CN113604472B - CRISPR/Cas gene editing system applied to Trichoderma reesei - Google Patents
CRISPR/Cas gene editing system applied to Trichoderma reesei Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
Abstract
The invention provides a gRNA expression frame applied to a CRISPR/Cas gene editing system, wherein the gRNA expression frame has the following structure from 5 '-3': A-B-C-D, wherein A is a viral sequence having the function of initiating transcription of gRNA, B is gRNA, C is a gRNA backup, D is a hepatitis virus ribozyme (HDV) sequence, and the gRNA expression cassette is recognized and transcribed by the RNA polymerase II promoter in Trichoderma reesei. The invention realizes the simultaneous in vivo transcription of Cas9 and gRNA in Trichoderma reesei for the first time, and avoids the defects of complexity, instability and the like of in vitro transcription and reconversion of gRNA.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a gRNA expression frame for a Trichoderma reesei CRISPR/Cas gene editing system and the CRISPR/Cas gene editing system comprising the gRNA expression frame.
Background
The development of global economic systems has been dependent on the development of fossil energy, however, fossil energy is limited and is a non-renewable, disposable resource for a short period of time. The dependence on fossil energy not only brings potential energy crisis to human society, but also causes a series of ecological and environmental problems. Accordingly, more and more research is beginning to find raw materials capable of replacing fossil energy and methods of using the same. Lignocellulosic biomass is the largest renewable resource on earth and can be used as a substitute material of fossil energy, and is increasingly and widely applied to the development of a biorefinery system which is emerging worldwide. The necessary key steps needed for converting the lignocellulose material into biomass energy and new materials are that the lignocellulose material is enzymatically hydrolyzed into fermentable sugar by cellulase, and then the fermentable sugar can be fermented and processed to produce bioethanol and other energy sources and chemicals. Therefore, it is very important for the development of cellulases. At present, the most common strain for producing cellulase is Trichoderma reesei, but the enzyme producing capability of the strain is far from the industrial enzyme producing requirement, and further targeted modification is needed.
CRISPR is an immune weapon in which bacteria and viruses fight against each other in the genome of prokaryotes, in short, viruses integrate their own genes into bacteria and serve their own gene replication by means of bacterial cellular tools. Bacteria in order to clear foreign invasive genes of the virus, a CRISPR/Cas9 system has evolved, with which the bacteria can immittably excise viral genes from their own genome, which is the bacterial specific immune system.
CRISPR/Cas9 is a novel, powerful gene editing tool, which is rapidly applied to the field of eukaryotic genome editing after being developed in 2012, and realizes editing of mammalian cells, yeasts and filamentous fungi. The principle is as follows: the Cas9 protein can cut and edit a target site under the guidance of guide RNA (gRNA) so as to achieve the aim of species genetic modification. For this system, there are three expression modes of gRNA and Cas9, one in which both are expressed in the target species at the same time, the other in which Cas9 is expressed in the species, gRNA is additionally transformed after in vitro transcription, and the third in which both are additionally transformed after in vitro synthesis of ribonucleoprotein complex. The in vivo expression of the two has great advantages, and the defects of unstable in vitro transcription of the gRNA, additional independent transformation of Cas9 and the operation complexity of the gRNA and the like can be avoided.
At present, the CRISPR/Cas9 technology has not matured when a gene editing technology is applied to Trichoderma reesei, and although a Cas9 gene editing method for a filamentous fungus Crispr-Cas system is disclosed, the technology is that the CRISPR-Cas9 gene editing technology is applied to Trichoderma reesei by a method of expressing Cas9 in vivo and performing additional transformation after in vitro transcription of gRNA. For example, zhou Zhihua et al have had to resort to methods of in vitro transcription of guide RNAs followed by transformation into cells for the first time to test the feasibility of the CRISPR/Cas9 system in the filamentous fungus trichoderma reesei due to the lack of knowledge of the fungal microrna transcription machinery. Although guide RNAs can also be introduced into cells in the form of RNAs after in vitro transcription, leading to localization of Cas9 proteins, stability and conversion efficiency of guide RNAs can affect genome editing efficiency and increase operational difficulty.
The reason for this is that simultaneous in vivo transcription of gRNA and Cas9 remains a critical issue, since promoters such as U3 or U6, which are commonly used for gRNA transcription, are not found in trichoderma reesei. In order to overcome the defects of the prior art, the invention constructs a novel CRISPR-Cas9 gene editing system, realizes the simultaneous in-vivo transcription of Cas9 and gRNA in Trichoderma reesei for the first time, and avoids the defects of complexity, instability and the like of in-vitro transcription and reconversion of gRNA.
