CN107630041A - A kind of eukaryotic gene edit methods based on Virginia streptomycete IBL14 I Type B Cas systems - Google Patents

A kind of eukaryotic gene edit methods based on Virginia streptomycete IBL14 I Type B Cas systems Download PDF

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CN107630041A
CN107630041A CN201710847164.1A CN201710847164A CN107630041A CN 107630041 A CN107630041 A CN 107630041A CN 201710847164 A CN201710847164 A CN 201710847164A CN 107630041 A CN107630041 A CN 107630041A
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gene
plasmid
dna
eukaryotic
gene editing
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童望宇
王安静
孙焰
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Anhui University
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Anhui University
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Abstract

The invention discloses a kind of eukaryotic gene edit methods based on Virginia streptomycete IBL14 I Type B Cas systems, gene editing of the 1 class I types CRISPR Cas systems by 3 Cas albumen to eukaryotic gene group is realized first；Eukaryotic gene editor to be carried out based on 2 class II types and V-type CRISPR Cas systems provides new supplement and selection.Using the system to eucaryote saccharomyces cerevisiae genome can easily and fast, effectively carry out gene editing.The gene editing system after optimization is expected to be applied in other Eukaryotic gene editings.

Description

A kind of eukaryotic gene based on Virginia streptomycete IBL14 I-B type Cas systems is compiled The method of collecting
The present invention relates to the technique for gene engineering of biological technical field, is exactly that one kind is based on Virginia streptomycete The eukaryotic gene edit methods of IBL14 I-B type Cas systems.
Background technology
Effector of the 2 class CRISPR-Cas systems only containing a Cas is (such as：Cas9, cpf1) successful modification has been into the 3rd For artificial endonucleases, with zinc finger endonuclease (zinc finger endonuclease, ZEN) and class transcriptional activation because Sub- effector nuclease (transcription activator-like effector nuclease, TALEN) equally, can be used Edited in the complexity of various genes, such as：Rite-directed mutagenesis, gene insertion, gene knockout etc..Because its efficiency high, it is simple to operate and into The features such as this is low, 2 class II type CRISPR-Cas9 systems are widely used in eucaryon and the gene editing of prokaryotes by people In.
Lycopene, a Carotenoids, it is a kind of ethylenic unsaturation hydrocarbon compound, is deposited as a kind of natural pigment, extensively It is distributed in the fruit such as tomato, tomato product and watermelon, is the primary pigments in mature tomato.In human body, lycopene master It is distributed in serum and tissue, is concentration highest carotenoid in human serum.Lycopene biological action is main There is anti-oxidant, reduction risk of cardiovascular diseases, reduce genetic damage and suppress tumor development etc. (Bartley＆ Scolnik, 1995；Tracewell et al., 2001；Fraser＆Bramley, 2004).In saccharomyces cerevisiae with lycopene Synthesizing directly related key gene has crtE, crtI and crtB gene.Wherein, Mang ox base pyrophosphate synthetase (GGPS) By crtE gene codes, phytoene synthetase (PSY) is encoded by crtB, and phytoene dehydrogenase (PDB) and ζ- Carotene dehydrogenase (ZDS) is encoded by crtI.
So far, the effector for containing multiple Cas on 1 class CRISPR-Cas systems is used for prokaryotic gene editor Though report (Tong Wangyu, Xu Xin, Zhang Yan, Sun Yan, Cao Suli, a kind of Virginia streptomycete IBL14type I-B- by us Sv14 type CAS gene editing systems, application number：CN201611113137.3；Tong Wangyu, Qiu Caihua, Yang Xingwang, Wang Anjing, one Gene editing method of the kind based on Virginia streptomycete IBL14 genes cas7-5-3, application number：CN201611089333.1), But have no report applied to eukaryotic gene editor.
The present invention is by first public one with I-B type CRISPR-Cas systems 3 in the streptomycete IBL14 chromosomes of Virginia Designed based on gene cas7-5-3 by Cas7-5-3 expression vectors (plasmid-cas7-5-3) and gene editing carrier The gene editing system of (plasmid-t/g-gene abbreviation) composition.Due to eucaryote have nuclear membrane and only with The feature that monocistron is translated, therefore 3 cas genes in the present invention after base optimization are respectively provided with transcripting promoter, end Only son and nuclear localization sequence.This gene editing instrument realizes based on 3 Cas albumen pair in I type CRISPR-Cas systems first The gene editing of eukaryotic gene group.The successful application of the gene editing instrument is the gene based on Cas9 in eucaryote Editor provides a new selection approach, while digs novel gene edit tool for further hair and provide reference.
The content of the invention
The technical problem to be solved in the present invention is to provide one kind to be similar to 2 class CRISPR-Cas systems (such as：Cas9, Cpf1) The eukaryotic gene edit tool based on Virginia streptomycete IBL14 I-B type Cas systems.To be solved up to this purpose, the present invention Certainly technical scheme is used by its problem：
A kind of eukaryotic gene edit methods based on Virginia streptomycete IBL14 I-B type Cas systems, its feature exist In：Using in the streptomycete IBL14 genomes of Virginia containing 3 gene cas7-5-3 constructed by protein expressing plasmid and base Because editor's plasmid pair eukaryotic gene group carries out gene editing.
A kind of described eukaryotic gene edit methods based on Virginia streptomycete IBL14 I-B type Cas systems include The acquisition and inspection of the structure of protein expressing plasmid, the structure and recon of gene editing plasmid, are comprised the following steps that：
(1) structure of protein expressing plasmid
According to gene cas7-5-3 sequences in the streptomycete IBL14 of Virginia, the gene and phase of its codon optimization are synthesized Primer is closed, by one-step cloning by the cas7-5-3 genes directed cloning after optimization to plasmid plasmid, obtains Cas7-5-3 eggs White expression plasmid plasmid-cas7-5-3；
(2) structure of gene editing plasmid
According to eucaryote target gene DNA sequence dna information design primer, using eukaryotic gene group as masterplate, pass through Amplification obtains targeting of the end simultaneous with restriction enzyme restriction enzyme site and overlap PCR complementary series respectively for PCR reactions The upstream and downstream homology arm fragment of gene, and upstream and downstream homology arm is connected to by gene editing masterplate t- by overlap PCR On DNA, while designed according to eucaryote target gene sequence information and directly synthesize head and the tail and include galactose promoter respectively With the target gene fragment g-DNA of RNA terminators, according to the cohesive end on restriction enzyme site by gene editing masterplate t- DNA and target gene fragment g-DNA is connected on plasmid, obtains gene editing plasmid plasmid-t/g-DNA；
(3) acquisition and inspection of recon
Eukaryotic cells competence is prepared, the protein expressing plasmid plasmid-cas7-5-3 that will be obtained by step (1) The gene editing plasmid plasmid-t/g-DNA obtained with step (2) is transformed into target bacterium competence respectively, is obtained different Recon；PCR and gene sequencing are carried out to different restructuring daughter chromosomes, or carries out functional analysis, is obtained with screening correct Purpose recon.
