WO2021139779A1 - 一种优化的真菌双体外基因组编辑方法 - Google Patents

一种优化的真菌双体外基因组编辑方法 Download PDF

Info

Publication number
WO2021139779A1
WO2021139779A1 PCT/CN2021/070906 CN2021070906W WO2021139779A1 WO 2021139779 A1 WO2021139779 A1 WO 2021139779A1 CN 2021070906 W CN2021070906 W CN 2021070906W WO 2021139779 A1 WO2021139779 A1 WO 2021139779A1
Authority
WO
WIPO (PCT)
Prior art keywords
sgrna
transformation
concentration
inositol
gene
Prior art date
Application number
PCT/CN2021/070906
Other languages
English (en)
French (fr)
Inventor
周志华
邹根
肖美丽
柴顺星
朱志华
Original Assignee
中国科学院分子植物科学卓越创新中心
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院分子植物科学卓越创新中心 filed Critical 中国科学院分子植物科学卓越创新中心
Publication of WO2021139779A1 publication Critical patent/WO2021139779A1/zh

Links

Images

Classifications

    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H15/00—Fungi; Lichens
    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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
    • C12N9/10—Transferases (2.)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/0201—Orotate phosphoribosyltransferase (2.4.2.10)

Definitions

  • the present invention belongs to the field of biotechnology; more specifically, the present invention relates to an optimized method for in vitro genome editing of fungi, in particular to a method for using compound molecules to improve the efficiency of fungal genome editing and its application in filamentous fungi.
  • Genome editing technology is currently a commonly used biotechnology in life science research. Using this technology, scientists can perform targeted knockout and insertion of target genes, which is an important tool for functional genome research.
  • ZFNs zinc finger nucleases
  • TALEN transcription activator-like effector nucleases
  • CRISPR/Cas9 CRISPR/Cas9 technology has the advantages of simple operation, good specificity, and simultaneous multi-site editing, making it the main genome editing technology in the current laboratory.
  • the CRISPR/Cas (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system is discovered from the natural immune system of bacteria. Its main function is to recognize invading viruses or other foreign DNA and degrade them. This process mainly includes the synthesis of crRNA and the recognition and cutting of DNA sites under the guidance of crRNA, so this system is transformed into an effective genome editing system. According to the different functional elements of this genome editing system, the CRISPR/Cas system can be divided into three categories, of which category I and III require multiple CRISPR-related proteins (Cas proteins) to work together, while the category II system requires only one Cas protein That is to say, this provides convenient conditions for its wide application. At present, Cas9 from Streptococcus pyogenes is the most widely used in CRISPR/Cas genome editing technology.
  • the CRISPR/Cas9 system mainly includes three parts: crRNA (CRISPR RNA), tracrRNA (trans-activating RNA), and Cas9 (CRISPR associated system).
  • CrRNA and tracrRNA can form chimeric RNA, called sgRNA, which can recognize and target the 20bp sequence upstream of PAM (protospacer advanced motif, the sequence is 5'-NGG-3') and is guided by sgRNA , Cas9 cuts the DNA double strands at this site.
  • the sgRNA in the CRISPR/Cas9 system is now formed by folding a single nucleotide containing crRNA and tracrRNA, which can be formed by transcription in vitro or in vivo.
  • Fungi are a kind of microorganisms closely related to humans. In addition to their huge edible and medicinal value, they are also widely used in industrial development. Therefore, the establishment of an efficient genome editing system in fungi is beneficial to its research and application.
  • Liu successfully established the CRISPR/Cas9 system in Trichoderma reesei and achieved efficient gene knockout. This is the first time that the CRISPR/Cas9 system has been established in a filamentous fungus. Subsequently, the CRISPR/Cas9 system was successfully established in other filamentous fungi.
  • the main methods include: (1) expressing Cas9 protein in the starting strain, transcribing sgRNA in vivo or directly introducing sgRNA synthesized in vitro; (2) constructing Cas9 and sgRNA on a plasmid, and introducing them into the starting strain to edit the target gene; 3) The sgRNA synthesized in vitro and Cas9 are formed into a complex and then introduced into the starting strain for gene editing (dual in vitro CRISPR/Cas9 system). With the further development of CRISPR/Cas9 technology and the gradual deepening of people's understanding of it, it will be applied in more organisms and more research results will be obtained.
  • CRISPR/Cas9 there are still many problems in the application of CRISPR/Cas9 technology, such as low genome editing efficiency, off-target, PAM dependence, etc.; and for polykaryotes such as Aspergillus oryzae, ergot, etc., it needs to be passaged after genetic manipulation. Only by screening to obtain homozygous transformants, this is a time-consuming and laborious task. In order to enable such organisms or more fungi to be better used in scientific research and actual production, it is necessary to establish an efficient CRISPR/Cas gene editing system.
  • the purpose of the present invention is to provide an optimized method for in vitro genome editing of fungi, and reagents and kits for the method.
  • a method for improving fungal protoplast transformation efficiency or gene editing efficiency including: mixing sgRNA and Cas enzyme to obtain a sgRNA-Cas enzyme complex, and transforming the protoplast; During the transformation, add: TritonX-100 or its analogue, a mitotic inhibitor, or inositol or its analogue.
  • the mitosis-inhibiting bacteriostatic agent includes an agent selected from the group consisting of benomyl, carbendazim, thiophanate-methyl, or analogs thereof; or the inositol analog includes : Phosphatidylinositol.
  • the protoplast transformation before the protoplast transformation (preferably, for multinucleate filamentous fungi), it further includes: adding the mitotic inhibitor during the sporulation, spore germination stage and/or protoplast regeneration stage of the fungus Bacteriostatic agent, or the inositol or its analogue.
  • the concentration of the TritonX-100 or its analogue in the conversion system is 0.00001 to 0.1% (w/v); preferably 0.0005 to 0.05%; more preferably 0.001 to 0.02 %; such as 0.002%, 0.004%, 0.006%, 0.008%, 0.01%, 0.015%, etc.
  • the concentration of the mitosis-inhibiting bacteriostatic agent in the transformation system is: 0.008-10ug/ml, preferably 0.01-5ug/ml; such as 0.02ug/ml, 0.05ug/ml, 0.08ug/ml, 0.1ug/ml, 0.2ug/ml, 0.4ug/ml, 0.5ug/ml, 0.8ug/ml, 1ug/ml, 1.5ug/ml, 2ug/ml, 3ug/ml, 4ug/ml .
  • the concentration of the inositol or its analogue in the conversion system is: 0.006% to 0.8%, preferably 0.01% to 0.5%; such as 0.02%, 0.05%, 0.1%, 0.2 %, 0.3%, 0.4%.
  • sgRNA and Cas enzyme are used for gene editing, and the sgRNA is targeted to the site to be edited in the fungal genome; preferably, sgRNA and Cas enzyme are mixed Prepare sgRNA-Cas enzyme complex and transform protoplasts.
  • the concentration of the sgRNA-Cas enzyme complex in the transformation system is 80-300 nM; such as preferably 90-250 nM; more preferably 100-200 nM, such as 120, 140, 160, 180, 190nM.
  • sgRNA and Cas enzyme are mixed at a molar ratio of 1:1 to 120:1, preferably 5:1 to 100:1, more preferably 10:1 to 95:1, and more preferably 20: 1 to 90:1, such as 30:1, 40:1, 50:1, 60:1, 70:1, 80:1; preferably, the Cas enzyme is Cas9 enzyme.
