CN112646811B - CRISPR/Cas9 system suitable for multi-gene knockout of yeast and construction method and application thereof - Google Patents
CRISPR/Cas9 system suitable for multi-gene knockout of yeast and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a CRISPR/Cas9 system suitable for multi-gene knockout of yeast as well as a construction method and application thereof. The CRISPR/Cas9 vector suitable for multigene knockout of saccharomyces cerevisiae is constructed, toxin resistance genes pdr5, pdr10 and pdr15 in the CRISPR/Cas9 vector are knocked out, and the anti-virus effect of toxin-sensitive saccharomyces cerevisiae is verified through an anti-virus experiment. The invention provides a multigene knockout CRISPR/Cas9 system knockout method for yeast and even other fungi, and provides a reliable model strain for the verification of the anti-virus effect of other toxin resistance genes. The inactivation of the gene is realized by introducing a terminator in advance into the homologous repair template, so that the multi-gene knockout method provided by the invention has the advantages of simple vector construction, high knockout efficiency, low cost and the like. Therefore, the method can be widely applied to multi-gene knockout of yeast and filamentous fungi, thereby promoting genetic engineering modification of the yeast and the filamentous fungi.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a CRISPR/Cas9 system suitable for multigene knockout of yeast, and a construction method and application thereof.
Background
Saccharomyces cerevisiae is a single-cell eukaryotic cell factory which is widely applied and has the advantages of easy culture, clear genetic background, convenient and efficient gene operation and the like. The advantages of saccharomyces cerevisiae, which is the first eukaryotic organism to be completely sequenced, possessing the ability of post-translational modification of protein expression and being free of endotoxin, make saccharomyces cerevisiae widely used for the industrial production of recombinant proteins. Due to the proximity of the genetic background, s.cerevisiae is often used to synthesize secondary natural products of eukaryotic origin, or to identify single functional proteins. The production of the artemisinine (artemisinine) precursor artemisinine acid by Saccharomyces cerevisiae is the most well-known application in this field.
The saccharomyces cerevisiae is used as a eukaryotic microorganism with strong adaptability, and has a set of systemic and complete self-protection mechanisms. The self-protection mechanism of microorganisms involves the transport of intracellular toxic substances out and their transformation or decomposition into less toxic compounds. The previous studies revealed that genes encoding antitoxin such as pdr5, pdr10 and pdr15 are present in s.cerevisiae. The gene PDR5 encodes an ABC transporter protein (PDR5), and the transcription process is induced and regulated by PDR1 and PDR 3. Not only PDR5, but also a number of ABC transporters and pleiotropic anti-virus genes are regulated by PDR 1-3. The later identified proteins encoded by pdr10 and pdr15 also play an important role in improving the tolerance of Saccharomyces cerevisiae, and thus interfere with the functional verification of foreign genes in the research. In order to study the specificity of the exogenous gene in the Saccharomyces cerevisiae cells, it is usually necessary to knock out or silence the pdr series gene of Saccharomyces cerevisiae, and to construct a sensitive Saccharomyces cerevisiae with poor tolerance to toxins, otherwise the functional identification of the exogenous gene will be affected.
The CRISPR/Cas9 technology is widely applied to genome editing of eukaryotic cells such as mammalian cells, stem cells, yeasts and plants due to simple construction, high knockout efficiency and strong operability. With the continuous progress of molecular biology technology, nCas9, Cas12a, dCas9-VP64, dCas9-VPR and other technologies are continuously emerged, and the gene knockout efficiency and the transcription regulation efficiency of the CRISPR/Cas9 system are improved. The CRIPSR/Cas9 system has been widely used for gene knockout of yeast, but the CRIPSR/Cas9 system still needs further improvement in multi-gene knockout of yeast.
Disclosure of Invention
The invention aims to overcome the current situation that the multi-gene knockout efficiency in yeast is low at present, and provides a CRISPR/Cas9 system suitable for multi-gene knockout of yeast and a construction method and application thereof.