Described eucaryote refers to the eukaryotic of saccharomyces cerevisiae microorganism or animal and plant.
Described gene editing refer to the chromogene of eukaryotic is inserted, knocked out, seamless point mutation and any Combination.
The present invention is a kind of eukaryotic gene edit methods based on Virginia streptomycete IBL14 I-B type Cas systems, first The secondary gene editing for realizing 1 class I type CRISPR-Cas systems by 3 Cas albumen to eukaryotic gene group；For based on 2 classes The eukaryotic gene editor that II types are carried out with V-type CRISPR-Cas systems provides new supplement and selection.Using the system To eucaryote saccharomyces cerevisiae genome can easily and fast, effectively carry out gene editing.The gene editing system after optimization It is expected to be applied in other Eukaryotic gene editings.
Brief description of the drawings
Fig. 1 gene editing tools build schematic diagrames.(A) protein expressing plasmid pRS415-cas7-5-3：Ori/origin： DNA replication dna initiation site, f1ori：The replication initiation position of f1 bacteriophages origin, CEN/ARS：Centriole/autonomous replication, gal1promoter：Galactose promoter, combined with RNA polymerase and start DNA transcriptions, LEU2promoter：Leucine starts Son, AmpR/ampicillin resistance：Amicillin resistance；(B) gene editing plasmid pYES2/NTA-t/g- Δs crtE：Ori/origin:DNA replication dna starting point, f1ori：Phage replication initiation site, produce single stranded DNA, 2 μ ori：Replicate Starting point, URA3promoter：Uracil promoter, T7promoter:T7Promoter, starting DNA transcriptions, AmpR/ ampicillin resistance:Amicillin resistance.
The knockout result of Fig. 2 SCBY4741 genome crtE genes.(A) (red (being black in figure) be for erythroleukoplakia screening Original strain, white is recombinant bacterial strain)；(B) PCR electrophoresis (swimming lane M：5000bp DNA ladder, swimming lane 1：Blank control, swimming Road 2：Plasmid pRS415-cas7-5-3 and pYES2/NTA-t/g- Δ crtE transformants, swimming lane 3：PRS415-cas7-5-3 and PYES2/NTA-t- Δ crtE transformants, swimming lane 4：Plasmid pRS415-cas7-5-3 and t- Δ crtE transformants, swimming lane 5：Plasmid PRS415-cas7-5-3 and pYES2/NTA-g- Δ crtE and t- Δ crtE transformants, swimming lane 6：Host SCBY4741).
Embodiment
In order to more fully understand the technology contents of the present invention, technical scheme is made with reference to specific embodiment It is further described and illustrates, it is intended to preferably explains present disclosure, following examples do not limit the scope of the invention. In addition, use following material unless otherwise instructed in listed embodiment：
1) bacterial strain and plasmid
Escherichia coli Escherichia coli DH5 α/EC DH5 α, import the saccharomyces cerevisiae of lycopene Saccharomyces cerevisiae BY4741/SC BY4741, plasmid pRS415, pYES2/NTA, pMD18T.
2) culture medium
LB culture mediums
Dusty yeast 5g, peptone 10g, NaCl 10g, after adding the dissolving of appropriate running water, it is settled to 1L, pH is adjusted to 7.0~ 7.2, after packing wrapping, 121 DEG C/20min sterilizings, flat board such as processed adds 20g agar.
YNB culture mediums
Yeast extract 10g, peptone 20g, glucose 20g, after adding appropriate running water dissolving, it is settled to 1L, packing wrapping Afterwards, 115 DEG C/30min sterilizes, and flat board such as processed adds 20g agar.
SD-LEU Selective agar mediums (100mL)
0.17g YNB, 0.5g ammonium sulfate, 2g glucose, after adding appropriate distilled water dissolving, 99.6ml is settled to, is dispensed After wrapping, 115 DEG C/30min sterilizings.Add 400 μ l amino acid (methionine 0.5g/100ml, histidine 0.5g/ of filtration sterilization 100ml, uracil 0.5g/100ml), flat board such as processed adds 2g agar and 18.2g sorbierites.
SD-LEU/URA Selective agar mediums (100mL)
0.17g YNB, 0.5g ammonium sulfate, 2g glucose, after adding appropriate distilled water dissolving, 99.6ml is settled to, is dispensed After wrapping, 115 DEG C/30min sterilizings.Add 400 μ l amino acid (methionine 0.5g/100ml, histidine 0.5g/ of filtration sterilization 100ml), flat board such as processed adds 2g agar and 18.2g sorbierites.
SD-LEU inducing cultures (100ml)
0.17g YNB, 0.5g ammonium sulfate, 2g galactolipins, 1g raffinoses, after adding appropriate distilled water dissolving, it is settled to 99.6ml, after packing wrapping, 115 DEG C/30min sterilizings.Add filtration sterilization 400 μ l amino acid (methionine 0.5g/100ml, Histidine 0.5g/100ml, uracil 0.5g/100ml).
3) material
LiAc/DTT/TE buffer solutions (400ml)
4.088g 0.1M LiAc ﹒ H2O, 0.484g 10mM Tris (pH7.5), 0.117g 1mM EDTA, add 300ml distilled waters are dissolved, and pH to 7.5 is adjusted with HCl, are settled to 400ml, 115 DEG C/30min sterilizings；0.617g DTT filtration sterilizations Add afterwards in buffer solution.
