CN107058385A - One kind builds new technology based on solid diffusivity CRISPR/Cas9gRNA tandem expression vectors - Google Patents

One kind builds new technology based on solid diffusivity CRISPR/Cas9gRNA tandem expression vectors Download PDF

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CN107058385A
CN107058385A CN201710228205.9A CN201710228205A CN107058385A CN 107058385 A CN107058385 A CN 107058385A CN 201710228205 A CN201710228205 A CN 201710228205A CN 107058385 A CN107058385 A CN 107058385A
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grna
pcr
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expression vectors
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杨兴林
陈国泽
潘讴东
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Yuan Biotechnology (shanghai) Ltd By Share Ltd
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    • C12N2810/00—Vectors comprising a targeting moiety
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Abstract

New technology is built the invention discloses a kind of CRISPR/Cas9gRNA tandem expression vectors based on solid diffusivity, PCR Start Fragments, extension fragment and termination fragment that the technology is coupled using Streptavidin MagneSphere and biotin, by the restriction enzyme site of careful design, each fragment is set accurately to be connected by viscous end.By digestion, cleaning, circulation the step of connection and multiple fragments are efficiently connected to multiple gRNA expression vector establishments technologies in a short time.Construction schedule can be shortened in 1 day and completed by the technology from 15 20 days, the greatly cost-effective and time.

Description

One kind is built new based on solid diffusivity CRISPR/Cas9gRNA tandem expression vectors Technology
Technical field
New technology is built the present invention relates to a kind of CRISPR/Cas9 gRNA tandem expression vectors based on solid diffusivity, should Application of many gRNA expression vectors in the genome editing technique such as cell and animal model mediated used in CRISPR/Cas9.
Background technology
CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)/ Cas9 technologies are that one kind can be used for the brand-new of accurate modification genomic DNA after Zinc finger nuclease (ZFN) and TALEN technologies Genome editing technique.The characteristics of it has fast efficiency high, speed DEG C and simple economy, in cell and animal basic research and experiment Application prospect in terms of the structure of model is all boundless[1]。
At present, from Streptococcus pyogenes CRISPR/spCas9 systems and come from Staphylococcus aureus CRISPR/saCas9 is most widely used.Cas9 albumen contains nuclease domain, can With cutting DNA double-strand.Cas9 is combined into compound with guide RNA (gRNA) first, then passes through PAM (Protospacer Adjacent Motif) sequence combines and invades DNA, forms RNA-DNA composite constructions, and then target DNA double-strand is cut Cut, be broken DNA double chain[2,3。
GRNA is attached to the crucial recognition sequence of target DNA as Cas9, and gRNA design determines that Cas9-gRNA is combined The final cutting efficiency of body.Usual gRNA obtains maturation gRNA sequences by polymerase III type promoter such as U6, H1 transcriptions.But Single gRNA expression vectors have been difficult the demand for meeting current experiment, can fast and efficiently be built while expressing multiple gRNA's The demand of many gRNA expression vectors is very urgent.The demand and advantage of many gRNA expression vectors are mainly reflected in following Aspect：
1) optimal target dna target spot is screened, the gRNA sequences of design 3 or more than 3 are usually required for a target spot； 2) it is used for the high gRNA target spots of screening efficiency, single gRNA expression vectors need to build 3 or more than 3, and the cycle is long, tests numerous It is trivial；3) need with once for multiple target dna target spots；4) a plurality of gRNA is used simultaneously for a target spot, to improve most Whole cutting efficiency.
