOSE + 10% FBS + 1% double antibody (penicillin + streptomycin)) to continue culturing, observing the cell morphology and the growth condition under an inverted microscope every day, and beginning passage when the cells grow to about 90% of the bottom of a culture flask. Observing the growth condition of cells in a culture bottle, when the cells proliferate to 80-90% of the bottom of the culture bottle, absorbing the old culture solution in the bottle, washing the old culture solution for 1-2 times by using preheated DPBS, adding 1mL of 0.1% trypsin into the culture bottle, digesting the cells for 2min at 37 ℃, placing the culture bottle under a microscope for observation, when the cell forms become round and the cell gaps are increased, removing the trypsin and adding 3mL of DMEM to stop the digestion reaction, gently and repeatedly blowing the cells by using a pipettor, forming cell suspension after the cells are separated from the bottle wall, absorbing a proper amount of suspension, transferring the suspension into a new culture bottle, adding a fresh culture medium, uniformly mixing, and placing the culture bottle in CO2After the cells are cultured in a constant temperature incubator until the cells are 90% confluent, F2 cells are inoculated on a 24-well plate for 24 hours according to the passage method.
Transfection of RAW264.7 cells: 1.5. mu.g of each of pX458-Gfi1b-1 and pX458-Gfi1b-2 vector DNAs was added to 150. mu.L of serum-reduced medium (Opti-MEM) to dilute the plasmids, and 3. mu.g of the total was used as an experimental group; 0.75 μ L of liposome Lipofectamine 3000 was diluted in 150 μ L of reduced serum medium (Opti-MEM), and the liposome dilution was added to the DNA dilution, mixed well and incubated at room temperature for 20 min. The medium in the 24-well plate in which the cultured cells had been seeded in the above step was discarded, and then the complexes were added to the corresponding cells in order of 300. mu.L per well, and three additional wells were added as a parallel control experiment group and cells to which no transfection mixture was added as a negative control group. All cells were incubated at 37 ℃ with 5% CO2After incubation for 1.5h in the incubator, the medium was discarded and the culture continued with replacement of fresh medium.
(7) Sorting single cells: after transfection for 72h, the medium was discarded, the cells were resuspended in a centrifuge tube containing 500. mu.L of fresh medium by trypsinization, after centrifugation at 1350rpm for 5min, most of the medium supernatant was discarded, about 200. mu.L of the medium was left at the bottom of the tube, gently pipetted and mixed, and then 400. mu.L of fresh medium was added and mixed, and transferred to a flow tube. To obtain stable mutant monoclonal cells, single cells that were double positive for DsRed2 and ECFP fluorescence were sorted by flow cytometry into 96-well cell culture plates to which 150 μ L of fresh medium had been added. A schematic diagram of single cell sorting on DsRed2 and ECFP double positive cells by flow cytometry is shown in FIG. 3, in which the upper right round box represents the DsRed2 and ECFP fluorescent double positive cell population. After 14 days of culture, the single clones were transferred to 48-well cell culture plates for further expansion and subsequent analysis.
(8) Obtaining monoclonal cell genomic DNA by direct lysis method: taking a small amount of cells (no quantification is needed, about 100-.