The first purpose of the invention is to provide a construction method of CRISPR/Cas9 system suitable for multiple gene knockout of yeast, which comprises the following steps:
1) respectively analyzing and selecting gene knockout targets of a plurality of target genes by using a CRISPR website (http:// CRISPR. dbcls. jp /);
2) according to the selected knockout target, a target sequence is introduced into a CRISPR-Cas9 knockout vector p426-PSNR52-grna. can1.y-TSUP4 by utilizing a homologous recombination technology, and a plurality of single knockout vectors are respectively constructed;
3) on the basis of the constructed single knock-out plasmid, constructing a multi-knock-out vector for knocking out a plurality of genes simultaneously by utilizing a homologous recombination technology;
4) designing homologous recombination fragment Donor DNA for genome repair according to the selected knockout target, and introducing a termination codon TAA onto or near a PAM sequence NGG to destroy the NGG structure and avoid repeated knockout;
thereby obtaining the CRISPR/Cas9 system suitable for multiple gene knockout of yeast.
Preferably, the method specifically comprises the following steps:
1) the CRISPR website (http:// CRISPR. dbcls. jp /) is utilized to respectively analyze and select the gene knockout targets of three target genes pdr5, pdr10 and pdr 15;
2) according to a selected knockout target, a target sequence is introduced into a CRISPR-Cas9 knockout vector p426-PSNR52-gRNA. CAN1.Y-TSUP4 by utilizing a homologous recombination technology, and three single knockout vectors p 426-delta pdr5, p 426-delta pdr10 and p 426-delta pdr15 are respectively constructed;
3) on the basis of the constructed single knock-out plasmid, a triple knock-out vector p 426-delta pdr5-10-15 for knocking out three genes simultaneously is constructed by utilizing a homologous recombination technology;
4) designing a homologous recombination fragment Donor DNA for genome repair according to a selected knockout target, and introducing a termination codon TAA onto or near a PAM sequence NGG to destroy the structure of the NGG and avoid repeated knockout;
thereby obtaining the CRISPR/Cas9 system suitable for yeast three-gene knockout.
The second purpose of the invention is to provide a method for knocking out genes by using the CRISPR/Cas9 system suitable for yeast multi-gene knocking out, which is to transfer a multi-knocking vector and Donor DNA into a host cell to knock out the genes.
Preferably, the triple-knock-out vectors p 426-delta pdr5-10-15 and Donor DNA are transferred into Saccharomyces cerevisiae S.cerevisiae BJ5464, coated on an auxotrophic plate SD-Trp-Ura, and the saccharomyces cerevisiae strain S.cerevisiae BJ5464-D with three target genes knocked out simultaneously is screened by combining a yeast colony PCR and sequencing means - 。
The third purpose of the invention is that the gene knockout vector suitable for the saccharomyces cerevisiae, which is constructed by the construction method, comprises single knockout vectors p 426-delta pdr5, p 426-delta pdr10 and p 426-delta pdr 15; double knock-out vector p 426-delta pdr5-10 and triple knock-out vector p 426-delta pdr 5-10-15.
The fourth purpose of the invention is to provide the saccharomyces cerevisiae strain S - Application in verifying the function of a presumed antitoxic gene.
A fifth object of the present invention is to provide a method for verifying the function of a putative antiviral gene by transforming the putative antiviral gene into a strain of Saccharomyces cerevisiae BJ5464-D - Then culturing on a culture medium containing a target toxin, and verifying the saccharomyces cerevisiae strain S - Whether it has the function of resisting the target toxin.
The target toxin is deoxynivalenol.
The CRISPR/Cas9 vector suitable for multigene knockout of saccharomyces cerevisiae is constructed, toxin resistance genes pdr5, pdr10 and pdr15 in the CRISPR/Cas9 vector are knocked out, and the anti-virus effect of toxin-sensitive saccharomyces cerevisiae is verified through an anti-virus experiment. The invention provides a multigene-knockout CRISPR/Cas9 systematic knockout method for yeast and even other fungi, and provides a reliable model strain for verifying the anti-virus effect of other toxin resistance genes.
Compared with the prior art, the invention has the following beneficial effects:
at present, a CRISPR/Cas9 gene knockout system is widely applied to gene knockout of saccharomyces cerevisiae, but multiple gene knockout of saccharomyces cerevisiae generally involves the use of multiple selection markers, so that the defects of complex vector construction, low knockout efficiency, difficulty in introduction and elimination of the selection markers and the like exist. The inactivation of the gene is realized by introducing the terminator into the homologous repair template in advance, so that the multi-gene knockout method provided by the invention has the advantages of simple vector construction, high knockout efficiency, low cost and the like. Therefore, the method can be widely applied to multi-gene knockout of yeast and filamentous fungi, thereby promoting genetic engineering modification of the yeast and the filamentous fungi.
