CN109868288A - Cas9 transcription templates DNA and Plasmid DNA and in-vitro transcription method for drosophila CRISPR transgenosis - Google Patents
Cas9 transcription templates DNA and Plasmid DNA and in-vitro transcription method for drosophila CRISPR transgenosis Download PDFInfo
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- CN109868288A CN109868288A CN201910198850.XA CN201910198850A CN109868288A CN 109868288 A CN109868288 A CN 109868288A CN 201910198850 A CN201910198850 A CN 201910198850A CN 109868288 A CN109868288 A CN 109868288A
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Abstract
The present invention relates to the gene editing technical fields in genetic engineering, and in particular to a kind of cas9 transcription templates DNA and Plasmid DNA for drosophila CRISPR transgenosis and in-vitro transcription method.The cas9 transcription templates DNA has the base sequence as shown in SEQ ID NO:1.The present invention surrounds application of the CRISPR/Cas9 technology in model animal drosophila, provides the expression vector of the Cas9 of optimization a kind of, improves gene editing efficiency significantly, also lays a good foundation for its application in other field.
Description
Technical field
The present invention relates to the gene editing technical fields in genetic engineering, are used for drosophila more particularly, to one kind
The cas9 transcription templates DNA and Plasmid DNA of CRISPR transgenosis and application and in-vitro transcription method.
Background technique
Traditional transgenic technology is mainly to utilize the principle of swivel base, and exogenous dna fragment is randomly integrated into biology base
(i.e. gene editing) is accurately modified because in group, being difficult to accomplish.Occur after the transgenic technology ZFN, TALEN and
CRISPR/Cas9 technology is all based on the principle of endonuclease accurately target gene group DNA, is able to achieve to genome
Accurate modification, becomes three big gene editing technologies of current mainstream.Relative to ZFN and TALEN technology, CRISPR/Cas9 technology
Operation is simpler, cost is less expensive, more efficient, become the first choice of current gene editing.
CRISPR/Cas9 gene editing technology, be by bacterium exercise immune function the CRISPR/Cas9 system reform and
Come.Mainly by one section of guidance RNA (single guide RNA, sgRNA), Cas9 endonuclease is directed to target patch
Section, and cut, recycle the non-homologous end joining (NHEJ) of organism itself or homologous recombination (HR) repair mechanism real
Now to the modification of genome and editor, including small fragment inserts and delete (indel), larger piece segment DNA deletes (deletion), spy
Addition sequence label, single base replacement etc. are set in positioning.Using CRISPR/Cas9 technology, target gene function can be constructed and lacked
Lose (KO) mutant, for the tool strain of testing goal gene expression, for single alkali of Accurate Analysis single amino acids function
Base modifies strain etc., greatly accelerates the progress of biological study.In addition to being applied to scientific research field, CRISPR/Cas9 gene is compiled
Volume technology is also applied to cell therapy, improving domestic animal, crop improvement etc. and people and lives closely bound up health, environment and energy
Source domain has broad application prospects.
Currently, influence CRISPR/Cas9 technical efficiency principal element first is that the activity of Cas9.Therefore, one kind is needed
Method can be improved the activity of Cas9, to improve the efficiency of gene editing.
Summary of the invention
The object of the present invention is to provide the expression vector of Cas9 a kind of and method is transcribed in vitro, is imitated with high gene editing
Rate.
To achieve the goals above, the first aspect of the present invention provides the cas9 for drosophila CRISPR transgenosis turns a kind of
Template DNA is recorded, which has the base sequence as shown in SEQ ID NO:1.
The cas9 transcription templates DNA is the cas9 transcription templates DNA after codon optimization.It is specifically that source of people cas9 is close
Numeral is optimized for the codon of drosophila adaptation, improves cas9-mRNA in the intracorporal translation efficiency of drosophila, to improve cas9 albumen
Expression.
Tool web site used by optimizing: http://www.jcat.de/.
Optimization gained sequence is subjected to gene chemical synthesis, the cas9CDS transcription templates after finally obtaining drosophila codon optimization
DNA。
The second aspect of the present invention provides a kind of Plasmid DNA for drosophila CRISPR transgenosis, the Plasmid DNA include with
Lower element:
Cas9 open gene, the cas9 open gene have the base sequence as shown in SEQ ID NO:1；
First 40-nls enters nuclear signal, is located at after the cas9 open gene initiation codon ATG, cas9 open gene
Before；
2nd 40-nls enters nuclear signal, be located at the cas9 open gene stop codon before, cas9 open gene it
Afterwards；
Kozak sequence, the Kozak sequence are located at before the initiation codon ATG；
T7 promoter；
Sv40-3UTR, the sv40-3UTR are located at after the terminator codon；
Restriction enzyme site, the restriction enzyme site are located at after the sv40-3UTR.