1M sorbierites
18.217g sorbierites, add distilled water to 100ml, 115 DEG C/30min sterilizing.
50mg/ml ampicillins
The ampicillin for weighing 0.5g is placed in 10ml centrifuge tubes, is added 4ml distilled waters, after mixing, is settled to 10ml, After 0.2 μm of sterile water system membrane filtration, it is dispensed into 1.5ml EP pipes, is put in -20 DEG C of refrigerators and preserves.
HEPES buffer solution
Weigh 0.8g NaCl, 0.037g KCl, 0.0135g Na2HPO4﹒ H2O, 0.5g glucose, 0.5g HEPES, add 90ml distilled waters dissolve, and NaOH adjusts pH to 7.5, is settled to 100ml.
50mg/ml G418
Weigh 0.5g G418 to be placed in 10ml centrifuge tubes, add 4ml HEPES buffer solutions, after mixing, be settled to 10ml, After 0.2 μm of sterile water system membrane filtration, it is dispensed into 1.5ml EP pipes, is put in -20 DEG C of refrigerators and preserves.
Agents useful for same is commercially available product.
Embodiment 1pRS415-cas7-5-3 knocks out SC BY4741crtE with the conversion of the steps of pYES2/NTA-t/g- Δs crtE bis- Gene
(1) protein expressing plasmid pRS415-cas7-5-3 structure
(A) protein expressing plasmid pRS415-cas3 structure
According to SC BY4741 genome sequencings information and plasmid pRS415 sequence informations, design carries plasmid pRS415- Cas3 the gene-specific primers cas3-F/cas3-R and pRS415-cas3-F/pRS415-cas3-R of cas9 complementary series.It is logical The cas3 genes of Shanghai Jierui Biology Engineering Co., Ltd's synthesis base optimization are crossed, are given birth to using Quan Shijin Bioisystech Co., Ltd The TransStart FastPfu DNA Polymerase of production carry out cas3 gene PCR amplifications, reaction condition：95±1℃5± 1min, 95 ± 1 DEG C of 30s, 62 ± 3 DEG C of 30s, 72 DEG C of 2 ± 2min (50 μ l reaction systems), 30 circulations, 72 DEG C of 10min.PCR is produced Thing detects through 1% agarose electrophoresis, kit recovery, the cas3 full-length genes purified.Entered by same archaeal dna polymerase Performing PCR expands, the pRS415 plasmid backbones purified.Produced by Sangon Biotech (Shanghai) Co., Ltd. ClonExpress MultiS multiple clips one-step clonings are seamless, and Cloning Kit carries out quick clone, reaction system (10 μ l)： The μ l of 26 μ l, vector pRS415 of μ l, Exnase MultiS1 μ l, insert cas3 of 5xCE MultiS Buffer 1.Body After the completion of system prepares, gently blown and beaten up and down with pipettor and mix each component several times, avoid producing bubble.It is placed in 37 DEG C of reactions 30min.After the completion of question response, reaction tube is placed in ice-water bath cools down 5min immediately.Reaction product is added to 100 μ l DH5 α In competent cell, flick and mixed under tube wall number, 30min is placed on ice.42 DEG C of heat shock 90s, ice-water bath are incubated 2min, add 900 μ l LB culture mediums, 37 DEG C are incubated 10min and fully recover.37 DEG C are shaken bacterium 45min.Take 100 μ l bacterium solutions be spread evenly across containing Have on Amp flat board, flat board is inverted, in 37 DEG C of overnight incubations.It will be carried out by primer cas3-check-F/cas3-check-R Bacterium colony PCR, by overnight incubation in the remaining colony inoculation of PCR positive bacterium colonies to the LB culture mediums containing Amp, extract plasmid identification Obtain protein expressing plasmid pRS415-cas3.
(B) acquisition of the cas7 full-length genes with promoter, terminator and nuclear localization sequence
According to SC BY4741 genome sequencings information and plasmid pMD-18T-crtI sequence informations, design carries plasmid Cas7 the gene-specific primers cas7-F/cas7-R and pMD-18T-cas7-F/pMD- of pMD-18T-crtI complementary series 18T-cas7-R.The cas7 genes of base optimization are synthesized by Shanghai Jierui Biology Engineering Co., Ltd, use full formula gold biology The TransStart FastPfu DNA Polymerase of Technology Co., Ltd.'s production carry out cas7 gene PCR amplifications, expand bar Part is the same as embodiment 1 (1-A).PCR primer detects through 1% agarose electrophoresis, kit recovery, the cas7 total length bases purified Cause.Enter performing PCR by same archaeal dna polymerase to expand, the pMD-18T plasmid bones containing CDC19p and CDC19t purified Frame.It is seamlessly connected again by one-step cloning, reaction system, with the structure of pRS415-cas3 plasmids, is identified with step of converting PMD-18T-cas7 plasmids；Then TransStart FastPfu DNA Polymerase and primer pRS415- are passed through Cas7-F/pRS415-cas7-R enters performing PCR amplification, and the cas7 containing CDC19p, CDC19t and SV40NLS purified is complete Long gene.
(C) acquisition of the cas5 full-length genes with promoter, terminator and nuclear localization sequence
According to SCBY4741 genome sequencings information and plasmid pMD-18T-crtE sequence informations, design carries plasmid Cas5 the gene-specific primers cas5-F/cas5-R and pMD-18T-cas5-F/pMD- of pMD-18T-crtE complementary series 18T-cas5-R.The acquisition of cas5 full-length genes is consistent with cas7 gene steps, enters performing PCR by same archaeal dna polymerase and expands Increase, amplification condition is with embodiment 1 (1-A), the pMD-18T plasmid backbones containing ENO2p and ENO2t purified.Pass through again One-step cloning is seamlessly connected, and reaction system and step of converting are with the structure of pRS415-cas3 plasmids, the pMD-18T- identified Cas5 plasmids；Then TransStart FastPfu DNA Polymerase and primer pRS415-cas5-F/pRS415- are passed through Cas5-R enters performing PCR amplification, the cas5 full-length genes containing ENO2p, ENO2t and SV40NLS purified.
