CN105624187A - Site-directed mutation method for genomes of saccharomyces cerevisiae - Google Patents
Site-directed mutation method for genomes of saccharomyces cerevisiae Download PDFInfo
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 67
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 title claims abstract description 67
- 230000035772 mutation Effects 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 39
- 108091033409 CRISPR Proteins 0.000 claims abstract description 33
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- 238000006243 chemical reaction Methods 0.000 description 11
- 238000002703 mutagenesis Methods 0.000 description 11
- 231100000350 mutagenesis Toxicity 0.000 description 11
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- 241000699666 Mus <mouse, genus> Species 0.000 description 2
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Abstract
本发明涉及微生物领域，特别涉及酿酒酵母基因组定点突变的方法。本发明利用CRISPR/Cas9技术修复酿酒酵母基因组上待修复的单碱基突变位点。如果该突变位点位于PAM序列(NGG)中或者PAM序列前11bp内，则可利用guide？RNA引导Cas9蛋白在待修复位点附近发挥作用，将酿酒酵母基因组进行切割处双链切口，从而实现供体DNA的高效重组。本发明对比已有成果具有以下有益效果：高效、快速实现酿酒酵母基因组位点的突变；无需向酿酒酵母基因组整合筛选标记。The invention relates to the field of microbes, in particular to a method for site-directed mutation of the brewer's yeast genome. The invention utilizes CRISPR/Cas9 technology to repair the single base mutation site to be repaired on the Saccharomyces cerevisiae genome. If the mutation site is located in the PAM sequence (NGG) or within the first 11bp of the PAM sequence, you can use the guide? The RNA guides the Cas9 protein to play a role near the site to be repaired, and double-stranded nicks the Saccharomyces cerevisiae genome, thereby achieving efficient recombination of the donor DNA. Compared with the existing achievements, the present invention has the following beneficial effects: efficient and rapid realization of the mutation of the genome site of Saccharomyces cerevisiae; no need to integrate screening markers into the Saccharomyces cerevisiae genome.
Description
Technical field
The present invention relates to microorganism field, particularly to the method for genes of brewing yeast group rite-directed mutagenesis.
Background technology
As Eukaryotic type strain, saccharomyces cerevisiae is widely used in various fields such as medicine, food, and the rite-directed mutagenesis of genes of brewing yeast or chromosomal DNA sequence provides great reference significance for the research of eukaryotic gene function. The introducing of genes of brewing yeast group rite-directed mutagenesis completes generally by two-step method: 1. utilize the homologous recombination of saccharomyces cerevisiae to insert selection markers in the position of sudden change to be introduced; 2. utilize the DNA carrying sudden change to knock out the selection markers of introducing, it is achieved genome unit point mutation. But, the method needs to utilize PCR to build the expression cassette carrying selection markers, experimental period, complex operation, and generally a target can only be carried out rite-directed mutagenesis every time. Introduce rite-directed mutagenesis if, with the method simultaneously to multiple sites, then need to build multiple expression cassettes carrying selection markers, operate more loaded down with trivial details, inefficiency.
A kind of adaptive immunity defence that CRISPR/CasCRISPR/CasCRISPR/Cas is antibacterial and archeobacteria is formed in long-term evolution process, can be used to resist the virus of invasion and foreign DNA. CRISPR/Cas system is by being incorporated into the fragment of invasion phage and plasmid DNA in CRISPR, and utilizes corresponding CRISPRRNAs (crRNAs) to instruct the degraded of homologous sequence, thus providing immunity. The operation principle of this system is that crRNA (CRISPR-derivedRNA) combines formation tracrRNA/crRNA complex by base pairing with tracrRNA (trans-activatingRNA), and this complex guides nuclease Cas9 albumen shearing double-stranded RNA with the crRNA sequence target site matched. And by engineer both RNA, it is possible to transformation forms the sgRNA (shortguideRNA) with guiding function, it is sufficient to guide Cas9 that the fixed point of DNA is cut.
Gene Knock-Out Animal Model model is the important tool carrying out gene functional research, searching suitable drug action target on living animal all the time. But traditional gene knockout method requires over the series of steps such as the targeting vector structure of complexity, ES cell screening, allophenic mice selection-breeding, not only flow process is loaded down with trivial details, the requirement of technology is significantly high, and expense is big, consuming time longer, success rate is subject to the restriction of many factors. Even for the laboratory that Technical comparing is ripe, utilize conventional art to build the large and small Mus of gene knockout and be generally also required to more than 1 year. CRISPR/Cas technology is to can be used for fixed point after the technology such as Zinc finger nuclease (ZFN), ES cell targeting and TALEN to build the 4th kind of method of the large and small Mus animal of gene knockout, and effective percentage is high, speed is fast, system genitale transfer ability is strong and the feature of simple economy, the application prospect built at animal model is by boundless.