(9) Designing a primer, and detecting whether the selected monoclonal cell strain has base deletion or insertion. Specific primers were designed against the genomic sequence of the targeting site using the in-line primer design software primer3(http:// primer3.ut. ee /), Gfi1b-S PCR-F (SEQ ID NO. 7): 5'-ATCTGGGGAGCAAGGCCTAT-3' and Gfi1b-S PCR-R (SEQ ID NO. 8): 5'-CGATGCTCCCTCAACTCCAA-3', amplifying target fragments (with a theoretical length of 480bp) containing the target sites by PCR, wherein the system comprises: (Vazyme, P111)2 Taq Master PCR MIX: 10 μ L of each of Gfi1b-S PCR-F (5 μ M) and Gfi1b-S PCR-R (5 μ M); genomic DNA: 10 ng; ddH2O was supplemented to a total volume of 20 μ L; the procedure is as follows: 95 ℃ for 5 min; 95 ℃, 30s, 60 ℃, 30s, 72 ℃, 40 s; 72 ℃ for 10 min. The result of agarose gel electrophoresis detection of PCR products is shown in FIG. 4, and it can be clearly seen from the electrophoresis chart that a large number of sorted DsRed2 and ECFP fluorescence double-positive monoclonal cells have large fragment base deletion, wherein the single cell clones No.1, 2, 3, 15 and 17 are large fragment deletion homozygotes, the single cell clones No. 9, 21 and 22 are large fragment deletion heterozygotes, and the results show that the single cell clones No.1, 2, 3, 15 and 17 are Gfi1b gene knockout cell strains.
(10) And (4) carrying out amplification culture on the verified monoclonal cell strain, and freezing and storing liquid nitrogen for seed preservation.
Example 3
A method for quickly knocking out large-fragment deleted RAW264.7 cell line Pparg genes through a CRISPR/Cas9 system.
(1) Determining the specific target sites sgRNA1, sgRNA 2: the mouse Pparg gene DNA sequence (Transcript ID: ENSMUST00000171644.7) was found in the mouse genome database ensemble (http:// asia. ensemble. org), and then the online design software CRISPOR was used
(http:// crispor. for. net/crispor. cgi), 2 specific sites were determined to be selected within the target site intron2-3 of the mouse Pparg gene as target sequences for sgrnas, which are:
sgRNA1(SEQ ID NO.9)：5‘-GTATACCTAACAAGATACTA TGG-3’；
sgRNA2(SEQ ID NO.10)：5‘-GTGAAGCTGTGCGTCATTTC AGG-3’。
(2) designing a primer: according to the sgRNA target sequence in the step (1), 4 primers (Shanghai Bailey Biotechnology limited, and BbsI enzyme cutting site is added at the 5' end of the primer sequence:
M-Pparg-IVT-1(SEQ ID NO.11)：5‘-CACCGTATACCTAACAAGATACTA-3’；
M-Pparg-IVT-2(SEQ ID NO.12)：5‘-AAACTAGTATCTTGTTAGGTATAC-3’；
M-Pparg-IVT-3(SEQ ID NO.13)：5‘-CACCGTGAAGCTGTGCGTCATTTC-3’；
M-Pparg-IVT-4(SEQ ID NO.14)：5‘-AAACGAAATGACGCACAGCTTCAC-3’。
(3) obtaining cohesive powder through primer annealing and pairingDouble-stranded DNA fragment at the end: the 4 primers synthesized in step (2) are used for annealing pairing in a combination mode of M-PParg-IVT-1+ M-PParg-IVT-2 and M-PParg-IVT-3+ M-PParg-IVT-4 to obtain double-stranded DNA fragments with sticky ends, and the specific procedures are as follows: firstly, respectively phosphorylating two pairs of synthesized primers, wherein the phosphorylation reaction system is that 1 mu l (100 mu.M) of each primer M-PParg-IVT-1+ M-Pparg-IVT-2 and M-Pparg-IVT-3+ M-Pparg-IVT-4 is added, then 1 mu l of 10 XT 4Ligation buffer (NEB) is added, then 0.5 mu. l T4 polynuceotide Kinase (NEB M020 0201S) is added, and finally 6.5 mu.l of ddH is added2O to the total volume of 10 mu l, fully and uniformly mixing after preparing a reaction system, and incubating for 30min at 37 ℃; taking out the reaction product, transferring the reaction product to PCR for denaturation and annealing, and carrying out the following reaction procedures: 95 ℃ for 5 min; 95-25 ℃ at-5 ℃/min.