The saccharomyces cerevisiae BJ5464 of the present invention is a known strain, and can be purchased from commercial companies.
Drawings
FIG. 1 is a schematic diagram of the construction process of a three-gene knockout vector p426- Δ pdr 5-10-15;
FIG. 2 is an agarose gel examination of map (A) and (B) of p426- Δ pdr5 vectors constructed from a single knock-out plasmid;
FIG. 3 is an electrophoresis diagram of agarose gel detection of fragments PDR5-Fragment and PDR10-Fragment for constructing the double knock-out vector p 426-. DELTA.pdr 5-10; (B) a colony PCR electrophoretogram of knock-out vector p 426-delta pdr 5-10;
FIG. 4 is an electrophoresis chart of construction of triple-knock-out vector p426- Δ PDR5-10-15 agarose gel detection of fragments PDR5-10-Fragment and PDR15-Fragment of (A); (B) colony PCR electrophoretogram of knock-out vector p426- Δ pdr 5-10-15;
FIG. 5 is a PCR verification of a Saccharomyces cerevisiae colony using CRISPR-Cas9 technology gene knockout;
FIG. 6 shows PCR band sequencing verification of genetically modified Saccharomyces cerevisiae colonies;
FIG. 7 is a graph of the analysis of the tolerance of Saccharomyces cerevisiae BJ5464 (labeled p414) and BJ5464-D- (labeled p414+ p426) to DON toxin, wherein the left graph is DON 100. mu.M and the right graph is 0. mu.M.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1: construction of Saccharomyces cerevisiae BJ5464 multi-gene knockout vector
1. Selection of knockout targets
(1) The Gene sequences of pdr5(Gene ID:854324), pdr10(Gene ID:854506) and pdr15(Gene ID:852015) of the S.cerevisiae BJ5464 genome were first searched and obtained from the yeast genome nucleic acid database (https:// www.yeastgenome.org /).
(2) And uploading the target sequence by using a CRISPR tool (http:// CRISPR. dbcls. jp /), and analyzing to obtain all potential target sites of the target gene, namely all downstream 20bp sequences containing NGG characteristic bases. And selecting a sequence with higher evaluation score and closer to the initiation codon ATG as a gRNA knockout target.
2. Construction of Single knock-out vectors
Based on a CRISPR knockout vector p426-PSNR52-gRNA. CAN1.Y-TSUP4, 3 single gene knockout plasmids (single knockout plasmids) are constructed: p 426-. DELTA.pdr 5, p 426-. DELTA.pdr 10 and p 426-. DELTA.pdr 15. The construction work is briefly described by taking the construction of a vector p 426-delta pdr5 targeting a target gene pdr5 as an example.
Firstly, using CRISPR knockout vector p426-PSNR52-gRNA. CAN1.Y-TSUP4 purchased from Addgene company as a template, and respectively using Tong-F/PDR5-R and PDR5-F/Tong-R as a primer pair to carry out PCR amplification, so as to obtain two fragments PDR5-Fragment 1 and PDR5-Fragment 2, wherein the lengths of homologous arms of the two fragments are 20bp and 22bp respectively. Specific PCR amplification conditions are shown in Table 1.
TABLE 1 PCR program for PDR5-1 Gene fragment
When PDR5-Fragment 2 was amplified, the extension time of step 4 in the reaction program in Table 1 was changed to 25 s.
After the PCR is finished, agarose gel electrophoresis detection is carried out on the two amplified fragments, and after the two amplified fragments are purified by a DNA product purification kit, the concentrations of the two amplified fragments are respectively measured by a micro-spectrophotometer. Two-fragment recombination was performed using the Clonexpress II recombinant cloning kit, with the recombination conditions as shown in Table 2:
TABLE 2 two-fragment recombination reaction System
Immediately after the reaction was completed, the reaction tube was taken out and cooled on ice for 5min, and transformed into competent cells DH5 α: the reaction system (10. mu.L) was added to 50. mu.L of E.coli DH 5. alpha. competent cells, mixed by flicking the tube wall, and allowed to stand on ice for 30 min. The mixture is heat-shocked in a water bath at 42 ℃ for 90s and immediately frozen for 2-3 min. 950. mu.L of LB medium was added to the clean bench and cultured at 37 ℃ and 180rpm for 45 min. Taking out the bacterial liquid, centrifuging for 1min for a short time, discarding 900 microliter supernatant, suspending the bacteria, then coating all the bacteria on an LB/Amp + plate, and culturing for 16-20h at 30 ℃.
