CN110438141A - A kind of gene editing method based on SviCas6-SviCas3 - Google Patents
A kind of gene editing method based on SviCas6-SviCas3 Download PDFInfo
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- CN110438141A CN110438141A CN201910420998.3A CN201910420998A CN110438141A CN 110438141 A CN110438141 A CN 110438141A CN 201910420998 A CN201910420998 A CN 201910420998A CN 110438141 A CN110438141 A CN 110438141A
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Abstract
The gene editing method based on SviCas6-SviCas3 that the invention discloses a kind of.Using SviCas6 in conjunction with SviCas3 t-DNA and g-DNA, realize SviCas6-SviCas3 for the first time to the gene editing of biological cell genome, also the gene editing to carry out based on CRISPR-Cas system provides new supplement and multifarious selection.The system can carry out effective, accurate, stable gene editing to gene in protokaryon and eukaryotic gene group, it is expected to be applied in many fields relevant to biotechnology.
Description
The present invention relates to the genetic engineering fields in biotechnology, are exactly a kind of derived from Virginia streptomycete
The gene editing method of SviCas6 and SviCas3 in the I-B-Svi type CRISPR-Cas system of IBL14.
Background technique
Substantially, gene editing is exactly the template DNA (template DNA/t-DNA) by engineering design required function,
Restriction enzyme by being oriented to specificity cuts the DNA in genome, and then is repaired using the homologous of cell itself
Multiple (homology directed repair/HDR) mechanism achieve the purpose that gene editing [Giedrius Gasiunas,
Tomas Sinkunas, and Virginijus Siksnys (2014) Molecular mechanisms of CRISPR-
Mediated microbial immunity.Cell Mol Life Sci 71,449-465, doi:10.1007/s00018-
013-1438-6].The guide mechanism for being oriented to the restriction enzyme of specificity can be divided into three levels: (1) protein targeting,
Such as: zinc finger endonuclease (zinc-finger nucleases/ZFN)；(2) RNA is oriented to, such as CRISPR-Cas system；With
(3) DNA is oriented to, such as we have found that SviCas3 guiding [Tong Wangyu, Tang Yanyan, Xia Tingting (20170919) are a kind of to be based on I-B
The gene editing method of type CRISPR-Cas system gene cas3, application number: 201710847193.8.].With protein targeting phase
Than RNA guiding is because its is at low cost, easy, quick, is efficiently widely used to the fields such as biology, medicine, agricultural and food.
Based on the composition and structure of the immune middle Cas effector of prokaryote, CRISPR-Cas system is now divided into 2 classes six
Type [Eugene V Koonin, Kira S Makarova and Feng Zhang (2017) Diversity,
classification and evolution of CRISPR-Cas systems.Current opinion in
Microbiology 37,67-78, doi:10.1016/j.mib.2017.05.008].And 1 class I type is the most extensive, Zhan Yijian
60% or more of fixed cellular genome.It is well known that in 1 class I type CRISPR-Cas system, by Cse1, Cse2, Cas5,
The Cascade of the albumen such as Cas6, Cas7 composition is functional complex necessary to cellular immunity, and wherein Cas6 executes crRNA and hair
The processing of clamping structure forms function [Yibei Xiao, Min Luo, Adam E.Dolan, Maofu Liao, Ailong Ke
(2018).Structure basis for RNA-guided DNA degradation by Cascade and
Cas3.Science (New York, N.Y.) 361, doi:10.1126/science.aat0839；Prarthana
Mohanraju, Kira S.Makarova, Bernd Zetsche, Feng Zhang, Eugene V.Koonin, John van
der Oost(2016)Diverse evolutionary roots and mechanistic variations of the
CRISPR-Cas systems.Science (New York, N.Y.) 353, aad5147, doi:10.1126/
science.aad5147]。
Gene editing based on the single Cas9 of 2 class, two type has become mainstream, and based on 1 class I type CRISPR-Cas system into
Row gene editing is detected in I-B-Svi type CRISPR-Cas system [Tong Wangyu, Tang Yanyan, the Xia Tingting of our reports
(20170919) a kind of gene editing method based on I-B type CRISPR-Cas system gene cas3, application number:
201710847193.8.；Tong Wangyu, Xia Tingting, Tang Yanyan (20170919) are a kind of based on gene in I type CRISPR-Cas system
The eukaryotic gene edit methods of cas7-3, application number: 201710851388.X.；Tong Wangyu, Sun Yan, Wang Anjing (20170919)
A kind of prokaryotic gene edit methods of the gene cas5-3 in I type CRISPR-Cas system, application number:
201710846388.0.].The R- that we are formed using targeting g-DNA transcription and the crRNA after SviCas6 is processed and target DNA
Loop, guidance SviCas3 carries out the cutting of specificity, and then reaches base using the homologous repair mechanism of t-DNA and cell itself
Because of the purpose of editor.Due to different eucaryotes has strong cipher selectivity, there are nuclear membrane and only in a manner of monocistron
Protein translation is carried out, therefore the present invention, in eukaryotic gene editor, the base sequence of SviCas6 and SviCas3 gene is wanted
Optimization, and cas6 and cas3 gene must be respectively provided with nuclear localization sequence, transcripting promoter and terminator in expression vector.