Summary of the invention
In view of this, the method that the invention provides genes of brewing yeast group rite-directed mutagenesis. CRISPR/Cas9 technology and saccharomyces cerevisiae cotransformation technology are combined by the present invention, it is proposed to the brand-new technology that genes of brewing yeast group carries out rite-directed mutagenesis, consuming time short, simple to operate.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
A kind of method that the invention provides genes of brewing yeast group rite-directed mutagenesis, comprises the steps:
Step 1: select the target site in mutational site;
Step 2: yeast conversion, introduces genes of brewing yeast group point mutation;
Step 3: point mutation is verified.
In some specific embodiments of the present invention, in described method step 1 select mutational site be arranged in 11bp before PAM sequence (NGG) or PAM sequence as target site.
In some specific embodiments of the present invention, in described method, yeast conversion described in step 2 is the DNA fragmentation after converting Cas9 plasmid, guideRNA plasmid in brewing yeast cell and carrying sudden change.
In some specific embodiments of the present invention, guideRNA described in described method guides Cas9 albumen to play a role at location proximate to be repaired, and genes of brewing yeast group carries out cut place double-strand otch, it is achieved the efficient restructuring of donor dna.
In some specific embodiments of the present invention, point mutation described in described method is site 9, site 14 or site 16 on saccharomyces cerevisiae V chromosome.
In some specific embodiments of the present invention, described method is verified described in step 3 PCR and SangerDNA sequence verification.
In some specific embodiments of the present invention, point mutation described in described method is at least 1.
In some specific embodiments of the present invention, point mutation described in described method is that on saccharomyces cerevisiae V chromosome, the A in site 14 sports C.
In some specific embodiments of the present invention, point mutation described in described method is that on saccharomyces cerevisiae V chromosome, the A in site 16 sports C.
In some specific embodiments of the present invention, point mutation described in described method is that on saccharomyces cerevisiae V chromosome, the C in site 16 sports G.
Present invention also offers the saccharomyces cerevisiae mutant bacterial that described method builds.
The present invention utilizes CRISPR/Ca9 technology to repair single base mutation site to be repaired in genes of brewing yeast group. If this mutational site is arranged in 11bp before PAM sequence (NGG) or PAM sequence, then available guideRNA guides Cas9 albumen to play a role at location proximate to be repaired, genes of brewing yeast group is carried out cut place double-strand otch, thus realizing the efficient restructuring of donor dna.
Present invention contrast has been fruitful and has had the advantages that
Efficiently, the sudden change in genes of brewing yeast group site is quickly realized;
Without integrating selection markers to genes of brewing yeast group.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, the accompanying drawing used required in embodiment or description of the prior art will be briefly described below.
Fig. 1 shows that the embodiment of the present invention 1 utilizes the co-transformation method of particle based on CRISPR/Cas9 to induce the schematic diagram of S. cerevisiae chromosomal multisite mutation;
Fig. 2 shows and treats mutant gene group sequence and sequencing result in embodiment 1;
Fig. 3 shows that the embodiment of the present invention 2 utilizes the co-transformation method of particle based on CRISPR/Cas9 to induce the schematic diagram of S. cerevisiae chromosomal multisite mutation;
Fig. 4 shows and treats mutant gene group sequence and sequencing result in embodiment 2.
Detailed description of the invention
The method that the invention discloses genes of brewing yeast group rite-directed mutagenesis, those skilled in the art can use for reference present disclosure, is suitably modified technological parameter and realizes. Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are considered as including in the present invention. Method and the application of the present invention are described already by preferred embodiment, method described herein and application substantially can be modified or suitably change and combination by related personnel in without departing from present invention, spirit and scope, realize and apply the technology of the present invention.
The present invention utilizes CRISPR/Cas9 cotransformation technology to enter multiple mutational site in genes of brewing yeast group, and technical scheme is summarized as follows:
1. select mutational site be arranged in 11bp before PAM sequence (NGG) or PAM sequence as target site, the DNA fragmentation of sequence after then converting Cas9 plasmid, guideRNA plasmid in brewing yeast cell and carrying sudden change. GuideRNA guides Cas9 albumen to play a role at location proximate to be repaired, and genes of brewing yeast group carries out cut place double-strand otch, thus realizing the efficient restructuring of donor dna;
2., when carrying out yeast conversion, the DNA in all the other sites that suddenly change is transformed in yeast body simultaneously simultaneously;
PCR and SangerDNA order-checking is utilized to carry out the checking of point mutation.