After the culture, selecting a monoclonal scratch plate, continuing to culture for 10-12h at 37 ℃, and performing primary screening on whether the transformant is inserted into a target fragment by adopting a colony PCR method. The scheme is as follows:
selecting single clone (5-10 clones) from the transformation plate, dissolving in 20 μ L dd H2O, cracking at 95 deg.C for 10min, centrifuging at 13400rpm for 2min, and taking supernatant as template;
the band of interest was amplified using 2 × PCR Mix, the procedure was as follows:
TABLE 3 colony PCR System and procedure (5runs)
After the PCR was completed, the PCR products were detected by agarose gel electrophoresis and the fragment sizes were compared with a standard Marker ladder. And (3) selecting a suspected positive strain, inoculating the strain to an LB/Amp + liquid culture medium, culturing overnight at 37 ℃ and 220rpm, extracting the plasmid by using a plasmid extraction kit, sending the plasmid to Shenzhen Huada (Shenzhen) company for sequencing, and selecting the strain with correct sequencing to obtain a single knock-out vector p 426-delta pdr5 (figure 2).
P426-PSNR52-gRNA. CAN1.Y-TSUP4 is used as a template, Tong-F/PDR10-R and PDR10-F/Tong-R are used as a primer pair, the primers are verified to be PDR10-V-R and PDR10-V-F, and a single knock-out vector p 426-delta PDR10 for knocking out the PDR10 gene is constructed according to the same method.
A single knock-out vector p 426-delta PDR15 with a PDR15 gene knocked out is constructed according to the same method by taking p426-PSNR52-gRNA, CAN1, Y-TSUP4 as a template, Tong-F/PDR15-R and PDR15-F/Tong-R as primer pairs and verifying that the primers are PDR15-V-R and PDR 15-V-F.
3. Construction of double-and triple-knock vectors
After the successful construction of the single knock-out vectors p426- Δ pdr5 and p426- Δ pdr10, the present study uses the above plasmids as templates to construct a double knock-out vector p426- Δ pdr5-10 targeting both the target genes pdr5 and pdr10, and the specific experimental method is as follows:
firstly, taking p 426-delta PDR5 as a template and Tong-F/Tong3-R as a primer pair, and carrying out PCR amplification on a segment containing a PDR5 targeted knockout sequence to obtain a segment PDR5-Fragment 3; and then, using p 426-delta PDR10 as a template and Tong3-F/Tong-R as a primer pair, and carrying out PCR amplification on a Fragment containing the PDR10 targeted knockout sequence to obtain a Fragment PDR 10-Fragment. Finally, a knock-out vector p426- Δ pdr5-10 containing two targeting fragments simultaneously was constructed by homologous recombination, i.e., using Clonexpress II recombinant cloning kit to recombine plasmids with two fragments (FIG. 3).
The process for constructing the triple-knock-out vector p426- Δ pdr5-10-15 is similar to the process for constructing the double-knock-out vector: firstly, taking p 426-delta PDR5-10 as a template and Tong-F/Tong4-R as a primer pair, and carrying out PCR amplification on a segment containing a PDR5-10 targeted knockout sequence to obtain a segment PDR 5-10-Fragment; and then, using p 426-delta PDR15 as a template and Tong4-F/Tong-R as a primer pair, and carrying out PCR amplification on a Fragment containing the PDR15 targeted knockout sequence to obtain a Fragment PDR 15-Fragment. Finally, a knockout vector p426- Δ pdr5-10-15 (shown in FIG. 1 and FIG. 4) containing three targeting fragments simultaneously is constructed by a homologous recombination method, namely, a two-fragment recombinant plasmid is carried out by using a Clonexpress II recombinant cloning kit.
4. In the case of knocking out a specific gene in the genome of s.cerevisiae, it is necessary to provide a homologous recombination fragment (Donor DNA). According to the report of the literature, about 84bp is selected as the length of the homology arm in the research, and the Donor DNA with the length can be synthesized into an upstream primer and a downstream primer which are mutually used as templates and is obtained by annealing and extending. The sequences of the homology arms for the three genes pdr5, pdr10 and pdr15 are shown in Table 4, bold for the stop codon TAA.