The first public one kind of the present invention is based on SviCas6-SviCas3 in I-B-Svi subtype C RISPR-Cas system
Gene editing method.It has been observed that after SviCas6 and SviCas3 expression, it can be to biological cell base in conjunction with t-DNA and g-DNA
Because of a group progress efficient gene editor.The appearance of the gene editing tool to be based on SviCas3, SviCas5-SviCas3 and
SviCas7-SviCas3 gene editing provides multifarious selection and supplement, also for based on other types of CRISPR-Cas
The gene editing too development of system provides foundation.
Summary of the invention
The gene editing method based on SviCas6-SviCas3 that the technical problem to be solved in the present invention is to provide a kind of, with
Realize its gene editing to biological cell genome.For this purpose, the technical scheme adopted by the invention is that:
A kind of gene editing method based on SviCas6-SviCas3, it is characterised in that: Virginia streptomycete IBL14
Contain an I-B-Svi type CRISPR-Cas system, protein s viCas6 and the SviCas3 (amino in the system in genome
Acid sequence is seen attached list) combine template t-DNA (template DNA) and targeting g-DNA (guide-DNA) can be to biological cell base
Because the gene in group is effectively edited.
A kind of gene editing method based on SviCas6-SviCas3, which is characterized in that including edit tool
Building and the building of recon and checking procedure are as follows:
(1) building of carrier is edited
According to SviCas6 and SviCas3 amino acid sequence, the DNA sequence dna of the base optimization of corresponding biological cell is synthesized, and
In conjunction with t-DNA, g-DNA and expression vector dna primers, cuts and connect through primer synthesis, PCR amplification, restriction enzyme
Enzyme connection is connect, connects protein coding gene, t-DNA, g-DNA and expression vector to obtain Protein S viCas6-SviCas3 respectively
The gene editing tool Vector-cas6-3-t/g-DNA of expression.
(2) building and inspection of recon
Biological cell competence is prepared, the edit tool Vector-cas6-3-t/g-DNA obtained by step (1) is passed through
The method of conversion or transfection imports in biological cell, and selection culture obtains potential gene editing recon；To potential recon
Function Identification, target sequence PCR amplification and sequencing analysis are carried out, correct gene editing recon is obtained.
The SviCas6 and SviCas3 refers to the SviCas6 and SviCas3 after original or Amino acid sequence mutants.
The t-DNA refer to synthesis by required genetic function design have the DNA fragmentation complementary with target sequence both ends,
It can separate and be individually present with edit tool.
The g-DNA refer to synthesis it is transcribed obtain crRNA/CRISPR-RNA DNA fragmentation (by transcripting promoter/
Promoter, repetitive sequence/repeat, intervening sequence/spacer and transcription terminator/terminator composition).
The DNA sequence dna of the base optimization of the corresponding biological cell refers to according to SviCas6 and SviCas3 amino acid sequence
And the cDNA sequence of the base optimization for being suitble to the protein expression in different biological cells optimized.
The gene editing recon refer to because the DNA in genome by the knockout of specificity, insertion, seamless point mutation and
Lead to the recon of the changed inheritance stability of phenotype, function and physiology of host cell after the editor of any combination.
The biological cell refers to any one of prokaryote and eukaryotic cells, such as: Escherichia coli, paddy ammonia
Sour bar bacterium, saccharomyces cerevisiae, human embryonic kidney cells etc..
Beneficial effect
The present invention describes a kind of gene editing method based on SviCas6-SviCas3.Using SviCas6 with
SviCas3 combination t-DNA and g-DNA realize SviCas6-SviCas3 for the first time and compile to the gene in biological cell genome
Volume, also the gene editing to be carried out based on CRISPR-Cas system provides new supplement and multifarious selection.The system can
Effective, accurate, stable gene editing is carried out to gene in protokaryon and eukaryotic gene group, it is expected to be applied to it is many with
In the relevant field of biotechnology.
Specification subordinate list, attached drawing
The amino acid sequence of subordinate list, SviCas6 and SviCas3
Fig. 1, gene editing carrier pRS415-cas6-3-t/g- Δ crtE::egfp structure figures.Ori: height copy
The DNA replication dna initiation site of ColE1, pMB1, pBR322 and pUC；CEN/ARS: it is fused to autonomously replicating sequence
S.cerevisiae CEN6 centriole, LEU2 promoter: leucine promoter；AmpR: amicillin resistance.
The PCR electrophoretogram of Fig. 2, saccharomyces cerevisiae Δ crtE::egfp gene editing.Swimming lane L:5000bp DNA ladder；
1: host's control: target sequence in wild type SC LYC4 genome；2-4: gene editing mutant strain SC LYC4- Δ crtE::egfp
(the DNA band of crtE-vF/vR primer extension product is smaller about than original strain crtE gene target sequence for target sequence in genome
1000bp shows gene knockout success)；5: water control.
Specific embodiment
In order to more fully understand technology contents of the invention, technical solution of the present invention is made combined with specific embodiments below
It is further described and illustrates, it is intended to preferably explain that the contents of the present invention, following embodiment do not limit the scope of the invention.