Present invention contrast has been fruitful and has had the advantages that
Efficiently, the sudden change in genes of brewing yeast group site is quickly realized;
Without integrating selection markers to genes of brewing yeast group.
In the method for genes of brewing yeast group rite-directed mutagenesis of the present invention, bacterial strain uses therefor and raw material all can have market to buy.
Below in conjunction with embodiment, the present invention is expanded on further:
Embodiment 1
Utilizing CRISPR/Cas9 technology, on saccharomyces cerevisiae V chromosome, site 14 and 16 place introduces single-site mutant (Fig. 1), comprises the following steps:
1. site 14 place sequence is " ctggagatgAaccaatgattTGG " (as shown in SEQIDNo.1), and wherein TGG is PAM sequence, and A is for treating mutational site; Introducing the sequence after single-site mutant is " ctggagatgCaccaatgattTGG " (as shown in SEQIDNo.2), and wherein TGG is PAM sequence, and C is the site after sudden change. Site 16 place sequence is " ttagctagcaaAcccttagaac " (as shown in SEQIDNo.3), wherein A is band mutational site, introducing the sequence after single-site mutant is " ttagctagcaaCcccttagaac " (as shown in SEQIDNo.4), and wherein C is the site after sudden change. Selecting site 14 to cut target spot for Cas9, site 16 is cotransformation mutational site.
2. building the guide-RNA plasmid in site 14 to be repaired, its construction step is as follows:
A) selecting protospacer is ctggagatgAaccaatgatt;
B) synthetic primer
" GCAGTGAAAGATAAATGATCctggagatgAaccaatgattGTTTTAGAGCTAGAAA TAGC " (as shown in SEQIDNo.5) and
" GCTATTTCTAGCTCTAAAACaatcattggtTcatctccagGATCATTTATCTTTCA CTGC " (as shown in SEQIDNo.6);
C) two primers of annealing bonding, obtain double-stranded DNA;
D) restricted enzyme NotI and CIP digested plasmid pRS426+SNR52p-gRNA is utilized, so as to linearisation;
E) utilize Gibson to assemble linearization plasmid and double-stranded DNA to be assembled;
F) reaction system is converted in DH5 �� competent escherichia coli cell, coat on LB+Carb flat board, cultivate 12h for 37 DEG C;
G) 5 single colony inoculations of picking are in 5mLLB+Carb fluid medium, after 37 DEG C of incubated overnight, extract plasmid, carry out Sanger order-checking;
H) the plasmid called after pRS426+SNR52P-gRNA.14 checking order correct.
3. Saccharomyces cerevisiae transformant, its step is as follows:
A) utilize conversion pRS415+Cas9 plasmid in LiOAC normal direction saccharomyces cerevisiae, be coated with flat board and SC-Leu culture plate top sieve menu bacterium colony;
B) picking carries the saccharomyces cerevisiae list bacterium colony of pRS415+Cas9 plasmid in 5mLSC-Leu fluid medium, 30 DEG C of incubated overnight;
C) the saccharomyces cerevisiae culture fluid OD of incubated overnight is measured600, inoculate overnight culture fluid to (0.1250D in 5mLYPD600/ ml), 30 DEG C, 220rpm when be cultured to OD600Reach 0.5 (about needing 3.5 4.5hrs);
D) drawing 1.5mL saccharomyces cerevisiae culture fluid to 1.5mLEP pipe, 5000rpm is centrifuged 1min, collects cell; With 1mL sterilized water re-suspended cell, ibid it is centrifuged, collects cell; Use 1mL0.1MLiOAc re-suspended cell, be ibid centrifuged, collect cell; Absorb 900 �� L of supernatant, remaining 100 �� LLiOAc re-suspended cells with pipettor, be placed on ice, obtain competent cell.
E) preparing transformation system, wherein DNA is pRS426+SNR52p-gRNA.14,50ng; Site 14 point mutation fragment, 200ng; Site 16 point mutation fragment, 200ng:
F) adding to transformation system in 100 �� L competent cells, pressure-vaccum is uniform, most high speed vortex 10s; 30 DEG C of incubators hatch 30min; Add 90 �� LDMSO, vortex concussion 10s; 42 DEG C of heat shock 15min; 3600rpm is centrifuged 30s, collects cell; Sucking-off is asked, adds 400 �� L5mMCaCl2, re-suspended cell, stand 5min; 3600rpm is centrifuged 30s, and sucking-off is asked, and is coated with the screening of SC-Leu-Ura sifting motion cultivation plate after resuspended in sterilized water.