Disclosure of Invention
The invention aims to provide a gRNA expression cassette for a Trichoderma reesei CRISPR/Cas gene editing system, and another aim of the invention is to provide a CRISPR/Cas gene editing system comprising the gRNA expression cassette and a construction method thereof.
The aim of the invention is achieved by the following technical scheme.
In a first aspect, the invention provides a gRNA expression cassette for use in a CRISPR/Cas gene editing system, the gRNA expression cassette having the following structure from 5 '-3': A-B-C-D, wherein A is a viral sequence having an initiating gRNA transcription, B is gRNA, C is a gRNA backup, and D is a hepatitis virus ribozyme (HDV) sequence.
The gRNA expression frame provided by the invention can be recognized and transcribed by an RNA polymerase II promoter in trichoderma reesei.
The virus sequence with the function of initiating gRNA transcription is selected from tobacco ringspot virus (TRSV) sequence, chopsticks mustard mosaic virus (ARMV) sequence, chicory yellow spot virus (CYMVT) sequence and hammerhead virus (HH) sequence.
The tobacco circovirus sequence is (SEQ ID NO: 1):
tactgtcgtggcgagttagtagccagacgaccggagtaaaatc
the chopstick mustard mosaic virus sequence is (SEQ ID NO: 2):
tgatatagccaatccgttaagagcggaaacaaaatcaaaccgt
chicory yellow spot virus sequence (SEQ ID NO: 3):
ttcttggccaagatgcctcagctgggacacagtgtgacactgtgtc
hammerhead virus sequence is (SEQ ID NO: 4):
tcaatccctgatgagtccgtgaggacgaaacgagtaagctcgtc
the gRNA is used for guiding the Cas protein to carry out gene editing on a target gene, and the gRNA sequence is a complementary sequence synthesized according to one or more sites of the target gene. In a specific embodiment of the invention, the gRNA sequence is a complementary sequence of a gene locus of the Trichoderma reesei ace1 gene, in particular gattgagacctcgtgagagt (SEQ ID NO: 5).
The gRNA backbone sequence is (SEQ ID NO: 6):
gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgct。
the hepatitis virus ribozyme (HDV) sequence of the invention is (SEQ ID NO: 7):
ggccggcatggtcccagcctcctcgctggcgccggctgggcaacatgcttcggcatggcgaatgggac。
preferably, the viral sequence with initiation of gRNA transcription is selected from the group consisting of tobacco ringspot virus (TRSV) sequence, chopsticks mustard mosaic virus (ARMV) sequence, chicory yellow spot virus (CYMVT) sequence.
Most preferably, the viral sequence that initiates transcription of the gRNA is selected from the group consisting of tobacco ringspot virus (TRSV) sequences.
In a second aspect, the invention provides a CRISPR/Cas gene editing system comprising an expression cassette as shown above. The CRISPR/Cas gene editing system is CRISPR/Cas9, CRISPR/nCas9 or CRISPR/dCAs9.
Preferably, the CRISPR/Cas gene editing system is CRISPR/Cas9.
In a third aspect, the invention provides the use of a CRISPR/Cas gene editing system in trichoderma reesei gene editing, including one of gene knockout, gene insertion, and transcriptional regulation.
In a fourth aspect, the present invention provides a gene editing method for gene editing and transcriptional regulation of trichoderma reesei using the CRISPR/Cas gene editing system described above, the gene editing comprising one of gene knockout and gene insertion.
Preferably, the gene editing method is to knock out the gene of Trichoderma reesei by using a CRISPR/Cas gene editing system, the success rate of gene knockout is higher than 92%, and the optimal success rate is 100%.
In a fifth aspect, the present invention provides a trichoderma reesei prepared by the method of: (1) The CRISPR/Cas gene editing system is utilized to edit the genes of Trichoderma reesei, and transformants are coated into a screening culture medium for screening; (2) selecting transformants; (3) screening to obtain a positive transformant; (4) screening to obtain a positive transformant with successful gene knockout; (5) obtaining the high-yield trichoderma reesei through shake flask fermentation screening.
Preferably, the trichoderma reesei is a knock-out of the ace1 gene by the CRISPR/Cas gene editing system described in the present invention.
The components of the screening culture medium are as follows: 1M sorbitol, 20g/L glucose, 15g/L KH 2 PO 4 ，5g/L(NH 4 ) 2 SO 4 ，0.6g/L MgSO 4 ·7H 2 O，0.6g/L CaCl 2 ，0.005g/L FeSO 4 ·7H 2 O，0.0016g/L MnSO 4 ·H 2 O，0.0014g/L ZnSO 4 ·7H 2 O，0.002g/L CoCl 2 20g/L agar powder. Sterilizing in high pressure steam sterilizing pot (121deg.C, 25 min), cooling to 45-50deg.C, adding hygromycin B (final concentration 175 μg/mL) and ampicillin (final concentration 100 μg/mL) into the ultra-clean bench, and cooling.