(4) The vector (pX458-DsRed2 and pX458-ECFP) DNA was linearized with the restriction enzymes BbsI and then purified and recovered to obtain vector DNA fragments having cohesive ends, in the same manner as in step (4) of example 2.
(5) The whole sgRNA and Cas9 expression vectors are obtained through ligation, transformation and recombinant screening, the specific method is the same as the step (5) in the example 2, and finally expression vectors pX458-Pparg-1 and pX458-Pparg-2 expressing DsRed2 and ECFP respectively can be obtained, the sequencing peaks of the sgRNA expression vectors are shown in fig. 2, B: pX458-Pparg-1 and pX458-Pparg-2 sequencing peak plots.
(6) The constructed expression vectors are mixed in equal mass, and then a mouse macrophage system RAW264.7 is transfected by a liposome-mediated transfection mode. The detailed steps of cell culture and transfection were as in (6) in example 2.
(7) Sorting single cells: the procedure was the same as in (7) in example 2 above.
(8) Obtaining monoclonal cell genomic DNA by direct lysis method: the procedure was the same as in (8) in example 2 above.
(9) Designing a primer, and detecting whether the selected monoclonal cell strain has base deletion or insertion. Specific primers were designed against the genomic sequence of the targeting site using the in-line primer design software primer3(http:// primer3.ut. ee /), Pparg-S PCR-F (SEQ ID NO. 15): 5'-TGGCAGCAGCTAGTCTCTCA-3' and Pparg-S PCR-R (SEQ ID NO. 16): 5'-GGTGCAAGACTGTGCATACG-3', PCR amplification of the target fragment (theoretical length 378bp) containing the targeting site, in the following system: (Vazyme, P111)2 Taq Master PCR MIX: 10 μ L of each of Vav1-S PCR-F (5 μ M) and Vav1-S PCR-R (5 μ M); genomic DNA: 10 mu g of the mixture; ddH2O was supplemented to a total volume of 20 μ L; the procedure is as follows: 95 ℃ for 5 min; 95 ℃, 30s, 60 ℃, 30s, 72 ℃, 40 s; 72 ℃ for 10 min. The result of agarose gel electrophoresis detection of PCR products is shown in FIG. 5, and it can be clearly seen from the electrophoresis chart that a large number of sorted DsRed2 and ECFP fluorescence double-positive monoclonal cells have large fragment base deletion, wherein the single cell clones No.2, 3 and 9 are large fragment deletion homozygotes, the single cell clones No.1, 5, 6, 8 and 11 are large fragment deletion heterozygotes, and the results show that the single cell clones No.2, 3 and 9 are Pparg gene knockout cell strains.
(10) And (4) carrying out amplification culture on the verified monoclonal cell strain, and freezing and storing liquid nitrogen for seed preservation.
Example 4:
a method for quickly knocking out a large-fragment deleted HaCaT cell line DSG4 gene by a CRISPR/Cas9 system.
(1) Determining the specific target sites of the mouse Gene DSG4 to be knocked out (Gene ID:2661061), sgRNA1, sgRNA 2: the mouse DSG4 gene DNA sequence (Transcript ID: ENSMUST00000019426.4) was found in the mouse genome database ensemble (http:// asia. ensemble. org), and then 2 specific sites were determined to be selected within the target site exon12(exon ID: ENST00000308128.8) of the mouse DSG4 gene as the target sequences for sgRNAs using the online design software CRISPOR (http:// CRISPOR. for. net/CRISPOR. cgi): sgRNA1(SEQ ID No. 17): 5'-CTTAGCCGTAAGGATTGCCG AGG-3', sgRNA2(SEQ ID NO. 18): 5'-GTGGTTGTCATCGCAATCAC AGG-3' are provided.