  • the amount of protoplasts in the transformation system is 1 ⁇ 10 4 pieces/ml ⁇ 1 ⁇ 10 9 pieces/ml; preferably 1 ⁇ 10 5 pieces/ml ⁇ 1 ⁇ 10 8 pieces/ml ; Such as 5 ⁇ 10 5 pieces/ml, 1 ⁇ 10 6 pieces/ml, 5 ⁇ 10 6 pieces/ml, 1 ⁇ 10 7 pieces/ml or 5 ⁇ 10 7 pieces/ml, etc.
  • TritonX-100 or its analogue a mitotic inhibitory bacteriostatic agent, or the use of inositol or its analogue to: improve fungal protoplast transformation efficiency or gene editing efficiency; or A composition for improving the efficiency of fungal protoplast transformation or gene editing is prepared.
  • the transformation of the protoplasts into PEG mediates the transformation of protoplasts.
  • kits for fungal protoplast transformation which includes: sgRNA, Cas enzyme, or their complex targeted to the fungal genome; a compound selected from: TritonX-100 or Its analogs, mitotic inhibitors, or inositol or its analogs, or a combination thereof.
  • reagents selected from the following group: fungal culture medium (such as PDA medium, SDB medium, CD medium, etc.); protoplast solution (such as a solution containing sorbitol and KH 2 PO 4 ) , A solution containing sorbitol, CaCl 2 , Tris-HCl, a solution containing PEG6000, CaCl 2 , Tris-HCl, a solution containing NaCl, KH 2 PO 4 ); protoplast cleaning solution; trace elements; genome extraction reagents.
  • fungal culture medium such as PDA medium, SDB medium, CD medium, etc.
  • protoplast solution such as a solution containing sorbitol and KH 2 PO 4
  • protoplast cleaning solution such as trace elements; genome extraction reagents.
  • the concentration of the sgRNA-Cas enzyme complex is 80-300 nM.
  • the concentration of the TritonX-100 or its analogue is 0.00001 to 0.1%; preferably 0.0005 to 0.05%; more preferably 0.001 to 0.02%;
  • the concentration (w/v) of the inositol or its analogue is 0.006% to 0.8%, preferably 0.01% to 0.5%.
  • the concentration of the mitosis-inhibiting bacteriostatic agent is 0.008-10ug/ml, preferably 0.01-5ug/ml.
  • composition for transformation comprising: an sgRNA-Cas enzyme complex and an effective amount of TritonX-100 or its analogues, a mitotic inhibitory bacteriostatic agent, or inositol or its Analog; preferably, the concentration of the sgRNA-Cas enzyme complex is 80-300nM; or the concentration of TritonX-100 or its analog is 0.00001-0.1%; preferably 0.0005-0.05%; Preferably, it is 0.001 to 0.02%; the concentration of the inositol or its analog is 0.006% to 0.8%, preferably 0.01% to 0.5%; or the concentration of the mitotic inhibitor is 0.008 to 10ug/ ml, preferably 0.01-5ug/ml.
  • composition for transformation further includes: other transformation reagents, or cytologically acceptable carriers.
  • kit or the composition for transformation is provided for: improving fungal protoplast transformation efficiency or gene editing efficiency; or performing fungal protoplast transformation or Gene editing.
  • the fungus is a polynuclear fungus or a filamentous fungus; preferably, the fungus is a fungus of the phylum Ascomycota; preferably, the fungus includes (but not limited to): Trichoderma fungi, Cordyceps fungi, Aspergillus fungi; more preferably, the fungi include (but are not limited to): Trichoderma reesei, Cordyceps militaris, Aspergillus oryzae, Myceliophthora thermophila (Myceliophthora thermophila), Aspergillus niger (A.niger), Penicillium chrysogenum, Ganoderma lucidum, Lentinus edodes, Pleurotus ostreatus, Agaricus bisporus, Flammulina velutipes (Flammulina velutipes), Hypsizygus marmoreus (H
  • the sgRNA is targeted to: the ura5 gene of Trichoderma reesei; preferably, the target gene recognition site has the nucleotides shown at positions 355 to 374 in SEQ ID NO:1 Sequence; the ura5 gene of Cordyceps militaris; preferably, the target gene recognition site has the nucleotide sequence shown at positions 492 to 511 in SEQ ID NO: 2; the AowA gene of Aspergillus oryzae; preferably, the target gene recognition The site has the nucleotide sequence shown at positions 160 to 179 in SEQ ID NO: 3.
  • Lane M in the figure is the result of DS-2000 DNA Marker electrophoresis (the fragments are 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom); lanes 1 and 2 are sgRNA in vitro transcription products.
  • FIG. 4 Schematic diagram of PCR verification results of CRISPR/Cas9 mutant wA transformants of Aspergillus oryzae in vitro.
  • Lane M in the figure is 1kb DNA Ladder (fragments are 12000bp, 8000bp, 6000bp, 5000bp, 4000bp, 3000bp, 2500bp, 2000bp, 1500bp, 1000bp, 500bp from top to bottom)
  • lane 1 is the ⁇ wA homozygous mutant transformant ( There is only one band of about 3000 bp)
  • lane 2 is the ⁇ wA heterozygous mutant transformant (there are 2 bands, respectively the mutant 3000 bp and the wild type 300 bp)
  • the lane 3 is the wild type (about 300 bp).
  • Figure 5 The genetic operation process of Aspergillus oryzae with the addition of inositol or benomyl. That is, in the process of protoplast-mediated transformation of Aspergillus oryzae, inositol or benomyl is added at the three stages of sporulation, spore germination, and protoplast regeneration to increase the probability of Aspergillus oryzae monocytes, thereby increasing homozygous transformants. Probability.
  • TritonX-100 or its analogues are added during or before the transformation of protoplasts for the purpose of gene editing, a mitotic inhibitor, or inositol or its analogues; more preferably, it also regulates sgRNA-
  • concentration of Cas enzyme effectively improves the fungal protoplast transformation efficiency and gene editing efficiency, saves a lot of time for obtaining transformants or gene editing products, and increases the yield.
  • the present invention provides a method for improving fungal protoplast transformation efficiency or gene editing efficiency, including: using CRISPR/Cas gene editing method to target the site to be edited in the fungal genome to perform gene editing; wherein CRISPR/Cas gene editing method is used for gene editing;
  • CRISPR/Cas gene editing method is used for gene editing;
  • the Cas gene editing reagent performs protoplast transformation, add: TritonX-100 or its analogue, a mitotic inhibitor, or inositol or its analogue.
  • the fungus is a polynucleated fungus.
  • the fungus is a filamentous fungus.
  • the fungus is a fungus of the phylum Ascomycota; preferably, the fungus includes: Trichoderma fungi, Cordyceps fungi, and Aspergillus fungi.
  • the fungi include: Trichoderma reesei, Cordyceps militaris, Aspergillus oryzae, Myceliophthora thermophila, Aspergillus niger, Penicillium chrysogenum, Ganoderma lucidum, Lentinus edodes, Pleurotus ostreatus, Agaricus bisporus, Flammulina velutipes, Hypsizygus marmoreus and so on. It should be understood that although these strains are preferably used as examples in the embodiments of the present invention, the technical solution of the present invention can also be applied to other fungi.
  • the present inventors also found that adding inositol or benomyl to the medium during the sporulation, spore germination or protoplast regeneration stages of fungi, especially polykaryon fungi, can improve the efficiency of obtaining homozygous transformants.