In a preferred embodiment of the present invention, the screening medium in the step (1) is an enhanced screening medium, and the enhanced screening medium is obtained by adding 0.5-1.5g/L betaine and 10-15mL/L dandelion extract to the components of the screening medium.
Specifically, the components of the enhanced screening culture medium are as follows: 1M sorbitol, 20g/L glucose, 15g/L KH 2 PO 4 ，5g/L(NH 4 ) 2 SO 4 ，0.6g/L MgSO 4 ·7H 2 O，0.6g/L CaCl 2 ，0.005g/L FeSO 4 ·7H 2 O，0.0016g/L MnSO 4 ·H 2 O，0.0014g/L ZnSO 4 ·7H 2 O，0.002g/L CoCl 2 20g/L of agar powder, 0.5-1.5g/L of betaine and 10-15mL/L of dandelion extract. Sterilizing in high pressure steam sterilizing pot (121deg.C, 25 min); after cooling to 45-50deg.C (preferably without scalding hands), hygromycin B (final concentration 175 μg/mL) and ampicillin (final concentration 100 μg/mL) are added into the ultra-clean bench, and the mixture is poured into a plastic culture dish and cooled for use.
In a most preferred embodiment of the present invention, the enhanced screening medium is prepared by adding 1g/L betaine and 10mL/L dandelion extract to the components of the screening medium.
Preferably, the preparation method of the dandelion extract comprises the following steps: 100g of dandelion dried whole herb is added into 1L of boiling water to be extracted for 3-4 hours, cooled and centrifuged to obtain supernatant, thus obtaining dandelion leaching liquid.
The technical scheme provided by the invention has the following advantages: (1) the gRNA in vitro transcription in the prior art is very unstable and is easy to cause deletion of transcription fragments. Moreover, the transcription of Cas9 and then the transcription of gRNA require two operations and two screens, which have great damage to Trichoderma reesei, affect growth and also affect enzyme production. The CRISPR/Cas gene editing system provided by the application realizes simultaneous in-vivo transcription of Cas9 and gRNA in Trichoderma reesei for the first time, and avoids the defects of complexity, instability and the like of in-vitro transcription and reconversion of gRNA.
(2) By optimizing the gRNA expression frame, the gene knockout success rate of the CRISPR/Cas gene editing system on Trichoderma reesei is higher than 92%, and the enzyme production capacity of Trichoderma reesei is obviously improved, so that the CRISPR/Cas gene editing system provided by the invention can provide technical support for modifying high-yield strains of cellulase.
(3) The invention screens and optimizes the Trichoderma reesei after gene knockout through hygromycin enhanced screening culture medium to obtain Trichoderma reesei with good resistance and high enzyme production efficiency.
Drawings
FIG. 1 shows a plasmid map of the Cas9 protein expression vector pCRISPR-ace 1;
FIG. 2 shows a schematic representation of the gRNA expression cassette in the pCRISPR-acel plasmid map;
FIG. 3 is a flow chart for obtaining high yield Trichoderma reesei;
FIG. 4 is a graph showing trends in pilot enzyme yields of 4 high-yield Trichoderma reesei obtained in example 3;
FIG. 5 shows a trend of the enzyme yield of 3 high-yield Trichoderma reesei pilot tests obtained by enhancing the screening medium.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The composition and preparation method of the culture medium related by the invention are as follows:
the trichoderma agar medium comprises the following components: 10g/L glucose, 2g/L corn steep liquor, 2.8g/L KH 2 PO 4 ，3.92g/L(NH 4 ) 2 SO 4 ，0.84g/L MgSO 4 ·7H 2 O,0.84g/L Urea, 2g/L Tween-80, 0.014g/L FeSO 4 ·7H 2 O，0.00436g/L MnSO 4 ·H 2 O，0.00392g/L ZnSO 4 ·7H 2 O，0.0056g/L CoCl 2 20g/L agar; after formulation, the pH was adjusted to 4.8 with 2M NaOH solution. Sterilizing in a high-pressure steam sterilizing pot (121 ℃ for 25 min); after cooling to 45-50deg.C (preferably without scalding hands), ampicillin (final concentration 100 μg/mL) is added into the ultra-clean bench, and the mixture is poured into a plastic culture dish and cooled for use.
