CN110564774B - Method for improving fixed-point modification efficiency of cell genome by using modified ssODN - Google Patents

Abstract

The invention discloses a method for improving the fixed-point modification efficiency of a cell genome by using modified ssODN. The method is realized by the CRISPR/Cas9 system and the modified ssODN acting on the cell together. Specifically, the CRISPR/Cas9 system comprises a target gene gRNA fragment, can recognize a target gene at a fixed point, and enables the target gene to generate double-strand break, and the ssODN and the target gene are subjected to base complementary pairing at the break, so that the fixed-point modification of a genome is efficiently completed, and the modified ssODN can remarkably improve the efficiency of the fixed-point modification.

Description

Method for improving fixed-point modification efficiency of cell genome by using modified ssODN

Technical Field

The invention relates to the technical field of genetic engineering. In particular, the invention relates to a method for improving the efficiency of site-directed modification of a cellular genome using a modified ssODN.

Background

Currently, the most widely used gene editing tools mainly include Zinc finger endonuclease (ZFN), Transcription activator-like effector nuclease (TALEN), regularly Clustered short spaced palindromic repeats (CRISPR), wherein the CRISPR/Cas9 system consists of an exogenous single-stranded guide rna (sgRNA) and Cas9 protein, and the sgRNA recognizes a genome sequence by the base complementary pairing principle and guides the Cas9 protein to generate a genome Double-stranded break (DSB) at a binding target. Meanwhile, cells activate two different repair mechanisms, namely non-Homologous end joining (NHEJ) or Homologous-directed repair (HDR) in vivo, so that endogenous genes are knocked out or exogenous genes are knocked in at a fixed point. Although the efficiency of generating DSB by CRISPR/Cas9 is guaranteed to a certain extent, the efficiency of site-directed modification mediated by HDR is still quite low, so that a new way for improving the efficiency of site-directed modification of genome is urgently needed to be searched.

Disclosure of Invention

According to one aspect of the present disclosure, a method is provided for increasing the efficiency of site-directed modification of a genome of a cell using a modified ssODN by acting on the cell with the modified ssODN through a CRISPR/Cas9 system. Specifically, the CRISPR/Cas9 system comprises a target gene gRNA fragment, can recognize a target gene at a fixed point, and enables the target gene to generate double-strand break, at the break, the ssODN and the target gene are subjected to base complementary pairing to carry out homologous recombination and repair, the fixed-point modification of a genome is efficiently completed, and the modified ssODN can remarkably improve the efficiency of the fixed-point modification.

In certain embodiments, the modified ssODN comprises a BIO modification or a PHO modification or a THS modification or a THO modification or an AMN modification. Therefore, compared with unmodified ssODN, the modified ssODN can significantly improve the fixed-point modification efficiency of the cell genome by 2.8 times.

In certain embodiments, the modified ssODN is a THO modification. Thus, the efficiency of site-directed modification can be increased by 2.8-fold compared to unmodified ssODN.

In certain embodiments, the THO-modified ssODN has the gene sequence shown in SEQ ID No. 9. Thus, the efficiency of site-directed modification can be increased by 2.8-fold compared to unmodified ssODN.

In certain embodiments, the modified ssODN is a THS modification. Thus, the efficiency of site-directed modification can be increased by 2.5-fold compared to unmodified ssODN.

In certain embodiments, the THS-modified ssODN has the gene sequence shown in SEQ ID No. 8. Thus, the efficiency of site-directed modification can be increased by 2.5-fold compared to unmodified ssODN.

In certain embodiments, a method for increasing the efficiency of site-directed modification of a genome of a cell, the method comprising the steps of:

constructing a target gene CRISPR/Cas9 system expression vector;

synthesis of ssODN reporter vector;

synthesizing the modified ssODN;

screening cell lines that can express the ssODN reporter vector;

the CRISPR/Cas9 system expression vector co-transfects with the modified ssODN a cell line that can express an ssODN reporter vector;

and co-treating the screened effectively modified ssODN and the CRISPR/Cas9 system to improve the efficiency of genome site-directed modification.