(2) Designing a primer: designing 4 primers (Shanghai Bailey biotechnology, Inc.) with 2 pairs according to the sgRNA target sequence in the step (1), and adding BbsI enzyme cutting site at the 5' end of the primer sequence:
M-DSG4-IVT-1(SEQ ID NO.19)：5‘-CACCGCTTAGCCGTAAGGATTGCCG-3’；
M-DSG4-IVT-2(SEQ ID NO.20)：5‘-AAACCGGCAATCCTTACGGCTAAG C-3’；
M-DSG4-IVT-3(SEQ ID NO.21)：5‘-CACCGTGGTTGTCATCGCAATCAC-3’；
M-DSG4-IVT-4(SEQ ID NO.22)：5‘-AAACGTGATTGCGATGACAACCAC-3’。
(3) obtaining double-stranded DNA fragments with sticky ends through primer annealing and pairing: the 4 primers synthesized in step (2) were used for annealing and pairing in the combination of M-DSG4-IVT-1+ M-DSG4-IVT-2 and M-DSG4-IVT-3+ M-DSG4-IVT-4 to obtain double-stranded DNA fragments with sticky ends, and the specific procedure was as follows: firstly, respectively phosphorylating two pairs of synthesized primers, wherein the phosphorylation reaction system is that primers M-DSG4-IVT-1+ M-DSG4-IVT-2 and M-DSG4-IVT-3+ M-DSG4-IVT-4 are respectively added with 1 mul (100 mu.M), then 1 mul 10 XT 4Ligation Buffer (NEB) is added, then 0.5 mu. l T4 polynuceotide Kinase (NEB M020 0201S) is added, and finally 6.5 mul ddH is added2O to the total volume of 10 mu l, fully and uniformly mixing after preparing a reaction system, and incubating for 30min at 37 ℃; taking out the reaction product, transferring the reaction product to PCR for denaturation and annealing, and carrying out the following reaction procedures: 95 ℃ for 5 min; 95-25 ℃ at-5 ℃/min.
(4) The vector (pX458-DsRed2 and pX458-ECFP) DNA was linearized with the restriction enzymes BbsI and then purified and recovered to obtain vector DNA fragments having cohesive ends, in the same manner as in step (4) of example 2.
(5) The whole sgRNA and Cas9 expression vectors are obtained through ligation, transformation and recombinant screening, the specific method is the same as the step (5) in the example 2, and finally expression vectors pX458-DSG4-1 and pX458-DSG4-2 expressing DsRed2 and ECFP respectively can be obtained, the sequencing peaks of the sgRNA expression vectors are shown in fig. 2, C: pX458-Dsg4-1 and pX458-Dsg4-2 sequencing peak plots.
(6) The constructed expression vectors are mixed in equal mass, and then the human immortalized epidermal cell line HaCaT is transfected by a transfection mode mediated by liposome. The detailed steps of cell culture and transfection are as follows:
culture and passage of HaCaT cells: the cells were purchased from an ATCC cell bank,bring the cells back to the laboratory in CO2Culturing in an incubator, after the cells are completely attached to the wall, replacing the DPBS with a liquid to wash out dead cells and cell metabolites, adding a fresh culture solution (MEM/GLUCOSE + 10% FBS + 1% double antibody + 1% sodium pyruvate + 1% non-essential amino acid) to continue culturing, observing the cell morphology and growth condition under an inverted microscope every day, and beginning passage when the cells grow to about 90% of the bottom of a culture flask. The passaging method is the same as that of Raw264.7 cells. In contrast, HaCaT cells were digested at 37 ℃ for 10min during subculture digestion, and the flasks were observed under a microscope, and when the cell morphology became round and the cell space increased, trypsin was discarded and 3ml of MEM was added to terminate the digestion reaction.
Transfection of HaCaT cells: the vector DNA pX458-DSG4-1 and pX458-DSG4-2 were transfected into HaCaT cells as in Raw264.7 cells.
(7) Sorting single cells: the procedure was the same as in (7) in example 2 above.
(8) Obtaining monoclonal cell genomic DNA by direct lysis method: the procedure was the same as in (8) in example 2 above.