  • the inositol is a growth factor.
  • the benomyl is a bactericide, which can inhibit the filamentation of cells.
  • the TritonX-100 or its analogues include reagents selected from the group consisting of: TritonX-100 or its analogues, among which TritonX-100 is preferred; and the mitotic inhibitory bacteriostatic agent Including reagents selected from the group consisting of benomyl, carbendazim, thiophanate-methyl, or their analogs, preferably benomyl; or the inositol or its analogs include reagents selected from the following : Inositol, Phosphatidylinositol.
  • the present invention also includes isomers, solvates, precursors or salts of the above-mentioned compounds, as long as they also have the same or substantially the same function as the compound.
  • the “salt” refers to the salt formed by the reaction of a compound with inorganic acid, organic acid, alkali metal or alkaline earth metal.
  • These salts include (but are not limited to): (1) salts formed with the following inorganic acids: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts formed with the following organic acids, such as acetic acid, oxalic acid, succinic acid, and tartaric acid , Methanesulfonic acid, maleic acid, or arginine.
  • salts include those formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium), as well as esters and carbamates.
  • the compound has one or more asymmetric centers. Therefore, these compounds can exist as racemic mixtures, individual enantiomers, individual diastereomers, mixtures of diastereomers, cis or trans isomers.
  • the "precursor of the compound” means that when added by a suitable method, the precursor of the compound is converted into the compound or its active form (such as an active salt or isomer) in a transformation system or a culture system. .
  • the compound of the present invention can be obtained by a variety of methods well known in the art and using known raw materials, such as chemical synthesis or from biological (such as animal or plant) The methods of extraction or modification on the basis of extraction in ), these methods are all included in the present invention.
  • the synthesized compound can be further purified by column chromatography, high performance liquid chromatography and other methods.
  • the compound of the present invention can also be obtained commercially.
  • the present invention also provides a composition for transformation, containing an effective amount of TritonX-100 or its analogs, a mitotic inhibitor or inositol, or their isomers , Solvates, precursors, and sgRNA-Cas enzyme complexes.
  • the present invention also provides a composite medium, which includes: a fungal culture medium and an effective amount of TritonX-100 or its analogs, a mitotic inhibitor or inositol; preferably
  • the fungal culture medium is a culture medium used for fungal sporulation, spore germination and protoplast regeneration stages.
  • the culture medium for the fungal sporulation or spore germination stage may be a medium known in the art.
  • the "contains”, “having” or “including” includes “including”, “mainly composed of”, “essentially composed of”, and “consisted of”; Mainly composed of", “basically composed of” and “consisted of” belong to the subordinate concepts of "containing", “having” or “including”.
  • the inventors mixed the sgRNA-Cas enzyme complex obtained by mixing sgRNA and Cas enzyme with protoplasts, but found that the transformation efficiency is extremely low, and in some cases it is even difficult to obtain effective Transformants.
  • the inventors analyzed and tested the possible factors affecting the transformation and found that the concentration of the sgRNA-Cas enzyme complex has a greater impact on the transformation efficiency.
  • the present inventors optimized the concentration of the sgRNA-Cas enzyme complex in the transformation system.
  • the concentration of the sgRNA-Cas enzyme complex in the transformation system is 80-300 nM, preferably 90%. ⁇ 250 nM, more preferably 100 ⁇ 200 nM, such as 120, 140, 160, 180, 190 nM.
  • sgRNA and Cas enzyme are mixed at a molar ratio of 1:1 to 120:1.
  • the applicable Cas enzymes include Cas9, Cas9n, Cas13a, Cas13d, CasRx, etc., and these Cas can be applied in the present invention.
  • the Cas enzyme is Cas9.
  • variants, derivatives, active fragments or fusion forms of these enzymes can also be applied to the present invention.
  • the sgRNA is a sequence designed for the site to be edited in the genome.
  • the sgRNA is designed for a sequence region with a length of about 15-30 bp (for example, about 20 bp) in the site region of the target gene to be edited. According to the specific needs of targeted editing, those skilled in the art can also design sgRNAs with different sequence structures and different lengths.
  • a donor sequence (Donor) that matches the sgRNA can also be designed.
  • the present invention also provides a kit for fungal protoplast transformation, which includes: sgRNA targeting the fungal genome, Cas enzyme, or their complex; a compound selected from: TritonX-100 or its analogues, mitosis inhibiting bacteriostatic agent, or inositol or its analogues, or a combination thereof.
  • the kit also includes reagents selected from the following group: fungal culture medium (such as PDA medium, SDB medium, CD medium, etc.); protoplast solution (such as containing sorbitol, KH 2 PO 4 solution, solution containing sorbitol, CaCl 2 , Tris-HCl, solution containing PEG6000, CaCl 2 , Tris-HCl, solution containing NaCl, KH 2 PO 4 ); protoplast cleaning solution; trace element ; Gene extraction reagents. It should be understood that other reagents conventionally used for protoplast transformation, cleaning, stabilization, etc., reagents used for fungal culture, preparation of protoplasts, etc. may also be included in the kit.
  • fungal culture medium such as PDA medium, SDB medium, CD medium, etc.
  • protoplast solution such as containing sorbitol, KH 2 PO 4 solution, solution containing sorbitol, CaCl 2 , Tris-HCl, solution containing PEG6000, CaCl 2
  • kit of the present invention may also include instructions for use that explain the usage and dosage, operation steps, etc. of each reagent therein.
  • sgRNAs targeting the following genes are designed: the ura5 gene of Trichoderma reesei, the ura5 gene of Cordyceps militaris, and the AowA gene of Aspergillus oryzae.
  • concentration regulation of the sgRNA-Cas enzyme complex in the transformation system and the regulation using TritonX-100 inositol or benomyl, all show significant technical effects of improving transformation efficiency and improving gene editing efficiency.
  • the Trichoderma reesei is a fungus of the genus Trichoderma in the phylum Ascomycota. As an industrial strain, it is used as an enzyme for decomposing plant materials, including cellulase, hemicellulase, protease, amylase and the like.
  • the Cordyceps militaris belongs to the genus Cordyceps of Ascomycota and has important medicinal value.
  • Said Aspergillus oryzae is a fungus of the Aspergillus genus Ascomycota.
  • As a food-safe strain commonly used in food brewing it has multinucleate characteristics, which enables it to maintain genetic stability, but also makes genome editing changes. It is more difficult to get a homozygous transformant.
  • Ura5 is a gene encoding orotate phosphoribosyltransferase in organisms and is involved in uracil synthesis and metabolism. Because of its bidirectional selection (ura5 cannot grow under conditions containing 5-FOA; ura5 - cannot grow on minimal medium, additional uridine or uracil needs to be added), it is often used as a selection marker in the process of genetic manipulation.
  • the wA gene is a polyketide synthase gene, which is involved in the synthesis of Aspergillus oryzae conidia pigment.
  • the argB gene is involved in the biosynthesis of arginine. In the present invention, it is used as an auxotrophic screening marker to knock out the wA gene.
  • the sequence of Trichoderma reesei ura5 has the nucleotide sequence shown in SEQ ID NO:1; the sequence of Cordyceps militaris ura5 (Cmura5) has the nucleotide sequence shown in SEQ ID NO: 2; Aspergillus oryzae wA( The AowA) sequence has the nucleotide sequence shown in SEQ ID NO: 3; the Aspergillus oryzae argB sequence has the nucleotide sequence shown in SEQ ID NO: 4.