In addition, being all made of following material unless otherwise instructed in listed embodiment:
1) bacterial strain and carrier
Bacterial strain: Virginia streptomycete Streptomyces virginiae IBL14/SV IBL14, Escherichia coli
Escherichia coli DH5 α/EC DH5 α, Escherichia coli JM109 (DE3)/EC JM109 (DE3), paddy ammonia
Sour corynebacteria Corynebacterium glutamicum B253/CG B253, Saccharomyces Cerevisiae in S accharomyces
293 cells/HEK293T of cerevisiae LYC4/SC LYC4, human embryonic kidney cells human embryonic kidney；It carries
Body: pCas, pRS415, pEC-XK99E, pMD-18T, pAIO.
2) buffer and culture medium
HEPES buffer solution (100mL)
Weigh 0.8g NaCl, 0.0135g Na2HPO4.H2O, 0.037g KCl, 0.5g glucose, 0.5g HEPES [4-
(2-hydroxyethyl) -1-piperazineethanesulfonic acid], add 90mL ddH2O dissolution, NaOH tune pH is extremely
7.5, ddH2O is settled to 100mL.
LiAc solution (100mM)
The LiAc solid powder for weighing 0.66g, adds 90mL ddH2O dissolves and is settled to 100mL.
LiAc/DTT/TE buffer (400mL)
0.48456g Tris (10mM, pH7.5), 4.088g LiAc2H2O (0.1M), 0.1169g EDTA (1mM),
300mL ddH2O dissolution, HCl tune pH to 7.5, ddH2O is settled to 400mL (being added after 0.617g DTT filtration sterilization).
Sorbierite (1M)
18.217g sorbierite adds ddH2O to 100mL, 120 DEG C/25min sterilizing.
Ampicillin (50mg/mL)
The ampicillin for weighing 0.5g is placed in 10mL centrifuge tube, and 4mL ddH is added2O after mixing, is settled to 10mL,
It after 0.2 μm of sterile water system membrane filtration, is dispensed into the EP pipe of 1.5mL, is put in -20 DEG C of refrigerators and saves.
EPO culture medium (1000mL)
Yeast extract 5g, peptone 10g, NaCl 10g, glycine 30g, Tween80 1g, ddH2O constant volume, 115 DEG C/
30min sterilizing.
LB liquid medium (1000mL)
Constant volume after appropriate tap water dissolution is added in yeast powder 5g, peptone 10g, NaCl 10g, and pH is adjusted to 7.0~7.3,
After packing wrapping, 115 DEG C/25min sterilizes (plate adds agar 15g).
LBG culture medium (1000mL)
Yeast extract 5g, peptone 10g, NaCl 10g, glucose 5g, are added constant volume after appropriate tap water dissolution, and pH is adjusted to
7.0, after packing wrapping, 115 DEG C/30min sterilizing.
LBGT culture medium (1000mL)
Yeast extract 5g, peptone 10g, NaCl 10g, glycine 30g, Tween80 1g, after appropriate tap water dissolution is added
Constant volume, pH are adjusted to 7.0-7.2,115 DEG C/30min sterilizing.
LBHIS culture medium (1000mL)
Sorbierite 91g, yeast extract 2.5g, peptone 5g, NaCl 5g, brain heart infusion 18.5g, addition are distilled water-soluble in right amount
15g agar is added in constant volume after solution, 121 DEG C/20min sterilizing, plate such as processed.
LBPET culture medium (1000mL)
Add in LB culture medium: 20mM MgCl2·6H2O, 40mg/l arabinose, 50mg/l X-gal, 50mg/l IPTG,
100mg/l kanamycins and 100mg/l apramycin (being protected from light).
SCCM culture medium (1000ml)
900mL DMEM in high glucose, 100mL fetal bovine serum, 100mg penicillin, 100mg streptomysin, 10g glutamine are (high
Sugared DMEM culture medium/GibcoTM: Thermo Fisher Scientific Inc., 168 Third Avenue are purchased from,
Waltham, MA USA 02451).
SC-raffinose induced medium (100mL)
0.17g YNB (Yeast Nitrogen Base without Amino Acids), 0.5g ammonium sulfate, 2g gala
Sugar, 1g gossypose, adds ddH2(400 μ L SD-AA, SD-AA formulas are added such as using preceding in O to 99.6mL, 115 DEG C of sterilizing 30min
Under).
SD-LEU Selective agar medium (100mL)
0.5g ammonium sulfate, 2g glucose, 0.17gYNB (Yeast Nitrogen Base without Amino
Acids), ddH299.6mL is settled to after O dissolution, after packing wrapping, 115 DEG C/30min sterilizing.400 μ L of filtration sterilization are added
2g agar is added in amino acid (methionine 0.5g/100mL, histidine 0.5g/100mL, uracil 0.5g/100mL), plate such as processed
With 18.2g sorbierite.
SD-Ura Selective agar medium (100mL)
0.5g ammonium sulfate, 2g glucose, 0.17gYNB (Yeast Nitrogen Base without Amino
Acids), ddH is added299.6mL is settled to after O dissolution, after packing wrapping, 115 DEG C/30min sterilizing.Filtration sterilization is added
400 μ L amino acid (methionine 0.5g/100mL, histidine 0.5g/100mL, leucine 0.5g/100mL), plate such as processed are added
2g agar and 18.2g sorbierite.