The trichoderma seed culture medium comprises the following components: 12g/L glucose, 0.7g/L corn steep liquor, 1.96g/L KH 2 PO 4 ，1.372g/L(NH 4 ) 2 SO 4 ，0.294g/L MgSO 4 ·7H 2 O,0.294g/L urea, 0.0049g/L FeSO 4 ·7H 2 O，0.0015g/L MnSO 4 ·H 2 O，0.0014g/L ZnSO 4 ·7H 2 O，0.002g/L CoCl 2 . After preparation, the mixture was sterilized in an autoclave (121 ℃ C., 25 min).
The trichoderma fermentation medium comprises the following components: 40g/L of steam explosion miscanthus, 1g/L of glucose, 2g/L of corn steep liquor and 2.8g/L of KH 2 PO 4 ，3.92g/L(NH 4 ) 2 SO 4 ，0.84g/L MgSO 4 ·7H 2 O,0.84g/L urea, 2g/L Tween-80, 0.014g/L FeSO 4 ·7H 2 O，0.00436g/L MnSO 4 ·H 2 O，0.00392g/L ZnSO 4 ·7H 2 O，0.0056g/L CoCl 2 . Sterilizing at 121deg.C under 0.1MPa for 30min before use.
EXAMPLE 1 construction of gRNA expression cassette for in vivo transcription of Trichoderma reesei
Ace1 is a DNA binding protein containing 3 Cys (2) -His (2) -like zinc finger structures, involved in regulating the expression of cellulases and hemicellulases in trichoderma reesei. After the ace1 gene is knocked out from the Trichoderma reesei, the expression level of the cellulase gene and the hemicellulase gene of the Trichoderma reesei are improved under the same culture conditions.
The invention constructs a Trichoderma reesei CRISPR-Cas9 gene editing system by taking a knock-out ace1 gene as an example, takes one position of the ace1 gene as a target sequence, and designs a gRNA sequence gRNA ace1 (SEQ ID NO: 5) by utilizing an online tool http:// gRNA. The gRNA ace1 sequence is synthesized to synthesize the gRNA backbond sequence, and then the viral sequence and the hepatitis virus (HDV) sequence with the function of initiating gRNA transcription are respectively designed and synthesized at the 5 'end and the 3' end of the gRNA and the gRNA backbond sequence. The techniques for synthesizing and ligating the above-described gene fragments are methods commonly used by those skilled in the art.
In the first case, the invention constructs the 5'TRSV-gRNA ace1-gRNA backup-3' HDV as a gRNA expression frame by the method, and the sequence is shown as SEQ ID NO: 8:
tactgtcgtggcgagttagtagccagacgaccggagtaaaatcgattgagacctcgtgagagtgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctggccggcatggtcccagcctcctcgctggcgccggctgggcaacatgcttcggcatggcgaatgggac
in the second case, the invention constructs the 5'ARMV-gRNA ace1-gRNA backup-3' HDV as a gRNA expression frame by the method, and the sequence is shown as SEQ ID NO: 9:
tgatatagccaatccgttaagagcggaaacaaaatcaaaccgtgattgagacctcgtgagagtgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctggccggcatggtcccagcctcctcgctggcgccggctgggcaacatgcttcggcatggcgaatgggac
in the third case, the invention constructs the 5'CYMVT-gRNA ace1-gRNA backup-3' HDV as a gRNA expression frame by the method, and the sequence is shown as SEQ ID NO: 10:
ttcttggccaagatgcctcagctgggacacagtgtgacactgtgtcgattgagacctcgtgagagtgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctggccggcatggtcccagcctcctcgctggcgccggctgggcaacatgcttcggcatggcgaatgggac
in the fourth case, the invention constructs 5'HH-gRNA ace1-gRNA backup-3' HDV as gRNA expression frame by the method, and the sequence is shown as SEQ ID NO: 11:
tcaatccctgatgagtccgtgaggacgaaacgagtaagctcgtcgattgagacctcgtgagagtgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctggccggcatggtcccagcctcctcgctggcgccggctgggcaacatgcttcggcatggcgaatgggac
EXAMPLE 2 construction of CRISPR/Cas9 Gene editing System for Trichoderma reesei
The gRNA expression cassette prepared in example 1 is inserted into an expression plasmid of Cas9 gene to obtain a knock-out plasmid pCRISPR-ace1 of ace1 gene, and a CRISPR/Cas9 gene editing system for Trichoderma reesei gene modification is completed. The sequence arrangement of part of key elements in the CRISPR/Cas9 gene editing system is shown in figure 1. The Cas9 gene to which the present invention pertains is a conventional sequence that is not optimized, and plasmid construction methods are also well known to those skilled in the art.