  • the technical scheme of the present invention can be modified for the above-mentioned useful strains in industrial, pharmaceutical or food sciences, and reflects its good application value.
  • the present invention also provides a preferred target gene recognition site for the ura5 gene of Trichoderma reesei, which has the nucleotide sequence shown in SEQ ID NO:1; for the ura5 gene of Cordyceps militaris
  • the preferred target gene recognition site which has the nucleotide sequence shown in SEQ ID NO: 1; and the preferred target gene recognition site for the AowA gene of Aspergillus oryzae, which has the nucleotide sequence shown in SEQ ID NO: 4 Nucleotide sequence.
  • the sgRNA designed for the recognition sites of these target genes after being complexed with the Cas enzyme, presents an ideal gene editing effect.
  • the transformation and gene editing method of the present invention can obtain more transformants at one time and the gene editing efficiency is very high, saves a lot of time, and may reduce the probability of off-target. It is useful for fungal genome editing, gene function research and molecular breeding. Here comes a great convenience.
  • the sgRNA-Cas enzyme complex is a mixture of nucleic acid and protein (double in vitro CRISPR/Cas9 system). There are problems of low efficiency and unsatisfactory gene editing effect during transformation.
  • the present invention adds appropriate compounds And/or optimize the concentration of sgRNA-Cas9, which effectively improves the efficiency of genome editing in vitro, and can make the genome editing efficiency reach 100%.
  • sgRNA-F TAATACGACTCACTATA-sgRNA site-GTTTTAGAGCTAGAAAT AGCAA.
  • the sgRNA-F sequence designed for Trichoderma reesei RutC30 orotate phosphoribosyltransferase ura5 (SEQ ID NO:1) is:
  • sgRNA-Trura5-F TAATACGACTCACTATA GGCGAGGGCGGCAACAT CGT GTTTTAGAGCTAGAAATAGCAA (SEQ ID NO: 5).
  • the sgRNA-F sequence designed for ura5 (SEQ ID NO: 2) of Cordyceps militaris Cm01 is:
  • sgRNA-Cmura5-F TAATACGACTCACTATA GGACCGGATGGAGAAG CTTC GTTTTAGAGCTAGAAATAGCAA (SEQ ID NO: 6).
  • the sgRNA-F sequence designed for the polyketide synthase wA of Aspergillus oryzae NSAR1 is:
  • sgRNA-wA-F TAATACGACTCACTATA GGGAAGATCTTCCGTTCAGA GTTTTAGAGCTAGAAATAGCAA (SEQ ID NO: 7).
  • sgRNA-F primers are PCR amplified with sgRNA-R primers, and the sgRNA template is recovered; the sequence of the sgRNA-R primers is as follows:
  • sgRNA-R AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAA (SEQ ID NO: 8).
  • the purified DNA as a template, use the in vitro transcription kit (HiScribe TM Quick T7 High Yield RNA Synthesis Kit, NEB#E2050S, refer to the kit instructions for specific methods) overnight at 37°C to obtain the crude sgRNA product, which is subjected to DNA gel electrophoresis After verification ( Figure 1), the sgRNA was purified using the same method as the purified sgRNA template, and the dried sgRNA was dissolved in 5ul Nuclease-free water and stored at -80°C.
  • HiScribe TM Quick T7 High Yield RNA Synthesis Kit NEB#E2050S, refer to the kit instructions for specific methods
  • the purified sgRNA and commercialized Cas9 were added to the reaction buffer at a ratio of 70:1 (moles).
  • the reaction buffer was 2 ⁇ protoplast solution 2 (2M sorbitol, 100mM CaCl) 2 , 2M Tris-HCl, pH 7.5) and 10 ⁇ active buffer (200mM HEPES free acid, 1.5M KCl, 5mM DTT, 1mM EDTA, 100mM MgCl 2 ⁇ 6H 2 O, pH 7.5), mix well and incubate at 37°C for 15min .
  • the sgRNA-Cas9 assembly method of the ura5 gene of Trichoderma reesei The purified sgRNA-Trura5 and Cas9 were added to the reaction system at a ratio of 70:1, and incubated at 37°C for 15 minutes. And by controlling the amount of sgRNA and Cas9 added, 20nM, 60nM, 100nM, and 180nM sgRNA-Cas9 complexes were prepared respectively.
  • the sgRNA-Cas9 assembly method of Cordyceps militaris ura5 gene The purified sgRNA-Cmura5 and Cas9 are added into the reaction system at a ratio of 70:1 (Cas9 is 6ug), and incubated at 37°C for 15min.
  • the sgRNA-Cas9 assembly method of Aspergillus oryzae wA gene The purified sgRNA-AowA and Cas9 are added to the reaction system at a ratio of 70:1 (Cas9 is 6ug), and the 2 ⁇ protoplast solution 2 in the reaction buffer is replaced with 2 ⁇ solution 1 (1.6M NaCl, 20mM CaCl 2 , 20mM Tris-HCl, pH 8.0), incubate at 37°C for 15 min.
  • Trichoderma reesei Rut C30 as the chassis cell, the orotate phosphoribosyl transferase ura5 gene (SEQ ID NO:1; positions 355 to 374 are the sgRNA target sequence) using the dual in vitro CRISPR/Cas9 technology to mutate, using The sgRNA-Cas9 complex (sgRNA template is formed by PCR amplification of sgRNA-Trura5-F and sgRNA-R) is used to transform, and methods to increase the number of transformants obtained from gene transformation and improve the efficiency of gene editing are studied.
  • the sgRNA-Cas9 complex sgRNA template is formed by PCR amplification of sgRNA-Trura5-F and sgRNA-R
  • the preparation of protoplasts was modified according to the literature (Rantasalo, A.et al. A universal gene expression system for fungi. Nucleic Acids Res, 2018 46, e111).
  • the hyphae for preparing protoplasts were cultured in a cellophane-covered PDA at 28°C. Obtained in 15h, the concentration of lyase (sigma) is 5mg/ml.
  • Transformation system collect protoplasts by filtration and centrifugation. After washing 4ml protoplasts, 200ul protoplast solution 2 (1M sorbitol, 50mM CaCl 2 , 1M Tris-HCl, pH 7.5) is resuspended, and sgRNA-Cas9 complex is added, 74 ⁇ l protoplasts are added.
  • the genome is extracted, amplified with ⁇ Trura5-F/ ⁇ Trura5-R primers, and then sequenced and analyzed. A total of 12 samples of protoplast transformants (numbered 1-12) that had undergone the aforementioned transformation operations were tested, and wild-type Trura5 was used as a control.
  • Trichoderma reesei Rut C30 as the chassis cell, the orotate phosphoribosyl transferase ura5 gene (SEQ ID NO:1; positions 355 to 374 are the sgRNA target sequence) using the dual in vitro CRISPR/Cas9 technology to mutate, using sgRNA-Cas9 complex (sgRNA template formed by sgRNA-Trura5-F and sgRNA-R PCR amplification) was transformed, and the effect of sgRNA-Cas9 concentration on gene editing efficiency was detected.