TABLE 4 homologous recombination fragments
Using the construction of the homologous recombinant fragment PDR5-M as an example, the primers PDR5-M-F and PDR5-M-R were amplified by PCR using the system shown in Table 5, and after agarose gel detection, the PCR product was purified with an ultra-thin DNA product purification kit and the concentration was determined.
TABLE 5 PCR procedure for homologous recombination fragment pdr5-M
This gave a homologous recombination fragment (Donor DNA) pdr 5-M.
A homologous recombination fragment (Donor DNA) PDR10-M was constructed in the same manner using the primers PDR10-M-F and PDR 10-M-R.
A homologous recombination fragment (Donor DNA) PDR15-M was constructed in the same manner using the primers PDR15-M-F and PDR 15-M-R.
According to the selection of the knockout target, the Donor DNA fragment for homologous recombination repair of the knockout site is synthesized, and the specific method is to synthesize two partially complementary primers to anneal to form a double strand, as described above. The Donor DNA fragment is introduced with a termination codon TAA, which can terminate the translation of the target gene in advance and achieve the purpose of blocking gene expression.
The invention discloses primers as follows:
TABLE 6 primers used in the present invention
Example 2: knockout of saccharomyces cerevisiae BJ5464 toxin resistance gene:
2.1 preparation of Yeast competent cells
Competent cells of yeast strain s.cerevisiae BJ5464 were prepared using yeast Transformation Kit s.c.easycop Transformation Kit (Invitrogen) as follows:
(1) selecting yeast single colony, inoculating to 5mL YPD medium, culturing at 30 deg.C and 220rpm overnight until OD600 value is 3.0-5.0;
(2) and (3) diluting thalli: inoculating the strain in 5mL YPD medium to make initial OD600 value between 0.2-0.4, culturing at 30 deg.C and 220rpm for 4-6 hr to obtain OD 600 The value reaches 0.6 to 1.0;
(3) and (3) collecting thalli: placing 5mL of bacterial liquid in a 50mL centrifuge tube, centrifuging for 5min at 4000g at normal temperature, and discarding the supernatant;
(4) washing residual culture medium: taking 5mL Solution I (Wash Solution) to re-suspend the thalli, centrifuging for 5min at 4000g at normal temperature, and discarding the supernatant;
(5) preparing competence: taking 1mL of Solution II re-suspended bacteria, wherein the bacteria cells are competent cells of S.cerevisiae BJ5464, subpackaging the competent cells in a specification of 20 mu L per tube, and immediately transforming or freezing at-80 ℃.
2.2 Yeast strains capable of expressing Cas9 protein
A p414-TEF1p-Cas9-CYC1t plasmid containing a Cas9 expression element is transformed into S.cerevisiae BJ5464 competent cells by using a yeast Transformation Kit S.c. EasyComp Transformation Kit, so that the Cas9 protein can be continuously expressed. The method comprises the following specific steps: one tube of the competent cells prepared at 2.1 was taken, returned to room temperature, added with 400ng of p414-TEF1p-Cas9-CYC1t plasmid and 200. mu.L of Solution III (Transformation Solution), vortexed, shaken and mixed, placed in an incubator at 30 ℃ for 45min, and then shaken two to three times. After the completion of the culture, 50. mu.L of the cells were plated on corresponding auxotrophic plates (SD-Trp) and cultured at 30 ℃ for 2 to 4 days. The SD-Trp plate formula is as follows: SD medium: preparing triple-deficient SD medium (100mL) of delta Leu-delta Trp-delta Ura: 2g (2%, w/v) of anhydrous glucose is weighed, the volume is adjusted to 80mL by distilled water, and the mixture is sterilized at 115 ℃ for 20min under high temperature and high pressure. Sterilizing, cooling, adding 10 Xyeast nitrogen source (YNB) solution and 10 XDO Supplement solution at a ratio of 1:10 in a superclean bench, and storing at 4 deg.C for use. If a single-deficiency or double-deficiency culture medium containing certain amino acid is required to be prepared, 100 times of corresponding amino acid mother liquor is added into a triple-deficiency SD culture medium according to the proportion of 1:100 (if Leu is deleted in the culture medium, the culture medium is marked as SD-Leu culture medium).
After single colony grows out, single colony is picked up and cultured in SD-Trp (Trp defect type culture medium) liquid culture medium, the strain is made into competent cell according to 2.1 steps, and then the competent cell is frozen and preserved at minus 80 ℃ after being subpackaged with 20 mu L of each tube.