List gRNA expression vectors are contrasted, many gRNA expression vectors need to only be built once, can equally be screened by sequencing Optimal gRNA, while importing efficiency can be improved, improves the positive rate of cell or animal model.In addition, current mainstream scheme All application virus technology expresses Cas9 and gRNA, and multiple carriers mean to pack more viruses, financial cost and time Cost can all be multiplied.Answering for CRISPR/Cas9 will be greatly improved by expressing multiple gRNA simultaneously in single carrier as can be seen here With space, cost is reduced, efficiency is improved.The constructing plan of many gRNA expression vectors main at present is structure one every time GRNA, builds complete several times, and the whole construction schedule of the carrier so comprising 3 gRNA is up to more than 15d.In order to solve structure Periodic problem is built, the Policy Table for having researcher to employ tRNA shearings reaches multiple gRNA[4], but program complex operation, not There is the root problem for solving construction schedule, so not being used widely.
Solid phase connecting method of the invention based on Streptavidin MagneSphere walks the connection for building 3-4 gRNA to single carrier Rapid shorten in 1d can just complete, and drastically increase efficiency, save plenty of time and financial cost.
Bibliography
1.Estrela R,Cate JH.Energy biotechnology in the CRISPR-Cas9 era.Curr Opin Biotechnol.2016Feb 10；38:79-84.
2.Mali P,Yang L,Esvelt KM,Aach J,Guell M,DiCarlo JE,Norville JE,Church GM.RNA-guided human genome engineering via Cas9.Science.2013 Feb 15；339 (6121):823-6.
3.Cong L,Ran FA,Cox D,Lin S,Barretto R,Habib N,Hsu PD,Wu X,Jiang W, Marraffini LA,Zhang F.Multiplex genome engineering using CRISPR/Cas systems.Science.2013 Feb 15；339(6121):819-23.
4.Xie K,Minkenberg B,Yang Y.Boosting CRISPR/Cas9multiplex editing capability with the endogenous tRNA-processing system.Proc Natl Acad Sci U S A.2015 Mar 17；112(11):3570-5.
The content of the invention
Many gRNA expression vectors have structure complexity, cycle length, cost necessary to be currently used in CRISPR/Cas9 technologies High shortcoming.Main reason is that current method need each step will confirm could to carry out after successfully constructing after building it is next Step is built.The cycle for many gRNA expression vectors that can express 3 gRNA simultaneously is built in more than 15d, 4 gRNA's of construction expression The cycle of many gRNA expression vectors is in more than 20d.
In order to solve the above technical problems, construction method of the present invention using solid diffusivity, either builds 3 gRNA still 4 gRNA many gRNA expression vectors, can complete to connect construction work within 1 working day, drastically increase structure effect Rate.
The first aspect of the present invention, the PCR Start Fragments being coupled using Streptavidin MagneSphere and biotin, extension fragment With termination fragment, by the restriction enzyme site of careful design, each fragment is set accurately to be connected by viscous end.By digestion, clearly Wash, circulation the step of connection and many gRNA expression vector establishments methods for efficiently connecting multiple fragments in a short time.
Second aspect of the present invention there is provided a kind of template sequence being made up of gRNA-spacer-U6promoter modules, its In, the corresponding templates of spCas9 contain the base sequence shown in SEQ.NO.1, and the corresponding templates of saCas9 contain SEQ.NO.2 institutes The base sequence shown.
A kind of third aspect present invention, the primer for providing base sequence as shown in containing SEQ.NO.3 and SEQ.NO.4 is used Initial segment is obtained in PCR.
Fourth aspect present invention, provides a kind of base sequence as shown in containing SEQ.NO.5, SEQ.NO.6 and SEQ.NO.7 Row.Wherein SEQ.NO.5 or SEQ.NO.6 primers can be a plurality of according to gRNA to be introduced quantitative design.SEQ.NO.7 coordinates root It is used for PCR according to SEQ.NO.5 or SEQ.NO.6 design primers and obtains extension fragment.
Fifth aspect present invention, provide it is a kind of with containing the base sequence shown in SEQ.NO.8 according to SEQ.NO.5 or SEQ.NO.6 design primers are used for PCR and obtain termination fragment.
Brief description of the drawings
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.