It is described to refer to " before " positioned at the upstream 5 ' of target sequence end in the present invention, it is described to refer to " later " positioned at target
It holds in the downstream 3 ' of sequence.
Each element schematic of the Plasmid DNA is as shown in Figure 1, the complete map of Plasmid DNA is as shown in Figure 2.
In accordance with the present invention it is preferred that the restriction enzyme site is Xba I restriction enzyme site.
According to the present invention, sv40-nls is entered into nuclear signal (its amino acid sequence are as follows: Pro-Lys-Lys-Lys-Arg-Lys-
Val, SEQ ID NO:3) it is added to the two sides of cas9CDS (behind ATG and before terminator codon).Both ends are put into simultaneously believes into core
Number, the passability of cas9 protein on cells core is increased, genomic gene editor can be more efficiently carried out.
Kozak sequence " GCCACC " is added before initiation codon ATG, increases cas9 in the intracorporal expression water of drosophila
It is flat.
Cas9DNA sequence is transcribed in vitro in T7 promoter, has higher transcriptional efficiency, can be under the same conditions
It is cas9mRNA higher that promoters more other than sp6 etc. obtain yield.
3UTR of the sv40 3UTR as cas9mRNA is added behind terminator codon, is saved than tradition plus the method for polyA
Transcription synthesis mRNA cost and time, meanwhile, sv40-3UTR can increase as the drosophila gene common 3UTR of expression
Cas9mRNA plays facilitation in the intracorporal stability of drosophila, to expression cas9 albumen.
By the optimization of the various aspects to cas9 transcription templates DNA sequence dna, it is endogenous in drosophila body to improve cas9-mRNA
The efficiency of expression, to improve the efficiency to drosophila gene group gene editing.
According to the present invention, using the Plasmid DNA with said elements can be realized improve cas9-mRNA in drosophila body in
The efficiency of source expression, it will be apparent that, which can not influence said elements function or can assist above-mentioned member containing other
Other DNA sequence dnas of part, for example, the segment between T7 promoter and Kozak sequence, the total length of such DNA sequence dna is not
More than 100nt.
A kind of preferred embodiment according to the present invention, the cas9 transcription templates DNA only includes function element, is free of
There is other function element.Most preferably, the cas9 transcription templates DNA has the base sequence as shown in SEQ ID NO:2.
The third aspect of the present invention provides the application of at least one of above-mentioned cas9 transcription templates DNA and above-mentioned Plasmid DNA.
The application is for example as gene editing tool.
Specifically, the present invention also provides a kind of cas9mRNA, and method is transcribed in vitro, this method comprises:
(1) above-mentioned Plasmid DNA is subjected to amplification and linearization for enzyme restriction, and obtained linearisation template is purified；
(2) template after purification that step (1) obtains is transcribed in vitro and is purified.
In this method, conventional molecular biological method is can be used in above-mentioned amplification and linearization for enzyme restriction.
In step (1), the step of purifying, is preferably included: adding SDS to remove the restriction endonuclease of digestion system using Proteinase K
It removes, then passes through ethanol precipitation.The simple purification method, yield are high, and purity is also very high.
According to the present invention, conventional molecular biological method can also be used in the in-vitro transcription, and uses conventional commercial body
Outer transcript reagent.Preferably, when the dosage of GTP is 20-50mM in the in-vitro transcription, the efficiency of transcription can be greatly improved,
Improve the yield of cas9mRNA.
According to the present invention, this method further include: the concentration of cas9mRNA is detected, the detection method is electrophoresis ratio
To method.That is, the multiple for not waiting synthetic mRNA dilution, the electrophoresis together with DNA standard are determined according to the brightness of band
The actual concentrations of cas9mRNA.
The present invention surrounds application of the CRISPR/Cas9 technology in model animal drosophila, provides a kind of Cas9 of optimization
Expression vector, improve gene editing efficiency significantly.Also it lays a good foundation for its application in other field.
In addition, the present invention also provides cas9mRNA, and method is transcribed in vitro, this method is had the advantage that
(1) add the purification process of SDS using Proteinase K, simplicity, yield are high, and purity is also very high.
(2) dosage for increasing GTP in transcription system greatly improves the efficiency of transcription, improves the yield of cas9mRNA.