(9) Designing a primer, and detecting whether the selected monoclonal cell strain has base deletion or insertion. Specific primers were designed for the genomic sequence of the targeting site using the in-line primer design software primer3(http:// primer3.ut. ee /), DSG4-S PCR-F (SEQ ID NO.23): 5'-GTATTAGGGAGAGTTAACCACCCC-3' and DSG4-S PCR-R (SEQ ID NO.24): 5'-TTCAGTGACAGGCCCATACG-3', and the fragment of interest (296 bp theoretical length) containing the targeting site was amplified by PCR using the system: (Vazyme, P111)2 Taq Master PCR MIX: 10 μ L of each of DSG4-S PCR-F (5 μ M) and DSG4-S PCR-R (5 μ M); genomic DNA: 10 mu g of the mixture; supplemental ddH2O to the total volume of 20 mu L; the procedure is as follows: 95 ℃ for 5 min; 95 ℃, 30s, 60 ℃, 30s, 72 ℃, 40 s; 72 ℃ for 10 min. The result of detecting PCR products by agarose gel electrophoresis is shown in FIG. 6, and it can be clearly seen from the electrophoresis chart that a large part of DsRed2 and ECFP fluorescence double-positive monoclonal cells obtained by sorting all have large fragment base deletion, wherein the No.2, 3, 4, 6, 8 and 10 single cell clones are large fragment deletion homozygotes, and the No.1, 5, 7 and 9 single cell clones are large fragment deletion homozygotesThe single cell clone is a large fragment deletion heterozygote, and the results show that the single cell clones No.2, 3, 4, 6, 8 and 10 are the DSG4 gene knockout cell strains.
(10) And (4) carrying out amplification culture on the verified monoclonal cell strain, and freezing and storing liquid nitrogen for seed preservation.
<110> New countryside medical college
<120> cell line gene knockout method for rapidly obtaining large fragment deletion through CRISPR/Cas9 system
<160>29
<170>PatentIn version 3.5
<211>23
<212>DNA
<213> sequence
<221> Gfi1b Gene target site 1
<400>1
agtgacaagc gctagtcctt tgg 23
<211>23
<212>DNA
<213> sequence
<221> Gfi1b Gene target site 2
<400>2
ttaccaccag ccccgggcac agg 23
<211>25
<212>DNA
<213> sequence
<221>M-Gfi1b-IVT-1
<400>3
caccgagtga caagcgctag tcctt 25
<211>25
<212>DNA
<213> sequence
<221>M-Gfi1b-IVT-2
<400>4
aaacaaggac tagcgcttgt cactc 25
<211>25
<212>DNA
<213> sequence
<221>M-Gfi1b-IVT-3
<400>5
caccgttacc accagccccg ggcac 25
<211>25
<212>DNA
<213> sequence
<221>M-Gfi1b-IVT-4
<400>6
aaacgtgccc ggggctggtg gtaac 25
<211>20
<212>DNA
<213> sequence
<221>Gfi1b-S PCR-F
<400>7
atctggggag caaggcctat20
<211>20
<212>DNA
<213> sequence
<221>Gfi1b-S PCR-R
<400>8
<211>23
<212>DNA
<213> sequence
<221> Pparg Gene target site 1
<400>9
gtatacctaa caagatacta tgg 23
<211>23
<212>DNA
<213> sequence
<221> Pparg Gene target site 2
<400>10
gtgaagctgt gcgtcatttc agg 23
<211>24
<212>DNA
<213> sequence
<221>M-Pparg-IVT-1
<400>11
caccgtatac ctaacaagat acta 24
<211>24
<212>DNA
<213> sequence
<221>M-Pparg-IVT-2
<400>12
aaactagtat cttgttaggt atac24
<211>24
<212>DNA
<213> sequence
<221>M-Pparg-IVT-3
<400>13
caccgtgaag ctgtgcgtca tttc 24
<211>24
<212>DNA
<213> sequence
<221>M-Pparg-IVT-4
<400>14
aaacgaaatg acgcacagct tcac 24