Accompanying drawing 1:PcDNA3.1 (+) carrier
Accompanying drawing 2：The many gRNA expression vectors (HY-multi-gRNA-Express) of general eukaryotic
Accompanying drawing 3：The many gRNA expression vectors (HY-multi-gRNA-ADV-Express) of adenovirus
Accompanying drawing 4：The many gRNA expression vectors (HY-multi-gRNA-LV-Express) of slow virus
Accompanying drawing 5：The many gRNA expression vectors (HY-multi-gRNA-AAV-Express) of adeno-associated virus
Accompanying drawing 6：Each junction fragment and carrier segments electrophoretogram
Accompanying drawing 7：The many gRNA built using solid phase connecting method are overexpressed adenovirus vector collection of illustrative plates
Accompanying drawing 8：Positive clone identification electrophoretogram (1490bp).
Embodiment
The chemical reagent and biological products being related to below, are all commercially produced product if not otherwise specified.In addition, other are not noted Bright experimental implementation is carried out according to conventional molecular biological operating method.
Embodiment 1
1. it is as follows to design 3 spCas9 gRNA sequences for people's gene PDCD1 (gene I/D 5133)：GRNA target spots 1： GCTCTCTTTGATCTGCGCCTTGG；GRNA target spots 2:GTGTCACACAACTGCCCAACGGG；GRNA target spots 3: GCTTGTCCGTCTGGTTGCTGGGG；
2. coordinate SEQ.NO.5 to design 3 PCR primers for these three sites
sp01-pdcd1-F:5’GAGAAGACAACACCGCTCTCTTTGATCTGCGCCTTGGGTTTTAGAGCTAGAAATAGCA AG 3’
sp02-pdcd1-F:5’GAGAAGACAACACCGTGTCACACAACTGCCCAACGGGGTTTTAGAGCTAGAAATAGCA AG 3’
sp03-pdcd1-F:5’GAGAAGACAACACCGCTTGTCCGTCTGGTTGCTGGGGGTTTTAGAGCTAGAAATAGCA AG 3’
One, experimental procedures are (to build 4 spCas9 gRNA expressing in series to adenovirus vector HY-multi-gRNA-Adv- Exemplified by Express)
(1) experiment material prepares
1. use equipment
96 hole magnetic bases, Streptavidin MagneSphere Dynabeads MyOne Streptavidin C1 (Invitrogen, goods Number:650.01), restriction endonuclease BbsI (NEB article No.s:), R0539S restriction endonuclease BsaI (NEB, article No.:), R0535V restriction endonuclease Hind III (NEB article No.s:), R0104S restriction endonuclease Xho I (NEB article No.s:), R0146S T4DNA Ligase (Rapid) (NEB article No.s: L6030-HC-L)
2.PCR template plasmids are built
Different gRNA sequences for spCas9 and saCas9 build two pcr template plasmids respectively.Two fragments of chemical synthesis SEQ.NO.1spCas9 gRNA+spacer+U6promoter and SEQ.NO.2saCas9 gRNA+spacer+U6promoter, It is building up between Hind III and the Xho I sites of pcDNA3.1 (+) carrier (see accompanying drawing 1).For subsequent PCR amplification template, It is respectively designated as pcDNA3.1-spCas9-gRNA-U6 and pcDNA3.1-saCas9-gRNA-U6
3. more than gRNA expression skeleton carrier
Different carriers can be selected as needed
3. primer is synthesized
SEQ.NO.3：Start-F:Biotin-5 ' GAGAAGACAAGAACGAGGGCCTATTTCCCATG3 ' (5 ' end biotin marks Note)