Therefore, the gRNA in the expression vector of the CRISPR/Cas9 system recognizes the sequence on the ssODN report vector by the base complementary pairing principle, so that the report gene generates double-strand break, the modified ssODN and the report gene are subjected to homologous recombination and repair, the report gene is subjected to site-directed modification, an effective ssODN modification mode can be screened, the screened effectively modified ssODN and the CRISPR/Cas9 system co-process a target cell, and the genome site-directed modification efficiency of the cell can be remarkably improved.

In some embodiments, the construction of the CRISPR/Cas9 system expression vector comprises selecting a target gene, designing and synthesizing a corresponding gRNA, and constructing into a PX330 plasmid to construct a PX330-ROSA26 expression vector, wherein the gene sequence of the PX330-ROSA26 expression vector is shown in SEQ ID No: 3.

In some embodiments, the synthesis of the ssODN reporter vector comprises selecting a ssODN template sequence, and linking the selected ssODN template sequence with EGFP to form the ssODN-GFP reporter vector, wherein a sequence homologous to the target gene is inserted into the EGFP gene of the reporter vector, and the ssODN reporter gene sequence is shown in SEQ ID No: 4. Therefore, the target gene gRNA and the Cas9 protein are matched to form a CRISPR/Cas9 system, the homologous sequence of the target gene inserted between EGFP can be identified in a fixed-point manner, the double-strand break of the genome is generated, the accurate positioning and the double-strand break of the reporter gene EGFP can be realized, and the basis is provided for the subsequent realization of the fixed-point modification of the gene.

In certain embodiments, the method of increasing the efficiency of site-directed modification of a cellular genome comprises co-transfecting the obtained PX330-ROSA26 expression vector with a modified ssODN into a cell line stably expressing a reporter vector, thereby increasing the efficiency of site-directed modification of a genome.

Drawings

FIG. 1 is a schematic diagram of a ssODN-GFP reporter vector;

FIG. 2 is a graph showing the effect of different modifications of ssODN on the efficiency of site-directed modification of human cells;

FIG. 3 is a graph showing the results of different modifications of ssODN on the efficiency of site-directed hamster cell modification;

FIG. 4 is a graph showing the effect of different modifications of ssODN on the efficiency of site-directed modification of porcine-derived cells.

Detailed Description

The present disclosure is described in further detail below with reference to specific examples.

The first embodiment is as follows: method for improving human source cell genome fixed-point modification efficiency

This example was tested using human cells (293T) as a tool cell line.

1. Plasmid construction

The gRNA sequence of the porcine ROSA26 gene (the gene sequence is shown in SEQ ID No:1 and SEQ ID No: 2) was designed using an online website (https:// crispr. cos. uni-heidelberg. de/index. html) and was artificially synthesized by Huada Gene. The synthetic primer is annealed and then connected with a linearized PX330 plasmid which is cut by BpiI enzyme, and the successfully constructed plasmid is named PX330-ROSA26 (the gene sequence of the plasmid is shown as SEQ ID No: 3). Meanwhile, the ssODN-GFP report vector is designed and synthesized by Nanjing Kingsler Biotech Co., Ltd (the gene sequence of the report vector is shown as SEQ ID No: 4): a sequence homologous to ROSA26 gene, a stop codon and a BamHI enzyme cutting site are inserted in the middle of the EGFP sequence of the report vector. The unmodified ssODN and the BIO, PHO, THS, THO, AMN modified ssODN were artificially synthesized by Yinxie Jie (Shanghai) trade Co., Ltd., and the sequences are shown in SEQ ID No. 5-SEQ ID No. 10. When DSBs are generated, cells undergo homologous repair with 140nt ssODN as a homology template, EGFP repair is completed, cells can detect green fluorescence signals, and expression is terminated without homologous repair (the mode diagram of ssODN-GFP reporter vector is shown in fig. 1).