YNB culture medium (1000mL)
Appropriate ddH is added in peptone 20g, yeast extract 10g, glucose 20g2Constant volume after O dissolution, after packing wrapping, 115
DEG C/30min sterilizing, plate addition 15g agar such as processed.
Agents useful for same is commercially available product.
The knockout of lacI gene in embodiment 1, EC JM109 (DE3)
(1) building of gene editing tool pCas-cas6-3-t/g- Δ lacI
(A) building of pCas-cas6-3
According to the DNA sequence dna information of SV IBL14 gene cas6, cas3 and carrier pCas, design is with mutual with carrier pCas
Primer pCas-cas6-F/R and pCas-cas3-F/R (table mend, can be used for overlap PCR, gene cas6 and cas3
1).SV IBL14 genomic DNA is extracted, Standard PCR difference amplification gene cas6 and cas3 [50 μ L PCR reactants are first passed through
System: 5 μ 10 × Pfu of L buffer, 5 μ L dNTPs (2.5mM each), 1 10 μM of μ L cas6-F/R or cas3-F/R, 5 μ L
DMSO, 0.5 μ L Pfu DNA Polymerase, 0.5 μ L SV IBL14 genomic DNA, 32 μ L ddH2O(nuclease-
Free), reaction condition: 95 DEG C of 5min, 94 DEG C of 60 ± 30s, 55 ± 3 DEG C of 30s, 72 DEG C of 90 ± 30s, 2.5 ± 0.5U DNA
Polymerase (TransStart FastPfu DNA Polymerase, Quan Shijin Bioisystech Co., Ltd), 30 circulations, 72
℃ 10min].PCR product is detected through 1% agarose electrophoresis, and kit recycling obtains cas6 and the cas3 full-length gene of purifying.It is pure
Cas6 and the cas3 full-length gene of change connect [50 μ L overlap PCR reaction systems: 5 10 × Pfu of μ L through overlap PCR
Buffer, 5 μ L dNTPs (2.5mM each), 10 μM of pCas-cas6-F/R (1 μ L each), 10 μM of pCas-cas3-F/R
(1 μ L each), 5 μ L DMSO, 0.5 μ L Pfu DNAPolymerase, 31.5 μ L ddH2O (nuelease-free), reaction
Condition: 95 DEG C of 5min, 94 DEG C of 60 ± 30s, 55 ± 3 DEG C of 30s, 72 DEG C of 90 ± 30s, 2.5 ± 0.5U archaeal dna polymerase (one
Primer UF and each 1 μ L of DR are added after circulation), continue 30 circulations, 72 DEG C of 10min], after product purification gene cas6-
cas3.The gene cas6-cas3 of purifying is connect with carrier pCas (pCas-F/R linearisation) by one-step cloning, Cas6- is obtained
Cas3 expression vector pCas-cas6-3.
(B) building of pCas-cas6-3-t/g- Δ lacI
According to pcas-cas6-3 and lacI gene sequence information, design and chemical synthesis sequence include transcripting promoter,
The molecular target gene segment g-lacI of crDNA (being made of repeat and spacer), tanscription termination, passes through g-lacI-F/
G-lacI-R primer PCR amplifies erDNA segment.Simultaneously according to EC JM109 (DE3) gene order-checking information, design and synthesize
Gene lacI verifies primer lacI-vF/vR, can be used for the upstream lacI homology arm (UHA) primer of overlap PCR connection
LacI-UF/UR and downstream homology arm (DHA) primer lacI-DF/DR (table 1), extraction EC JM109 (DE3) genomic DNA are mould
Plate, standard PCR amplification and purifying by same step (1-A), obtains UHA and DHA is spare；Obtained UHA is passed through with DHA synchronous
Suddenly the overlap PCR ligation amplification of (1-A) obtains gene editing template t- Δ lacI after purification.By obtained t- prepared above
Carrier pCas-cas6-3 (pCas-g-F/ is connected to by one-step cloning after Δ lacZ and g-lacZ segment overlap PCR
PCas-g-R linearisation) on, it is transformed into E.coli DH5 α and obtains gene editing tool pCas-cas6-3-t/g- Δ lacI.
Primer used in the present embodiment and the g-lacI and t- Δ lacI sequence of design synthesis are as shown in table 1.It is empty in table
Line represents complementary series；Single underscore represents promoter；Double underline is metabolized terminator；Tilted letter represents spacer；Runic
Letter represents repeat.
1 primer of table and editor's element sequences table
(2) gene editing recon EC JM109 (DE3)-Δ lacI building and verifying
(A) preparation of EC JM109 (DE3) competence
With sterilized toothpick from picking EC JM109 (DE3) monoclonal on LB plate in 50mL LB liquid medium,
37 ± 5 DEG C, 200 ± 50rpm is incubated overnight；100 ± 50 μ L are incubated overnight liquid and are forwarded in new LB liquid medium, and 37 ± 3
DEG C, 200 ± 50rpm cultivates about 3 ± 2h, until bacterium solution OD600Value is 0.5 ± 0.1；Take the above-mentioned bacterium solution of 1mL into 1.5mL EP pipe, 4
5000rpm is centrifuged 5min at DEG C, removes supernatant, is blown and beaten bacterial sediment uniformly, i.e., with the SSCS solution that 50 μ L are pre-chilled on ice
The competent cell of EC JM109 (DE3) is obtained, saves backup (process carries out on ice entirely) at -80 DEG C.