(3) traditional method needs to carry out polyA tailing to the cas9mRNA that transcription is completed, due to introducing in advance
Sv40-3UTR simplifies experiment flow so not needing tailing.
(4) in the prior art using the method for nanodrop detection nucleic acid concentration, have in the system completed due to transcription big
The interference for measuring dNTP causes measurement concentration more excessively high than actual concentrations, if measuring concentration according to nanodrop to prepare drosophila note
Sample is penetrated, the amount that will lead to cas9mRNA in sample is very few, largely effects on transgene efficiency；The present invention using electrophoresis Comparison Method come
The concentration of Cas9mRNA is measured, the concentration that this method determines is more acurrate, and then improves transgene efficiency.
It is most ultimate present invention incorporates a variety of improvement of transcription templates DNA, expression vector and dubbing method to Cas9
The earth improves the efficiency of transgenosis.
Other features and advantages of the present invention will then part of the detailed description can be specified.
Detailed description of the invention
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings, it is of the invention above-mentioned and its
Its purpose, feature and advantage will be apparent.
Fig. 1 is each element schematic of Plasmid DNA for drosophila CRISPR transgenosis of the invention.
Fig. 2 is the complete map of the Plasmid DNA for drosophila CRISPR transgenosis of the invention.
Fig. 3 is the testing result figure that electrophoresis Comparison Method determines cas9mRNA concentration.
Fig. 4 is the electrophoresis result figure of sample identification.
Specific embodiment
The preferred embodiment that the present invention will be described in more detail below with reference to accompanying drawings.Although showing the present invention in attached drawing
Preferred embodiment, however, it is to be appreciated that may be realized in various forms the present invention without the embodiment party that should be illustrated here
Formula is limited.
The person that is not specified actual conditions in embodiment, all carries out according to conventional conditions or manufacturer's recommended conditions.Examination used
Production firm person is not specified in agent or instrument, is the conventional products that can be obtained by commercially available purchase.
Embodiment 1
According to the building of conventional molecular biological method there is the Plasmid DNA of the sequence as shown in SEQ ID NO:2 (to be named as
T7-cas9-sv40 plasmid).Complete map is as shown in Figure 2.
1.Cas9mRNA is transcribed in vitro
1) linearisation and purifying of transcription templates
The good T7-cas9-sv40 plasmid of above-mentioned optimization is expanded, extraction obtains sufficient amount.
1. linearization for enzyme restriction
System (50 μ l):
Condition: 37 DEG C are reacted 1 hour
2. linear die purifies
The restriction endonuclease of digestion system is got rid of using Proteinase K plus SDS, then ethanol precipitation DNA obtains pure DNA
Linear die, this method is relatively simple, and yield is very high, and purity is also very high, the method is as follows:
The good system total amount of digestion is 50 μ l,
2.5 μ l of 10%SDS is added to final concentration 0.5%,
Proteinase K (20 μ g/ μ l) 1.3 μ l to about 0.5 μ g/ μ l of final concentration are added,
50 DEG C of reaction 30min,
Isometric Tris saturated phenol/chloroform 55 μ l, 14000rpm is added and is centrifuged 5min, draws 45 μ l of supernatant to new centrifugation
Pipe,
1/10 volumes of acetic acid sodium, 5 μ l is added, mixes well,
2 times of volume dehydrated alcohols, 100 μ l is added, is sufficiently mixed, -20 DEG C of placement 30min,
4C, 14000rpm are centrifuged 10min, siphon away supernatant,
70% ethyl alcohol 400 μ l, 14000rpm, 5min is added, siphons away supernatant, drying to be precipitated,
It is dissolved in suitable ddH2O (DEPC+),
After surveying concentration, DNA solution is diluted to 1 μ g/ μ l.
2) it is transcribed in vitro and purifies
1. transcription:
It is transcribed in vitro using mMESSAGE mMACHINE T7Kit (@ambion, product number AM1344)
System (100 μ l):
Compared to the protocol provided in kit, the amount of GTP is increased, substantially increases the efficiency of transcription, is improved
The yield of cas9mRNA.
Condition: 37 DEG C are reacted 1.5 hours
The time of transcription is optimized, transcription in 1.5 hours is basically completed, and preventing from excessively transcribing causes mRNA to degrade.
2. LiCl precipitates RNA
Totally 100 μ l system adds 150 μ l ddH2O (DEPC+), 150 μ l LiCl, -80 DEG C of precipitating 2h.It is carried out at -80 DEG C
Precipitating improves yield and ensure that the purity and quality of mRNA.
