CN109777829A - A kind of construction method of the sgRNA expression component of gene editing U6 promoter driving - Google Patents
A kind of construction method of the sgRNA expression component of gene editing U6 promoter driving Download PDFInfo
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Abstract
The invention discloses a kind of construction methods of the sgRNA expression component of gene editing U6 promoter driving, belong to biological gene engineering, target gene of the present invention using the IDH1 gene of human genome as CRISPR/Cas9 gene editing, pass through gene specific sequence in design sgRNA, it obtains the completely sgRNA containing U6 promoter and expresses component sequence, it expands U6-sgRNA and expresses component, synthesize the PCR primer of U6-sgRNA expression component, and carry out PAGE purifying, it finally carries out the step of over-lap PCR obtains the double chain DNA fragment of complete U6-sgRNA expression component and completes building, the success rate of entire construction method gene editing, not only facilitate and at low cost.
Description
Technical field
The present invention relates to technical field of biological genetic engineering, a kind of gene editing U6 promoter driving is particularly related to
The construction method of sgRNA expression component.
Background technique
Gene editing is current most popular one of scientific research and treatment means, can be modified to specific gene order
Editor, gene functional research, model animal building and the clinical treatment of disease etc. being widely used in scientific research at present.
Main system includes ZFN, TALEN and CRISPR/Cas9 system to gene editing at present.Since ZFN and TALEN system needs root
Rebuilding for albumen is carried out according to the aim sequence difference of gene editing, the time of consuming and cost are more, and CRISPR/Cas9
System it is only necessary to rebuild Short interfering RNA sequence for the sequence difference of target gene, use with timeliness and
The advantage of cost.In CRISPR/Cas9 system, Cas9 nuclease needs the assistance identifying purpose sequence of crRNA and tracrRNA
Column carry out shearing to aim sequence and form double-strand break.For convenience, people later integrate crRNA and tracrRNA
Form a RNA molecule-sgRNA.
It is current to carry out gene editing two significant components of needs, i.e. sgRNA and Cas9 nucleic acid using CRISPR/Cas9 system
Enzyme.When carrying out gene editing, sgRNA identifying purpose DNA sequence dna instructs Cas9 to be sheared in particular sequence, forms double-strand
Fracture, the sequence reparation of subsequent dependent cells itself or the homologous recombination of foreign gene realize gene editing.Gene editing
The different designs of specificity and site selection dependent on unique identification sequence in sgRNA, different sgRNA specific recognition sequences
The corresponding different target spot DNA sequence dna of the design of column, it is seen that the design and building pair of sgRNA, especially its unique identification sequence
Gene editing is most important.Meanwhile sgRNA needs type III RNA polymerase to close by Transcript patterns as a kind of non-coding RNA
At.So how rapidly to construct the sgRNA expression component containing type III RNA polymerase promoter (such as U6 promoter) extremely
It closes important.
For at present, it may be implemented to construct the sgRNA table containing type III RNA polymerase promoter there are mainly two types of approach
Up to component.The first technical solution, i.e., by synthesizing sgRNA full length sequence and being inserted into containing type III rna polymerase promoter
Method in the vector plasmid of son, to obtain the sgRNA expression component containing type III RNA polymerase promoter.Though the method than
It is relatively quick, but since the segment of synthesis is free of type III RNA polymerase promoter, the type III RNA polymerase relied on carrier opens
Mover, thus the carrier containing type III RNA polymerase promoter need to be used when selecting carrier, lack in the selection of carrier
Flexibility, replaces carrier and system is relatively complicated, and when connection enters vector plasmid, in fact it could happen that the possibility of Opposite direction connection,
It needs further to screen and examine, efficiency is lower.The synthesis of second of technical solution, i.e. full genome is opened containing type III RNA polymerase
The sgRNA of mover expresses component, and the method is more direct, but sufficiently expensive, needs if encountered for a large amount of different locis
The case where building sgRNA expression component, will expend a large amount of financial resources.
