CN115058451A - Double-reporting plasmid for homologous recombination and single base editing and construction method and application thereof - Google Patents
Double-reporting plasmid for homologous recombination and single base editing and construction method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and discloses a double-reporting plasmid for homologous recombination and single base editing, and a construction method and application thereof. The double-reporting plasmid for homologous recombination and single base editing comprises a framework sequence and an insertion sequence, wherein the insertion sequence sequentially comprises a blue fluorescent sequence BFP, a connecting element T2A, a red fluorescent sequence mCherry, an incomplete green fluorescent sequence GFPHDR and an STgRNA sequence. When single base editing and homologous recombination occur in the double-reporting plasmid, the fluorescence of the reporting plasmid changes, so that the gene editing vector still has high editing activity after entering cells, and meanwhile, the application range of the reporting plasmid is improved because the reporting can be performed on both the homologous recombination and the single base editing.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a double-reporting plasmid for homologous recombination and single base editing and a construction method and application thereof.
Background
Gene editing refers to a technique for site-directed modification of a genome. By using the technology, a certain position of a genome can be accurately positioned, a target DNA fragment is cut off at the position, and a new gene fragment is inserted. The gene editing technology reserves the characteristic of site-directed modification, can be applied to more species, and has higher efficiency, shorter construction time and lower cost. Currently, the main gene editing technologies are an artificial nuclease-mediated Zinc Finger Nucleases (ZFNs) technology, a transcription activator-like effector nucleases (TALENs) technology and an RNA-guided CRISPR-Cas nuclease technology (CRISPR-Cas RGNs), respectively.
In the prior art, the cell screening strategy of homologous recombination and single base editing is mainly based on the screening marker of a gene editing vector, and gene editing detection is carried out after drug or fluorescence screening. However, if several vectors are transferred simultaneously, the screened cells can only ensure that the gene editing vectors enter the cells, and cannot ensure that the screened cells meet the expected editing effect, if the cells have higher editing activity, and the efficiency of gene editing at one site of the genome after gene editing at another site is greatly improved, so that the screened cells are still negative.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a double-reporting plasmid for homologous recombination and single-base editing and a construction method thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a dual-reporter plasmid for homologous recombination and single-base editing, comprising a backbone sequence and an insertion sequence, wherein the insertion sequence comprises, in order, a blue-fluorescent sequence BFP, a linker element T2A, a red-fluorescent sequence mCherry, an incomplete green-fluorescent sequence GFPHDR, and a gRNA sequence.
When single base editing and homologous recombination occur in the double-reporting plasmid, the fluorescence of the reporting plasmid changes, so that the gene editing vector still has high editing activity after entering cells, and meanwhile, because both homologous recombination and single base editing (such as C-to-T) can be reported, the application range of the reporting plasmid is improved.
As a preferred embodiment of the dual reporter plasmid for homologous recombination and single base editing according to the present invention, the sequence of the target gene of the STgRNA is shown in SEQ ID No. 1; the first part of the BFP sequence is shown as SEQ ID No. 2; the second part of the BFP sequence is shown as SEQ ID No. 3; the sequence of the mCherry is shown as SEQ ID No. 4; the GFPHDR is shown as SEQ ID No. 5.
As a preferred embodiment of the dual reporter plasmid for homologous recombination and single base editing according to the present invention, the backbone sequence is derived from an expression vector pCEP 4.
In a second aspect, the present invention provides a method for constructing the dual-reporter plasmid for homologous recombination and single-base editing, comprising the following steps:
(1) carrying out double enzyme digestion on the skeleton expression vector;
(2) respectively carrying out enzyme digestion on the BFP gene, the mCherry gene, the incomplete GFPHDR gene and the target gene after PCR amplification;
(3) and (3) connecting the fragments recovered after the enzyme digestion in the step (1) with the fragments recovered after the enzyme digestion in the step (2).
As a preferred embodiment of the construction method of the double-reporting plasmid for homologous recombination and single base editing, the primer sequence of PCR amplification is shown as SEQ ID No. 6-16.
In a third aspect, the invention provides an expression host bacterium comprising said dual reporter plasmid for homologous recombination and single base editing.
In a fourth aspect, the invention provides the double-report plasmid, the preparation method and the application of the expression host bacterium in homologous recombination and single base editing in a simultaneous report cell.
As a preferred embodiment of the use according to the invention, the double reporter plasmid is co-transfected with plasmids px459 and/or pCMV-BE4max-P2A-Puro into the cells.