The invention uses 4 gRNA expression cassettes prepared in example 1,5'TRSV-gRNA ace1-gRNA backbone-3'HDV、/>5'ARMV-gRNA ace1-gRNA backbone-3'HDV、/>5'CYMVT-gRNA ace1-gRNA backbone-3'HDV、/>the 5'HH-gRNAace1-gRNA backbond-3' HDV is respectively inserted into the expression plasmids of Cas9 genes, so that 4 plasmids pCRISPR-ace1 (a), pCRISPR-ace1 (b), pCRISPR-ace1 (c) and pCRISPR-ace1 (d) for knocking out the Trichoderma reesei ace1 genes are obtained.
Example 3 method for knock-out of Trichoderma reesei ace1 Gene Using CRISPR/Cas9 System
(1) Plasmid transformation
The invention uses Trichoderma reesei RUT-C30 as an initial strain, uses pCRISPR-ace1 (a) plasmid constructed in example 2 to transform protoplast of Trichoderma reesei RUT-C30 (the operation method is well known to the skilled in the art, the steps are omitted here), coats the transformant into a screening culture medium, picks up 50 strains of the transformant after growing for 5-7 days, transfers the 50 strains to trichoderma agar culture medium for propagation, and scrapes spores to obtain spore suspension. The spore suspension was inoculated to trichoderma seed medium for overnight culture to obtain hyphae.
The components of the screening medium described in this example are: 1M sorbitol, 20g/L glucose, 15g/L KH 2 PO 4 ，5g/L(NH 4 ) 2 SO 4 ，0.6g/L MgSO 4 ·7H 2 O，0.6g/L CaCl 2 ，0.005g/L FeSO 4 ·7H 2 O，0.0016g/L MnSO 4 ·H 2 O，0.0014g/L ZnSO 4 ·7H 2 O，0.002g/L CoCl 2 20g/L agar powder. Sterilizing in high pressure steam sterilizing pot (121deg.C, 25 min), cooling to 45-50deg.C, adding hygromycin B (final concentration 175 μg/mL) and ampicillin (final concentration 100 μg/mL) into the ultra-clean bench, and cooling.
(2) Detecting gene knockout success rate
The DNA of the hypha of the overnight cultured transformant was extracted as a template DNA, and related primers were designed, and PCR amplification and agarose gel electrophoresis were performed using the related primers designed in the following table, to verify whether the transformation of the above transformant was successful and whether the ace1 gene was knocked out was successful.
TABLE 1
The PCR amplification and agarose gel electrophoresis program and flow are as follows:
(1) PCR amplification
The PCR reaction system (20. Mu.L) was as follows:
| 2×Taq PCR MasterMix | 10μL |
| forward primer (10. Mu.M) | 0.5μL |
| Reverse primer (10. Mu.M) | 0.5μL |
| Template DNA (10 ng/. Mu.L) | 3μL |
| ddH 2 O | 6μL |
The PCR amplification procedure was as follows:
(2) Agarose gel electrophoresis
A1% agarose gel was prepared with running buffer TAE (1X) and heated in a microwave oven until completely dissolved. Taking out, shaking, adding nucleic acid dye (Genegreen, tiangen Biochemical technology Co., ltd.) when cooling to avoid scalding hands, and lightly pouring onto electrophoresis tank horizontal plate with sample comb inserted. After the agarose gel had solidified, the comb was carefully pulled out, the horizontal plate of the electrophoresis tank with the gel was placed in the electrophoresis tank, and electrophoresis buffer TAE (1×) was added.
Marker and PCR samples of appropriate size were spotted in the sample well at a spot dose of 5-10. Mu.L and the sample spot order was recorded. And (3) adjusting the voltage to 100-150V, carrying out electrophoresis for 20-30min, taking out gel, placing the gel in a G-box for blue light irradiation, checking and analyzing electrophoresis bands by using GeneSys software, and carrying out sequencing identification on the PCR products with the bands.
Wherein, PCR amplification and agarose gel electrophoresis are carried out by using a primer Cas9-F, cas9-R designed on a plasmid Cas9 fragment, and 30 target bands exist.
The 30 PCR products with bands by agarose gel electrophoresis are sent to sequence, and the sequencing results can be completely compared with the target fragments on the plasmid. These 30 transformants were confirmed to be positive transformants (positive rate up to 60%), and the pCRISPR-ace1 (a) knockout plasmid in this example was successfully transferred into Trichoderma reesei RUT-C30. The present invention marks these 30 positive transformants as A1-A30.