2.3 construction of Saccharomyces cerevisiae toxin-sensitive Strain
The triple knock-out vector p 426-delta pdr5-10-15 and three homologous recombination fragments (Donor DNA) pdr5-M, pdr10-M, pdr15-M are simultaneously transferred into saccharomyces cerevisiae competent cells prepared in advance, spread on an auxotrophic plate SD-Trp-Ura and cultured at 30 ℃ until a single colony is grown. 4 single colonies were selected for PCR validation of yeast colonies, and the three target fragments were validated using the validation primer pairs (Table 1) respectively, as shown in FIG. 5.
Three PCR products of the 3# and 4# bacterial colonies are simultaneously selected and sequenced, the result shows that the target targets are knocked out successfully (figure 6), the accurate blocking of the target genes is realized, the genetic engineering yeast is obtained, the strain is named as BJ5464-D-, and the tolerance of the strain to toxin compounds is obviously reduced compared with the original strain.
Example 3: research on self-resistance of saccharomyces cerevisiae to exogenous toxin addition
After the genes pdr5, pdr10 and pdr15 of saccharomyces cerevisiae are knocked out to obtain a toxin-sensitive strain BJ5464-D-, the tolerance of the strain to addition of exogenous toxin DON needs to be investigated, and the main purposes are two: (1) is it verified that knocking out three genes of pdr series reduces or weakens the resistance of s.cerevisiae against exogenous toxins? (2) And a foundation is laid for selecting proper toxin concentration and verifying the function of the suspected antitoxic gene in the later period. Thus, the study investigated the tolerance of Saccharomyces cerevisiae to DON before and after knockdown.
On YPD plates at 100. mu.M DON toxin concentration, Saccharomyces cerevisiae colonies were visibly sparse and blurry with increasing dilution gradient, with OD values of about 0.01 (10) -2 ) The colony can maintain a round shape and is full in shape; OD of about 10 -3 And 10 -4 The bacterial colony is obviously sparse, and part of the bacterial colony is in a single bacterial colony state; OD of about 10 -5 The colonies of (2) are often not produced because of excessive dilution.
Meanwhile, DON toxins with different concentrations have larger influence on the saccharomyces cerevisiae cells, and the colony growth of BJ 5464-D-under different dilution gradients is obviously inhibited along with the increase of the DON toxin concentration. This inhibition was at an OD of about 10 -3 Is particularly significant at the following concentrations: on the DON toxin-free plates, yeast grew well and formed single colonies, but on the DON toxin plates of 80 and 100. mu.M, the diameter of the single colonies became significantly smaller, the number of colonies became smaller, and it was difficult to observe colony formation even on the plates of 150. mu.M. This indicates that the DON toxin, which is a trichothecene, has a significant viability pressure on Saccharomyces cerevisiae. Finally, 100 μ M DON toxin was selected for this study as the experimental concentration for subsequent screening.
Subsequently, under the screening pressure of 100 μ M DON toxin, the present study verified whether the tolerance of saccharomyces cerevisiae to DON toxin before and after knockout was changed, as shown in fig. 7.
The experimental results show that the high-temperature-resistant steel wire,on YPD plates (0. mu.M) without DON toxin, the growth of the Saccharomyces cerevisiae colonies before and after knockout was almost identical at different dilutions, of which 10 -3 And 10 -4 The dilution gradient of (4) shows that BJ 5464-D-may cause a slight decrease in the number of colonies due to poor culture conditions or a large dilution degree. In contrast, BJ5464 and BJ 5464-D-exhibited significant growth differences on YPD plates of 100. mu.M DON toxin. After three transporter-related genes of pdr5, pdr10 and pdr15 are knocked out, the growth of BJ 5464-D-is obviously inhibited by DON toxin, colonies under different dilution gradients are smaller and sparser than original bacteria BJ5464 before knocking out, and the diameter of a single colony is obviously smaller. In addition, the primitive bacteria BJ5464 grew almost without difference on the DON toxin-added/not-added plates, indicating that 100 μ M DON toxin does not constitute growth stress for the primitive bacteria. The above results indicate that knocking out pdr series gene can weaken the resistance of Saccharomyces cerevisiae BJ5464 to exogenous toxin DON, and the Saccharomyces cerevisiae becomes more sensitive to toxin, so that whether the assumed antitoxic gene can exert antitoxic function can be observed more easily in subsequent experiments.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