Summary of the invention
In view of this, it is an object of the invention to propose that a kind of sgRNA of gene editing U6 promoter driving expresses component
Construction method, it is all or part of insufficient in the prior art to overcome.
Based on a kind of above-mentioned purpose building of the sgRNA expression component of gene editing U6 promoter driving provided by the invention
Method includes the following steps:
1) it designs gene specific sequence in sgRNA: being compiled using the IDH1 gene of human genome as CRISPR/Cas9 gene
The target gene collected carries out sequence design, obtains the sgRNA gene specific sequence of IDH1 gene editing；
2) it obtains the completely sgRNA containing U6 promoter and expresses component sequence: the sgRNA gene that step (1) is obtained
Specific sequence is placed in the template of sgRNA expression component, obtains U6-sgRNA component；
3) it obtains the template plasmid of U6-sgRNA expression component: being contained by what molecular biology method obtained for mouse
The U6-sgRNA-X component of non-coding region is connected into pGEM-T carrier on X chromosome, obtains plasmid pGEM-T/U6-sgRNA-X, and
Sequence is SEQ ID NO.7；
4) PCR primer of synthesis U6-sgRNA expression component, and carry out PAGE purifying, wherein the primer sequence is as follows,
F1 and F4 is forward primer, and F2 and F3 are reverse primer；
F1:5 '-GGGGCTCGAGGAATTCACGCGTTGTACAAAAAAGCAGGCTT
TAAAG-3'；
F2:5 '-AAGCGGCCGCAAGCTTACGCGTTAATGCCAACTTTGTACAAG
AAAG-3'；
F3:5 '-AGTTTTAGCACTGGCACACCGGTGTTTCGTCCTTTCCACA-3 '；
F4:5 '-GGTGTGCCAGTGCTAAAACTGTTTTAGAGCTAGAAATAGCAAG-3 '；
5) double chain DNA fragment that over-lap PCR obtains complete U6-sgRNA expression component is carried out.
In some optional embodiments, editor's purpose section of the IDH1 gene is IDH1 ENSG00000138413,
17230bp-17350bp。
In some optional embodiments, the sequence design designs website Benchlin using CRISPR, and chooses wherein
" On-Target Score " it is higher and " Off-Target Score " it is lower with " G " be initiation nucleotide sequence.
In some optional embodiments, the sgRNA gene specific sequence is 5 '-GGTGTGCCAGTGCTAAAACT-
3’。
In some optional embodiments, the step (5) includes the following steps:
A) it is utilized respectively the F1/F3 primer and F2/F4 primer synthesized in step (4), with pGEM- described in step (3)
T/U6-sgRNA-X is that template carries out PCR amplification；
B) agarose gel electrophoresis is carried out to the reaction product in step (a), and carries out gel recycling size respectively and is
The A segment and size of 360bp is the B segment of 159bp, and A, B segment is carried out PCR amplification；
C) step (b) reaction system is taken out from PCR instrument, primers F 1 and F2 is added thereto, then carry out PCR amplification；
D) agarose gel electrophoresis is carried out to the reaction product in step (c), and carries out gel recycling size respectively and is
The DNA fragmentation of the U6-sgRNA expression component of 499bp；
E) sequencing identification is carried out to the DNA fragmentation of the U6-sgRNA expression component recycled in (d).
In some optional embodiments, the PCR amplification condition of the step (a) is 98 DEG C of initial denaturation 30s；98 DEG C of 10s, 64
DEG C 30s, 72 DEG C of 30s are recycled 30 times；72 DEG C sufficiently extend 10min；4 DEG C of holdings.
In some optional embodiments, the PCR amplification condition of the step (b) is 98 DEG C of initial denaturation 30s；98 DEG C of 10s, 63
DEG C 30s, 72 DEG C of 30s are recycled 10 times；72 DEG C sufficiently extend 10min；4 DEG C of holdings.
In some optional embodiments, the PCR amplification condition of the step (c) is 98 DEG C of initial denaturation 30s；98 DEG C of 10s, 63
DEG C 30s, 72 DEG C of 30s are recycled 25 times；72 DEG C sufficiently extend 10min；4 DEG C of holdings.