4 DEG C, 14000rpm is centrifuged 10min,
Supernatant is siphoned away, is washed once with 70% ethyl alcohol, 400 μ l, 4 DEG C, 14000rpm is centrifuged 5min,
Supernatant is siphoned away, it is drying precipitated,
It is dissolved in suitable ddH2O。
2.Cas9mRNA Concentration Testing
0.6 μ l of cas9mRNA is taken to add ddH2O (DEPC+) is diluted to 6 μ l, takes out 1.5 μ l and 0.5 μ l respectively and runs electrophoresis (phase
When in the cas9mRNA of 0.15 μ l and 0.05 μ l progress electrophoresis), 5 μ l of marker (DL5000) applied sample amount, electrophoresis result such as Fig. 3 institute
Show, wherein M represents marker.
The Luminance Analysis that electrophoretic band is carried out using software I mageJ, is obtained:
0.15 μ l band brightness are as follows: 141120
0.05 μ l band brightness are as follows: 47520
Marker 3kb band brightness are as follows: 9194
Known marker 3kb band is 50ng, and the band that 0.15 μ l is calculated is 767ng, and the band of 0.05 μ l is
258ng, therefore, concentration are about 5 μ g/ μ l.
Test case 1
Drosophila embryos are injected respectively with the cas9mRNA of the cas9mRNA of embodiment 1 and conventional method, by the effect of transgenosis
Rate does lateral comparison, the method is as follows:
Transgenosis project name: X-gene-KO (to protect customer privacy, does not write Gene Name)
Project purpose: the overall length (1.5kb or so) of X-gene in drosophila gene group is knocked out
Project process:
1. two gRNA are at a distance of about 1.5kb at gene 5 ' end and 3 ' end one gRNA of each design；
2. after two gRNA transcription synthesis, the cas9mRNA and conventional cas9mRNA of difference mix embodiment 1, preparation
Two groups of samples, it may be assumed that
Sample X-gene-KO-1:gRNA1+gRNA2+cas9mRNA (embodiment 1)
Sample X-gene-KO-2:gRNA1+gRNA2+cas9mRNA (comparative example)
3. injecting drosophila w1118 embryo；
4. after one day, extracting first-instar young genome, Molecular Identification is carried out；
Specific method:
In 5 ' end gRNA upstream design, one forward primer F, a reverse primer R, distance 2kb are designed in 3 ' the end downstreams gRNA
Left and right carries out PCR by template of larvae-gene group, runs electrophoresis, if it is the KO positive, it should obtain the bis- bands of 2kb+500bp.
As a result:
Each sample identifies 8 larvas respectively, is respectively as follows: X-gene-KO-1-1~1-8 (embodiment) and X-gene-
KO-2-1~2-8 (comparative example), electrophoresis result is as shown in Figure 4.As shown in Figure 4: 8 drosophilas of X-gene-KO-1 are the positive,
It is the positive that 8 drosophilas of X-gene-KO-2, which only have 4,.
It can be seen that cas9mRNA transgene efficiency of the invention is doubled.
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and
It is not limited to disclosed each embodiment.Without departing from the scope and spirit of illustrated each embodiment, for this skill
Many modifications and changes are obvious for the those of ordinary skill in art field.