(B) pCas-cas6-3-t/g- Δ lacI is converted
10 ± 5 μ L plasmid pCas-cas6-3-t/g- Δ lacI are added in 100 μ L EC JM109 competence, are mixed
Afterwards in 30 ± 5min on ice, 45 ± 2 DEG C of water-bath 90s, then at 15 ± 10min on ice, it is uniformly coated on LBPET plate, 30 ± 5 DEG C
It is incubated overnight 24-36h, obtains candidate transformant EC JM109 (DE3)-Δ lacI.
(C) daughter chromosome PCR and gene sequencing analysis are recombinated
It is incubated overnight, extracts from picking white monoclonal candidate transformant on LBPET plate by the method for step (2-A)
DNA genome is template, then is carried out with lacI gene verifying primer (table 1) lacI-vF/lacI-v by the method for step (2-A)
PCR amplification.PCR product is as a result consistent with the t- Δ lacI sequence of design through 1% agarose electrophoresis and sequencing, shows that gene is compiled
It collects successfully.
The insertion of egfp in embodiment 2, SC LYC4 genome
(1) building of gene editing tool pRS415-cas6-3-t/g- Δ crtE::egfp
(A) building of pRS415-cas6-3
(a) it is respectively provided with the preparation of the cas6-3 genetic fragment of promoter, terminator and nuclear localization sequence
Using the cas6 gene of yeast base optimization and pMD-18T plasmid as template, with what is optimized according to pMD-18T and base
The cas6-yF/yR and pMD-18T-cas6-yF/yR (table 2) that cas6 gene order designs and synthesizes specificity are primer, with same
The conventional PCR method of embodiment 1 (1-A) expands the pMD-18T skeleton and gene that plasmid contains TEF2p and TEF2t respectively
Cas6 sequence.The cas6 of purification and recovery and pMD-18T skeleton is seamless by same 1 (1-A) method of embodiment progress one-step cloning
Connection.Connection product is converted into EC DH5 α competence, and the monoclonal plasmid cultivated is detected through PCR and sequencing analysis, just
True bacterium colony passes through overnight incubation in the LB culture medium containing Amp again, and extracting and purifying must succeed the pMD-18T-cas6 plasmid constructed.With
PMD-18T-cas6 is template, and pRS415-cas6-F/R is the conventional PCR method that primer (table 2) uses embodiment 1 (1-A) again
It expands, DNA fragmentation TEF2p-cas6-TEF2t is obtained after product purification.Except what is optimized according to CDC19p, CDC19t and yeast base
The corresponding primer cas3-yF/yR and pMD-18T-cas3-yF/yR of cas3 gene order, design is outer (table 2), DNA fragmentation
The same TEF2p-cas6-TEF2t of the preparation step of CDC19p-cas3-CDC19t.With TEF2p-cas6-TEF2t after purification and
CDC19p-cas3-CDC19t is template, and pRS415-cas6-F/pRS415-cas3-R is primer (table 2), passes through same embodiment 1
(20 μ L amplification systems include: 4 μ L 5 × CE II buffer, 1 μ L for the overlap PCR method amplification connection of (1-C)
TEF2p-cas6-TEF2t, 1 μ L CDC19p-cas3-CDC19t, 2 μ L Exnase II, the 12 sterile ddH of μ L2O it) obtains and contains respectively
There is the DNA fragmentation cas6-3 of promoter, terminator and nuclear localization signal.
(b) preparation of pRS415-cas6-3
Using pRS415 as template, pRS415-cas6-3-F/R is primer (table 2), with the Standard PCR of same embodiment 1 (1-A)
Method expands plasmid pRS415 skeleton.One step of cas6-3 segment and pRS415 plasmid backbone embodiment 1 (1-C) after purification
After cloning process connection, convert into EC DH5 α competence.Plasmid is extracted after Colony Culture, it is correct through PCR and sequence verification
Afterwards, plasmid pRS415-cas6-3 is obtained after extraction purification.
(B) building of pRS415-cas6-3-t/g- Δ crtE::egfp
Except according to crtE target sequence in SC LYC4 and pRS415-cas6-3 sequence design and synthesizing g- Δ crtE and pass through
Primer g-crtE-F/R expands spare (table 2).It is to draw by template Δ crtE-UF/R, Δ crtE-DF/R of SC LYC4 genome
Object distinguishes PCR amplification and goes out UHA and DHA, then amplifies egfp segment (UHA-egfp-F/DHA- by template of pEGFP-N1 plasmid
Resulting g- Δ crtE, UHA, egfp, DHA is linked together by way of overlap PCR and is obtained by egfp-R) (table 2)
To t- Δ crtE::egfp segment.By plasmid pRS415-cas6-3 and the t- Δ after the linearisation of pRS415-g-F/R (table 2) primer
CrtE::egfp segment links together to obtain plasmid pRS415-csa6-3-t/g- Δ crtE::egfp by one-step cloning method
(Fig. 1).
Primer used in the present embodiment and the g-crtE and t- Δ crtE::egfp sequence of design synthesis are as shown in table 2.