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Claims (5)
1. A method for utilizing and applying to saccharomyces cerevisiaeSaccharomyces cerevisiae) BJ5464 multigene knockout CRISPRThe method for knocking out the genes of the/Cas 9 system is characterized by comprising the following steps:
construction of CRISPR/Cas9 system with one-gene and three-gene knockout
1) The CRISPR website is utilized to analyze and select gene knockout targets of three target genes pdr5, pdr10 and pdr15 respectively;
2) according to a selected knockout target, a target sequence is introduced into a CRISPR-Cas9 knockout vector p426-PSNR52-gRNA, CAN1.Y-TSUP4 by utilizing a homologous recombination technology, and three single knockout vectors p 426-delta pdr5, p 426-delta pdr10 and p 426-delta pdr15 are respectively constructed;
3) on the basis of the constructed single knock-out plasmid, a triple knock-out vector p 426-delta pdr5-10-15 for knocking out three genes simultaneously is constructed by utilizing a homologous recombination technology;
4) designing homologous recombination fragment Donor DNA for genome repair according to the selected knockout target, and introducing a termination codon TAA onto or near a PAM sequence NGG;
thereby obtaining a CRISPR/Cas9 system suitable for yeast three-gene knockout;
two, knock-out gene
The Donor DNA sequences for the three genes pdr5, pdr10 and pdr15 are shown below:
pdr5-M：GCGTCTTCTTCTACTGAAAACGCTGCTGATCTACACAATTATAATTAATTCGATGAGCATACAGAAGCTCGAATCCAAAAACT；
pdr10-M：CGCCCTCAAGTTCAAACTCGGGTTTGAATCAAGGAAATGCTGCGTAAGACGGCCCACCTAACGAAACACAGCCGTACGAAGGCC；
pdr15-M ：GAGCTCGAGCTCAAGCTCGAGCTCGAACTCTGCCGCCCAATCCATTTAACAGCATCCATACCGCGGTTTCGACAGCGAAGCCGC；
the construction method comprises the following steps:
carrying out PCR amplification on primers PDR5-M-F and PDR5-M-R, thereby obtaining a homologous recombination fragment Donor DNA PDR 5-M;
carrying out PCR amplification by using primers PDR10-M-F and PDR10-M-R to construct a homologous recombination fragment Donor DNA PDR 10-M;
carrying out PCR amplification by using primers PDR15-M-F and PDR15-M-R to construct a homologous recombination fragment Donor DNA PDR 15-M;
primers used
Transferring the three-gene knocked-out CRISPR/Cas9 system and the Donor DNA into a saccharomyces cerevisiae BJ5464 cell, knocking out genes pdr5, pdr10 and pdr15 to obtain the saccharomyces cerevisiae strain with three target genes knocked out simultaneouslyS. cerevisiae BJ5464-D - 。
2. The method of claim 1, wherein a triple-knockout CRISPR/Cas9 system, Donor DNA pdr5-M, Donor DNA pdr10-M and Donor DNA pdr15-M are transferred into Saccharomyces cerevisiaeS. cerevisiaeCoating the BJ5464 on an auxotrophic flat plate SD-Trp-Ura, and screening out the saccharomyces cerevisiae strains with three target genes knocked out simultaneously by utilizing a mode of combining yeast colony PCR and sequencing meansS. cerevisiae BJ5464-D - 。
3. A strain of Saccharomyces cerevisiae constructed according to the method of claim 1 or 2S. cerevisiae BJ5464-D - 。
4. The Saccharomyces cerevisiae strain of claim 3S. cerevisiae BJ5464-D - The use of a putative anti-virus gene for verifying its function by transferring the putative anti-virus gene into a strain of Saccharomyces cerevisiae according to claim 3S. cerevisiae BJ5464-D - Then culturing on a culture medium containing the target toxin, and verifying the saccharomyces cerevisiae strainS. cerevisiae BJ5464-D - Whether it has a function against the target toxin; the target toxin is deoxynivalenol.
5. A method for verifying the function of a putative anti-viral gene comprising transferring the putative anti-viral gene into a strain of Saccharomyces cerevisiae as claimed in claim 3S. cerevisiae BJ5464-D - Is expressed in a medium containing a target virusCulturing on the culture medium of the element, and verifying the saccharomyces cerevisiae strainS. cerevisiae BJ5464-D - Whether it has an anti-target toxin function; the target toxin is deoxynivalenol.
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