(D) protein expressing plasmid pRS415-cas7-5-3 structure
Pass through primer pRS415-cas7-5-3-F/pRS415-cas7-5-3-R and TransStart FastPfu DNA Polymerase, template plasmid pRS415-cas3 is entered into performing PCR amplification, the pRS415-cas3 skeletons purified.Pass through one Step clone is seamlessly connected, and reaction system (10 μ l) is 2 ﹡ transgene MIX 5 μ l, pRS415-cas3 3 μ l, ENO2p- The 1 μ l of μ l, CDC19p-cas7-CDC19t of cas5-ENO2t 1.Step of converting is consistent with the structure of pRS415-cas3 plasmids.Sun Property bacterium colony upgrading grain, the pRS415-cas7-5-3 plasmids identified (Fig. 1 (A)).
(2) gene editing plasmid pYES2/NTA-t/g- Δs crtE structure
(A) preparation of upstream and downstream homology arm
It is homologous according to crtE genes complete sequence design crtE upstream region of gene homology arm primers crtE-UF and crtE-UR, downstream Arm primer crtE-DF and crtE-DR (black matrix overstriking is overlap PCR complementary series), and upper homology arm sense primer contains HindIII restriction enzyme digestion sites, lower homology arm anti-sense primer restriction enzyme digestion sites containing BamHI.With pure The crtE gene DNAs of change are template, first expand upstream and downstream homology arm respectively, amplification condition is the same as embodiment 1 (1-A).PCR primer Detected through 1.5% agarose electrophoresis, kit recovery, the upstream and downstream homology arm DNA fragmentation obtained after purification is standby.
(B) gene editing masterplate carrier pYES2/NTA-t- Δs crtE structure
Take homology arm purified product to be mixed with each 0.5 μ l of lower homology arm purified product and be used as template, 25 μ l reaction systems Overlap PCR are carried out, reaction condition is：94 DEG C of 5min, 94 DEG C of l min, 62 DEG C of 30s, 72 DEG C of 1min, are added after a circulation Each 1 μ l of primer crtE-UF and crtE-DR, continue PCR, and reaction condition is：94 DEG C of 5min, 94 DEG C of 30s, 62 DEG C of 30s, 72 DEG C 1min, carry out 30 circulations, 72 DEG C of 10min.1.5% agarose gel electrophoresis detects amplified production and purified, and obtains gene editing Masterplate t- Δs crtE；The t- Δs crtE of acquisition is cut out by HindIII restriction enzymes enzyme, BamHI restriction enzymes Cohesive end, the T4 ligases then produced by Quan Shijin Bioisystech Co., Ltd are connected to pYES2/NTA plasmids On, obtain gene editing masterplate carrier pYES2/NTA-t- Δs crtE.
(C) gene editing plasmid pYES2/NTA-t/g- Δs crtE structure
Target gene fragment containing galactose promoter and g- Δ crtE connection products is direct by the general biotech firm in Chuzhou Synthesis, head and the tail add EcoRI and XhoI restriction enzyme sites respectively, and centre is promoter successively, repetitive sequence (repeat), is spaced sequence Arrange (spacer), repetitive sequence (repeat) and terminator；The target gene fragment of synthesis is restricted by EcoRI and XhoI Restriction endonuclease cuts out cohesive end, is then connected to obtain gene editing masterplate carrier pYES2/NTA-t- Δs by T4 ligases CrtE it is upper gene editing plasmid pYES2/NTA-t/g- Δs crtE (Fig. 1 (B)).
The g- Δ crtE sequences (PAM of synthesis:ttc)：
(3) acquisition and inspection of recon
(A) prepared by SCBY4741 competence
In super-clean bench, the monoclonal on the sterilized toothpick picking SCBY4741 flat boards of use is trained in 50ml YPD liquid Support in base, 30 DEG C, 200rpm is incubated overnight；It will first be cultivated in 50ml YPD to OD600For 1.3-1.5 saccharomycete, ice is placed Upper 10min, ddH2O and sorbierite shift to an earlier date precooling (ice is put)；5000rpm centrifuges 5min, abandons supernatant；Add ice 25ml ddH2O is washed Wash once, 5000rpm centrifugation 5min, abandon supernatant；Add 25ml ice sorbierite (1M) washed once, 5000rpm centrifugation 5min, abandon Supernatant；2ml LiAc/DTT/TE buffer solutions are first added, suspends, 23ml buffer solutions is added, with ParafilmTM, 30 degree of shaking tables 30min, the thalline of 5000rpm collections afterwards；Add 1ml ddH2O suspends, then is forwarded in 1.5ml EP pipes, 4000rpm centrifugations 3min, abandon supernatant；1ml ice sorbierites are added, washing, 4000rpm centrifugation 3min, supernatant is abandoned and (uses the small-sized centrifugation with temperature Machine)；Add 100-200 μ l sorbierites and obtain SCBY4741 competent cells, convert or be placed in -80 DEG C of preservations for lower step.
(B) plasmid pRS415-cas7-5-3 step conversion
5 μ l plasmid pRS415-cas7-5-3 are added into 100 μ lSCBY4741 competence, are transferred in electric revolving cup, on ice Stand 5min；It is 1.5kV, 5ms that electricity, which turns condition, and electricity has turned to add the 900 μ l sorbierites for shifting to an earlier date precooling immediately；100 μ l are taken to be coated in On the selection flat board of SD-LEU defects, 30 DEG C are cultivated 3 days.
(C) daughter chromosome PCR and gene sequencing analysis are recombinated
Picking red monoclonal verifies primer cas3-check-F/cas3- as template, then with cas7-5-3 genes Check-R carries out pcr amplification reaction, and amplification condition is the same as embodiment 1 (1-A).PCR primer detects through 1% agarose electrophoresis, observation Recon cas3 peripheries amplified band is consistent with expected results, it was demonstrated that pRS415-cas7-5-3 plasmids are successfully transferred to yeast In.
(D) it is prepared by the SC BY4741 competence for successfully importing pRS415-cas7-5-3 plasmids
Except the above-mentioned BY4741 for being successfully transferred to pRS415-cas7-5-3 plasmids of picking monoclonal, lured in 50ml SC-LEU Culture medium China and foreign countries are led, competence preparation method is the same as embodiment 1 (3-A).