As a preferred embodiment of the application of the invention, the ratio of the double-reporting plasmid to the plasmid px459 or pCMV-BE4max-P2A-Puro is 1: 0.8-1.2.
Compared with the prior art, the invention has the beneficial effects that:
when single base editing and homologous recombination occur in the double-reporting plasmid, the fluorescence of the reporting plasmid changes, so that the gene editing vector still has high editing activity after entering cells, and meanwhile, the application range of the reporting plasmid is improved because the reporting can be performed on both homologous recombination and single base editing (such as C-to-T).
Drawings
FIG. 1 is a flow chart of the construction of pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA expression vector;
FIG. 2 is a schematic structural diagram of pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA expression vector;
FIG. 3 is a fluorescent emission test of 293T cells after transfection and at 24h dosing;
fig. 4 is a schematic diagram of the conversion of blue light to green light.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.
Example 1: selection of target sites and design of sgrnas
A sequence on a non-eukaryotic genome is selected as a target site, 1 sgRNA is constructed according to the design principle of the target sequence of the sgRNA (the sequence of the target gene is shown as SEQ ID No. 1: actcacggggtgcagtgctt), the base length of the target site is 20bp, 3 bases adjacent to the 3' end of the target site form a PAM region, and the sequence is NGG (N is any base).
Two oligos are designed aiming at sgRNA of the sequence on the genome, and are respectively as follows: CACCGactcacggggtgcagtgctt for ST gRNA-F; ST gRNA-R: AAACaagcactgcaccccgtgagtC.
Example 2: construction of pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA expression vector
The construction flow chart of pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA expression vector is shown in figure 1. A blue fluorescence sequence (BFP), a red fluorescence sequence (mCherry), an incomplete green fluorescence sequence (GFPHDR) and a section of gRNA containing a target site sequence are connected into a plasmid pCEP4, and an expression vector pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA which can report homologous recombination and single base editing is constructed, and the specific steps are as follows:
(1) construction of plasmid pCEP4-BFP-T2A-mCherry
1) Plasmid pCEP4(invitrogen, V044-50) was digested simultaneously with Kpn I and Hind III as shown in Table 1, and after 3 hours at 37 ℃ fragment 1(10390bp) was recovered using the gel recovery kit (TIANGEN) for use.
TABLE 1 pCEP4 enzyme cleavage System
| pCEP4 | 3μg |
| Kpn I(Takara，1618) | 2μl |
| Hind III(Takara，1615) | 2μl |
| 10×Buffer | 10ul |
| ddH 2 O | Supplement to 100ul |
2) Taking a synthetic plasmid BFP-T2A-EGFP as a template, and using primers F1 and R1 (the first part of the sequence of an amplified fragment BFP is shown as SEQ ID No. 2: atggtcagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtcaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctcaccctcaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtcaccaccctcACTCACGGGGTGCAGTGCTTCGGccgcta), primer F2 and R2 (the second part of the sequence of the amplified fragment BFP is shown in SEQ ID No. 3: ccccgaccacatgcgacagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtcaagttcgagggcgacaccctggtcaaccgcatcgagctcaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtcaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctcagcacccagtccgccctcagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtcaccgccgccgggatcactctcggcatggacgagctgtacaaggaattctaaCGAGGGCAGAGGAAGTCTGCTCACATG), performing PCR amplification under 98 deg.C (1min) as shown in Table 2; [98 ℃ (10s), 58 ℃ (15s), 72 ℃ (20s) ]. times.30 Cycles; 72 ℃ (5 min); 16 ℃ (∞). Fragment 2(245bp) and fragment 3(558bp) were gel recovered separately for use.
TABLE 2 PCR System Using BFP-T2A-EGFP as template
| Plasmid BFP-T2A-EGFP | 100ng |
| 2×Primer Star Max Premix(Takara) | 2μl |
| Primer F1 | 1μl |
| Primer R1 | 1μl |
| ddH 2 O | Make up to 50 μ l |
TABLE 2 PCR System Using BFP-T2A-EGFP as template
3) Plasmid donor-mCherry-lx is used as a template, and primers F3 and R3 (the sequence of the amplified fragment mCherry is shown as SEQ ID No. 4: CGGCGACGTCGAGGAGAATCCTGGCCCAATGgtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagtAA), performing PCR amplification under 98 deg.C (1min) as shown in Table 3; [98 ℃ (10s), 58 ℃ (15s), 72 ℃ (20s) ] x 30 Cycles; 72 ℃ (5 min); 16 ℃ (∞). Fragment 4(780bp) was recovered from the gel and used.