TABLE 1 primer sequence information

2. Cell recovery and culture

And opening the water bath kettle, setting the temperature to be 37 ℃, taking out the cell cryopreservation tube from the liquid nitrogen tank after the temperature is reached, and immediately putting the tube into a water bath at 37 ℃ for rapid thawing. Transferring the thawed cell suspension into a centrifugal tube in an ultraclean workbench, adding 3 times of culture solution, centrifuging at the room temperature for 5min, removing supernatant, adding culture solution containing 10% fetal calf serum to suspend and precipitate cells, diluting the cells to a required concentration, inoculating the cells into a culture dish with the diameter of 6cm, standing and culturing in a 5% CO2 incubator at the temperature of 39 ℃, changing the solution the next day, and performing subculture when the confluence degree of the cells reaches 80-90%.

3. Tool cell line screening

The ssODN-GFP reporter plasmid was digested with BglII restriction endonuclease and transfected into cells, which were then replaced with 15% FBS complete medium after 6 h. After 24h the selection was started with 400. mu.g/mL of G418, after which the G418 concentration was adjusted with the cell density. After 6d, the maintenance medium is replaced by 200 mu g/mL according to the cell growth condition, until a monoclonal cell mass is selected after 14d, and the cell mass is subcultured to a complete medium 24-well culture dish containing 15% FBS after digestion. The cells are subcultured in a 6-well plate, when the cells grow to reach 80% confluence, the frozen stock solution added with 10% DMSO is digested and stored overnight at-80 ℃, and then stored in liquid nitrogen for later use.

4. 293 cells Co-transfection of PX330-ROSA26 plasmid and ssODN Donor results

Transfection was initiated when 293 cell lines in 24-well plates grew to 60% -80%. Transfection procedures refer to Thermo fisher

LTX&Plus Reagent protocol, 1. mu.g/well PX330-ROSA26 plasmid and 400ng ssODN were transfected into each well. Wherein, the transfected ssODN is a blank group without modification and an experimental group modified by BIO, PHO, THS, THO and AMN. After 4-6h, the culture medium containing 10% fetal calf serum was replaced with fresh culture medium, and the percentage of the fluorescence number of the cells was measured by flow cytometry after 48 hours of continuous culture.

The results show that: the ssODN modified by THO and THS can significantly improve the efficiency of 293 cell site-directed modification, wherein the THO modification is improved by about 2 times, and the THS modification is improved by 1.8 times, and the results are shown in fig. 2.

Example two: method for improving fixed-point modification efficiency of mouse-derived cell genome

This example was tested using hamster kidney cells (BHK cells) as the tool cell line.

In the embodiment, the steps of plasmid construction, cell recovery and culture and tool cell line screening are specifically implemented according to the steps 1-3 in the first embodiment.

4. Results of cotransfection of Bingo Kidney cells with PX330-ROSA26 plasmid and ssODN Donor

Transfection was started when hamster kidney cells grew 60% -80% in 24-well plates. Transfection procedures refer to Thermo fisher

LTX&Plus Reagent protocol, 1. mu.g/well PX330-ROSA26 plasmid and 400ng ssODN were transfected into each well. Wherein, the transfected ssODN is a blank group without modification and an experimental group modified by BIO, PHO, THS, THO and AMN. After 4-6h, the culture medium containing 10% fetal calf serum was replaced with fresh culture medium, and the percentage of the fluorescence number of the cells was measured by flow cytometry after 48 hours of continuous culture.

The results show that: the modification of THO and THS can obviously improve the modification efficiency, wherein the THO obtains the highest modification efficiency which is about 2.3 times of that of a control group; while other modifications had no effect on the efficiency of site-directed modification, the results are shown in FIG. 3.