The 30 positive transformants were further verified to verify that the ace1 gene was knocked out successfully. The PCR amplification and agarose gel electrophoresis are carried out by taking Trichoderma reesei RUT-C30 as a control, using Ace1-F and Ace1-R primers, taking the DNA of an A1-A30 positive transformant and Trichoderma reesei RUT-C30 as a template (Trichoderma reesei RUT-C30 is taken as a negative control and marked as WT), and sequencing PCR amplification products, wherein the sequencing result shows that the Ace1 genes of 29 transformants except A11 are knocked out successfully, and the knocked-out success rate is 97%.
Similarly, the procedures for transformation of Trichoderma reesei RUT-C30 with the pCRISPR-ace1 (b), pCRISPR-ace1 (C) and pCRISPR-ace1 (d) plasmids were as described above, and the number of positive transformants and the number of successful transformants in gene knockout were examined by the PCR amplification and agarose gel electrophoresis methods described above, and the statistics of the results are shown in the following table.
TABLE 2
From the result of converting 4 kinds of pCRISPR-ace1 plasmids into Trichoderma reesei, the CRISPR/Cas9 gene editing system prepared by the invention has the highest gene knockout success rate of pCRISPR-ace1 (a) up to 97%, and the lowest gene knockout success rate of pCRISPR-ace1 (d) of 90%. The analytical reasons may be that the pCRISPR-ace1 (a) plasmid has the highest expression level of gRNA in Trichoderma reesei, and the expression level of gRNA has a significant effect on the positioning of Cas9 and the gene knockout efficiency. Therefore, the pCRISPR-ace1 plasmid gene knockout efficiency is generally high, and it is confirmed that the expression level of gRNA is high.
The viruses with the function of initiating gRNA transcription, such as tobacco ringspot virus (TRSV), chopsticks mustard mosaic virus (ARMV), chicory yellow spot virus (CYMVT) and hammerhead virus (HH), have the function of improving the expression level of gRNA, wherein TRSV, ARMV, CYMVT mediated pCRISPR-ace1 plasmid gene knockout efficiency is higher than HH mediated pCRISPR-ace1 plasmid gene knockout efficiency, and TRSV is highest. Because TRSV, ARMV, CYMVT is hairpin-shaped sequence, capping gRNA by hairpin sequence enhances stability of gRNA and binding capacity of gRNA and Cas9, and improves transcription activity. In addition, 6 bases in the hammerhead virus (HH) sequence need to be complementarily paired with a target sequence in the gRNA, which directly leads to frequent construction of the gRNA expression system and relatively low transcriptional activity, so that the HH-mediated pCRISPR-ace1 plasmid gene knockout efficiency is relatively low.
Effect example Positive transformants enzyme Activity detection
(1) Enzyme yield of shake flask
In order to further screen the gene knockout strain with higher enzyme yield for expansion culture, positive transformants which were successfully subjected to gene knockout using the pCRISPR-ace1 (a) plasmid were subjected to shake flask fermentation (A1-A30 strains except A11), the actual influence of ace1 gene knockout on fermentation was searched for by using the original strain Trichoderma reesei RUT-C30 (WT) as a control, and the gene knockout strain with the highest enzyme yield was screened.
Fermentation culture: the optical density of the liquid of the overnight cultured Trichoderma strain was measured, and when the optical density reached 7-8, the liquid was transferred to Trichoderma fermentation medium (100 mL) for fermentation culture (the transfer amount was 10%), the fermentation culture was carried out for 8 days, the filter paper enzyme activities were measured from the 2 nd day, and the filter paper enzyme activities of the respective gene knockout strains were compared on the 8 th day.
As a result, the final enzyme activity of Trichoderma reesei RUT-C30 on day 8 of fermentation was 4.07FPU/mL, the highest enzyme activity in the positive transformant was A8, and the final enzyme activity was 6.7FPU/mL. The results show that the filter paper enzyme activity of the ace1 gene knockout strain A8 prepared by the invention is higher than that of the original strain, and the enzyme activity improvement rate is 64.6%. The CRISPR/Cas9 gene editing system prepared by the invention can be used for successfully knocking out the Trichoderma reesei gene, and a gene mutant strain A8 with stronger cellulase production capability is obtained.
(2) Amount of enzyme produced in the middle trial
Further, the strain A8 with good identification result of shake flask fermentation enzyme production is subjected to 500L pilot fermentation tank enzyme production, the effect of enzyme production test in engineering strain factories is searched, and meanwhile, the original strain Trichoderma reesei RUT-C30 is used as a control.
Seed culture: spore suspension (concentration 10) 8 cell/mL) was inoculated into a 50L seed tank containing 35L of Trichoderma seed medium, 10-seed 9 Culturing at 30deg.C, pH 4.8, and 210rpm for about 30 hr, monitoring mycelium Optical Density (OD) value at 650nm wavelength with spectrophotometer (JH-13-12, shanghai Beijing technology and instruments Co., ltd.) every 2-3 hr until OD reaches 7-8, and inoculating to fermentation medium.