4. treating that yeast grows 2 days on sifting motion cultivation plate, picking list bacterium colony divides pure in the flat lining out of SC-Leu-Ura.
5. use the forward primer 14-test-F " TTGTTTGGATTTCCAAATCATTGGTG " (as shown in SEQIDNo.7) and downstream primer 14-test-R " CGTCGTCCTCTTCAGTAGTAGCC " (as shown in SEQIDNo.8) of specificity verification site 14 point mutation, and the bacterial strain in step 4 is carried out yeast colony PCR checking (Annaluru by the forward primer 16-test-F " AATCCTCGTGCAAGCCATCA " (as shown in SEQIDNo.9) and downstream primer 6-test-R " TGATAGTTCAAGAGGTTCTAAGGGT " (as shown in SEQIDNo.10) of specificity verification site 16 point mutation, N., etal.Science (2014), 344, 55-8). use the PCR reaction system of 15 �� L: GoTaqGreenMasterMix7.5 �� L, forward primer (10 ��Ms) 0.1 �� L, downstream primer (10 ��Ms) 0.1 �� L, ddH2O6.3 �� L, template DNA 1 �� L. PCR reaction condition: 98 DEG C of denaturation 5min; 98 DEG C of degeneration 30s; 59 DEG C of annealing 30s; 72 DEG C extend 30s; 30 circulations; 72 DEG C extend 10min, 4 DEG C of preservations.
6. select PCR checking and occur that the bacterial strain of band extracts genome as template, utilize forward primer 14-F " CCTATCCCTTATCCAGAACAACC " (as shown in SEQIDNo.11) and downstream primer 14-R " CTAACCTTTGGAGCACCACTGAC " (as shown in SEQIDNo.12) and forward primer 16-F " AGGAACTGGGACGCTCAAGACGC " (as shown in SEQIDNo.13) and downstream primer 16-R " TTTAGTCCACATCCACCAACCAC " (as shown in SEQIDNo.14) to carry out pcr amplification. Use Phusion high-fidelity DNA polymerase to carry out PCR and obtain DNA fragmentation, and carry out sepharose electrophoresis recovery. Use same primers that DNA glue recovery product is carried out Sanger order-checking confirmation form site and repair successfully (Fig. 2).
7. the Wine brewing yeast strain after pair point mutation carries out plasmid loss.
Embodiment 2
Utilize CRISPR/Cas9 technology, saccharomyces cerevisiae synthesis type V chromosomal foci 9 place (Fig. 3) introduced single-site mutant, comprises the following steps:
1. the sequence at single-site mutant place to be introduced is " aaatacgaagaacCattttgCGG " (as shown in SEQIDNo.15), and wherein CGG is PAM sequence, and C is site to be repaired; Introducing the sequence after single-site mutant is " aaatacgaagaacGattttgCGG " (as shown in SEQIDNo.16), and wherein CGG is PAM sequence, and G is the site after repairing. Selecting site 9 to cut target spot for Cas9, site 15-2 is cotransformation mutational site.
2. building the guide-RNA plasmid in site 9 to be repaired, its construction step is as follows:
A) selecting protospacer is aaatacgaagaacCattttg;
B) synthetic primer " GCAGTGAAAGATAAATGATCaaatacgaagaacCattttgGTTTTAGAGCTAGAAA TAGC " (as shown in SEQIDNo.17) and " GCTATTTCTAGCTCTAAAACcaaaatGgttcttcgtatttGATCATTTATCTTTCA CTGC " (as shown in SEQIDNo.18);
C) two primers of annealing bonding, obtain double-stranded DNA;
D) restricted enzyme NotI and CIP digested plasmid pRS426+SNR52p-gRNA is utilized, so as to linearisation;
E) utilize Gibson to assemble linearization plasmid and double-stranded DNA to be assembled;
F) reaction system is converted in DH5 �� competent escherichia coli cell, coat on LB+Carb flat board, cultivate 12h for 37 DEG C;
G) 5 single colony inoculations of picking are in 5mLLB+Carb fluid medium, after 37 DEG C of incubated overnight, extract plasmid, carry out Sanger order-checking;
H) the plasmid called after pRS426-SNR52P+gRNA.9 checking order correct.