TABLE 3 PCR System Using donor-mCherry-lx as template
| Plasmid donor-mCherry-lx | 100ng |
| 2×Primer Star Max Premix(Takara) | 2μl |
| Primer F3 | 1μl |
| Primer R3 | 1μl |
| ddH 2 O | Make up to 50 μ l |
4) These four fragments were used with recombinase (Clonexpress)
One Step Cloning Kit) was performed under the recombination conditions of 37 ℃ for 30min, and the recombination system is shown in Table 4.
TABLE 4 recombination System
| Fragment 1 | 100ng |
| Fragment 2 | 10ng |
| Fragment 3 | 10ng |
| Fragment 4 | 10ng |
| 5×CE MultiS Buffer | 2μl |
| Exnase Multis | 1μl |
| ddH 2 O | Make up to 10 μ l |
5) After the recombination is finished, transferring the recombination product into a competent cell (DH5 alpha) for transformation, carrying out ice bath for 30min, carrying out heat shock for 45s at 42 ℃, then placing on ice for cooling for 2min, taking the LB agar solid culture medium containing the antibiotic out of a 4 ℃ refrigerator, placing in a room temperature, and coating the competent cell on a culture medium plate in a clean bench when the temperature is raised to the room temperature. In order to prevent the condensed water on the cover of the bacteria culture dish from dripping on the culture medium to cause pollution, the plate is placed upside down when the bacteria are cultured, and is placed in a bacteria culture box at 37 ℃ for overnight culture.
6) Picking a single colony, carrying out enzyme digestion identification by KpnI and BamH I, selecting several plasmids with correct enzyme digestion, sending the plasmids to a sequencing company for sequencing, and obtaining a sequencing result to show that the 5 plasmid is successfully connected with the fragments to obtain the plasmid pCEP 4-BFP-T2A-mCherry.
The primer sequences used were:
F1：gatctctagaagctgggtaccATGGTCAGCAAGGGCGAGGAGCT；
R1：tagcggccgaagcactgcaCCCCGTGAGTGAGGGTGG；
F2：actcacggggTGCAGTGCTTCGGCCGCTA；
R2：CATGTGAGCAGACTTCCTCTGCCCTCGttagaattccttgtacagctcgtc；
F3：AgaggaagtctgctcacatgCGGCGACGTCGAGGAGAATCCTGGCCCAAT
GGTGAGCAAGGGCGAGGAGGAT；
R3：cgagcggccgctagcaagcttTTACTTGTACAGCTCGTCCATGCC。
(2) construction of plasmid pU6-ST-gRNA
1) The plasmid pU6-sgRNA was digested with Bbs I as shown in Table 5, and after 3h at 37 ℃, fragment 5(3950bp) was recovered with gel for use.
TABLE 5 pU6-sgRNA cleavage System
| pU6-sgRNA | 3μg |
| BbsI(NEB，R3539S) | 2μl |
| 10×Buffer | 10ul |
| ddH 2 O | Supplementing to 100ul |
2) The primer ST gRNA-F/R was synthesized, and Oligos annealing was performed in a PCR apparatus at 95 ℃ for 10min as shown in Table 6, followed by immediate removal and cooling to room temperature (about room temperature for 30 min).
TABLE 6 annealing System
3) Fragment 5 was ligated with the annealed product using solution I at 16 ℃ for 2h, the ligation system is shown in Table 7.
TABLE 7 connection system
| Fragment 5 | 1μl |
| Annealed product | 4μl |
| SolutionI(Takara) | 5μl |
| In total | 10μl |
4) After the connection is completed, a single colony is transformed and selected for sequencing identification, and the sequencing result shows that the No.1 plasmid is successfully connected with a fragment, so that the plasmid pU6-ST-gRNA is obtained.
The primer sequences used were:
ST gRNA-F：CACCGactcacggggtgcagtgctt；
ST gRNA-R：AAACaagcactgcaccccgtgagtC。
(3) construction of plasmid pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA
1) Plasmid pCEP4-BFP-T2A-mCherry was digested with both AfeI and AgeI as shown in Table 8, and after 3h of digestion at 37 ℃, fragment 6(11814bp) was recovered with gel for use.