Sequence table
<110>tension
Li Zheng
<120>for the cas9 transcription templates DNA of drosophila CRISPR transgenosis and Plasmid DNA and in-vitro transcription method
<130> 1700207
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4104
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atggacaaga agtactccat cggcctggac atcggcacca actccgtggg ctgggccgtg 60
atcaccgacg agtacaaggt gccctccaag aagttcaagg tgctgggcaa caccgaccgc 120
cactccatca agaagaacct gatcggcgcc ctgctgttcg actccggcga gaccgccgag 180
gccacccgcc tgaagcgcac cgcccgccgc cgctacaccc gccgcaagaa ccgcatctgc 240
tacctgcagg agatcttctc caacgagatg gccaaggtgg acgactcctt cttccaccgc 300
ctggaggagt ccttcctggt ggaggaggac aagaagcacg agcgccaccc catcttcggc 360
aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgcgcaag 420
aagctggtgg actccaccga caaggccgac ctgcgcctga tctacctggc cctggcccac 480
atgatcaagt tccgcggcca cttcctgatc gagggcgacc tgaaccccga caactccgac 540
gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggagaacccc 600
atcaacgcct ccggcgtgga cgccaaggcc atcctgtccg cccgcctgtc caagtcccgc 660
cgcctggaga acctgatcgc ccagctgccc ggcgagaaga agaacggcct gttcggcaac 720
ctgatcgccc tgtccctggg cctgaccccc aacttcaagt ccaacttcga cctggccgag 780
gacgccaagc tgcagctgtc caaggacacc tacgacgacg acctggacaa cctgctggcc 840
cagatcggcg accagtacgc cgacctgttc ctggccgcca agaacctgtc cgacgccatc 900
ctgctgtccg acatcctgcg cgtgaacacc gagatcacca aggcccccct gtccgcctcc 960
atgatcaagc gctacgacga gcaccaccag gacctgaccc tgctgaaggc cctggtgcgc 1020
cagcagctgc ccgagaagta caaggagatc ttcttcgacc agtccaagaa cggctacgcc 1080
ggctacatcg acggcggcgc ctcccaggag gagttctaca agttcatcaa gcccatcctg 1140
gagaagatgg acggcaccga ggagctgctg gtgaagctga accgcgagga cctgctgcgc 1200
aagcagcgca ccttcgacaa cggctccatc ccccaccaga tccacctggg cgagctgcac 1260
gccatcctgc gccgccagga ggacttctac cccttcctga aggacaaccg cgagaagatc 1320
gagaagatcc tgaccttccg catcccctac tacgtgggcc ccctggcccg cggcaactcc 1380
cgcttcgcct ggatgacccg caagtccgag gagaccatca ccccctggaa cttcgaggag 1440
gtggtggaca agggcgcctc cgcccagtcc ttcatcgagc gcatgaccaa cttcgacaag 1500
aacctgccca acgagaaggt gctgcccaag cactccctgc tgtacgagta cttcaccgtg 1560
tacaacgagc tgaccaaggt gaagtacgtg accgagggca tgcgcaagcc cgccttcctg 1620
tccggcgagc agaagaaggc catcgtggac ctgctgttca agaccaaccg caaggtgacc 1680
gtgaagcagc tgaaggagga ctacttcaag aagatcgagt gcttcgactc cgtggagatc 1740
tccggcgtgg aggaccgctt caacgcctcc ctgggcacct accacgacct gctgaagatc 1800
atcaaggaca aggacttcct ggacaacgag gagaacgagg acatcctgga ggacatcgtg 1860
ctgaccctga ccctgttcga ggaccgcgag atgatcgagg agcgcctgaa gacctacgcc 1920
cacctgttcg acgacaaggt gatgaagcag ctgaagcgcc gccgctacac cggctggggc 1980
cgcctgtccc gcaagctgat caacggcatc cgcgacaagc agtccggcaa gaccatcctg 2040
gacttcctga agtccgacgg cttcgccaac cgcaacttca tgcagctgat ccacgacgac 2100
tccctgacct tcaaggagga catccagaag gcccaggtgt ccggccaggg cgactccctg 2160
cacgagcaca tcgccaacct ggccggctcc cccgccatca agaagggcat cctgcagacc 2220
gtgaaggtgg tggacgagct ggtgaaggtg atgggccgcc acaagcccga gaacatcgtg 2280
atcgagatgg cccgcgagaa ccagaccacc cagaagggcc agaagaactc ccgcgagcgc 2340
atgaagcgca tcgaggaggg catcaaggag ctgggctccc agatcctgaa ggagcacccc 2400
gtggagaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaacggccgc 2460
gacatgtacg tggaccagga gctggacatc aaccgcctgt ccgactacga cgtggaccac 2520
atcgtgcccc agtccttcct gaaggacgac tccatcgaca acaaggtgct gacccgctcc 2580
gacaagaacc gcggcaagtc cgacaacgtg ccctccgagg aggtggtgaa gaagatgaag 2640