SEQ.NO.4：Start-R:5 ' CCGGTCTCTGGTGTTTCGTCCTTTCCACAAGATA 3 ' are as built spCas9 gRNA Following primer is built, according to circumstances selection builds 3-4 bars：
SEQ.NO.5：
sp-F:(target spot is designed spCas9 to 5 ' GAGAAGACAACACC [target spot] GTTTTAGAGCTAGAAATAGCAAG3 ' GRNA target sequences, 20 bases)
Such as build saCas9 gRNA and build following primer, according to circumstances selection builds 3-4 bars：
SEQ.NO.6：
sa-F:(target spot is design saCas9 to 5 ' GAGAAGACAACACC [target spot] GTTTTAGTACTCTGGAAACAGAATC3 ' GRNA target sequences, 20 bases)
SEQ.NO.7：Extend-R:5’CCGGTCTCTGGTGTTTCGTCCTTTCCAC 3’
SEQ.NO.8：Final-R:5’CCGGTCTCTGGTGTAATGCCAACTTTGTA3’
4. specific experiment step
1) it is as follows for 3 spCas9 gRNA sequences of people's gene PDCD1 (gene I/D 5133) designs：
GRNA target spots 1：GCTCTCTTTGATCTGCGCCTTGG；GRNA target spots 2:GTGTCACACAACTGCCCAACGGG；gRNA Target spot 2:GCTTGTCCGTCTGGTTGCTGGGG
SEQ.NO.5 is coordinated to design 3 PCR primers for these three sites
sp01-pdcd1-F:
5’GAGAAGACAACACCGCTCTCTTTGATCTGCGCCTTGGGTTTTAGAGCTAGAAATAGCAAG 3’
sp02-pdcd1-F:5’GAGAAGACAACACCGTGTCACACAACTGCCCAACGGGGTTTTAGAGCTAGAAATAGCA AG 3’
sp03-pdcd1-F:5’GAGAAGACAACACCGCTTGTCCGTCTGGTTGCTGGGGGTTTTAGAGCTAGAAATAGCA AG 3’
2) prepared by PCR fragment
A) using SEQ1 as template, performing PCR is entered with Start-F and Start-R primer pairs and obtains the long initial segment (Biotin- of 353bp ), Star-Cut after using Bsa I digestions, glue reclaim obtains endonuclease bamhi Biotin-Start-Cut, and wherein PCR system is constituted For 34 μ l ddH2O, 4 μ l dNTPs (10mM；2.5mM each), 5 μ l Buffer, 2 μ l SEQ1 templates (1ng/ μ l), 1 μ l Enzyme (3.5U/ μ l), 2 μ l Start-F (10pmol/ μ l), 2 μ l Start-R (10pmol/ μ l)；PCR reaction conditions are 95 DEG C 5min, 95 DEG C of 30s, 53 DEG C of 30s | 20cycles, 72 DEG C of 30s/, 72 DEG C of 5min
B) using SEQ1 as template, with sp01-pdcd1-F, sp02-pdcd1-F primers are respectively cooperating with Extend-R primers and enter performing PCR It is 486bp to obtain (Extend-01, Extend-02) length DEG C, and PCR fragment is used after Bbs I digestions, and glue reclaim obtains digestion Fragment Extend-01-Cut, Extend-02-Cut, wherein PCR system composition are 34 μ l ddH2O, 4 μ l dNTPs (10mM； 2.5mM each), 5 μ l Buffer, 2 μ l SEQ1 templates (1ng/ μ l), 1 μ l Enzyme (3.5U/ μ l), 2 μ l sp01-F/ Sp02-F/sp03-F (10pmol/ μ l), 2 μ l Extend-R (10pmol/ μ l)；PCR reaction conditions are 95 DEG C of 5min, 95 DEG C 30s, 53 DEG C of 30s | 30cycles, 72 DEG C of 30s/, 72 DEG C of 5min；The following 10 μ l PCR productions of PCR primer digestion condition (50 μ l) Thing, 30 μ l water, the μ l BbsI (5U/ μ l) of 5 μ l NEBuffer 2,5
C) using SEQ1 as template, so that sp03-pdcd1-F and Final-R primer pairs enter performing PCR and obtain 168bp long segments (Final-03), PCR fragment is used after Bbs I digestions, glue reclaim acquisition endonuclease bamhi Final-03-Cut (see accompanying drawing 6, wherein PCR system constitutes 34 μ l ddH2O, 4 μ l dNTPs (10mM；2.5mM each), 5 μ l Buffer, 2 μ l SEQ1 templates (1ng/ μ l), 1 μ l Enzyme (3.5U/ μ l), 2 μ l sp04-F (10pmol/ μ l), 2 μ l Final-R (10pmol/ μ l).Wherein PCR Reaction condition:95 DEG C of 5min, 95 °C of 30s, 53 DEG C of 30s | 30cycles, 72 DEG C of 30s/, 72 DEG C of 5min；PCR primer digestion bar Part (50 μ l):10 μ l PCR primers, 30 μ l water, the μ l BbsI (5U/ μ l) of 5 μ l NEBuffer 2,5