(E) the two steps conversion of pYES2/NTA-t/g- Δs crtE plasmids
5 μ l plasmid pYES2/NTA-t/g- Δs are added into the freshly prepd 100 μ l Saccharomyces cerevisiae competents of above-mentioned steps (D) CrtE, it is transferred in electric revolving cup, in standing 5min on ice；It is 1.5kV, 5ms that electricity, which turns condition, and electricity, which has turned to add immediately, shifts to an earlier date precooling 900 μ l sorbierites；100 μ l are taken to be coated on the selection flat board of SD-LEU/URA defects, 30 DEG C are cultivated 3 days.
(F) daughter chromosome PCR and gene sequencing analysis are recombinated
Picking white monoclonal enters performing PCR amplification instead as template, then with crtE genes checking primer crtE-F/crtE-R Should, amplification condition is the same as embodiment 1 (1-A).PCR primer detects through 1% agarose electrophoresis, observes recon chromosome DNA amplification Band reduces, and proof crtE gene knockout successes are sequenced through general biosystem (Anhui) Co., Ltd, as a result sees Fig. 2.
Primer and its sequence involved by each step are shown in Table 1, and capitalization is restriction enzyme site in table, and black matrix overstriking is complementation Area.
The primer of table 1 and its sequence
Embodiment 2pRS415-cas7-5-3 and pYES2/NTA-t- Δ crtE two-step methods knock out SC BY4741crtE Gene
(1) protein expressing plasmid pRS415-cas7-5-3 structure
With embodiment 1 (1)
(2) gene editing masterplate carrier pYES2/NTA-t- Δs crtE structure
(A) preparation of upstream and downstream homology arm
With embodiment 1 (2-A)
(B) gene editing masterplate carrier pYES2/NTA-t- Δs crtE structure
With embodiment 1 (2-B)
(3) acquisition and inspection of recon
(A) prepared by SCBY4741 competence
With embodiment 1 (3-A)
(B) plasmid pRS415-cas7-5-3 step conversion
With embodiment 1 (3-B)
(C) daughter chromosome PCR and gene sequencing analysis are recombinated
With embodiment 1 (3-C)
(D) it is prepared by the SC BY4741 competence for successfully importing pRS415-cas7-5-3 plasmids
With embodiment 1 (3-D)
(E) gene editing masterplate carrier pYES2/NTA-t- Δs crtE two steps conversion
5 μ lpYES2/NTA-t- Δ crtE are added into the freshly prepd 100 μ l Saccharomyces cerevisiae competents of above-mentioned steps (D), It is transferred in electric revolving cup, in standing 5min on ice；It is 1.5kV, 5ms that electricity, which turns condition, and electricity has turned to add the 900 μ l for shifting to an earlier date precooling immediately Sorbierite；100 μ l are taken to be coated on the selection flat board of SD-LEU/URA defects, 30 DEG C are cultivated 3 days.
(F) daughter chromosome PCR and gene sequencing analysis are recombinated
With embodiment 1 (3-F), Fig. 2 is as a result seen.
Embodiment 3pRS415-cas7-5-3 knocks out SC BY4741crtE genes with the conversion of the steps of t- Δs crtE bis-
(1) protein expressing plasmid pRS415-cas7-5-3 structure
With embodiment 1 (1).
(2) gene editing masterplate t- Δs crtE structure
(A) preparation of upstream and downstream homology arm
With embodiment 1 (2-A).
(B) gene editing masterplate t- Δs crtE structure
With embodiment 1 (2-B).
(3) acquisition and inspection of recon
(A) prepared by SCBY4741 competence
With embodiment 1 (3-A).
(B) plasmid pRS415-cas7-5-3 step conversion
With embodiment 1 (3-B).
(C) daughter chromosome PCR and gene sequencing analysis are recombinated
With embodiment 1 (3-C).
(D) it is prepared by the SCBY4741 competence for successfully importing pRS415-cas7-5-3 plasmids
With embodiment 1 (3-D).
(E) gene editing masterplate t- Δs crtE two steps conversion
5 μ lt- Δ crtE are added into the freshly prepd 100 μ l Saccharomyces cerevisiae competents of above-mentioned steps (D), are transferred to electric revolving cup In, in standing 5min on ice；It is 1.5kV, 5ms that electricity, which turns condition, and electricity has turned to add the 900 μ l sorbierites for shifting to an earlier date precooling immediately；Take 100 μ l are coated on the selection flat board of SD-LEU defects, and 30 DEG C are cultivated 3 days.
(F) daughter chromosome PCR and gene sequencing analysis are recombinated
With embodiment 1 (3-F), Fig. 2 is as a result seen.
The conversion of the steps of embodiment 4pRS415-cas7-5-3, pYES2/NTA-g- Δ crtE and t- Δ crtE bis- knocks out SC BY4741crtE genes
(1) protein expressing plasmid pRS415-cas7-5-3 structure
With embodiment 1 (1).
(2) t- Δs crtE and pYES2/NTA-g- Δ crtE structure
(A) preparation of upstream and downstream homology arm
With embodiment 1 (2-A).
(B) gene editing masterplate t- Δs crtE structure
With embodiment 1 (2-B).
(C) gene editing oriented carrier pYES2/NTA-g- Δs crtE structure
In addition to being joined directly together and connecing with plasmid pYES2/NTA, remaining step is identical with the method for embodiment 1 (2-C).
(3) acquisition and inspection of recon
(A) prepared by SCBY4741 competence
With embodiment 1 (3-A).
(B) plasmid pRS415-cas7-5-3 step conversion
With embodiment 1 (3-B).
(C) daughter chromosome PCR and gene sequencing analysis are recombinated
With embodiment 1 (3-C).
(D) it is prepared by the SC BY4741 competence for successfully importing pRS415-cas7-5-3 plasmids
With embodiment 1 (3-D).
(E) pYES2/NTA-g- Δs crtE and t- Δ crtE cotransformation
Added into the freshly prepd 100 μ l Saccharomyces cerevisiae competents of step (D) 5 μ l plasmids pYES2/NTA-g- Δs crtE and 5 μ l t- Δ crtE, are transferred in electric revolving cup, in standing 5min on ice；It is 1.5kV, 5ms that electricity, which turns condition, and electricity, which has turned to add immediately, to be carried 900 μ l sorbierites of preceding precooling；100 μ l are taken to be coated on the selection flat board of SD-LEU/URA defects, 30 DEG C are cultivated 3 days.