Fermentation culture: the optical density of the liquid of the overnight cultured Trichoderma strain was measured, and when the optical density reached 7-8, the culture was transferred to Trichoderma fermentation medium (500L) for fermentation culture (the transfer amount was 10%), the fermentation culture was carried out for 8 days, the filter paper enzyme activity was measured from day 2, and the filter paper enzyme activity was recorded for 2-8 days, as shown in FIG. 4, and the feed was continuously fed during the fermentation culture.
As a result, the final filter paper enzyme activity result of the gene knockout strain A8 is 15.5FPU/mL, the final enzyme activity result of Trichoderma reesei RUT-C30 is 10.30FPU/mL, and the enzyme activity is improved by 50.5%.
According to the same method, positive transformants with successful gene knockout of pCRISPR-ace1 (B), pCRISPR-ace1 (C) and pCRISPR-ace1 (D) plasmids are subjected to shake flask fermentation, mutant strains with highest enzyme activities in the positive transformants are respectively B20 (6.3 FPU/mL), C9 (6.1 FPU/mL) and D10 (5.5 FPU/mL), the strains are subjected to amplified pilot fermentation to produce enzymes, the final filter paper enzyme activities are respectively 14.9FPU/mL, 14.3FPU/mL and 13.5FPU/mL, the enzyme activity improvement rates are respectively 44.7%, 38.8% and 31.1%, and the filter paper enzyme activities on days 2-8 of fermentation are shown in FIG. 4.
The unexpected discovery of the technical staff in the experiment is that the enzyme production effect of the trichoderma reesei RUT-C30 ace1 gene knockout mutant strain obtained by the final screening is better by adding 0.5-1.5g/L betaine and 10-15mL/L dandelion extract on the basis of the conventional screening culture medium. In order to optimize the optimal addition ratio, the present invention sets the following 3 concentrations of the enhancement screening medium.
| Enhanced selection of culture medium species | Adding components into screening culture medium |
| Culture medium I | 0.5g/L betaine, 10mL/L dandelion extract |
| Medium II | 1g/L betaine, 10mL/L dandelion extract |
| Culture medium III | 1.5g/L betaine, 15mL/L dandelion extract |
Taking the plasmid pCRISPR-ace1 (a) prepared in example 2 as an example, taking Trichoderma reesei RUT-C30 as an original strain, transforming protoplast of Trichoderma reesei RUT-C30 with the pCRISPR-ace1 (a) plasmid, respectively coating the transformants in the 3 enhanced screening media, growing for 5-7 days, respectively picking 50 transformants, transferring to a Trichoderma agar medium for propagation, and scraping spores to obtain spore suspension. The spore suspension was inoculated to trichoderma seed medium for overnight culture to obtain hyphae.
Using the obtained DNA of the mycelium as a template DNA, the success rate of gene knockout was examined by the same method as in example 3, and the target bands were found by PCR amplification and agarose gel electrophoresis, the amounts of which are shown in the following table. And sequencing the PCR products on the bands, wherein the product sequences detected as a result can be completely compared with the target fragments on the plasmids, and the transformants corresponding to the target bands are positive transformants. And then verifying whether the ace1 gene is knocked out successfully for the positive transformants, and obtaining positive transformants with successful gene knockout by the same method as that of example 3, wherein the number of the positive transformants with successful gene knockout is shown in the following table.
TABLE 3 Table 3
As can be seen from the data in the table, compared with the original screening culture medium, positive conversion rate is improved when betaine and dandelion leaching solutions with different concentrations are added into the screening culture medium, mainly because part of false positive hygromycin resistant strains can be eliminated due to sterilization effect along with the increase of the addition amount of the betaine and the dandelion, more strains containing hygromycin resistant genes are selected from the selected transformants, and the detected positive conversion rate is higher. The subsequent gene detection of the positive transformant shows that the success rate of gene knockout is unchanged. It was demonstrated that the addition of betaine and dandelion extract to the screening medium did not affect the expression level of the gRNA expression cassette, as well as the binding capacity to Cas9.
The positive transformant with successful gene knockout is subjected to shake flask fermentation culture, the culture method is shown by shake flask enzyme yield detection in the above effects, fermentation culture lasts for 8 days, filter paper enzyme activity is measured from day 2, and the highest filter paper enzyme activity on day 8 is recorded. Wherein, the best positive transformant corresponding to the enhanced screening medium I is I2, and the final enzyme activity result is 6.8FPU/mL. The best positive transformant corresponding to the enhanced selection medium II was II 18, and the final enzyme activity was 7.0FPU/mL. The best positive transformant corresponding to the enhanced selection medium III was III 9, and the final enzyme activity was 6.6FPU/mL.