3) prepared by skeleton plasmid
HY-multi-gRNA-Adv-Express carriers use glue reclaim after Bsm BI digestions, obtain Vector-Cut fragments.Its Middle endonuclease reaction condition:5 μ l BsmBI (10U/ μ l), 5 μ l NEBuffer 3,20 μ l HY-multi-gRNA-Adv- Express (5 μ g), 20 μ l H255 DEG C of digestion 8h of O
4) initial segment is connected
2 μ l purifying and the Biotin-Start-Cut fragments of digestion, 27 μ 2 × QLB of l Buffer, 2.5 μ l T4DNA connections The μ l Extend-01-Cut of enzyme (400U/ μ l) 22.5, mix room temperature connection 15min
5) magnetic bead prepares
5 μ l magnetic beads (Dynabeads MyOne Streptavidin C1) are added to 50ul 1 × B＆W Buffer (5.0mM Tris-HCl pH 7.5,0.5mM EDTA, 1.0M NaCl, 0.005%Tween 20), 5 cleaning magnetic beads are blown and beaten with rifle, 3min is placed on base, supernatant is sucked and repeats finally to be resuspended in 54ul 2 × B＆W with 1 × B＆W of 50ul above-mentioned steps, 3 Buffer (10.0mM Tris-HCl pH 7.5,1.0mM EDTA, 2.0M NaCl)
6) initial segment is connected on magnetic bead
Plus 54 obtain in the magnetic bead that reaction solution is prepared to 54 μ l steps 4 in μ l steps 3, mixed 5 times with above and below rifle, incubation at room temperature 15min (is wherein mixed once) per 5min, is placed 3min on magnetic bases, is sucked supernatant plus 100 μ 1 × B＆W of l buffer are anti- Even 10 times of compound, is placed into magnetic bases 1min. and sucks supernatant, 100 1 × BSA of μ l of addition mix 10 and are placed into magnetic bottom repeatedly Seat 3min, sucks supernatant；It is resuspended in the μ l BsaI reaction solutions of magnetic bead 50, mixes up and down 20 times, 50 DEG C of incubation 10min, plus 50 μ l 1 × B＆W buffer are placed into magnetic bases 3min, suck supernatant, plus 100 μ l 1 × B＆W buffer, mix 10 times repeatedly, Magnetic bases 1min is placed into, supernatant is sucked, 100 μ 1 × BSA of l are added, mixes 10 times repeatedly, is placed into magnetic bases 3min, Suck supernatant
7) connection of fragment is extended
22.5 μ l extension fragment mixtures are added in 1ml PE pipes or fragment mixture and 27.5 μ l master are terminated Mix, is mixed 10 times with above and below liquid-transfering gun, is incubated at room temperature 15min, and per 5min, mixing once, adds 50 1 × B＆W of μ l Buffer, is placed into magnetic bases and stands 3min, suck supernatant, add 100 μ l 1 × B＆W buffer, mix 10 times repeatedly, It is placed into magnetic bases and stands 1min, suck supernatant, add 100 μ 1 × BSA of l, mixes 10 times repeatedly, be placed into magnetic bottom Seat stands 3min, sucks supernatant.It is resuspended in the μ l BsaI reaction solutions of magnetic bead 50,20 50 DEG C of incubation 10min is mixed up and down, plus 50 μ l 1 × B＆W buffer, are placed into magnetic bases and stand 3min, suck supernatant standby