(F) daughter chromosome PCR and gene sequencing analysis are recombinated
With embodiment 1 (3-F), Fig. 2 is as a result seen.
Embodiment 5pRS415-cas7-5-3 and pYES2/NTA-t/g- Δ crtI::The conversion of the steps of G418 mono- knocks out SC BY4741crtI genes
(1) protein expressing plasmid pRS415-cas7-5-3 structure
With embodiment 1 (1).
(1) gene editing plasmid pYES2/NTA-t/g- Δs crtI::G418 structure
(A) LoxP-G418-LoxP fragments and the preparation of crtI upstream and downstream homology arm
According to SC BY4741 genome sequencing information, design and LoxP-G418-LoxP fragment-specific primers crtI- G418-F/crtI-G418-R and crtI-UF/crtI-UR and crtI-DF/crtI-DR is expanded and is detected purifying LoxP- respectively G418-LoxP fragments and crtI upstream and downstream homology arm, amplification condition is the same as embodiment 1 (2-A).
(B) gene editing masterplate carrier pYES2/NTA-t- Δs crtI::G418 structure
Take homology arm purified product to be mixed with each 0.5 μ l of LoxP-G418-LoxP purified products and be used as template, 25 μ l are anti- System is answered to carry out overlap PCR, reaction condition is：94 DEG C of 5min, 94 DEG C of 30s, 65 DEG C of 30s, 72 DEG C of 3min, after a circulation Primer each 1 μ l of crtI-UF and crtI-G418-R are added, continue PCR, reaction condition is：94 DEG C of 5min, 94 DEG C of 30s, 65 DEG C 30s, 72 DEG C of 3min, carry out 30 circulations, 72 DEG C of 10min, and 1.5% agarose gel electrophoresis detection amplified production simultaneously purifies；Take Obtained purified product crtI-UHA-LoxP-G418-LoxP is mixed with each 0.5 μ l of lower homology arm purified product and is used as template, and 25 μ l reaction systems carry out overlap PCR, and reaction condition is：94 DEG C of 5min, 94 DEG C of 30s, 62 DEG C of 30s, 72 DEG C of 4min, one is followed Primer each 1 μ l of crtI-UF and crtI-DR are added after ring, continue PCR, reaction condition is：94 DEG C of 5min, 94 DEG C of 30s, 59 DEG C 30s, 72 DEG C of 4min, 1.5% agarose gel electrophoresis detection amplified production simultaneously purify to obtain gene editing masterplate t- Δs crtI:: G418；By the t- Δs crtI of acquisition::G418 is cut out viscous by HindIII restriction enzymes enzyme, BamHI restriction enzymes Property end, the T4 ligases then produced by Quan Shijin Bioisystech Co., Ltd are connected on pYES2/NTA plasmids, Obtain gene editing masterplate carrier pYES2/NTA-t- Δs crtI::G418.
(C) gene editing plasmid pYES2/NTA-t/g- Δs crtI::G418 structure
In addition to the target gene fragment of g- Δ crtI connection products is different, the method phase of remaining step and embodiment 1 (2-C) Together, gene editing plasmid pYES2/NTA-t/g- Δs crtI is obtained::G418.
The g- Δ crtI sequences (PAM of synthesis：ttc)
(3) acquisition and inspection of recon
(A) prepared by SCBY4741 competence
With embodiment 1 (3-A)
(B) plasmid pRS415-cas7-5-3 and plasmid pYES2/NTA-t/g- Δs crtI::G418 step conversion
5 μ l plasmids pRS415-cas7-5-3 and 5 μ l are added into the freshly prepd 100 μ l Saccharomyces cerevisiae competents of step (A) Plasmid pYES2/NTA-t/g- Δs crtI::G418, it is transferred in electric revolving cup, in standing 5min on ice；Electricity turn condition for 1.5kV, 5ms, electricity have turned to add the 900 μ l sorbierites for shifting to an earlier date precooling immediately；100 μ l are taken to be coated in containing G418 resistances (the μ g/ of final concentration 600 Ml on YPD flat boards), 30 DEG C are cultivated 3 days.
(C) daughter chromosome PCR and gene sequencing analysis are recombinated
In addition to application crtI genes checking primer crtI-F/crtI-R carries out pcr amplification reaction, remaining step and embodiment 1 (3-F) method is identical.
The primer and its sequence that each step is related to are shown in Table 2, and capitalization is restriction enzyme site in table, and black matrix overstriking is complementation Area.
The primer of table 2 and its sequence
The technology contents described above that the present invention is 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.