TABLE 8 pCEP4-BFP-T2A-mCherry enzyme digestion system
| pCEP4-BFP-T2A-mCherry | 3μg |
| AfeI(Thermo，FD0324) | 2μl |
| AgeI(Thermo，ER1461) | 2μl |
| 10×Buffer | 10μl |
| ddH 2 O | Make up to 100 mul |
2) Taking a synthetic plasmid BFP-T2A-EGFP as a template, and using primers F4 and R4 (an amplified fragment is incomplete green fluorescence sequence GFPHDR, and the sequence of the amplified fragment is shown as SEQ ID No. 5: gtcagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtcaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctcaccctcaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtcaccaccctcacctacggcgtgcagtgcttcagccgctaccccgaccacatgcgacagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtcaagttcgagggcgacaccctggtc), performing PCR amplification under 98 deg.C (1min) as shown in Table 9; [98 ℃ (10s), 58 ℃ (15s), 72 ℃ (20s) ]. times.30 Cycles; 72 ℃ (5 min); 16 ℃ (∞). Fragment 7(430bp) was recovered from the gel for use.
TABLE 9 PCR System Using BFP-T2A-EGFP as template
| Plasmid BFP-T2A-EGFP | 100ng |
| 2×Primer Star Max Premix(Takara) | 2μl |
| Primer F4 | 1μl |
| Primer R4 | 1μl |
| ddH 2 O | Make up to 50 μ l |
3) Carrying out PCR amplification by using a plasmid pU6-ST-gRNA as a template and primers R4 and R5, wherein the PCR system is shown in Table 10, and the amplification conditions are 98 ℃ (1 min); [98 ℃ (10s), 58 ℃ (15s), 72 ℃ (20s) ] x 30 Cycles; 72 ℃ (5 min); 16 ℃ (∞). Fragment 8(392bp) was recovered from the gel for use.
TABLE 10 PCR System Using plasmid pU6-ST-gRNA as template
| Plasmid pU6-ST-gRNA | 100ng |
| 2×Primer Star Max Premix(Takara) | 2μl |
| Primer R4 | 1μl |
| Primer R5 | 1μl |
| ddH 2 O | Make up to 50 μ l |
4) The three fragments were used with a recombinase kit (Clonexpress)
One Step Cloning Kit) was performed under the recombination conditions of 37 ℃ for 30min, and the recombination system is shown in Table 11.
TABLE 11 recombination System
| Fragment 6 | 100ng |
| Fragment 7 | 10ng |
| Fragment 8 | 10ng |
| 5×CE MultiS Buffer | 2μl |
| Exnase Multis | 1μl |
| ddH 2 O | Make up to 10 μ l |
5) After the recombination is completed, transformation is carried out, a single colony is selected and subjected to enzyme digestion identification by AfeI and AgeI, the plasmid with a correct band is sent to a company for sequencing, the sequencing result shows that the plasmid No.3 is successfully connected with the fragments, and the plasmid pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA with the total length of 12569bp is obtained, as shown in figure 2.
The primer sequences used were:
F4：cggggcgcagccatgaccggtGTCAGCAAGGGCGAGGAGC；
R4：atccagtcgacGACCAGGGTGTCGCCCTC；
R5：cgaatacaaaacaaaagcgctCTAGAAAAAAAGCACCGACTCGG。
example 3: transfection and results analysis of 293T cells
(1) The frozen 293T cells were removed from liquid nitrogen for resuscitation and cultured in high-sugar DMEM (HyClone) medium supplemented with 10% FBS (Gibco), 1% L-glutamine (Gibco), 1% Non-essentia amino acids (Gibco) and 1% Sodium pyruvate (Gibco), and when the cell status was good, cell passaging and cell transfection were performed, as follows:
1) observing the growth speed of 293T cells, removing the culture solution in a culture dish when the cell growth density reaches about 80%, washing twice by PBS (Jinuo) without calcium and magnesium ions, adding 0.25% pancreatin (NCM Biotech) digestive juice, digesting for 2-3min in an incubator at 37 ℃, adding a DMEM culture medium to stop digestion when cell shedding is observed under a microscope, transferring the cells into a 15mL centrifuge tube, centrifuging for 5min at 1000rpm, removing the supernatant, adding about lmL cell culture solution, and gently blowing and beating the cells to mix uniformly.
2) A portion of 293T cells was seeded into a 24-well plate, and the remaining portion was frozen and seeded into a 10cm dish for further culture.
3) After the cells are adherent, Lipo8000 is used TM (Byunshi, C0533) transfection reagents were used for cell transfection, plasmid pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA and plasmid px459 (vast Ling, providing Cas9 protein) were transfected into 293T cells in a 1:1 ratio, respectively, plasmid pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA and plasmid pCMV-BE4max-P2A-Puro (available from addgene,112093, providing cytosine base editor) in a 1:1 ratio.