8) junction fragment departs from magnetic bead
Plus 100 μ 1 × B＆W of l buffer in the supernatant of above-mentioned gained, repeatedly mix 10 times, be placed into magnetic bases standing 1min, sucks supernatant, adds 100 μ 1 × BSA of l, and 10 are mixed repeatedly, is placed into magnetic bases and stands 3min, sucks, In the BbsI reaction solutions for being resuspended in the μ l of magnetic bead 50,37 DEG C of incubation 2h, while 1500rpm shakes 2min, are placed into magnetic bases quiet 3min is put, retains supernatant, fragment carries out glue reclaim
9) junction fragment is connected (see accompanying drawing 7) with skeleton plasmid
The DNA of 3 μ l purifying, 1 μ l Vector-Cut skeleton plasmids, 1 μ l T4DNA ligases (400U/ μ l), 5 μ l 2 × quick Ligase buffer, 37 DEG C of incubation 15min
10) transformed competence colibacillus cell
4 μ l connection products and 50 μ l competent cells, which are mixed, to be converted
11) identification is (see accompanying drawing 8)
Synthesis identification primer, PCR obtains 1490bp purpose bands, confirms that structure is correct after sequencing；Forward direction identification primer： TTTTAGGCGGATGTTGTA reversely identifies primer：ATGGGCTATGAACTAATGAC, electrophoresis result meets it is contemplated that experimental result Confirm that this method can fast and effectively carry out the structure of many gRNA expression vectors.
<110>With first biotechnology (Shanghai) limited company
<120>A kind of CRISPR/Cas9 gRNA tandem expression vector construction methods based on solid diffusivity
<160> 8
<210> 1
<211> 435
<212> DNA
<213>Artificial sequence
<223> spCas9 gRNA+spacer+U6 promoter
<400> 1
GTTTTAGAGC TAGAAATAGC AAGTTAAAAT AAGGCTAGTC CGTTATCAAC TTGAAAAAGT 60
GGCACCGAGT CGGTGCTTTT TTTCTAGACC CAGCTTTCTT GTACAAAGTT GGCATTATGT 120
ACAAAAAAGC AGGCTTTAAA GGAACCAATT CAGTCGACTG GATCCGGTAC CAAGGTCGGG 180
CAGGAAGAGG GCCTATTTCC CATGATTCCT TCATATTTGC ATATACGATA CAAGGCTGTT 240
AGAGAGATAA TTAGAATTAA TTTGACTGTA AACACAAAGA TATTAGTACA AAATACGTGA 300
CGTAGAAAGT AATAATTTCT TGGGTAGTTT GCAGTTTTAA AATTATGTTT TAAAATGGAC 360
TATCATATGC TTACCGTAAC TTGAAAGTAT TTCGATTTCT TGGCTTTATA TATCTTGTGG 420
AAAGGACGAA ACACC 435
<210> 2
<211> 435
<212> DNA
<213>Artificial sequence
<223> saCas9 gRNA+ spacer +U6 promoter
<400> 2
GTTTTAGTAC TCTGGAAACA GAATCTACTA AAACAAGGCA AAATGCCGTG TTTATCTCGT 60
CAACTTGTTG GCGAGATTTT TTTCTAGACC CAGCTTTCTT GTACAAAGTT GGCATTATGT 120
ACAAAAAAGC AGGCTTTAAA GGAACCAATT CAGTCGACTG GATCCGGTAC CAAGGTCGGG 180
CAGGAAGAGG GCCTATTTCC CATGATTCCT TCATATTTGC ATATACGATA CAAGGCTGTT 240
AGAGAGATAA TTGGAATTAA TTTGACTGTA AACACAAAGA TATTAGTACA AAATACGTGA 300
CGTAGAAAGT AATAATTTCT TGGGTAGTTT GCAGTTTTAA AATTATGTTT TAAAATGGAC 360
TATCATATGC TTACCGTAAC TTGAAAGTAT TTCGATTTCT TGGCTTTATA TATCTTGTGG 420