Sequence table
<110>University of Anhui
<120>A kind of eukaryotic gene edit methods based on Virginia streptomycete IBL14 I-B type Cas systems
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1020
<212> DNA
<213> S. virginiae IBL14
<400> 1
atggttgccg gtgcaccaaa caatggtgaa ggtgaagata atacaggtag agttaaaaaa 60
ttaagggttg gtagggaaga atttccctac gtttccgcac aagcctttag acgttggttg 120
agggattcct tacctgccca agaacctagg tccgttgtta ctagatccgg ttcaggtgct 180
aaacaacaag ctcatacagc aggtagacct gatttgcatt tggatgatga cctctttggt 240
tatatggttg ctgttaaagg taaaggtggt tcatatcaaa gagatacagt tttagcaact 300
ggtacattag tttccgttgt tcctcaaagg ccaactttgg attttggtac tatgtctagg 360
gattttccag ccggtgaaca tcctgttatt catagtcatg aattgtatag tgccactttg 420
gccggcgatg ttttattgga cctccctagg gccggtgttt ttgaaactga tggtaatggt 480
ttaagagttg caatttcacc agccgttgct gaagaagccg ctaaaaatgg tgccgaagtt 540
actactttga ggggttctgc tgctattaga ttaccattga cagaaagaca tcggcggatt 600
ggtacattat tgaggacttt agcatccgtt aggggtggtg ccaaacaagc cttgcattat 660
ggcgaccgag caccatcctt agttttatta gctccattaa aaggtggtgt taatccgttt 720
actagagttt tgggtgccag agatggtaaa cctgttttct tgtccgatgt tttgagggaa 780
gaattggaag cctgggccga tgaattggat ggtccagttt tattgggttg ggcaccaggg 840
ttcttggggg accaaaggga acaagttaga agggaattaa aagaccttat tgatgaaggt 900
agagttgttt tgtcacatcc tagagtttta ttgactcaat tagcagatag gattgaacaa 960
ggtgaccatg atgcctggtt tgaagatagc gctgctccca agaagaagag gaaggtgtga 1020
<210> 2
<211> 684
<212> DNA
<213> S. virginiae IBL14
<400> 2
atgacaggta ctgaagttac tgccttgcaa attactgtta cagctccagt tgtttctttt 60
agaaatcctt tgtatgcagg tgttcaagtt acattaccat gtccaccacc tgccactgtt 120
ggtggtttat tagctgcggc tgccggtggt tgggaacaag ttaatccaga attaagattt 180
gctatggcct ttcatgcggg aggtaaagcc gttgatttgg aaacttatca tccattggat 240
gcctccggga aaaaagcatc acctgcccct aggaatagag aatttttaac agccgccgaa 300
ttgacagttt ggttagttga tgatcctgaa ggttggcaac ggcgtttacg ccgtccagtt 360
tggccattga gattgggtcg gtcacaagac ttggttggta ttaggactgg tttagttcca 420
ttgagagcag aaccaggtga acaacgatcg gctgttgttc ctgaaaccgc tggtagaatg 480
ggtactttat tgagattgcc tacagccgtt tccggtggta gggataggac tagatgggat 540
tcatatagat ttgattcttc cggtaggtcc gatcatgttg ttgttggtgg ttggagtaca 600
gcaggtggtc aagccgttat tttattacct tccgcacatc ctgatacagt tgctagatct 660
cccaagaaga agaggaaggt gtga 684
<210> 3
<211> 2337
<212> DNA
<213> S. virginiae IBL14
<400> 3
atgggtagat tggatgccgt tgaagatgtt tttggtggta gattttggcc agttgttgaa 60
ttggccggtt tgacacatga tgccggaaag attccagaag gttttcaaag aatgttagcc 120
ggttatagta gagcatgggg tgaaagacat gaagttgctt ccttggggtt cttacctgcc 180
ttaattgggg accctgatgt tttattgtgg gttgctacag ctgttgcaac acatcataga 240
ccattaactg gtcaaaatgg tagggacctc caaactttgt atagtggtgt tactattaca 300
gaattggctc ataggtttgg tccatttgat ccaagggccg ttcctgcctt ggaagcctgg 360
ttaagggctt cagcaattag agttggttta cctgccgccg ccgttcctga tgatggtact 420
ttaactgata caggtgttgt tgctggtgct catcaattat tggaagaaat tttggatagg 480
tgggccgata gagttagacc agaagttggt ttagcagcag ttttattaca aggtgccgtt 540
actttggccg atcatttgtc ttccgcccat caagccttac caacagttca accattgggt 600
gcaggttttc gtagtaggtt agaaaaagaa tttgcagaaa ggggtaggac attaagggca 660
catcaattgg aagccgccac tgttacaggt catttgttat tgagaggtcc tacaggttca 720
ggaaagacag aagcagcctt gttgtgggct gcttcacaag ttgaagcctt aaaagccgaa 780
ggtagaggtg ttcctagagt tttctttaca ttaccatacc ttgcttctat taatgctatg 840
gccactagat tgggagacac tttgggggat ggtgaagctg ttggtgttgc ccattcgcgc 900
gctgcttctt atcatttggc tcaagctatt gccccacaag atggcgacga agaagatgaa 960
catggtgcac catgtagagt tgatgcagct gctaaagcct tgtcacgggc agctgctact 1020
aaactcttta gggaatccgt tagagttgcc actccatatc aattattaag ggccgcctta 1080
gccggtccag cacattccgg tattttaatt gatgcagcta attccgtttt tattttggat 1140
gaattacatg cttacgatgc acggaggttg ggttatattt tagcttccgc cagattgtgg 1200
gaaagattgg gtggtaggat tactgttttg tccgcaactt taccaagggc cttagccgat 1260
ttgtttgaat ctactttaac agcacctatt acgtttttgg atactcctga tttgggttta 1320
ccagccaggc atttgttaca tactaggggt catcatttga ctgatcctgc aactttggaa 1380
gaaattaggt tgagattgtc cagggatgaa tccgttttag ttattgctaa caacgtttct 1440
caagctattg ccttgtatga acaattagca cctgatgttt gtgaaaggtt tggtcaagat 1500
gcagccttgt tattacattc tcgctttaga cggatggatc gcagcaggat tgaacaaaaa 1560
attgccgata gatttgccac agttgctcct gatgctcaaa atagtaggaa accaggttta 1620
gttgttgcta cacaagttgt tgaagtttcc ttggatgttg attttgatgt tttgtttaca 1680
ggtgctgccc cattggaagc cttattgcaa agatttggta ggactaatag agttggtgcc 1740
agacctccag ccgatgttat tgttcatcat ccagcatgga ctacacgtcg tagacaacct 1800
ggtgaatatg ccgatggtat ctatccaagg gaacctgttg aatccgcttg gcatatttta 1860
actagaaatc atggtagagt tattgatgaa gccgatgcca ctgcatggtt ggatgaagtt 1920
tatgcaactg attggggtag gcaatggcat agggaagttt tggaaagacg tgaaagattt 1980
gatagagcct ttttgcagtt tagataccca tttgaagata ggactgatct cgcagatact 2040
tttgatgaat tatttgatgg ttccgaagca attttagcag aagatcaaga tgcctatagt 2100
gccgccttag ctgccccaga tggagaccat ccaggtgccg gtaggttatt agccgaagaa 2160
tatttgattc cagttcctca ttgggcttca ccattatcta ggtatgaaaa acaattaaaa 2220
gttagagtta ttaatggcga ctatcatcca gatcatggtt taatggccgt taggggttta 2280
cctcaacctg catatagggc cggtgaagtt ttacccaaga agaagaggaa ggtgtga 2337
<210> 4
<211> 807
<212> DNA
<213>Artificial sequence ()
<400> 4