After prepared in the proportions shown in Table 12, the mixture was gently and uniformly dropped into a 24-well plate.
TABLE 12 transfection System
4) After transfection, the 24-well plate was placed at 37 ℃ and 5% CO 2 The incubator of (2).
(2) After 24h, the medium was removed and fresh fetal calf serum-containing medium and fresh fetal calf serum-and homologous recombination-promoting drug Farrerol (Selleck) (10. mu.M) medium were added, grouped as in Table 13, respectively.
Table 13 grouping
| Grouping | Plasmids | Whether to add medicine or not |
| 1 | pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA | Whether or not |
| 2 | pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA+px459 | Whether or not |
| 3 | pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA+px459 | Is that |
| 4 | pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA+pCMV-BE4max-P2A-Puro | Whether or not |
(3) After 24h of dosing, the fluorescence of the cells was observed under a fluorescence microscope and recorded by photography.
As a result, as shown in FIG. 3, the fluorescence emission of the cells was analyzed, and compared with the first group, it was found that:
the second and third groups of transfection plasmids pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA and px459 theoretically play a role in Cas9-gRNA and restore normal expression of a red fluorescence expression frame, and the experimental result shows that the two groups have red fluorescence expression. Meanwhile, the third group is added with 10 mu M Farrerol, theoretically, an incomplete green fluorescence sequence can recover a complete green fluorescence expression frame through homologous recombination, and green light expression is realized. Compared with the second group, the third group has more green fluorescence expression, which indicates that homologous recombination plays a role in changing part of blue light into green light. The expression of green fluorescence in the fourth group indicates that the cytosine base editor is functioning to turn blue light into green light.
The constructed plasmid pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA can report homologous recombination and single base editing.
The pCEP4-BFP-T2A-mCherry-GFPHDR-STgRNA plasmid of the invention has the following action principle:
1) the codons CAC (coding for histidine formation) on the exon 66 of the blue fluorescent sequence form new codons TAC and TAT (coding for tyrosine formation) and CAT (coding for histidine formation) under the action of a cytosine editor, so that the conversion of blue light to green light is realized, as shown in fig. 4.
2) The blue fluorescence sequence contains a partial sequence for expressing green fluorescence, and the incomplete green fluorescence sequence can form a complete sequence for expressing the green fluorescence through homologous recombination, so that the expression of green light is realized.
3) Because the red fluorescent sequence generates frame shift and does not express red light, the target gene is cut by nuclease to cause DNA double strand break through the CRIPSR/Cas9 and the gRNA action of the target to the target site, and at the moment, the cell can start a series of repair modes such as NHEJ, thereby recovering the normal red fluorescent expression sequence.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> university of Wuyi
<120> double-reporting plasmid for homologous recombination and single base editing and construction method and application thereof
<130> 2022
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Synthetic
<400> 1
actcacgggg tgcagtgctt 20
<210> 2
<211> 224
<212> DNA
<213> Synthetic
<400> 2
atggtcagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtca acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctca ccctcaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtcacca ccctcactca cggggtgcag tgcttcggcc gcta 224
<210> 3
<211> 529
<212> DNA
<213> Synthetic
<400> 3
ccccgaccac atgcgacagc acgacttctt caagtccgcc atgcccgaag gctacgtcca 60
ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtcaagtt 120
cgagggcgac accctggtca accgcatcga gctcaagggc atcgacttca aggaggacgg 180
caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc 240
cgacaagcag aagaacggca tcaaggtcaa cttcaagatc cgccacaaca tcgaggacgg 300
cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct 360
gctgcccgac aaccactacc tcagcaccca gtccgccctc agcaaagacc ccaacgagaa 420
gcgcgatcac atggtcctgc tggagttcgt caccgccgcc gggatcactc tcggcatgga 480
cgagctgtac aaggaattct aacgagggca gaggaagtct gctcacatg 529
<210> 4
<211> 739
<212> DNA
<213> Synthetic
<400> 4
cggcgacgtc gaggagaatc ctggcccaat ggtgagcaag ggcgaggagg ataacatggc 60
catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga 120
gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct 180
gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat 240
gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc 300
cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac 360
cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg 420
caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc 480
ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa 540
gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc 600
cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga 660
ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat 720
ggacgagctg tacaagtaa 739
<210> 5
<211> 360
<212> DNA
<213> Synthetic
<400> 5
gtcagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60
gacgtcaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120
aagctcaccc tcaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180
gtcaccaccc tcacctacgg cgtgcagtgc ttcagccgct accccgacca catgcgacag 240
cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300
aaggacgacg gcaactacaa gacccgcgcc gaggtcaagt tcgagggcga caccctggtc 360
<210> 6
<211> 25
<212> DNA
<213> Synthetic
<400> 6
caccgactca cggggtgcag tgctt 25
<210> 7
<211> 25
<212> DNA
<213> Synthetic
<400> 7
aaacaagcac tgcaccccgt gagtc 25
<210> 8
<211> 44
<212> DNA
<213> Synthetic
<400> 8
gatctctaga agctgggtac catggtcagc aagggcgagg agct 44
<210> 9
<211> 37
<212> DNA
<213> Synthetic
<400> 9
tagcggccga agcactgcac cccgtgagtg agggtgg 37
<210> 10
<211> 29
<212> DNA
<213> Synthetic
<400> 10
actcacgggg tgcagtgctt cggccgcta 29
<210> 11
<211> 51
<212> DNA
<213> Synthetic
<400> 11
catgtgagca gacttcctct gccctcgtta gaattccttg tacagctcgt c 51
<210> 12
<211> 72
<212> DNA
<213> Synthetic
<400> 12
agaggaagtc tgctcacatg cggcgacgtc gaggagaatc ctggcccaat ggtgagcaag 60
ggcgaggagg at 72
<210> 13
<211> 45
<212> DNA
<213> synthetic
<400> 13
cgagcggccg ctagcaagct tttacttgta cagctcgtcc atgcc 45
<210> 14
<211> 40
<212> DNA
<213> Synthetic
<400> 14
cggggcgcag ccatgaccgg tgtcagcaag ggcgaggagc 40
<210> 15
<211> 29
<212> DNA
<213> Synthetic
<400> 15
atccagtcga cgaccagggt gtcgccctc 29
<210> 16
<211> 44
<212> DNA
<213> Synthetic
<400> 16
cgaatacaaa acaaaagcgc tctagaaaaa aagcaccgac tcgg 44
Claims (9)
1. A dual reporter plasmid for homologous recombination and single base editing comprising a backbone sequence and an insertion sequence, wherein the insertion sequence comprises in order a blue fluorescent sequence BFP, a linker element T2A, a red fluorescent sequence mCherry, an incomplete green fluorescent sequence GFPHDR and a STgRNA sequence.
2. The dual-reporter plasmid for homologous recombination and single base editing according to claim 1, wherein the sequence of the target gene of STgRNA is shown in SEQ ID No. 1; the first part of the BFP sequence is shown as SEQ ID No. 2; the second part of the BFP sequence is shown as SEQ ID No. 3; the sequence of the mCherry is shown as SEQ ID No. 4; the GFPHDR is shown as SEQ ID No. 5.
3. The dual reporter plasmid for homologous recombination and single base editing according to claim 1, characterized in that the backbone sequence is derived from an expression vector pCEP 4.
4. The method for constructing a double-reporter plasmid for homologous recombination and single base editing according to any one of claims 1 to 3, comprising the steps of:
(1) carrying out double enzyme digestion on the skeleton expression vector;
(2) respectively carrying out enzyme digestion on the BFP gene, the mCherry gene, the incomplete GFPHDR gene and the target gene after PCR amplification;
(3) and (3) connecting the fragments recovered after the enzyme digestion in the step (1) with the fragments recovered after the enzyme digestion in the step (2).
5. The dual-reporter plasmid for homologous recombination and single base editing according to claim 1, wherein the primer sequences for PCR amplification are shown in SEQ ID Nos. 6-16.
6. An expression host bacterium comprising the double reporter plasmid for homologous recombination and single base editing according to any one of claims 1 to 3.
7. Use of the dual-reporter plasmid according to any one of claims 1 to 3, the method of producing the dual-reporter plasmid according to claim 4 or 5, or the expression host bacterium according to claim 6 for simultaneous homologous recombination and single-base editing in a reporter cell.
8. The use according to claim 7, characterized in that said double reporter plasmid is co-transfected with plasmids px459 and/or pCMV-BE4max-P2A-Puro into said cells.
9. The use of claim 8, wherein the ratio of the dual reporter plasmid to plasmid px459 or pCMV-BE4max-P2A-Puro is 1: 0.8-1.2.
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