<211>20
<212>DNA
<213> sequence
<221>Pparg -S PCR-F
<400>15
<211>20
<212>DNA
<213> sequence
<221>Pparg -S PCR-R
<400>16
<211>23
<212>DNA
<213> sequence
<221> DSG4 Gene target site 1
<400>17
cttagccgta aggattgccg agg 23
<211>23
<212>DNA
<213> sequence
<221> DSG4 Gene target site 2
<400>18
gtggttgtca tcgcaatcac agg 23
<211>25
<212>DNA
<213> sequence
<221>M-DSG4-IVT-1
<400>19
caccgcttag ccgtaaggat tgccg 25
<211>25
<212>DNA
<213> sequence
<221>M-DSG4-IVT-2
<400>20
aaaccggcaa tccttacggc taagc 25
<211>24
<212>DNA
<213> sequence
<221>M-DSG4-IVT-3
<400>21
caccgtggtt gtcatcgcaa tcac 24
<211>24
<212>DNA
<213> sequence
<221>M-DSG4-IVT-4
<400>22
aaacgtgatt gcgatgacaa ccac 24
<211>24
<212>DNA
<213> sequence
<221>DSG4-S PCR-F
<400>23
gtattaggga gagttaacca cccc 24
<211>20
<212>DNA
<213> sequence
<221>DSG4-S PCR-R
<400>24
<211>753
<212>DNA
<213> sequence
<221> synthetic DsRed2 sequence
<400>25
gaattcggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat 60
cctggcccaa tggcctcctc cgagaacgtc atcaccgagt tcatgcgctt caaggtgcgc 120
atggagggca ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc 180
tacgagggcc acaacaccgt gaagctgaag gtgaccaagg gcggccccct gcccttcgcc 240
tgggacatcc tgtcccccca gttccagtac ggctccaagg tgtacgtgaa gcaccccgcc 300
gacatccccg actacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg 360
aacttcgagg acggcggcgt ggcgaccgtg acccaggact cctccctgca ggacggctgc 420
ttcatctaca aggtgaagttcatcggcgtg aacttcccct ccgacggccc cgtgatgcag 480
aagaaaacca tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg 540
aagggcgaga cccacaaggc cctgaagctg aaggacggcg gccactacct ggtggagttc 600
aagtccatct acatggccaa gaagcccgtg cagctgcccg gctactacta cgtggacgcc 660
aagctggaca tcacctccca caacgaggac tacaccatcg tggagcagta cgagcgcacc 720
gagggccgcc accacctgtt cctgtaggaa ttc 753
<211>792
<212>DNA
<213> sequence
<221> Synthesis of ECFP sequences
<400>26
gaattcggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat 60
cctggcccag tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 120
ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 180
acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg 240
cccaccctcg tgaccaccct gacctggggc gtgcagtgct tcagccgcta ccccgaccac 300
atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 360
atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 420
accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 480
gggcacaagc tggagtacaa ctacatcagc cacaacgtct atatcaccgc cgacaagcag 540
aagaacggca tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag 600
ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac 660
aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac 720
atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac 780
aagtaagaat tc 792
<211>480
<212>DNA
<213> sequence
<221> Gfi1b Gene PCR assay fragment (wild type)
<400>27
atctggggag caaggcctat cttatcttca ggcacatctt aacttgatgt gatattcaca 60
ggtgtcaggt ggatgtgaac tttggggaac gttatacaag ccagcatggg atagtgtgcc 120
tttgatccaa aggactagcg cttgtcactc ttagtcactc ccattgcccc tcctcccccg 180