aactactggc gccagctgct gaacgccaag ctgatcaccc agcgcaagtt cgacaacctg 2700
accaaggccg agcgcggcgg cctgtccgag ctggacaagg ccggcttcat caagcgccag 2760
ctggtggaga cccgccagat caccaagcac gtggcccaga tcctggactc ccgcatgaac 2820
accaagtacg acgagaacga caagctgatc cgcgaggtga aggtgatcac cctgaagtcc 2880
aagctggtgt ccgacttccg caaggacttc cagttctaca aggtgcgcga gatcaacaac 2940
taccaccacg cccacgacgc ctacctgaac gccgtggtgg gcaccgccct gatcaagaag 3000
taccccaagc tggagtccga gttcgtgtac ggcgactaca aggtgtacga cgtgcgcaag 3060
atgatcgcca agtccgagca ggagatcggc aaggccaccg ccaagtactt cttctactcc 3120
aacatcatga acttcttcaa gaccgagatc accctggcca acggcgagat ccgcaagcgc 3180
cccctgatcg agaccaacgg cgagaccggc gagatcgtgt gggacaaggg ccgcgacttc 3240
gccaccgtgc gcaaggtgct gtccatgccc caggtgaaca tcgtgaagaa gaccgaggtg 3300
cagaccggcg gcttctccaa ggagtccatc ctgcccaagc gcaactccga caagctgatc 3360
gcccgcaaga aggactggga ccccaagaag tacggcggct tcgactcccc caccgtggcc 3420
tactccgtgc tggtggtggc caaggtggag aagggcaagt ccaagaagct gaagtccgtg 3480
aaggagctgc tgggcatcac catcatggag cgctcctcct tcgagaagaa ccccatcgac 3540
ttcctggagg ccaagggcta caaggaggtg aagaaggacc tgatcatcaa gctgcccaag 3600
tactccctgt tcgagctgga gaacggccgc aagcgcatgc tggcctccgc cggcgagctg 3660
cagaagggca acgagctggc cctgccctcc aagtacgtga acttcctgta cctggcctcc 3720
cactacgaga agctgaaggg ctcccccgag gacaacgagc agaagcagct gttcgtggag 3780
cagcacaagc actacctgga cgagatcatc gagcagatct ccgagttctc caagcgcgtg 3840
atcctggccg acgccaacct ggacaaggtg ctgtccgcct acaacaagca ccgcgacaag 3900
cccatccgcg agcaggccga gaacatcatc cacctgttca ccctgaccaa cctgggcgcc 3960
cccgccgcct tcaagtactt cgacaccacc atcgaccgca agcgctacac ctccaccaag 4020
gaggtgctgg acgccaccct gatccaccag tccatcaccg gcctgtacga gacccgcatc 4080
gacctgtccc agctgggcgg cgac 4104
<210> 2
<211> 5137
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
taatacgact cactataggg gaatacaagc tacttgttct ttttgcagga tccgacacta 60
tagaatacaa gctacttgtt ctttttgcag gatctgccac catggctcca aagaaaaaac 120
gaaaagttat ggacaagaag tactccatcg gcctggacat cggcaccaac tccgtgggct 180
gggccgtgat caccgacgag tacaaggtgc cctccaagaa gttcaaggtg ctgggcaaca 240
ccgaccgcca ctccatcaag aagaacctga tcggcgccct gctgttcgac tccggcgaga 300
ccgccgaggc cacccgcctg aagcgcaccg cccgccgccg ctacacccgc cgcaagaacc 360
gcatctgcta cctgcaggag atcttctcca acgagatggc caaggtggac gactccttct 420
tccaccgcct ggaggagtcc ttcctggtgg aggaggacaa gaagcacgag cgccacccca 480
tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc 540
tgcgcaagaa gctggtggac tccaccgaca aggccgacct gcgcctgatc tacctggccc 600
tggcccacat gatcaagttc cgcggccact tcctgatcga gggcgacctg aaccccgaca 660
actccgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg 720
agaaccccat caacgcctcc ggcgtggacg ccaaggccat cctgtccgcc cgcctgtcca 780
agtcccgccg cctggagaac ctgatcgccc agctgcccgg cgagaagaag aacggcctgt 840
tcggcaacct gatcgccctg tccctgggcc tgacccccaa cttcaagtcc aacttcgacc 900
tggccgagga cgccaagctg cagctgtcca aggacaccta cgacgacgac ctggacaacc 960
tgctggccca gatcggcgac cagtacgccg acctgttcct ggccgccaag aacctgtccg 1020
acgccatcct gctgtccgac atcctgcgcg tgaacaccga gatcaccaag gcccccctgt 1080
ccgcctccat gatcaagcgc tacgacgagc accaccagga cctgaccctg ctgaaggccc 1140
tggtgcgcca gcagctgccc gagaagtaca aggagatctt cttcgaccag tccaagaacg 1200
gctacgccgg ctacatcgac ggcggcgcct cccaggagga gttctacaag ttcatcaagc 1260
ccatcctgga gaagatggac ggcaccgagg agctgctggt gaagctgaac cgcgaggacc 1320
tgctgcgcaa gcagcgcacc ttcgacaacg gctccatccc ccaccagatc cacctgggcg 1380
agctgcacgc catcctgcgc cgccaggagg acttctaccc cttcctgaag gacaaccgcg 1440