Example three: method for improving fixed-point modification efficiency of porcine cell genome

This example uses porcine cells (PK-15/PEF cell line) as a tool cell line for the experiments.

In the embodiment, the steps of plasmid construction, cell recovery and culture and tool cell line screening are specifically implemented according to the steps 1-3 in the first embodiment.

4. Results of Co-transfection of porcine derived cells with PX330-ROSA26 plasmid and ssODN donors

Transfection was initiated when 60% -80% of the porcine cells (PK-15/PEF cell line) in the 24-well plates grew. Transfection procedures refer to Thermo fisher

LTX&Plus Reagent protocol, 1. mu.g/well PX330-ROSA26 plasmid and 400ng ssODN were transfected into each well. Wherein, the transfected ssODN is a blank group without modification and an experimental group modified by BIO, PHO, THS, THO and AMN. After 4-6h, the culture medium containing 10% fetal calf serum was replaced with fresh culture medium, and the percentage of the fluorescence number of the cells was measured by flow cytometry after 48 hours of continuous culture.

The results show that: the efficiency of the THS-modified ssODN for improving PK-15 and PEF cells by about 1.1 times and 2.5 times respectively, while the efficiency of the THO-modified ssODN for improving PK-15 and PEF cells by 1.4 times and 2.8 times respectively is significantly better than that of the THS-modified ssODN, and the result is shown in FIG. 4.

Example four: the PK15 cell cotransfected by the THS modified ssODN and the CRISPR/Cas9 system can improve the site-directed modification efficiency of the ROSA26 gene

1. Plasmid construction

The gRNA sequence of the porcine ROSA26 gene (the gene sequence is shown in SEQ ID No:1 and SEQ ID No: 2) was designed using an online website (https:// crispr. cos. uni-heidelberg. de/index. html) and was artificially synthesized by Huada Gene. The synthetic primer is annealed and then connected with a linearized PX330 plasmid which is cut by BpiI enzyme, and the successfully constructed plasmid is named PX330-ROSA26 (the gene sequence of the plasmid is shown as SEQ ID No: 3).

2. Cell recovery and culture

And opening the water bath kettle, setting the temperature to be 37 ℃, taking out the cell cryopreservation tube from the liquid nitrogen tank after the temperature is reached, and immediately putting the tube into a water bath at 37 ℃ for rapid thawing. Transferring the thawed cell suspension into a centrifugal tube in an ultraclean workbench, adding 3 times of culture solution, centrifuging at the room temperature for 5min, removing supernatant, adding culture solution containing 10% fetal calf serum to suspend and precipitate cells, diluting the cells to a required concentration, inoculating the cells into a culture dish with the diameter of 6cm, standing and culturing in a 5% CO2 incubator at the temperature of 39 ℃, changing the solution the next day, and performing subculture when the confluence degree of the cells reaches 80-90%.

3. THS modified ssODN and CRISPR/Cas9 system cotransfected 293T cells

Transfection was initiated when 60% -80% of the PK15 cell lines grew in the 24-well plates. Transfection procedures refer to Thermo fisher

LTX&Plus Reagent protocol, 1. mu.g/well of PX330-ROSA26 plasmid and 400ng of unmodified ssODN or 400ng of THS-modified ssODN were transfected into each well, respectively. After 4-6h, the culture medium containing 10% fetal calf serum was replaced with fresh culture medium, and the percentage of the fluorescence number of the cells was measured by flow cytometry after 48 hours of continuous culture.

The results show that: the THS modified ssODN can obviously improve the site-directed modification efficiency of PK15 cells, and the THS modification efficiency can be improved by 1.5 times.

In other examples, the use of 293T cells as a tool cell line, the site-directed modification efficiency of PK15 cells was significantly improved by THS-modified ssODN, which was improved by 1.8-fold.

In other embodiments, BHK cells are used as a tool cell line, the site-directed modification efficiency of BHK cells can be significantly improved by THS-modified ssODN, and the THS modification efficiency can be improved by 2.1 times.