caggtgtggc gtgcacgcag aaaaatgcca cggtcctttc tagtgaagag taagaaggca 240
cacacttacc accagccccg ggcacagggt gatgagctgg tctggcctcc tgctgtaatt 300
cctggtgagt ctgagcttag gctatatggc ccacctcatg cctgcttggc actgtcttga 360
tgatgctgtg gcatttcttc tgtatctaac tgtcactgaa tgaatgaatg gacaagtgaa 420
taaatgaatg aatgaaataa atactcatct cctctagcct ttggagttga gggagcatcg 480
<211>378
<212>DNA
<213> sequence
<221> PCR detection fragment of Pparg gene (wild type)
<400>28
tggcagcagc tagtctctca aagtgctaaa atacatccat ctctctaata ttgtcaggtc 60
agtgtgtcta taagtgtact ccctaagtta taattttacc ctgaagaagt aatgctagag 120
aaactctaaa accatagtat cttgttaggt atacatttct tgtctcagag gaaatctggg 180
ctgagaaatt ttcttcactg acttagggga cctgaaatga cgcacagctt cactatgtta 240
gcttccttgg acacaccaga atgtcaaaga atttagtgaa tacttcctga gtacttgtgt 300
tggagtcatt taaatagaat tgggtttctg tacctttcta ctgcactatt ctttccttcg 360
tatgcacagt cttgcacc 378
<211>296
<212>DNA
<213> sequence
<221> PCR detection fragment of DSG4 gene (wild type)
<400>29
gtattaggga gagttaacca cccccctagc ccaccaagga atttccattt attttctgtt 60
tcctctcttc catttcagct acctcggcaa tccttacggc taagcaggtt ttatctccag 120
gattttatga aatcccaatc ctggtgaagg acagctataa cagagcatgt gaattggcac 180
aaatggtgca gttatatgcc tgtgattgcg atgacaacca catgtgcctg gactctggtg 240
ccgcgggcat ctacacagag gacataactg gtgacacgta tgggcctgtc actgaa 296
Claims (10)
1. A cell line gene knockout method for rapidly obtaining large fragment deletion through a CRISPR/Cas9 system is characterized by comprising the following steps: the method comprises the following steps:
1) selecting a target gene to be knocked out, and using online sgRNA design software to obtain two sgRNA sequences, wherein the interval between the sgRNA sequences is 50-100 bp;
2) adding enzyme cutting sites into the sgRNA sequence obtained in the step 1) and synthesizing single-stranded primers, annealing the paired primers to obtain corresponding double-stranded DNA fragments with sticky ends, respectively connecting the double-stranded DNA fragments into CRISPR/Cas9 system expression vectors with different fluorescent reporter genes, correspondingly connecting one double-stranded DNA fragment with one CRISPR/Cas9 system expression vector with the fluorescent reporter genes, equivalently mixing the constructed expression vectors, and transferring the mixed expression vectors into cells; the different fluorescent reporter genes are DsRed2 and ECFP;
3) after the cells are cultured, a flow cytometer is used for positive screening, and monoclonal cells containing all fluorescent reporter gene signals are separated;
4) carrying out amplification culture on the obtained monoclonal cells, taking the cells after 10-15 days, and obtaining genome DNA by a direct lysis method;
5) designing a PCR detection primer containing a target site, carrying out PCR amplification by taking the genomic DNA obtained in the step 4) as a template, carrying out gel electrophoresis detection on an amplification product, and selecting a large fragment-deleted homozygote cell line.