  • sgRNA-Cas9 complex sgRNA template formed by sgRNA-Trura5-F and sgRNA-R PCR amplification
  • the ura5 gene mutation efficiency of Trichoderma reesei at different sgRNA-Cas9 complex concentrations is shown in Table 1. It can be seen that the gene editing efficiency increases with the increase of the sgRNA-Cas9 complex concentration, reaching 100% at 100nM and 180nM. It shows that the relatively high concentration of sgRNA-Cas9 complex is beneficial to improve the gene editing efficiency of the CRISPR/Cas9 technology in vitro.
  • Example 5 The compound TritonX-100 improves the efficiency of CRISPR/Cas9 gene editing in vitro
  • the ura5 gene (SEQ ID NO: 1 (Trura5, positions 355 to 374 are sgRNA target sequences), SEQ ID NO: 2 (Cmura5, where the 492 ⁇ 511th positions are the sgRNA target sequence) are used for mutation, respectively using sgRNA-Cas9 complex 1 (sgRNA template is formed by sgRNA-Trura5-F and sgRNA-R PCR amplification), sgRNA-Cas9 complex Substance 2 (sgRNA template formed by PCR amplification of sgRNA-Cmura5-F and sgRNA-R) was transformed, and the gene editing efficiency was improved by adding TritonX-100.
  • sgRNA-Cas9 complex 1 sgRNA template is formed by sgRNA-Trura5-F and sgRNA-R PCR amplification
  • sgRNA-Cas9 complex Substance 2 sgRNA template formed by PCR amplification of sgRNA-Cmur
  • Transformant verification The transformant genome was extracted, and the verification method for the ura mutant transformant of Trichoderma reesei was the same as in Example 3.
  • the genome of the ura5 mutant transformant of Cordyceps militaris was amplified by PCR ( ⁇ Cmura5-F: ATGTCGGATCTCGCTCAGTAC (SEQ ID NO: 12) ), ⁇ Cmura5-R: CTAATCTGCATCTTTCT CC (SEQ ID NO: 13)), all PCR products are sent to the company for sequencing.
  • TritonX-100 The effect of TritonX-100 on the mutation efficiency of Cordyceps militaris ura5 gene is shown in Table 3, which indicates that no TritonX-100 was added during the transformation of pupal grassland biotopes, and no transformants were obtained. However, after TritonX-100 was added, 12 transformants were obtained. , Each transformant has a mutation at the sgRNA recognition site, as shown in Figure 3.
  • Cmura5 is wild-type. Twelve samples (numbered 1-12) were sequenced, and it was found that all transformants were mutated at the target.
  • the target sequence was 5'-GGACCGGATG GAGAAGCTTC cgg -3' (underlined Is the PAM sequence) (SEQ ID NO: 18).
  • Example 6 Inositol or benomyl effectively increase the probability of homozygous transformants of the multinucleated filamentous fungus Aspergillus oryzae
  • the polyketide synthase wA (SEQ ID NO: 3; the 160th to 179th positions are the sgRNA target sequence) gene was mutated using the dual in vitro CRISPR/Cas9 technology, and the sgRNA- Cas9 complex (sgRNA template is formed by PCR amplification of sgRNA-wA-F and sgRNA-R) for transformation.
  • the sgRNA- Cas9 complex sgRNA template is formed by PCR amplification of sgRNA-wA-F and sgRNA-R
  • Protoplast preparation the CD medium ((NH 4 ) 2 SO 4 4.66g/l, KCl 0.52g/l, KH 2 PO 4 1.52g/l, MgSO 4 ⁇ 7H 2 O 1mol/l, glucose 10g/l 1, trace element 2 1/1000, pH 6.5, and add 0.1% (w/v) inositol or 1 ⁇ g/ml benomyl). Wash the spores with 0.85% NaCl-0.1% Tween 80 and take 1ml of spores Receive 100ml of CD liquid medium (containing 0.1% (w/v) inositol or 1 ⁇ g/ml benomyl) and cultivate at 30°C.
  • CD medium ((NH 4 ) 2 SO 4 4.66g/l, KCl 0.52g/l, KH 2 PO 4 1.52g/l, MgSO 4 ⁇ 7H 2 O 1mol/l, glucose 10g/l 1, trace element 2 1/1000, pH 6.5, and add 0.1% (w/v) in
  • Protoplast transformation 200ul Solution1 (0.8M NaCl, 10mM CaCl 2 , 10mM Tris-HCl, pH 8.0) resuspend the protoplasts, add sgRNA-Cas9 complex (final concentration is 180nM), donor-DNA and 20% volume of Solution2 (40% PEG4000 , 50mM CaCl 2 , 50mM Tris-HCl, pH 8.0.), stand on ice for 30 min. Add 1ml Solution2, mix well, let it stand at room temperature for 30min, add 9ml native Solution1, mix well and collect the protoplasts by centrifugation.
  • Solution1 0.8M NaCl, 10mM CaCl 2 , 10mM Tris-HCl, pH 8.0
  • 200ul Solution1 was resuspended and added to 50ml CD upper layer medium (CD medium containing 0.8M NaCl, and inositol (312.5 ⁇ l 160g/l inositol, final concentration of 0.1% (w/v)) or benomyl (10 ⁇ l 5mg/ml benomyl, the final concentration is 1 ⁇ g/ml), mix gently, pour it on the lower medium (2% agar), culture at 30°C for about 3 days, you can observe the growth of hyphae (m The whole process of Aspergillus protoplast transformation is shown in Figure 5).
  • Transformant verification pick the transformant and culture in SDB, extract the genome, PCR amplification ( ⁇ wA-F: AGACCAGACAAGTGATTTCG (SEQ ID NO: 16), ⁇ wA-R: CGTTTCCAAGGTCTCCGTAG (SEQ ID NO: 17)) DNA agarose gel electrophoresis detection (Figure 4), and sequencing verification.
  • Example 7 Other mitotic inhibitors effectively increase the efficiency of homozygous transformants of the multinucleated filamentous fungus Aspergillus oryzae
  • the polyketide synthase wA (SEQ ID NO: 3; the 160th to 179th positions are the sgRNA target sequence) gene was mutated using the dual in vitro CRISPR/Cas9 technology, and the sgRNA- Cas9 complex (sgRNA template is formed by PCR amplification of sgRNA-wA-F and sgRNA-R; final concentration is 180nM) for transformation.
  • the sgRNA- Cas9 complex sgRNA template is formed by PCR amplification of sgRNA-wA-F and sgRNA-R; final concentration is 180nM
  • mitotic inhibitors such as carbendazim and thiophanate-methyl
  • 2ug/ml thiophanate-methyl can also significantly promote the production of more mononuclear protoplasts by Aspergillus oryzae. Therefore, other mitotic inhibitors can also increase the homozygous positive transformants of the genome editing of multinucleated filamentous fungi, and accelerate the speed of gene editing of multinucleated filamentous fungi.
  • Example 8 The effect of other surfactants on the efficiency of CRISPR/Cas9 gene editing in vitro
  • Trichoderma reesei Rut C30 the orotate phosphoribosyltransferase ura5 gene (SEQ ID NO:1; positions 355 to 374 are the sgRNA target sequence) was mutated using dual in vitro CRISPR/Cas9 technology.
  • sgRNA-Cas9 complex sgRNA template is formed by PCR amplification of sgRNA-Trura5-F and sgRNA-R; the final concentration is 180nM
  • surfactants such as Tween-80, NP-40, PEG4000
  • the protoplast preparation, transformation, transformation system, and transformant verification method are the same as those in Example 3. The difference is that TritonX-100 added during protoplast transformation is replaced with 0.006% NP-40 or 0.1% Tween-80.