agaagatcga gaagatcctg accttccgca tcccctacta cgtgggcccc ctggcccgcg 1500
gcaactcccg cttcgcctgg atgacccgca agtccgagga gaccatcacc ccctggaact 1560
tcgaggaggt ggtggacaag ggcgcctccg cccagtcctt catcgagcgc atgaccaact 1620
tcgacaagaa cctgcccaac gagaaggtgc tgcccaagca ctccctgctg tacgagtact 1680
tcaccgtgta caacgagctg accaaggtga agtacgtgac cgagggcatg cgcaagcccg 1740
ccttcctgtc cggcgagcag aagaaggcca tcgtggacct gctgttcaag accaaccgca 1800
aggtgaccgt gaagcagctg aaggaggact acttcaagaa gatcgagtgc ttcgactccg 1860
tggagatctc cggcgtggag gaccgcttca acgcctccct gggcacctac cacgacctgc 1920
tgaagatcat caaggacaag gacttcctgg acaacgagga gaacgaggac atcctggagg 1980
acatcgtgct gaccctgacc ctgttcgagg accgcgagat gatcgaggag cgcctgaaga 2040
cctacgccca cctgttcgac gacaaggtga tgaagcagct gaagcgccgc cgctacaccg 2100
gctggggccg cctgtcccgc aagctgatca acggcatccg cgacaagcag tccggcaaga 2160
ccatcctgga cttcctgaag tccgacggct tcgccaaccg caacttcatg cagctgatcc 2220
acgacgactc cctgaccttc aaggaggaca tccagaaggc ccaggtgtcc ggccagggcg 2280
actccctgca cgagcacatc gccaacctgg ccggctcccc cgccatcaag aagggcatcc 2340
tgcagaccgt gaaggtggtg gacgagctgg tgaaggtgat gggccgccac aagcccgaga 2400
acatcgtgat cgagatggcc cgcgagaacc agaccaccca gaagggccag aagaactccc 2460
gcgagcgcat gaagcgcatc gaggagggca tcaaggagct gggctcccag atcctgaagg 2520
agcaccccgt ggagaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga 2580
acggccgcga catgtacgtg gaccaggagc tggacatcaa ccgcctgtcc gactacgacg 2640
tggaccacat cgtgccccag tccttcctga aggacgactc catcgacaac aaggtgctga 2700
cccgctccga caagaaccgc ggcaagtccg acaacgtgcc ctccgaggag gtggtgaaga 2760
agatgaagaa ctactggcgc cagctgctga acgccaagct gatcacccag cgcaagttcg 2820
acaacctgac caaggccgag cgcggcggcc tgtccgagct ggacaaggcc ggcttcatca 2880
agcgccagct ggtggagacc cgccagatca ccaagcacgt ggcccagatc ctggactccc 2940
gcatgaacac caagtacgac gagaacgaca agctgatccg cgaggtgaag gtgatcaccc 3000
tgaagtccaa gctggtgtcc gacttccgca aggacttcca gttctacaag gtgcgcgaga 3060
tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtggtgggc accgccctga 3120
tcaagaagta ccccaagctg gagtccgagt tcgtgtacgg cgactacaag gtgtacgacg 3180
tgcgcaagat gatcgccaag tccgagcagg agatcggcaa ggccaccgcc aagtacttct 3240
tctactccaa catcatgaac ttcttcaaga ccgagatcac cctggccaac ggcgagatcc 3300
gcaagcgccc cctgatcgag accaacggcg agaccggcga gatcgtgtgg gacaagggcc 3360
gcgacttcgc caccgtgcgc aaggtgctgt ccatgcccca ggtgaacatc gtgaagaaga 3420
ccgaggtgca gaccggcggc ttctccaagg agtccatcct gcccaagcgc aactccgaca 3480
agctgatcgc ccgcaagaag gactgggacc ccaagaagta cggcggcttc gactccccca 3540
ccgtggccta ctccgtgctg gtggtggcca aggtggagaa gggcaagtcc aagaagctga 3600
agtccgtgaa ggagctgctg ggcatcacca tcatggagcg ctcctccttc gagaagaacc 3660
ccatcgactt cctggaggcc aagggctaca aggaggtgaa gaaggacctg atcatcaagc 3720
tgcccaagta ctccctgttc gagctggaga acggccgcaa gcgcatgctg gcctccgccg 3780
gcgagctgca gaagggcaac gagctggccc tgccctccaa gtacgtgaac ttcctgtacc 3840
tggcctccca ctacgagaag ctgaagggct cccccgagga caacgagcag aagcagctgt 3900
tcgtggagca gcacaagcac tacctggacg agatcatcga gcagatctcc gagttctcca 3960
agcgcgtgat cctggccgac gccaacctgg acaaggtgct gtccgcctac aacaagcacc 4020
gcgacaagcc catccgcgag caggccgaga acatcatcca cctgttcacc ctgaccaacc 4080
tgggcgcccc cgccgccttc aagtacttcg acaccaccat cgaccgcaag cgctacacct 4140
ccaccaagga ggtgctggac gccaccctga tccaccagtc catcaccggc ctgtacgaga 4200
cccgcatcga cctgtcccag ctgggcggcg acccaaagaa gaagcgtaag gtatgatgtt 4260
ggcatcaggt aggcatcaca cacgattaac aacccctaaa aatacacttt gaaaatattg 4320