2. The method for rapidly knocking out a cell line gene with a large fragment deletion by a CRISPR/Cas9 system according to claim 1, which comprises the following steps:
1) the EGFP fluorescent reporter gene in pX458 was replaced by DsRed2 and ECFP, which were designated as: pX458-DsRed2 and pX 458-ECFP; selecting a target gene to be knocked out, and using online sgRNA design software to obtain two sgRNA sequences, wherein the interval between the two sgRNA sequences is 50-100 bp;
2) adding enzyme cutting sites to the two sgRNA sequences obtained in the step 1) and synthesizing 4 single-stranded primers, annealing the paired primers to obtain two double-stranded DNA fragments with sticky ends, and respectively connecting the double-stranded DNA fragments into vectors pX458-DsRed2 and pX458-ECFP to obtain expression vectors pX458-DsRed2-sgRNA1 and pX458-ECFP-sgRNA 2;
3) transferring expression vectors of pX458-DsRed2-sgRNA1 and pX458-ECFP-sgRNA2 into cells by means of lipofection or electrotransformation, and performing double-positive single cell sorting on DsRed2 and ECFP fluorescent reporter genes by using a flow cytometer after culturing;
4) carrying out amplification culture on the obtained monoclonal cells, taking the cells after 10-15 days, and obtaining genome DNA by a direct lysis method;
5) designing a PCR detection primer containing two target sites, carrying out PCR amplification by taking the genomic DNA obtained in the step 4) as a template, carrying out gel electrophoresis detection on an amplification product, and selecting a large fragment-deleted homozygote cell line.
3. The method for rapidly knocking out a cell line gene with a large fragment deletion by a CRISPR/Cas9 system according to claim 2, wherein the method comprises the following steps: when the target gene is Gfi1b in step 1), the two sgRNA sequences are respectively: 5'-AGTGACAAGCGCTAGTCCTTTGG-3', 5 '-TTACCACCAGCCCCGGGCACAGG 3'.
4. The method for rapidly knocking out a cell line gene with a large fragment deletion by a CRISPR/Cas9 system according to claim 2, wherein the method comprises the following steps: when the target gene is Pparg in step 1), the two sgRNA sequences are respectively: 5'-GTATACCTAACAAGATACTATGG-3', 5'-GTGAAGCTGTGCGTCATTTCAGG-3' are provided.
5. The method for rapidly knocking out a cell line gene with a large fragment deletion by a CRISPR/Cas9 system according to claim 2, wherein the method comprises the following steps: when the target gene is DSG4 in step 1), the two sgRNA sequences are: 5'-CTTAGCCGTAAGGATTGCCGAGG-3', 5'-GTGGTTGTCATCGCAATCACAGG-3' are provided.
6. The method for rapidly knocking out a cell line gene with a large fragment deletion by a CRISPR/Cas9 system according to claim 2, wherein the method comprises the following steps: in designing the primer in step 2), if the 5' starting base of the upstream primer is not G, one additional base G is added.
7. The method for rapidly knocking out a cell line gene with a large fragment deletion by a CRISPR/Cas9 system according to claim 2, wherein the method comprises the following steps: culturing for 40-80h in the step 3), and then carrying out single cell sorting by using a flow cytometer.
8. The method for rapidly knocking out a cell line gene with a large fragment deletion by a CRISPR/Cas9 system according to claim 2, wherein the method comprises the following steps: the formula of the cracking solution used in the direct cracking method in the step 4) is as follows: 100mmol/L KCl, 20mmol/L Tris-HCl pH =9.0, 0.3% Triton X-100, 1.0mg/mL proteinase K.
9. The method for rapidly knocking out a cell line gene with a large fragment deletion by a CRISPR/Cas9 system according to claim 8, wherein the method comprises the following steps: the direct cracking method in the step 4) comprises the following specific operations: incubate at 55 ℃ for 15min, then incubate at 95 ℃ for 10 min.
10. The method for rapidly knocking out a cell line gene with a large fragment deletion by a CRISPR/Cas9 system according to claim 2, wherein the method comprises the following steps: and 5) comparing the amplification product to be detected with a wild type amplification product, and selecting a homozygote cell line with two chromosomes both suffering from large fragment base deletion.
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| US9163284B2 (en) | 2013-08-09 | 2015-10-20 | President And Fellows Of Harvard College | Methods for identifying a target site of a Cas9 nuclease |
| US9359599B2 (en) | 2013-08-22 | 2016-06-07 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
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