  • Trichoderma reesei ura5 gene The effect of these surfactants on the editing efficiency of Trichoderma reesei ura5 gene is shown in Table 6.
  • the number of transformants is relative to that without any surfactant.
  • Triton X-100 can increase the number of gene-edited transformants, but Tween80 and NP-40 have no effect on the number of gene-edited transformants at this concentration.
  • Trichoderma reesei Trura5 sequence (underlined indicates sgRNA target sequence) (SEQ ID NO: 1)
  • Cordyceps militaris Cmura5 sequence (underlined indicates sgRNA target sequence) (SEQ ID NO: 2)
  • Aspergillus oryzae AowA sequence (underlined indicates sgRNA target sequence) (SEQ ID NO: 3)

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Botany (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

本发明提供了优化的真菌原生质体转化技术以及基因编辑技术。通过在以基因编辑为目的的原生质体转化过程中或之前，添加TritonX-100或其类似物，抑制有丝分裂的抑菌剂，或肌醇或其类似物，有效地提高了真菌的原生质体转化效率及基因编辑效率。

Description

一种优化的真菌双体外基因组编辑方法 技术领域
本发明属于生物技术领域；更具体地，本发明涉及优化的真菌双体外基因组编辑方法，特别是涉及应用化合物分子提高真菌进行基因组编辑的效率的方法及其在丝状真菌中的应用。
背景技术
基因组编辑技术是当前生命科学研究中常用的生物技术，利用该技术，科学家们能够对目标基因进行定点敲除和插入，是功能基因组研究的重要工具。经过多年的发展，目前主要有锌指核酸酶(ZFNs)、类转录激活因子效应物核酸酶(TALEN)、CRISPR/Cas9三种基因组编辑技术。以上所述3种基因组编辑技术的原理都是相同的，即借助核酸酶使得基因组上特定位点的DNA双链发生断裂，从而激活细胞的天然修复机制(包括非同源末端连接和同源重组修复)，使得该位点发生各种突变。相较于ZFNs和TALEN技术，CRISPR/Cas9技术具有操作简单、特异性好、能同时进行多位点编辑等优势，使它成为当下实验室主要的基因组编辑技术。
CRISPR/Cas(Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein)系统是从细菌的天然免疫系统中发现的，它的主要作用是识别入侵的病毒或其他外源DNA，并对其降解。这一过程主要包括crRNA的合成与crRNA引导下的DNA位点识别与剪切，因此这个系统被改造成一个有效的基因组编辑系统。根据这个基因组编辑系统的功能元件不同，CRISPR/Cas系统可以分为3类，其中Ⅰ类和Ⅲ类需要多种CRISPR相关蛋白(Cas蛋白)共同发挥作用，而Ⅱ类系统只需要一种Cas蛋白即可，这为其能够广泛应用提供了便利条件。目前，来自Streptococcus pyogenes的Cas9在CRISPR/Cas基因组编辑技术中应用最为广泛。
CRISPR/Cas9系统主要包括crRNA(CRISPR RNA)、tracrRNA(trans-activating RNA)、Cas9(CRISPR associated system)三个部分。CrRNA和tracrRNA可以形成嵌合RNA，被称为sgRNA，它能识别并靶向PAM(protospacer adjacent motif，序列为5’-NGG-3’)位点上游的20bp序列，并在sgRNA的引导作用下，Cas9对该位点的DNA双链进行切割。经过CRISPR系统的不断改进，现在CRISPR/Cas9系统中的sgRNA是由一条包含crRNA和tracrRNA的核苷酸经折叠形成，它可以通过体外或体内转录形成。
真菌是一类与人类密切相关的微生物，除了具有巨大的食用和药用价值之外，还被广泛应用于工业开发，所以在真菌中建立高效的基因组编辑系统有利于它的研究和应用。2015年，Liu在里氏木霉中成功建立CRISPR/Cas9系统并实现对基因的高效敲除，这是首次在丝状真菌中建立CRISPR/Cas9系统。随后，CRISPR/Cas9系统在其他 丝状真菌中也被成功建立。主要方法包括：(1)在出发菌株中表达Cas9蛋白，体内转录sgRNA或直接导入体外合成的sgRNA；(2)将Cas9和sgRNA构建到质粒上，导入到出发菌株中对目的基因进行编辑；(3)将体外合成的sgRNA和Cas9形成复合物之后再导入出发菌株中进行基因编辑(双体外CRISPR/Cas9系统)。随着CRISPR/Cas9技术的进一步发展以及人们对其认知的逐渐深入，它将会在更多的生物体中应用，获得更多的研究成果。
但是，CRISPR/Cas9技术目前在应用中仍然存在许多问题，如基因组编辑效率低、脱靶、PAM依赖等；而对于多核生物如米曲霉、麦角菌等，对其进行遗传操作后还需要进行传代与筛选才能得到纯合转化子，这是一项费时费力的工作。为了能使此类生物体或更多的真菌更好的应用于科学研究和实际生产，建立一个高效的CRISPR/Cas基因编辑系统是必要的。
发明内容
本发明的目的在于提供优化的真菌双体外基因组编辑方法，用于该方法的试剂及试剂盒。
在本发明的第一方面，提供一种提高真菌的原生质体转化效率或基因编辑效率的方法，包括：将sgRNA与Cas酶混合获得sgRNA-Cas酶复合物，转化原生质体；其中，在进行原生质体转化时，添加：TritonX-100或其类似物，抑制有丝分裂的抑菌剂，或肌醇或其类似物。
在一个优选例中，所述的抑制有丝分裂的抑菌剂包括选自以下的试剂：苯菌灵、多菌灵、甲基硫菌灵，或其类似物；或所述的肌醇类似物包括：磷酸酰肌醇。
在另一优选例中，进行原生质体转化前(较佳地，对于多核丝状真菌)，还包括：在真菌的孢子形成、孢子萌发阶段和/或原生质体再生阶段，加入所述抑制有丝分裂的抑菌剂，或所述肌醇或其类似物。
在另一优选例中，所述的TritonX-100或其类似物在转化体系中的浓度为0.00001～0.1％(w/v)；较佳地为0.0005～0.05％；更佳地为0.001～0.02％；如0.002％，0.004％，0.006％，0.008％，0.01％，0.015％等。
在另一优选例中，所述的抑制有丝分裂的抑菌剂在转化体系中的浓度为：0.008～10ug/ml，较佳地0.01～5ug/ml；如0.02ug/ml，0.05ug/ml，0.08ug/ml，0.1ug/ml，0.2ug/ml，0.4ug/ml，0.5ug/ml，0.8ug/ml，1ug/ml，1.5ug/ml，2ug/ml，3ug/ml，4ug/ml。