cagccattcc attggagttt actccacaag atgatattgt tttgttggaa ccatatcatt 60
atttgggaaa aaatcctggt aaagaaattc gttcacaatt gattgaagcc tttaattatt 120
ggttggatgt taaaaaagag gaccttgaag ttattcaaaa tgttgttggt atgttgcata 180
ctgcttcttt gctcatggat gatgttgaag atagttccgt tttacgtcgg ggttcaccag 240
ttgctcattt gatctacggt attccacaaa ctattaatac agccaattat gtttactttt 300
tagcctatca agaaattttt aaattgcgtc caacacctat tcctatgcca gttggtggtt 360
tgtttcgtat tgccgttcgt ttgatgatgg ccaaatctga atgtgatatt gattttgttc 420
aattagttaa tttgattagt atctattttc aaattcgtga tgattatatg aatcttcaat 480
ctagtgaata tgcacataac aaaaattttg ccgaggacct tactgaaggt aaattttcct 540
ttccaactat tcatagtatt catactaatc caagtagtcg tttagttatt aatacattac 600
agaaaaaatc tacatcccca gaaattttgc atcattgtgt taattatatg cgtactgaaa 660
cacattcctt tgaatatact cgtgaagttt tgaatacttt gtccggtgcc ttggaacgtg 720
aattgggtcg tttgcaagaa gaatttgccg aagctaatag tcgtatggat ttgggcgacg 780
ttgaatccga aggtcgtaca gggaaaa 807
<210> 5
<211> 920
<212> DNA
<213>Artificial sequence ()
<400> 5
atcgtttgcg tcgggtgttc tcctttgccg ttatgtatat gggtcaatca ccatatagtg 60
cccctggtac atattcctta ttacaatata cagaattaac agaaggtatt tggtatcctc 120
gtggtggttt ttggcaagtt cctaatacat tgttgcaaat tgttaaacgt aacaatccat 180
ccgccaagtt taattttaat gcaccagttt cacaagtttt attatcacct gccaaagatc 240
gtgccacagg tgttcgttta gaatccggtg aagaacatca tgccgatgtt gttattgtta 300
atgccgattt ggtttatgcc tccgaacatc tcattccaat tgatccatca gcagctccag 360
aaggtaaaga tgctattgtt attttagttc catgtggtca tattgatact tctaatccac 420
aagattataa caagttagtt gctcgtgctc gtaaatttgt tattcaaaca ttaagtgcta 480
aattaggttt acctgatttt gaaaaaatga ttgttgccga aaaagttcat gatgcaccat 540
catgggaaaa agagtttaat ttgaaagatg gtagtatttt gggtttagca cataatttta 600
tgcaagtttt gggttttcgt cctagtactc gtcatcctaa atatgataaa ttgtttttcg 660
ttggtgcttc tactcatcca ggtacaggtg ttccaattgt tttggccggt gccaaattaa 720
ctgccaatca agttttggaa tcctttaatc gttctccagc acctgatcct aatatgtcct 780
tgtccgttcc atacggtaaa ccattgaaat ctaatggtac aggtattgat tctcaagttc 840
aattgaagtt tatggatttg gaaaaatggg tttatctttt agttttgtta attggtgccg 900
ttattgctcg ttcagttggt 920

Claims (4)

  1. A kind of 1. eukaryotic gene edit methods based on Virginia streptomycete IBL14 I-B type Cas systems, it is characterised in that： Using containing 3 genes in the streptomycete IBL14 genomes of VirginiacasProtein expressing plasmid and gene constructed by 7-5-3 Edit plasmid pair eukaryotic gene group and carry out gene editing.
  2. 2. a kind of eukaryotic gene based on Virginia streptomycete IBL14 I-B type Cas systems according to claim 1 is compiled The method of collecting, it is characterised in that：The acquisition and inspection of structure including protein expressing plasmid, the structure of gene editing plasmid and recon Test, comprise the following steps that：
    （1）The structure of protein expressing plasmid
    According to gene in the streptomycete IBL14 of Virginiacas7-5-3 sequences, the gene and correlation for synthesizing its codon optimization draw Thing, by one-step cloning by after optimizationcas7-5-3 genes directed cloning obtains Cas7-5-3 albumen tables on plasmid plasmid Up to plasmid plasmid-cas7-5-3；
    （2）The structure of gene editing plasmid
    It is anti-by PCR using eukaryotic gene group as masterplate according to eucaryote target gene DNA sequence dna information design primer End should be expanded to obtain respectively simultaneous with the target gene of restriction enzyme restriction enzyme site and overlap PCR complementary series Upstream and downstream homology arm fragment, and upstream and downstream homology arm is connected on gene editing masterplate t-DNA by overlap PCR, Designed simultaneously according to eucaryote target gene sequence information and directly synthesis head and the tail are whole comprising galactose promoter and RNA respectively Only sub target gene fragment g-DNA, according to the cohesive end on restriction enzyme site by gene editing masterplate t-DNA and target It is connected to genetic fragment g-DNA on plasmid, obtains gene editing plasmid plasmid-t/g-DNA；
    （3）The acquisition and inspection of recon
    Eukaryotic cells competence is prepared, will be by step（1）Obtained protein expressing plasmid plasmid-cas7-5-3 and step Suddenly（2）Obtained gene editing plasmid plasmid-t/g-DNA is transformed into target bacterium competence respectively, obtains different restructuring Son；PCR and gene sequencing are carried out to different restructuring daughter chromosomes or carries out functional analysis, correct purpose is obtained with screening Recon.
  3. A kind of 3. eucaryon base based on Virginia streptomycete IBL14 I-B type Cas systems according to claim 1 or 2 Because of edit methods, it is characterised in that：Described eucaryote refers to the eukaryotic of saccharomyces cerevisiae microorganism or animal and plant.
  4. 4. a kind of eukaryotic gene based on Virginia streptomycete IBL14 I-B type Cas systems according to claim 1 is compiled The method of collecting, it is characterised in that：Described gene editing refer to the chromogene of eukaryotic is inserted, knocked out, seamless point Mutation and any combination.
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