3. Saccharomyces cerevisiae transformant, its step is as follows:
A) utilize conversion pRS415+Cas9 plasmid in LiOAC normal direction saccharomyces cerevisiae, be coated with flat board and SC-Leu culture plate top sieve menu bacterium colony;
B) picking carries the saccharomyces cerevisiae list bacterium colony of pRS415+Cas9 plasmid in 5mLSC-Leu fluid medium, 30 DEG C of incubated overnight;
C) the saccharomyces cerevisiae culture fluid OD of incubated overnight is measured600, inoculate overnight culture fluid to (0.1250D in 5mLYPD600/ ml), 30 DEG C, 220rpm when be cultured to OD600Reach 0.5 (about needing 3.5 4.5hrs);
D) drawing 1.5mL saccharomyces cerevisiae culture fluid to 1.5mLEP pipe, 5000rpm is centrifuged 1min, collects cell; With 1mL sterilized water re-suspended cell, ibid it is centrifuged, collects cell; Use 1mL0.1MLiOAc re-suspended cell, be ibid centrifuged, collect cell; Absorb 900 �� L of supernatant, remaining 100 �� LLiOAc re-suspended cells with pipettor, be placed on ice, obtain competent cell.
E) preparing transformation system, wherein DNA is pRS426+SNR52p-gRNA.9,50ng; Site 9 point mutation fragment, 200ng; Site 15-2 point mutation fragment, 200ng:
F) adding to transformation system in 100 �� L competent cells, pressure-vaccum is uniform, most high speed vortex 10s; 30 DEG C of incubators hatch 30min; Add 90 �� LDMSO, vortex concussion 10s; 42 DEG C of heat shock 15min; 3600rpm is centrifuged 30s, collects cell; Sucking-off is asked, adds 400 �� L5mMCaCl2, re-suspended cell, stand 5min; 3600rpm is centrifuged 30s, and sucking-off is asked, and is coated with the screening of SC-Leu-Ura sifting motion cultivation plate after resuspended in sterilized water.
4. treating that yeast grows 2 days on sifting motion cultivation plate, picking list bacterium colony divides pure in the flat lining out of SC-Leu-Ura.
5. use the forward primer 9-test-F " CTGGAGGGCCGCAAAATG " (as shown in SEQIDNo.19) and downstream primer 9-test-R " TGGCTTGCTAATTTCATCTTATCCT " (as shown in SEQIDNo.20) of specificity verification site 9 point mutation, and the bacterial strain in step 4 is carried out yeast colony PCR checking (Annaluru by the forward primer 15-2-test-F " TTGAGCTGCGAGAGTGTGGG " (as shown in SEQIDNo.23) and downstream primer 15-2-test-R " GCGTAATTCCCTTCTGCTTACACT " (as shown in SEQIDNo.24) of the 15-2 point mutation of specificity verification site, N., etal.Science (2014), 344, 55-8). . use the PCR reaction system of 15 �� L: GoTaqGreenMasterMix7.5 �� L, forward primer (10 ��Ms) 0.1 �� L, downstream primer (10 ��Ms) 0.1 �� L, ddH2O6.3 �� L, template DNA 1 �� L. PCR reaction condition: 98 DEG C of denaturation 5min; 98 DEG C of degeneration 30s; 59 DEG C of annealing 30s; 72 DEG C extend 30s; 30 circulations; 72 DEG C extend 10min, 4 DEG C of preservations.
6. select PCR checking and occur that the bacterial strain of band extracts genome as template, utilize forward primer 9-F " AGTTCTTCCTGAGATTTAGCCTTTG " (as shown in SEQIDNo.21) and downstream primer 9-R " CGTCATCTTTCACTACCCTTTAC " (as shown in SEQIDNo.22) and forward primer 15-F " GCCTTCCCACCGCCTTGGTTGTT " (as shown in SEQIDNo.25) and downstream primer 15-R " AAACTTATCACAGCCGCTATTCA " (as shown in SEQIDNo.26) to carry out pcr amplification. Use Phusion high-fidelity DNA polymerase to carry out PCR and obtain DNA fragmentation, and carry out sepharose electrophoresis recovery. Use same primers that DNA glue recovery product is carried out Sanger order-checking confirmation form site and repair successfully (Fig. 4).
7. the Wine brewing yeast strain after pair point mutation carries out plasmid loss. Wine brewing yeast strain after loci 9 reparation carries out plasmid loss.
The above is only the preferred embodiment of the present invention; it should be pointed out that, for those skilled in the art, under the premise without departing from the principles of the invention; can also making some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.
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