Dotted line represents complementary series in table；Single underscore represents promoter；Double underline is metabolized terminator；Tilted letter represents
spacer；Bold-type letter represents repeat.
2 primer of table and editor's element sequences table
(2) building and verifying of gene editing recon SC LYC4- Δ crt::egfp
(A) preparation of SC LYC4 competence
SC LYC4 monoclonal is picked from the plate 30 DEG C in 50mL YPD fluid nutrient medium with sterilized toothpick
200rpm is cultivated to OD6001.3-1.5,8000rpm is centrifuged 5min after placing 10 ± 2min on ice, and 25mL is added in sediment fraction
ddH2O, 5000 ± 1000rpm 5min centrifuge washing, precipitating add 25mL sorbierite (1M) 5000 ± 1000rpm 5min from
Heart washing, the precipitating after washing are first added the suspension of 2mL LiAc/DTT/TE buffer, the 25mL buffer are added, with sealing
Film seals, and 30 DEG C of 35 ± 5min of culture, thalline were collected by centrifugation by rear 5000 ± 1000rpm 5min；Thallus 1mL ddH2O suspends
And it is forwarded in 1.5mL EP pipe, 5000 ± 1000rpm 5min；Precipitating is suspended with 1mL ice sorbierite, 5000 ± 1000rpm
5min centrifuge washing, the cell after washing are added 100-200 μ L sorbierite and suspend and dispense up to competent cell, are placed in -80
Degree saves stand-by (whole operation carries out under the conditions of 4 DEG C).
(B) conversion of pRS415-cas6-3-t/g- Δ crtE::egfp
The pRS415-cas6-3-t/g- Δ crtE::egfp of 10 ± 5 μ L is added in 100 μ L competent yeasts, is transferred to electricity
In revolving cup (2mm), in standing 5min on ice after mixing, 900 μ L ice sorbierites are added immediately after electric shock (1.5kV, 5S), after mixing
50-100 μ L coated plate (Selective agar medium SD-LEU) is taken, in 30 DEG C of insulating box culture 2-4d, obtains candidate transformant SC LYC4- Δ
Crt::egfp.
(C) daughter chromosome PCR and gene sequencing analysis are recombinated
Picking white monoclonal candidate transformant 30 DEG C of 200rpm in YPD fluid nutrient medium cultivate 36 ± 12h, to mention
The genomic DNA taken is template, and carries out PCR amplification with crtE gene verifying primer crtE-vF/vR, and product is through 1% agarose
As a result (Fig. 2) consistent with the t- Δ crtE::egfp sequence of design electrophoresis and sequence verification show gene editing success.
The knockout of ldhA gene in embodiment 3, CG B253
(1) building of gene editing tool pEC-XK99E-cas6-3-t/g- Δ ldhA
(A) building of pEC-XK99E-cas6-3
Except according to carrier pEC-XK99E sequence and gene cas6 and cas3 sequence information, design is had and carrier pEC-
The primer pEC-XK99E-cas6-F/R and pEC- of XK99E is complementary, can be used for overlap PCR gene cas6 and cas3
Outside XK99E-cas3-F/R (table 3) and linearized vector primer pEC-XK99E-F/R, remaining step is the same as 1 step (1- of embodiment
A)。
(B) building of pEC-XK99E-cas6-3-t/g- Δ ldhA
Except available according to ldh in CG B253 genome and carrier pEC-XK99E-cas6-3 sequence information design and synthesis
G- Δ ldhA (g-DNA-F/g-DNA-R), t- Δ ldhA (Δ ldhA-UF/ Δ ldhA-UR, Δ ldhA- in overlap PCR
DF/ Δ ldhA-DR) and linearized vector (pEC-XK99E-F1/R1) primer is outside (table 3), remaining step is the same as 1 step of embodiment
(1-B)。
Primer used in the present embodiment and the g-ldhA and t- Δ ldhA sequence of design synthesis are as shown in table 3.It is empty in table
Line represents complementary series；Single underscore represents promoter；Double underline represents terminator；Tilted letter represents spacer；Runic
Letter represents repeat.
3 primer of table and editor's element sequences table
(2) building and verifying of gene editing transformant CG B253- Δ-ldh
(A) preparation of CG B253 competence
The CG B253 monoclonal aseptically picked from the plate with sterilized toothpick is in 30 ± 20mL LBG
In seed culture medium, 30 ± 3 DEG C, 200 ± 100rpm is incubated overnight.300 ± 100 μ l of the bacterium solution being incubated overnight is taken to be forwarded to new
In 30 ± 20mL LBG seed culture medium, 30 ± 3 DEG C, 200 ± 100rpm cultivates about 4 ± 1h, until bacterium solution OD600Value about 0.6~
0.9 or so.Take the above-mentioned bacterium solution of 30mL into 50mL centrifuge tube, ice bath 20min, 4000rpm is centrifuged 10min at 4 DEG C, eliminates supernatant
After liquid, 10% glycerol being pre-chilled on ice is taken to blow and beat bacterial sediment uniformly, 4000rpm is centrifuged 10min, weight at 4 DEG C
It washes twice again, 10% glycerol being pre-chilled on ice with 200 μ L is uniformly thin up to CG B253 competence by bacterial sediment piping and druming
Born of the same parents save backup (this process whole process carries out on ice) at -80 DEG C.