In some optional embodiments, sequencing primer sequence is 5 '-in the step (e)
TGTACAAAAAAGCAGGCTTTAAAG-3’。
From the above it can be seen that a kind of sgRNA expression group of gene editing U6 promoter driving provided by the invention
The construction method of part designs sgRNA sequence, and resulting sgRNA sequence is under type III RNA polymerase promoter, can be with
The effective expression for guaranteeing sgRNA, guarantees the success rate of gene editing；And can efficiently it be contained using the reaction of PCR three times
The DNA fragmentation of the sgRNA expression component of type III RNA polymerase promoter, it is easy to operate；This design is only needed for different genes
Specific sgRNA sequence design synthesize F3 and F4 primer, F1, F2 primer and template plasmid are all general, thus this scheme
Not only facilitate and at low cost；Contain common restriction site in primers F 1 and F2, the carrier that sgRNA is carried out after convenient connects
It connects.
Detailed description of the invention
Fig. 1 is that the present invention carries out gene editing schematic diagram using CRISPR/Cas9 system in the prior art；
Fig. 2 designs gene specific sequence process schematic in sgRNA for the embodiment of the present invention；
Fig. 3 is the double chain DNA fragment mistake that the embodiment of the present invention carries out that over-lap PCR obtains complete U6-sgRNA expression component
Journey schematic diagram；
Fig. 4 is the DNA fragmentation agarose gel electrophoresis schematic diagram that U6-sgRNA of the embodiment of the present invention expresses component；
A segment after M-1kb plus ladder, 1-PCR, B segment after 2-PCR, A segment after the recycling of 3- glue, the recycling of 4- glue
B segment afterwards, U6-sgRNA after 5-PCR, U6-sgRNA after the recycling of 6- glue；
Fig. 5 is the DNA fragmentation sequencing result schematic diagram that U6-sgRNA of the embodiment of the present invention expresses component.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, and reference
Attached drawing, the present invention is described in more detail.
In order to solve to construct in the prior art containing type III RNA polymerase promoter sgRNA expression component efficiency compared with
Low and expensive problem, the embodiment of the invention provides a kind of sgRNA of gene editing U6 promoter driving to express component
Construction method, steps are as follows:
A kind of construction method of the sgRNA expression component of gene editing U6 promoter driving, includes the following steps:
1) it designs gene specific sequence in sgRNA: being compiled using the IDH1 gene of human genome as CRISPR/Cas9 gene
The target gene collected carries out sequence design, obtains the sgRNA gene specific sequence of IDH1 gene editing；
2) it obtains the completely sgRNA containing U6 promoter and expresses component sequence: the sgRNA gene that step (1) is obtained
Specific sequence is attached with U6 promoter sequence, obtains U6-sgRNA component；
3) amplification U6-sgRNA expresses component: U6-sgRNA component being connected into pGEM-T carrier, obtains plasmid pGEM-T/U6-
SgRNA-X, and sequence is SEQ ID NO.7；
4) PCR primer of synthesis U6-sgRNA expression component, and carry out PAGE purifying, wherein the primer sequence is as follows,
F1 and F4 is forward primer, and F2 and F3 are reverse primer；
F1:5 '-GGGGCTCGAGGAATTCACGCGTTGTACAAAAAAGCAGGCTTTAAAG-3 '；
F2:5 '-AAGCGGCCGCAAGCTTACGCGTTAATGCCAACTTTGTACAAGAAAG-3 '；
F3:5 '-AGTTTTAGCACTGGCACACCGGTGTTTCGTCCTTTCCACA-3 '；
F4:5 '-GGTGTGCCAGTGCTAAAACTGTTTTAGAGCTAGAAATAGCAAG-3 '；
5) double chain DNA fragment that over-lap PCR obtains complete U6-sgRNA expression component is carried out.