在另一优选例中，所述的肌醇或其类似物在转化体系中的浓度为：0.006％～0.8％，较佳地0.01％～0.5％；如0.02％，0.05％，0.1％，0.2％，0.3％，0.4％。
在另一优选例中，所述CRISPR/Cas基因编辑方法中，采用sgRNA与Cas酶进 行基因编辑，所述sgRNA靶向于真菌基因组中待编辑位点；较佳地，将sgRNA与Cas酶混合制备sgRNA-Cas酶复合物，转化原生质体。
在另一优选例中，所述sgRNA-Cas酶复合物在转化体系中的浓度为80～300nM；如较佳地90～250nM；更佳地100～200nM，如120，140，160，180，190nM。
在另一优选例中，sgRNA与Cas酶以摩尔比1:1～120:1混合，较佳地5:1～100:1，更佳地10:1～95:1，更佳地20:1～90:1，如30:1，40:1，50:1，60:1，70:1，80:1；较佳地，所述的Cas酶为Cas9酶。
在另一优选例中，转化体系中，原生质体的量为1×10 4个/ml～1×10 9个/ml；较佳地1×10 5个/ml～1×10 8个/ml；如5×10 5个/ml、1×10 6个/ml、5×10 6个/ml、1×10 7个/ml或5×10 7个/ml等。
在本发明的另一方面，提供TritonX-100或其类似物，抑制有丝分裂的抑菌剂，或肌醇或其类似物的用途，用于：提高真菌的原生质体转化效率或基因编辑效率；或制备提高真菌的原生质体转化效率或基因编辑效率的组合物。
在一个优选例中，所述的原生质体转化为PEG介导原生质体转化。
在本发明的另一方面，提供一种用于真菌的原生质体转化的试剂盒，其中包括：靶向真菌基因组的sgRNA，Cas酶，或它们的复合物；化合物，选自：TritonX-100或其类似物，抑制有丝分裂的抑菌剂，或肌醇或其类似物，或它们的组合。
在一个优选例中，其中还包括选自下组的试剂：真菌培养基(如PDA培养基，SDB培养基，CD培养基等)；原生质体溶液(如含有山梨醇、KH 2PO 4的溶液，含有山梨醇、CaCl 2、Tris-HCl的溶液，含有PEG6000、CaCl 2、Tris-HCl的溶液、含有NaCl、KH 2PO 4的溶液)；原生质体清洗液；微量元素；基因组抽提试剂。
在一个优选例中，所述sgRNA-Cas酶复合物的浓度为80～300nM。
在另一优选例中，所述TritonX-100或其类似物的浓度为0.00001～0.1％；较佳地为0.0005～0.05％；更佳地为0.001～0.02％；
在另一优选例中，所述肌醇或其类似物的浓度(w/v)为0.006％～0.8％，较佳地0.01％～0.5％。
在另一优选例中，所述抑制有丝分裂的抑菌剂的浓度为0.008～10ug/ml，较佳地0.01～5ug/ml。
在本发明的另一方面，提供一种用于转化的组合物，包括：sgRNA-Cas酶复合物和有效量的TritonX-100或其类似物，抑制有丝分裂的抑菌剂，或肌醇或其类似物；较佳地，所述sgRNA-Cas酶复合物的浓度为80～300nM；或所述TritonX-100或其类似物的浓度为0.00001～0.1％；较佳地为0.0005～0.05％；更佳地为0.001～0.02％；所述肌醇或其类似物的浓度为0.006％～0.8％，较佳地0.01％～0.5％；或所述抑制有丝分裂的抑菌剂的浓度为0.008～10ug/ml，较佳地0.01～5ug/ml。
在另一优选例中，所述用于转化的组合物中还包括：其它转化试剂，或细胞学上可接受的载体。
在本发明的另一方面，提供所述的试剂盒或所述的用于转化的组合物的用途，用于：提高真菌的原生质体转化效率或基因编辑效率；或进行真菌的原生质体转化或基因编辑。
在一个优选例中，所述真菌为多核真菌或丝状真菌；较佳地，所述真菌为子囊菌门的真菌；较佳地，所述真菌包括(但不限于)：木霉属真菌，虫草属真菌，曲霉属真菌；更佳地，所述真菌包括(但不限于)：里氏木霉(Trichoderma reesei)、蛹虫草(Cordyceps militaris)、米曲霉(Aspergillus oryzae)、嗜热毁丝菌(Myceliophthora thermophila)、黑曲霉(A.niger)、产黄青霉(Penicillium chrysogenum)、灵芝(Ganoderma lucidum)、香菇(Lentinus edodes)、平菇(Pleurotus ostreatus)、双孢蘑菇(Agaricus bisporus)、金针菇(Flammulina velutipes)、真姬菇(Hypsizygus marmoreus)。
在另一优选例中，所述的sgRNA靶向于：里氏木霉的ura5基因；较佳地，靶基因识别位点具有SEQ ID NO:1中第355～374位所示的核苷酸序列；蛹虫草的ura5基因；较佳地，靶基因识别位点具有SEQ ID NO:2中第492～511位所示的核苷酸序列；米曲霉的AowA基因；较佳地，靶基因识别位点具有SEQ ID NO:3中第160～179位所示的核苷酸序列。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、sgRNA体外转录产物在2％琼脂糖凝胶电泳下的验证结果。图中泳道M为DS-2000DNA Marker电泳结果(片段从上到下依次为2000bp、1000bp、750bp、500bp、250bp、100bp)；泳道1、2为sgRNA体外转录产物。
图2、里氏木霉双体外CRISPR/Cas9原生质体ura5突变转化子测序验证靶标是否发生突变；其中，上图为转化体系中添加TritonX-100时得到的ura5突变转化子测序结果(sgRNA-Cas9 180nM)，下图为未添加TritonX-100时得到的ura5突变转化子测序结果(sgRNA-Cas9 180nM)。
图3、蛹虫草双体外CRISPR/Cas9原生质体ura5突变转化子测序验证靶标是否发生突变。
图4、米曲霉双体外CRISPR/Cas9突变wA转化子PCR验证结果示意图。图中泳道M为1kb DNA Ladder(片段从上到下依次为12000bp、8000bp、6000bp、5000bp、4000bp、3000bp、2500bp、2000bp、1500bp、1000bp、500bp)，泳道1为△wA纯合突变转化子(只有一条约3000bp条带)，泳道2为△wA杂合突变转化子(有2条条带，分别为突变型 3000bp和野生型300bp)，泳道3为野生型(约300bp)。
图5、米曲霉在添加肌醇或苯菌灵下的遗传操作流程。即在米曲霉原生质体介导转化过程中，分别在孢子形成、孢子萌发、原生质体再生3个阶段都添加肌醇或苯菌灵，增加米曲霉单核细胞的概率，从而增加纯合转化子概率。
具体实施方式
本发明人经过深入的研究，提供了优化的真菌原生质体转化技术以及基因编辑技术。本发明通过在以基因编辑为目的的原生质体转化过程中或之前，添加TritonX-100或其类似物，抑制有丝分裂的抑菌剂，或肌醇或其类似物；更佳地还通过调节sgRNA-Cas酶的浓度，有效地提高了真菌的原生质体转化效率及基因编辑效率，大量节约了获得转化子或基因编辑产物的时间以及提高了获得量。
本发明提供了一种提高真菌的原生质体转化效率或基因编辑效率的方法，包括：以CRISPR/Cas基因编辑方法靶向于真菌基因组中待编辑位点，进行基因编辑；其中，在利用CRISPR/Cas基因编辑试剂进行原生质体转化时，添加：TritonX-100或其类似物，抑制有丝分裂的抑菌剂，或肌醇或其类似物。