(B) conversion of pEC-XK99E-cas6-3-t/g- Δ ldhA
5 μ L plasmid pEC-XK99E-cas6-3-t/g- Δ ldhA are added in 50 μ L CG B253 competence, pipe is flicked
Wall makes its mixing, is transferred in the electric revolving cup of precooled 2mm, and electricity turns (condition: 1.8kv, 5ms) after 10 ± 5min of ice bath, then
It being transferred in the test tube added with 800 ± 100 μ L LBHIS fluid nutrient mediums, 46 DEG C of water-baths 6min, 30 DEG C of 100rpm cultivate 1-2h,
Supernatant is abandoned under 4000rpm centrifugation 3min aseptic condition, and the fresh culture medium of 100 μ L is added and blows even, is uniformly coated in containing chloramphenicol
LBHIS plate on, 30 DEG C of 36 ± 12h of training obtain candidate transformant CG B253- Δ -/dh.
(C) recon clone PCR and gene sequencing analysis
The candidate CG B253- Δ ldhA monoclonal genome for extracting random picking is template, then is drawn with the verifying of ldhA gene
Object ldhA-vF/vR carries out PCR amplification, and product is as a result consistent with the Δ ldhA sequence of design through 1% agarose electrophoresis and sequencing,
Show gene editing success.
The insertion of egfp gene in embodiment 4, HEK293T cellular genome
(1) building of gene editing tool pAIO-cas6-3-t/g- Δ drosha::egfp
(A) building of pAIO-cas6-3
(a) it is respectively provided with the preparation of the cas6-3 genetic fragment of promoter, terminator and nuclear localization sequence
Using the cas6 gene of mammalian cell base optimization and pMD-18T plasmid as template, with excellent according to pMD-18T and base
The cas6-yF/yR and pMD-1gT-cas6-yF/yR (table 4) that the cas6 gene order of change designs and synthesizes specificity are primer,
The pMD-18T skeleton and base that plasmid contains TEF2p and TEF2t are expanded respectively with the conventional PCR method of same embodiment 1 (1-A)
Because of cas6 sequence.By the cas6 of purification and recovery and pMD-18T skeleton by same 1 (1-A) method of embodiment carry out one-step cloning without
Seam connection.Connection product is converted into EC DH5 α competence, and the monoclonal plasmid cultivated is detected through PCR and sequencing analysis,
Correct bacterium colony passes through overnight incubation in the LB culture medium containing Amp again, and extracting and purifying must succeed the pMD-18T-cas6 plasmid constructed.With
PMD-18T-cas6 is template, and pAIO-cas6-F/R is that primer (table 4) is expanded with the conventional PCR method of embodiment 1 (1-A) again
Increase, DNA fragmentation TEF2p-cas6-TEF2t is obtained after product purification；In addition to primer (table 4), DNA fragmentation CDC19p-cas3-
The preparation of CDC19t is identical as the preparation method of TEF2p-cas6-TEF2t.With TEF2p-cas6-TEF2t after purification and
CDC19p-cas3-CDC19t segment is template, and pAIO-cas6-F/pAIO-cas3-R is primer (table 4), passes through same embodiment 1
(20 μ L amplification systems include: 4 μ L 5 × CE II buffer, 1 μ L for the overlap PCR method amplification connection of (1-C)
TEF2p-cas6-TEF2t, 1 μ L CDC19p-cas3-CDC19t, 2 μ L Exnase II, the 12 sterile ddH of μ L2O) contained
Respective promoter terminator and the cas6-3 segment for calculating positioning signal.
(b) building of pAIO-cas6-3
Using pAIO as template, pAIO-F/R is primer (table 4), expands matter with the conventional PCR method of same embodiment 1 (1-A)
Grain pAIO skeleton.After the cas6-3 segment of purifying is connect with pAIO plasmid backbone with the one-step cloning method of embodiment 1 (1-C),
Conversion is into EC DH5 α competence.Plasmid is extracted after Colony Culture, after PCR and sequence verification are correct, purifies to obtain plasmid
pAIO-cas6-3。
(B) building of pAIO-cas6-3-t/g- Δ drosha::egfp
It can be used for overlap according to drosha and pAIO-cas6-3 sequence information design and synthesis in HEK293T genome
PCR g- Δ drosha (g-drosha-F/R) and t- Δ drosha::egfp (Δ drosha-UF/R, Δ drosha-DF/R,
UHA-egfp-F/DHA-egfp-R) primer (table 4) connects to obtain t/g- Δ drosha::egfp by overlap PCR mode
Segment.Linearization plasmid pAIO-cas6-3 and t/g- Δ drosha::egfp piece are digested respectively with restriction enzyme BamH I
Duan Hou connects to obtain pAIO-cas6-3-t/g- Δ drosha::egfp plasmid by T4 ligase overnight.
Primer used in the present embodiment and the g- Δ drosha and t- Δ drosha sequence of design synthesis are as shown in table 4.
Dotted line represents complementary series in table；Single underscore represents promoter；Double underline is metabolized terminator: tilted letter represents
spacer；Bold-type letter represents repeat.