In some optional embodiments, the sgRNA of a kind of gene editing U6 promoter driving provided in an embodiment of the present invention
Express the construction method of component, the specific steps are as follows:
Entire technical side is illustrated using the IDH1 gene of human genome as the target gene of CRISPR/Cas9 gene editing
Case.
1. designing gene specific sequence in sgRNA: design is directed to the sgRNA sequence of IDH1.Firstly, being set into CRISPR
It counts website Benchlin (benchling.com), selects IDH1 gene in human genome, and choose the mesh of wherein gene editing
Section (IDH1ENSG00000138413,17230bp-17350bp), use software default parameter carry out sequence design, choose
Wherein " On-Target Score " higher and " Off-Target Score " lower sequence.Simultaneously in view of finally using III
Type RNA polymerase synthesizes sgRNA, and need to be selected when selecting with " G " is the sequence of initiation nucleotide to improve expression efficiency.
Eventually by software design and screening, finally selecting gene specific sequence in the sgRNA of this IDH1 gene editing is 5 '-
GGTGTGCCAGTGCTAAAACT-3 ' (as shown in Fig. 2, this sequence is different because of different genes editor).
2. obtaining the completely sgRNA containing U6 promoter expresses component sequence: the sgRNA that above-mentioned steps are designed
Middle gene specific sequence information is put into the template sequence (as shown in Figure 3) of sgRNA, obtains complete U6 promoter
SgRNA expresses component sequence, this sequence is denoted as " U6-sgRNA " sequence by us.U6-sgRNA include U6 promoter sequence,
Gene specific sequence in sgRNA, the frame sequence of sgRNA, transcription terminator, sgRNA expression component Frame sequence and
The restriction enzyme site sequence of connection for carrier.In addition to gene specific sequence in sgRNA, other sequences are fixed sequence program,
(as shown in Figure 2) is remained unchanged in different genes editor.
3. obtaining the template plasmid of U6-sgRNA expression component: we designed and passed through molecular biology method early period
It obtains containing the U6-sgRNA component for non-coding region on mouse X-chromosome, and is connected into pGEM-T carrier, we
This plasmid is denoted as " pGEM-T/U6-sgRNA-X ", and as the template for synthesizing other U6-sgRNA later.pGEM-T/U6-
The sequence of sgRNA-X is SEQ IDNO.7.
4. the PCR primer of design synthesis U6-sgRNA expression component: as shown in Figure 2, synthesis F1, F2, F3, F4 tetra- is drawn
Object, and carry out PAGE purifying.Wherein F1 and F4 is forward primer, and sequence is consistent with selected areas in figure；F2 and F3 is reversely to draw
Selected areas reverse complemental in object, sequence and figure.The overlap of F3 and F4 is that gene is special in sgRNA designed in step 1
Anisotropic sequence, Yin Jiyin and target spot be different and different, overlap of this sequence as F3 and F4, as next step over-lap PCR
Lap.F1 and F2 primer is universal primer；F3 and F4 need to be according to the Bu Tong synthesis every time of gene editing sequence.
Wherein,
F1:5 '-GGGGCTCGAGGAATTCACGCGTTGTACAAAAAAGCAGGCTTTAAAG-3 '；
F2:5 '-AAGCGGCCGCAAGCTTACGCGTTAATGCCAACTTTGTACAAGAAAG-3 '；
F3:5 '-AGTTTTAGCACTGGCACACCGGTGTTTCGTCCTTTCCACA-3 '；
F4:5 '-GGTGTGCCAGTGCTAAAACTGTTTTAGAGCTAGAAATAGCAAG-3 '.
5. carrying out the double chain DNA fragment that over-lap PCR obtains complete U6-sgRNA expression component, whole flow process such as Fig. 3 institute
Show:
(a) it is utilized respectively the F1/F3 primer and F2/F4 primer synthesized in step 4, with pGEM-T/ described in step 3
U6-sgRNA-X is template, is prepared using Phusion PCR system (Thermo Scientific, Catalog No.:F531S)
Following PCR reaction system (50 μ L of total system), PCR amplification obtains DNA fragmentation A and B respectively:
Above-mentioned system is recycled as follows using PCR instrument:
Circulation should be controlled to be recycled at 30, in order to avoid introduce mutation.