aaaatatgtt tttgtataca tttttgatat tttcaaacaa tacgcagtta taaaactcat 4380
tagctaaccc attttttctt tgcttatgct tacagattgc aaagaactag aggatctttg 4440
tgaaggaacc ttacttctgt ggtgtgacat aattggacaa actacctaca gagatttaaa 4500
gctctaaggt aaatataaaa tttttaagtg tataatgtgt taaactactg attctaattg 4560
tttgtgtatt ttagattcca acctatggaa ctgatgaatg ggagcagtgg tggaatgcct 4620
ttaatgagga aaacctgttt tgctcagaag aaatgccatc tagtgatgat gaggctactg 4680
ctgactctca acattctact cctccaaaaa agaagagaaa ggtagaagac cccaaggact 4740
ttccttcaga attgctaagt tttttgagtc atgctgtgtt tagtaataga actcttgctt 4800
gctttgctat ttacaccaca aaggaaaaag ctgcactgct atacaagaaa attatggaaa 4860
aatatttgat gtatagtgcc ttgactagag atcataatca gccataccac atttgtagag 4920
gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat 4980
gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc 5040
atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 5100
ctcatcaatg tatcttatca tgtctggatc gtctaga 5137
<210> 3
<211> 7
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Pro Lys Lys Lys Arg Lys Val
1 5
Claims (9)
1. a kind of cas9 transcription templates DNA for drosophila CRISPR transgenosis, which is characterized in that cas9 transcription templates DNA
With the base sequence as shown in SEQ ID NO:1.
2. a kind of Plasmid DNA for drosophila CRISPR transgenosis, which is characterized in that the Plasmid DNA includes following elements:
Cas9 open gene, the cas9 open gene have the base sequence as shown in SEQ ID NO:1；
First 40-nls enters nuclear signal, be located at the cas9 open gene initiation codon ATG after, cas9 open gene it
Before；
2nd 40-nls enters nuclear signal, is located at before cas9 open gene suspension codon, after cas9 open gene；
Kozak sequence, the Kozak sequence are located at before the initiation codon ATG；
T7 promoter；
Sv40-3UTR, the sv40-3UTR are located at after the terminator codon；
Restriction enzyme site, the restriction enzyme site are located at after the sv40-3UTR.
3. the Plasmid DNA according to claim 2 for drosophila CRISPR transgenosis, wherein the restricted digestion position
Point is Xba I restriction enzyme site.
4. the Plasmid DNA according to claim 2 for drosophila CRISPR transgenosis, wherein the Plasmid DNA has such as
Base sequence shown in SEQ ID NO:2.
5. in Plasmid DNA described in any one of cas9 transcription templates DNA described in claim 1 and claim 2-4 extremely
A kind of few application.
6. method is transcribed in vitro in a kind of cas9mRNA, which is characterized in that this method comprises:
(1) Plasmid DNA described in any one of claim 2-4 is subjected to amplification and linearization for enzyme restriction, and the line that will be obtained
Property template is purified；
(2) template after purification that step (1) obtains is transcribed in vitro and is purified.
7. according to the method described in claim 6, wherein, in step (1), the step of purifying includes: to be added using Proteinase K
SDS removes the restriction endonuclease of digestion system, then passes through ethanol precipitation.
8. according to the method described in claim 6, wherein, the dosage of GTP is 20-50mM in the in-vitro transcription.
9. according to the method described in claim 6, wherein, this method comprises: the concentration to cas9mRNA detects, the inspection
Survey method is electrophoresis Comparison Method.
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