4 primer of table and editor's element sequences table
(2) building and verifying of gene editing mutant strain HEK293T- Δ drosha::egfp
(A) 293T bioblast transfects
4 × 10 are accessed in every hole of six hole Tissue Culture Dish5A good 293T cell of anabiotic state, culture dish is put
In 37 DEG C of 5%CO2Under the conditions of cultivate about 8-14 hours (convergence degree reaches 75-85%), 500 μ L Lip2000-Vector mixing
(contain 250 μ L Lip2000-DMEM mixtures: 10 μ L Lip2000+240 μ L DMEM and 250 μ L Vector-DMEM are mixed for object
Close object: 4 μ g AIO-mCherry-hCas3-t/g- Δ drosha::egfp+240 μ L DMEM) warm bath about 20 minutes at 37 DEG C
After be gradually added into each hole.In 37 DEG C of CO2About 8 hours in incubator, consumed in the fresh SCCM of same volume replacement ware
SCCM.The same terms are further cultivated about 3 days, replace the SCCM consumed in each hole with fresh SCCM once a day.Finally,
Every Kong Zhongyue 2mL cell liquid transfected is completely transferred in 25mL culture bottle, and supplements 3mL SCCM, is trained in the same terms
Support about 2-4 days (cell numbers about 2.0 × 107) after, by latent gene editor cell (HEK293T- Δ drosha::egfp) gene
Group carries out analysis verifying
(B) edited result is verified
Latent gene editor mutant strain HEK293T- Δ drosha::egfp genome is extracted using drosha-vF/R as primer
It carries out PCR verifying (table 4), product is as a result consistent with the tDNA sequence of design through 1% agarose electrophoresis and sequencing, shows gene
It edits successfully.
It is described above that technology contents of the invention are only further illustrated with embodiment, in order to which reader is easier to understand,
But embodiments of the present invention are not represented and are only limitted to this, any technology done according to the present invention extends or recreation, is sent out by this
Bright protection.
Claims (8)
1. a kind of gene editing method based on SviCas6-SviCas3, it is characterised in that: Virginia streptomycete IBL14 base
Protein s viCas6 and SviCas3 (amino acid because containing an I-B-Svi type CRISPR-Cas system in group, in the system
Sequence is seen attached list) combine template t-DNA (template DNA) and targeting g-DNA (guide-DNA) can be to biological cell gene
Gene in group is effectively edited.
2. a kind of gene editing method based on SviCas6-SviCas3 according to claim 1, which is characterized in that packet
It includes the building of edit tool and the building of recon and checking procedure is as follows:
(1) building of carrier is edited
According to SviCas6 and SviCas3 amino acid sequence, the DNA sequence dna of the base optimization of corresponding biological cell is synthesized, and is combined
T-DNA, g-DNA and expression vector dna primers, through primer synthesis, PCR amplification, restriction enzyme cutting and ligase
Connection connects protein coding gene, t-DNA, g-DNA and expression vector to obtain Protein S viCas6-SviCas3 expression respectively
Gene editing tool Vector-cas6-3-t/g-DNA.
(2) building and inspection of recon
Biological cell competence is prepared, the edit tool Vector-cas6-3-t/g-DNA obtained by step (1) is passed through into conversion
Or the method for transfection imports in biological cell, selection culture obtains potential gene editing recon；Potential recon is carried out
Function Identification, target sequence PCR amplification and sequencing analysis obtain correct gene editing recon.
3. a kind of gene editing method based on SviCas6-SviCas3 according to claim 1 or 2, it is characterised in that:
The SviCas6 and SviCas3 refers to the SviCas6 and SviCas3 after original or Amino acid sequence mutants.
4. a kind of gene editing method based on SviCas6-SviCas3 according to claim 1 or 2, it is characterised in that:
The t-DNA refer to synthesis by required genetic function design have the DNA fragmentation complementary with target sequence both ends, can be with editor
Tool is separated and is individually present.
5. a kind of gene editing method based on SviCas6-SviCas3 according to claim 1 or 2, it is characterised in that:
The g-DNA refer to synthesis it is transcribed obtain crRNA/CRISPR-RNA DNA fragmentation (by transcripting promoter/promoter,
Repetitive sequence/repeat, intervening sequence/spacer and transcription terminator/terminator composition).
6. a kind of gene editing method based on SviCas6-SviCas3 according to claim 1 or 2, it is characterised in that:
The DNA sequence dna of the base optimization of the corresponding biological cell refers to according to SviCas6 and SviCas3 amino acid sequence and optimizes
The cDNA sequence for being suitble to the base of the protein expression in different biological cells to optimize.
7. a kind of gene editing method based on SviCas6-SviCas3 according to claim 1 or 2, it is characterised in that:
The gene editing recon refers to because the DNA in genome is by the knockout of specificity, insertion, seamless point mutation and any combination
Editor after cause host cell the changed inheritance stability of phenotype, function and physiology recon.
8. a kind of gene editing method based on SviCas6-SviCas3 according to claim 1 or 2, it is characterised in that:
The biological cell refers to any one of prokaryote and eukaryotic cells, such as: Escherichia coli, glutamic acid rod
Bacterium, saccharomyces cerevisiae, human embryonic kidney cells etc..
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