(b) agarose gel electrophoresis that 1.5% is carried out to the reaction product in (a), carries out gel recycling, is separately recovered big
It is small be 360bp A segment and size be 159bp B segment.
The PCR being formulated as follows using Phusion PCR system (Thermo Scientific, Catalog No.:F531S)
Reaction system (20 μ L of total system) is expanded:
Above-mentioned system is recycled as follows using PCR instrument:
(c) above-mentioned reaction system is taken out from PCR instrument, primers F 1 (10 μM) and F2 (10 μM) each 1 μ L is added thereto,
It puts back in PCR instrument, is discussed below circulation:
(d) agarose gel electrophoresis that 1.5% is carried out to the reaction product in (c), carries out gel recycling, and recycling size is
The segment of 499bp, as U6-sgRNA express the DNA fragmentation of component, as a result see Fig. 4；
(e) sequencing identification is carried out to the DNA fragmentation of the U6-sgRNA expression component recycled in (d), sequencing primer sequence is
5 '-TGTACAAAAAAGCAGGCTTTAAAG-3 ', it is ensured that gene specific sequence (the i.e. overlapping of F3 and F4 in Fig. 2 in sgRNA
Sequence) correctness, this sequence determine gene editing target position (sequencing result is shown in Fig. 5).
The correct U6-sgRNA expression component of sequencing can also can use two in Fig. 2 in step (e) with direct transfection cell
The restriction enzyme site sequence access carrier at end transfects cell in turn, carries out gene editing.
It designs resulting sgRNA sequence in the embodiment of the present invention to be under type III RNA polymerase promoter, Ke Yibao
The effective expression for demonstrate,proving sgRNA, improves the success rate of gene editing.Carrying out PCR reaction three times simultaneously can efficiently be contained
The DNA fragmentation of the sgRNA expression component of type III RNA polymerase promoter, it is easy to operate.Design is only needed for different genes
Specific sgRNA specific sequence design synthesis F3 and F4 primer, F1, F2 primer and template plasmid are all general, thus this
Scheme is not only convenient and at low cost.Contain common restriction site in primers F 1 and F2, the load of sgRNA is carried out after convenient
Body connection.
It should be understood by those ordinary skilled in the art that: the discussion of any of the above embodiment is exemplary only, not
It is intended to imply that the scope of the present disclosure (including claim) is limited to these examples；Under thinking of the invention, above embodiments
Or can also be combined between the technical characteristic in different embodiments, step can be realized with random order, and be existed such as
Many other variations of the upper different aspect of the invention, for simplicity, they are not provided in details.