In other embodiments, site-directed modification is performed on the ACTB gene, and the THS-modified ssODN can significantly improve the site-directed modification efficiency by 2.3 times.

In other embodiments, the site-directed modification of GAPDH gene, the site-directed modification efficiency of THS-modified ssODN can be significantly increased by 2.7 times.

In other embodiments, where site-directed modification is performed on the SSA gene, the THS-modified ssODN can significantly improve the site-directed modification efficiency by 2.4 times.

Example five: the PK15 cell cotransfected by the ssODN modified by THO and the CRISPR/Cas9 system can improve the site-directed modification efficiency of the ROSA26 gene

1. Plasmid construction

The gRNA sequence of the porcine ROSA26 gene (the gene sequence is shown in SEQ ID No:1 and SEQ ID No: 2) was designed using an online website (https:// crispr. cos. uni-heidelberg. de/index. html) and was artificially synthesized by Huada Gene. The synthetic primer is annealed and then connected with a linearized PX330 plasmid which is cut by BpiI enzyme, and the successfully constructed plasmid is named PX330-ROSA26 (the gene sequence of the plasmid is shown as SEQ ID No: 3).

2. Cell recovery and culture

And opening the water bath kettle, setting the temperature to be 37 ℃, taking out the cell cryopreservation tube from the liquid nitrogen tank after the temperature is reached, and immediately putting the tube into a water bath at 37 ℃ for rapid thawing. Transferring the thawed cell suspension into a centrifugal tube in an ultraclean workbench, adding 3 times of culture solution, centrifuging at the room temperature for 5min, removing supernatant, adding culture solution containing 10% fetal calf serum to suspend and precipitate cells, diluting the cells to a required concentration, inoculating the cells into a culture dish with the diameter of 6cm, standing and culturing in a 5% CO2 incubator at the temperature of 39 ℃, changing the solution the next day, and performing subculture when the confluence degree of the cells reaches 80-90%.

3. THO-modified ssODN and CRISPR/Cas9 system cotransfected 293T cells

Transfection was initiated when 60% -80% of the PK15 cell lines grew in the 24-well plates. Transfection procedures refer to Thermo fisher

LTX&Plus Reagent protocol, 1. mu.g/well of PX330-ROSA26 plasmid and 400ng of unmodified ssODN or 400ng of THS-modified ssODN were transfected into each well, respectively. After 4-6h, the culture medium containing 10% fetal calf serum was replaced with fresh culture medium, and the percentage of the fluorescence number of the cells was measured by flow cytometry after 48 hours of continuous culture.

The results show that: the THO modified ssODN can obviously improve the site-directed modification efficiency of PK15 cells, and the THO modification efficiency can be improved by 2.2 times.

In other examples, the THO-modified ssODN can significantly improve the site-directed modification efficiency of PK15 cells by using 293T cells as a tool cell line, and the THO modification efficiency can be improved by 2.3 times.

In other embodiments, BHK cells are used as a tool cell line, the THO-modified ssODN can significantly improve the site-directed modification efficiency of BHK cells, and the THO modification efficiency can be improved by 2.5 times.

In other embodiments, site-directed modification is performed on the ACTB gene, and the THO-modified ssODN can significantly improve the site-directed modification efficiency by 2.7 times.

In other embodiments, the ssODN modified by THO can significantly improve the efficiency of site-directed modification by performing site-directed modification on GAPDH gene by 2.1 times.

In other embodiments, the SSA gene is site-directed modified, and the THO-modified ssODN can significantly improve the site-directed modification efficiency by 2.6 times.

What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept herein, and it is intended to cover all such modifications and variations as fall within the scope of the invention.