The best positive transformant is subjected to pilot fermentation, the specific method is shown in the test enzyme yield detection in the effect example, the result is shown in figure 5, the final filter paper enzyme activity result of I2 is 15.7FPU/mL, the final filter paper enzyme activity result of II 18 is 16.0FPU/mL, and the final filter paper enzyme activity result of III 9 is 15.6FPU/mL.
The skilled person considers that the enzyme production capacity of the positive transformant obtained by final screening is improved by adding betaine and dandelion leaching liquid into the screening culture medium, and compared with the enzyme production amount trend in fig. 4, the enzyme production increase trend of trichoderma reesei obtained by enhancing the screening culture medium is better in 2-5 days. In fact, at the beginning of the experiment, because betaine and dandelion have certain bactericidal effects, the inventor wants to play a long-acting and mild antibacterial role on a screening culture medium by adding betaine and dandelion leaching liquid. However, the inventors have unexpectedly found that betaine and dandelion leachate have the function of increasing trichoderma reesei enzyme production. The reason is that the antibacterial effect of dandelion can enhance the activity of trichoderma reesei, and the leaching solution contains various trace nutrients such as B vitamins, vitamin C, E, iron and the like which are lacking in the original culture medium, so that benign growth of trichoderma reesei is promoted. Meanwhile, the dandelion leaching solution contains a carbon source and a nitrogen source to provide nutrients for the growth of trichoderma reesei. Betaine also has bactericidal effect, enhances the stress resistance of Trichoderma reesei positive transformants, optimizes away Trichoderma reesei seeds with weak ability, and ensures that the screened Trichoderma reesei has better activity and stronger enzyme production capability. Wherein, when the concentration of betaine is 1g/L and the concentration of dandelion extract is 10mL/L, the screening effect on Trichoderma reesei is optimal.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Sequence listing
<110> institute of plant Material at national academy of sciences
SHANGHAI HANHE BIOLOGICAL NEW MATERIAL TECHNOLOGY Co.,Ltd.
<120> CRISPR/Cas Gene editing System for Trichoderma reesei
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<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
ctctccatga tggtgattcc 20
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
ggaaccgact gcgaacatgg 20
<210> 15
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
ccgtcatcag tcagaagtcg 20
Claims (2)
1. A screening method of trichoderma reesei for CRISPR/Cas gene editing, the method comprising: (1) Gene editing of trichoderma reesei using a CRISPR/Cas gene editing system, which is CRISPR/Cas9, CRISPR/nCas9 or CRISPR/dCas9, comprising a gRNA expression cassette having the following structure from 5'-3' and coating transformants into a screening medium for screening: A-B-C-D, wherein A is a viral sequence with an initial gRNA transcription function, B is gRNA, C is a gRNA backup, D is a hepatitis virus ribozyme sequence, and the viral sequence with the initial gRNA transcription function is one of a tobacco circovirus sequence, a chopsticks mustard mosaic virus sequence, a chicory yellow spot virus sequence and a hammerhead virus sequence; (2) selecting transformants; (3) screening to obtain a positive transformant; (4) screening to obtain a positive transformant with successful gene knockout; (5) obtaining high-yield trichoderma reesei through shake flask fermentation screening; the screening culture medium in the step (1) is an enhanced screening culture medium, wherein 0.5-1.5g/L betaine and 10-15mL/L dandelion extract are added into the components of the screening culture medium;
the tobacco circovirus sequence is shown as SEQ ID NO. 1; the sequence of the chopstick mustard mosaic virus is shown as SEQ ID NO. 2; the sequence of the chicory yellow spot virus is shown as SEQ ID NO. 3; the sequence of the hammerhead virus is shown as SEQ ID NO. 4.
2. The method according to claim 1, wherein the enhanced screening medium is prepared by adding 1g/L betaine and 10mL/L dandelion extract to the components of the screening medium.
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| EP3199632A1 (en) * | 2016-01-26 | 2017-08-02 | ACIB GmbH | Temperature-inducible crispr/cas system |
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| Title |
|---|
| Sonia Salazar-Cerezo等.CRISPR/Cas9 technology enables the development of the filamentous ascomycete fungus Penicillium subrubescens as a new industrial enzyme producer.《Enzyme Microb Technol》.2019,摘要和图1D. * |
| 顾赛红,孙建义,李卫芬,许梓荣.里氏木霉(Trichoderma reesei)产β-葡聚糖酶和木聚糖酶的条件研究.浙江大学学报(农业与生命科学版).2003,(05),摘要. * |
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