The embodiment of the present invention be intended to cover fall into all such replacements within the broad range of appended claims,
Modifications and variations.Therefore, all within the spirits and principles of the present invention, any omission, modification, equivalent replacement, the improvement made
Deng should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Anhui Normal University
<120>construction method of the sgRNA expression component of a kind of gene editing U6 promoter driving
<130> 2018.12.26
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 46
<212> DNA
<213>forward primer (F1)
<400> 1
ggggctcgag gaattcacgc gttgtacaaa aaagcaggct ttaaag 46
<210> 2
<211> 46
<212> DNA
<213>reverse primer (F2)
<400> 2
aagcggccgc aagcttacgc gttaatgcca actttgtaca agaaag 46
<210> 3
<211> 40
<212> DNA
<213>reverse primer (F3)
<400> 3
agttttagca ctggcacacc ggtgtttcgt cctttccaca 40
<210> 4
<211> 43
<212> DNA
<213>forward primer (F4)
<400> 4
ggtgtgccag tgctaaaact gttttagagc tagaaatagc aag 43
<210> 5
<211> 20
<212> DNA
<213>gene specific sequence (DNA) in sgRNA
<400> 5
ggtgtgccag tgctaaaact 20
<210> 6
<211> 24
<212> DNA
<213>sequencing primer sequence (DNA)
<400> 6
tgtacaaaaa agcaggcttt aaag 24
<210> 7
<211> 3472
<212> DNA
<213>mouse (Mus musculus)
<400> 7
gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 60
ttgtacaaaa aagcaggctt taaaggaacc aattcagtcg actggatccg gtaccaaggt 120
cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc 180
tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac 240
gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat 300
ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt 360
gtggaaagga cgaaacaccg gaccttatgg tggtcctgtg ttttagagct agaaatagca 420
agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 480
ttctagaccc agctttcttg tacaaagttg gcattaaatc actagtgaat tcgcggccgc 540
ctgcaggtcg accatatggg agagctccca acgcgttgga tgcatagctt gagtattcta 600
tagtgtcacc taaatagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 660
tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 720
gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 780
ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 840
cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 900
cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 960
aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 1020
gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 1080
tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 1140
agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 1200
ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 1260
taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 1320
gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 1380
gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 1440
ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 1500
ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 1560
gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 1620
caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 1680
taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 1740
aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 1800
tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 1860
tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 1920
gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 1980
gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 2040
aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 2100
gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 2160
ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 2220
tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 2280
atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 2340
ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 2400
ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 2460
ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 2520
atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 2580
gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 2640
tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 2700
ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 2760
acatttcccc gaaaagtgcc acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag 2820
aaaataccgc atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt 2880
tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca 2940
aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta 3000
aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta 3060
cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg 3120
aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga 3180
aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg 3240
ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc 3300
cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc 3360
agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc 3420
agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta ta 3472
Claims (9)
1. a kind of construction method of the sgRNA expression component of gene editing U6 promoter driving, which is characterized in that including walking as follows
It is rapid:
1) gene specific sequence in sgRNA is designed: using the IDH1 gene of human genome as CRISPR/Cas9 gene editing
Target gene carries out sequence design, obtains the sgRNA gene specific sequence of IDH1 gene editing；
2) it obtains the completely sgRNA containing U6 promoter and expresses component sequence: the sgRNA gene specific that step (1) is obtained
Property sequence with containing U6 promoter sequence sgRNA expression component template be attached, obtain U6-sgRNA component；
3) amplification U6-sgRNA expresses component: being contained by what molecular biology method obtained for non-volume on mouse X-chromosome
The U6-sgRNA-X component in code region is connected into pGEM-T carrier, obtains plasmid pGEM-T/U6-sgRNA-X, and sequence is SEQ ID
NO.7；
4) PCR primer of synthesis U6-sgRNA expression component, and carries out PAGE purifying, wherein the primer sequence is as follows, F1 and
F4 is forward primer, and F2 and F3 are reverse primer；
F1:5 '-GGGGCTCGAGGAATTCACGCGTTGTACAAAAAAGCAGGCTTTAAAG-3 '；
F2:5 '-AAGCGGCCGCAAGCTTACGCGTTAATGCCAACTTTGTACAAGAAAG-3 '；
F3:5 '-AGTTTTAGCACTGGCACACCGGTGTTTCGTCCTTTCCACA-3 '；
F4:5 '-GGTGTGCCAGTGCTAAAACTGTTTTAGAGCTAGAAATAGCAAG-3 '；
5) double chain DNA fragment that over-lap PCR obtains complete U6-sgRNA expression component is carried out.
2. the construction method of the sgRNA expression component of gene editing U6 promoter driving according to claim 1, feature
It is, editor's purpose section of the IDH1 gene is IDH1 ENSG00000138413,17230bp-17350bp.
3. the construction method of the sgRNA expression component of gene editing U6 promoter driving according to claim 1, feature
Be, the sequence design using CRISPR design website Benchlin, and choose wherein " On-Target Score " it is higher and
" Off-Target Score " it is lower with " G " be initiation nucleotide sequence.