Sequence listing

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tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 5040

aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 5100

tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 5160

cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 5220

ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 5280

atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 5340

gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 5400

gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 5460

ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa 5520

aagaaaaagt aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc 5580

agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca 5640

ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta 5700

ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc 5760

atgctgggga gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg 5820

ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg 5880

cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc 5940

tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc 6000

ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 6060

tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 6120

cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 6180

acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc 6240

ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 6300

gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat 6360

tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 6420

ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga 6480

tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc 6540

ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg 6600

tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct 6660

atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg 6720

gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 6780

gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 6840

tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 6900

tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 6960

gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 7020

acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat 7080

tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 7140

gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 7200

tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 7260

accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 7320

ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 7380

agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 7440

gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 7500

ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg 7560

tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 7620

ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 7680

gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 7740

acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 7800

aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 7860

atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 7920

gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 7980

tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca 8040

ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 8100

ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 8160

ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 8220

aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 8280

cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 8340

gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 8400

ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 8460

cagcaacgcg gcctttttac ggttcc 8486

<210> 4

<211> 6155

<212> DNA

<213> Artificial Synthesis ()

<400> 4

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat 960

cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga 1020

gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc 1080

cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta 1140

ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcta 1200

atgaaagctt ccttacggtc agataactct cacggatccc aggagcgcac catcttcttc 1260

aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 1320

aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 1380

ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 1440

atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 1500

cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 1560

ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 1620

ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaagaa 1680

ttcctgcaga tatccagcac agtggcggcc gctcgagtct agagggcccg tttaaacccg 1740

ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt 1800

gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat 1860

tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag 1920

caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc 1980

ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg 2040

cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 2100

cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 2160

ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 2220

cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac 2280

ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 2340

tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat 2400

ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg 2460

tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg 2520

caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca 2580

ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact 2640

ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta 2700

atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag 2760

tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc 2820

attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga 2880

ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa 2940

cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt 3000

ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg 3060

ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa 3120

gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac 3180

cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt 3240

gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact 3300

cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg 3360

ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg 3420

acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc 3480

atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt 3540

gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc 3600

gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg 3660

ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg 3720

attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct 3780

ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta 3840

ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 3900

ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 3960

gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt 4020

gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 4080

cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 4140

tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 4200

gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 4260

ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 4320

caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 4380

aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 4440

atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 4500

cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 4560

ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 4620

gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 4680

accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 4740

cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 4800

cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 4860

gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 4920

aaaccaccgc tggtagcggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 4980

gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 5040

cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 5100

attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 5160

accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 5220

ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 5280

gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 5340

agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 5400

ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 5460

ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 5520

gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 5580

ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 5640

tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 5700

tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 5760

cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 5820

tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 5880

gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 5940

tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 6000

ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 6060

attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 6120

cgcgcacatt tccccgaaaa gtgccacctg acgtc 6155

<210> 5

<211> 140

<212> DNA

<213> Artificial Synthesis ()

<400> 5

accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 60

aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 120

tcgagggcga caccctggtg 140

Claims (2)

1. A method for improving the fixed-point modification efficiency of a cell genome by using a modified ssODN is characterized in that the method is realized by the joint action of a CRISPR/Cas9 system and the modified ssODN on a cell, the modified ssODN is THO modified, and the THO modified ssODN gene sequence is shown as THO-ACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTG-THS-G-THS-T-THS-G; the method can improve the site-directed modification efficiency of the porcine ROSA26 gene, wherein the sequence of an expression vector of the ROSA26 gene is shown as SEQ ID No. 3.

2. A method for improving the fixed-point modification efficiency of a cell genome by using a modified ssODN is characterized in that the method is realized by the joint action of a CRISPR/Cas9 system and the modified ssODN on a cell, the modified ssODN is a THS modification, and the gene sequence of the THS modified ssODN is shown as A-THS-C-THS-C-THS-CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTG-THS-G-THS-T-THS-G; the method can improve the site-directed modification efficiency of the porcine ROSA26 gene, wherein the sequence of an expression vector of the ROSA26 gene is shown as SEQ ID No. 3.

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