AAAGGACGAA ACACC 435
<210> 3
<211> 32
<212> DNA
<213>Artificial sequence
<223>Start-F, 5' end Biotin are marked
<400> 3
GAGAAGACAA GAACGAGGGC CTATTTCCCA TG 32
<210> 4
<211> 34
<212> DNA
<213>Artificial sequence
<223> Start-R
<400> 4
CCGGTCTCTG GTGTTTCGTC CTTTCCACAA GATA 34
<210> 5
<211> 57
<212> DNA
<213>Artificial sequence
<223>Sp-F, N are the target sequence of design
<400> 5
GAGAAGACAA CACCNNNNNN NNNNNNNNNN NNNNGTTTTA GAGCTAGAAA TAGCAAG 57
<210> 6
<211> 59
<212> DNA
<213>Artificial sequence
<223>Sa-F, N are the target sequence of design
<400> 6
GAGAAGACAA CACCNNNNNN NNNNNNNNNN NNNNGTTTTA GTACTCTGGA AACAGAATC 59
<210> 7
<211> 28
<212> DNA
<213>Artificial sequence
<223> Extend-R
<400> 7
CCGGTCTCTG GTGTTTCGTC CTTTCCAC 28
<210> 8
<211> 29
<212> DNA
<213>Artificial sequence
<223> Final-R
<400> 8
CCGGTCTCTG GTGTAATGCC AACTTTGTA 29

Claims (6)

1. one kind builds new technology based on solid diffusivity CRISPR/Cas9gRNA tandem expression vectors, it is characterised in that：Use Streptavidin MagneSphere and the PCR Start Fragments of biotin couplings, extension fragment and termination fragment, pass through the digestion of careful design Site, enables each fragment accurately to be connected by viscous end；By digestion, cleaning, circulation the step of connection and by multiple Section efficiently connects multiple gRNA expression vector establishments technologies in a short time.
2. spCas9 the and saCas9 template sequences used according to claim 1 in method：It is characterized in that：By gRNA- The template sequence of spacer-U6 promoter modules composition, wherein, the corresponding templates of spCas9 contain shown in SEQ. NO.1 Base sequence, the corresponding templates of saCas9 contain the base sequence shown in SEQ. NO.2.
3. PCR primer sequence is used in method as claimed in claim 1：It is characterized in that：The primer sequence is to contain SEQ. Base sequence shown in NO.3 and SEQ. NO.4；SEQ. NO.3 and SEQ. NO.4 are used for PCR acquisition initial segments.
4. PCR primer sequence is used in method as claimed in claim 1：It is characterized in that：The primer sequence is to contain SEQ. Base sequence shown in NO.5, SEQ. NO.6 and SEQ. NO.7；Wherein SEQ. NO.5 or SEQ. NO.6 primers can bases The quantitative design for introducing gRNA is a plurality of；SEQ. NO.7 coordinates designs primer for PCR according to SEQ. NO.5 or SEQ. NO.6 Obtain extension fragment.
5. PCR primer sequence is used in method as claimed in claim 1：It is characterized in that：The primer sequence is to contain SEQ. Base sequence shown in NO.8；SEQ. NO.8 coordinates is used for PCR acquisitions eventually according to SEQ. NO.5 or SEQ. NO.6 design primers Limited step section.
6. many gRNA expression vectors of primer described in method and 2,3,4,5 are in cell and animal model according to claim 1 Deng the application in genome editing technique.
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