4. the construction method of the sgRNA expression component of gene editing U6 promoter driving according to claim 1, feature
It is, the sgRNA gene specific sequence is 5 '-GGTGTGCCAGTGCTAAAACT-3 '.
5. the construction method of the sgRNA expression component of gene editing U6 promoter driving according to claim 1, feature
It is, the step (5) includes the following steps:
A) it is utilized respectively the F1/F3 primer and F2/F4 primer synthesized in step (4), with pGEM-T/U6- described in step (3)
SgRNA-X is that template carries out PCR amplification；
B) agarose gel electrophoresis is carried out to the reaction product in step (a), and carrying out gel recycling size respectively is 360bp's
The B segment that A segment and size are 159bp, and A, B segment are subjected to PCR amplification；
C) step (b) reaction system is taken out from PCR instrument, primers F 1 and F2 is added thereto, then carry out PCR amplification；
D) agarose gel electrophoresis is carried out to the reaction product in step (c), and carrying out gel recycling size respectively is 499bp's
The DNA fragmentation of U6-sgRNA expression component；
E) sequencing identification is carried out to the DNA fragmentation of the U6-sgRNA expression component recycled in (d).
6. the construction method of the sgRNA expression component of gene editing U6 promoter driving according to claim 5, feature
It is, the PCR amplification condition of the step (a) is 98 DEG C of initial denaturation 30s；98 DEG C of 10s, 64 DEG C of 30s, 72 DEG C of 30s are recycled 30 times；
72 DEG C sufficiently extend 10min；4 DEG C of holdings.
7. the construction method of the sgRNA expression component of gene editing U6 promoter driving according to claim 5, feature
It is, the PCR amplification condition of the step (b) is 98 DEG C of initial denaturation 30s；98 DEG C of 10s, 63 DEG C of 30s, 72 DEG C of 30s are recycled 10 times；
72 DEG C sufficiently extend 10min；4 DEG C of holdings.
8. the construction method of the sgRNA expression component of gene editing U6 promoter driving according to claim 5, feature
It is, the PCR amplification condition of the step (c) is 98 DEG C of initial denaturation 30s；98 DEG C of 10s, 63 DEG C of 30s, 72 DEG C of 30s are recycled 25 times；
72 DEG C sufficiently extend 10min；4 DEG C of holdings.
9. the construction method of the sgRNA expression component of gene editing U6 promoter driving according to claim 5, feature
It is, sequencing primer sequence is 5 '-TGTACAAAAAAGCAGGCTTTAAAG-3 ' in the step (e).
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103388006A (en) * | 2013-07-26 | 2013-11-13 | 华东师范大学 | Method for constructing gene site-directed mutation |
| US20180280335A1 (en) * | 2017-03-28 | 2018-10-04 | University Of Pittsburgh-Of The Commonwealth System Of Higher Education | Retinoid compositions and methods of increasing immune cell-mediated killing of idh mutant cancer cells |
-
2019
- 2019-01-02 CN CN201910001943.9A patent/CN109777829A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103388006A (en) * | 2013-07-26 | 2013-11-13 | 华东师范大学 | Method for constructing gene site-directed mutation |
| US20180280335A1 (en) * | 2017-03-28 | 2018-10-04 | University Of Pittsburgh-Of The Commonwealth System Of Higher Education | Retinoid compositions and methods of increasing immune cell-mediated killing of idh mutant cancer cells |
Non-Patent Citations (2)
| Title |
|---|
| KATERINA DUKLESKA等: "Generation of CRISPR knockout of IDH1 in pancreatic ductal adenocarcinoma cell line: An optimal model to study pancreatic cancer metabolic reprogramming", 《HTTPS://JDC.JEFFERSON.EDU/SURGERYPOSTERS/8/》 * |
| 徐广军: "IFN-γ诱导的犬免疫相关GTP酶在弓形虫感染中的作用", 《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110331170A (en) * | 2019-07-06 | 2019-10-15 | 安徽师范大学 | The gene expression element and its construction method of a kind of dual gRNA and application |
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