作为本发明的优选方式，所述真菌为多核真菌。
作为本发明的优选方式，所述真菌为丝状真菌。
作为本发明的优选方式，所述真菌为子囊菌门的真菌；较佳地，所述真菌包括：木霉属真菌，虫草属真菌，曲霉属真菌。
在本发明的更为具体的实施方式中，所述真菌包括：里氏木霉(Trichoderma reesei)、蛹虫草(Cordyceps militaris)、米曲霉(Aspergillus oryzae)、嗜热毁丝菌(Myceliophthora thermophila)、黑曲霉(A.niger)、产黄青霉(Penicillium chrysogenum)、灵芝(Ganoderma lucidum)、香菇(Lentinus edodes)、平菇(Pleurotus ostreatus)、双孢蘑菇(Agaricus bisporus)、金针菇(Flammulina velutipes)、真姬菇(Hypsizygus marmoreus)等。应理解，尽管本发明的实施例中优选地以这些菌株作为实例，但是本发明的技术方案还可被应用于其它的真菌中。
本发明人研究了提高真菌原生质体转化效率以及基因编辑效率的方法。从一系列的化合物中，本发明人意外地发现，在转化体系中加入TritonX-100或其类似物，抑制有丝分裂的抑菌剂，或肌醇或其类似物，可以极为有效地提高转化效率和基因编辑效率。例如，在本发明人的一次具体的实验中，本发明人获得了12个转化子，且发现该12个转化子都发生了基因编辑。
本发明人还发现，在真菌，特别是多核真菌的孢子形成、孢子萌发或原生质体再生阶段的培养基中加入肌醇或苯菌灵，能提高获得纯合转化子的效率。所述肌醇是一种生长因子。所述苯菌灵是一种杀菌剂，能抑制细胞有丝分离。例如，在本发明人的 一次具体的实验中，不添加肌醇苯菌灵时，纯化转化子获得量为0，而当添加肌醇时转化子获得量为7个，当添加苯菌灵时转化子获得量为16个，可见肌醇和苯菌灵的添加，极大地增加了获得纯合转化子的比例。
在本发明的优选方式中，所述的TritonX-100或其类似物包括选自以下的试剂：TritonX-100或其类似物，其中优选的为TritonX-100；所述的抑制有丝分裂的抑菌剂包括选自以下的试剂：苯菌灵、多菌灵、甲基硫菌灵，或其类似物，其中优选的为苯菌灵；或所述的肌醇或其类似物包括选自以下的试剂：肌醇，磷酸酰肌醇。
本发明还包括上述化合物的异构体、溶剂合物、前体或盐，只要它们也具有与所述化合物具有相同或基本相同的功能。所述的“盐”是指化合物与无机酸、有机酸、碱金属或碱土金属等反应生成的盐。这些盐包括(但不限于)：(1)与如下无机酸形成的盐：如盐酸、硫酸、硝酸、磷酸；(2)与如下有机酸形成的盐，如乙酸、草酸、丁二酸、酒石酸、甲磺酸、马来酸、或精氨酸。其它的盐包括与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐，以酯、氨基甲酸酯。化合物具有一个或多个不对称中心。所以，这些化合物可以作为外消旋的混合物、单独的对映异构体、单独的非对映异构体、非对映异构体混合物、顺式或反式异构体存在。所述的“化合物的前体”指当用适当的方法添加后，该化合物的前体在转化体系或培养体系中转化成所述化合物或其活性形式(如具有活性的盐或异构体)。
本领域人员应理解，在得知了本发明化合物的结构以后，可通过多种本领域熟知的方法、利用公知的原料，来获得本发明的化合物，比如化学合成或从生物(如动物或植物)中提取或在提取基础上进行改造的方法，这些方法均包含在本发明中。合成的化合物可以进一步通过柱层析法、高效液相色谱法等方式进一步纯化。此外，也可以通过商购的方式获得本发明的化合物。
基于本发明人的新发现，本发明还提供了一种用于转化的组合物，含有有效量的TritonX-100或其类似物，抑制有丝分裂的抑菌剂或肌醇，或它们的异构体、溶剂合物、前体，以及sgRNA-Cas酶复合物。
基于本发明人的新发现，本发明还提供了一种复合培养基，其包括：真菌培养基和有效量的TritonX-100或其类似物，抑制有丝分裂的抑菌剂或肌醇；较佳地，所述的真菌培养基为用于真菌孢子形成、孢子萌发和原生质体再生阶段培养的培养基。所述的真菌孢子形成或孢子萌发阶段培养的培养基可以是本领域已知的培养基。
本发明中，所述的“含有”，“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”；“主要由……构成”、“基本上由……构成”和“由……构成”属于“含有”、“具有”或“包括”的下位概念。
对于真菌的原生质体转化，研究之初，本发明人将sgRNA与Cas酶混合获得的sgRNA-Cas酶复合物与原生质体混合转化，但发现其转化效率极为低下，在有些情况 下甚至难以获得有效的转化子。本发明人对于影响转化的可能因素进行分析、试验，发现sgRNA-Cas酶复合物的浓度对于转化效率具有较大的影响。在此基础上，本发明人优化了转化体系中sgRNA-Cas酶复合物的浓度，作为本发明的优选方式，sgRNA-Cas酶复合物在转化体系中的浓度为80～300nM，较佳地90～250nM，更佳地100～200nM，如120，140，160，180，190nM。
作为本发明的优选方式，所述的sgRNA-Cas酶复合物中，sgRNA与Cas酶以摩尔比1:1～120:1混合。
CRISPR介导的基因编辑中，可运用的Cas酶包括Cas9，Cas9n，Cas13a，Cas13d，CasRx等，这些Cas可被应用于本发明中。作为本发明的优选方式，所述的Cas酶为Cas9。此外，这些酶的变体、衍生物、活性片段或融合形式，也可被应用于本发明中。
所述的sgRNA(single-guided RNA)是针对基因组中待编辑的位点设计的序列。在本发明的优选实施方式中，所述的sgRNA针对于靶基因待编辑位点区域的约15～30bp(如约20bp)长度的序列区域进行设计。根据靶向编辑的特定需要，本领域技术人员还可以设计不同序列结构、不同长度的sgRNA。
此外，CRISPR介导的基因编辑中，根据靶向编辑的特定需要，还可以设计配合所述sgRNA的供体序列(Donor)。
基于本发明人的新发现，本发明还提供了一种用于真菌的原生质体转化的试剂盒，其中包括：靶向真菌基因组的sgRNA，Cas酶，或它们的复合物；化合物，选自：TritonX-100或其类似物，抑制有丝分裂的抑菌剂，或肌醇或其类似物，或它们的组合。
作为本发明的优选方式，所述的试剂盒中还包括选自下组的试剂：真菌培养基(如PDA培养基，SDB培养基，CD培养基等)；原生质体溶液(如含有山梨醇、KH 2PO 4的溶液，含有山梨醇、CaCl 2、Tris-HCl的溶液，含有PEG6000、CaCl 2、Tris-HCl的溶液、含有NaCl、KH 2PO 4的溶液)；原生质体清洗液；微量元素；基因抽提试剂。应理解，其它的常规用于原生质体转化、清洗、稳定等的试剂，用于真菌培养、制备原生质体等的试剂也可被包含在所述的试剂盒中。各种试剂可以被有效地配伍以方便本领域技术人员使用，或者可被分别置于不同的容器中备用。此外，本发明的试剂盒中还可包含说明其中各个试剂用法用量、操作步骤等的使用说明书。
在本发明的具体的实施例中，设计了靶向于下组基因的sgRNA：里氏木霉的ura5基因，蛹虫草的ura5基因，米曲霉的AowA基因。根据实施结果，对于sgRNA-Cas酶复合物在转化体系中的浓度调控，以及利用TritonX-100肌醇或苯菌灵的调节，均呈现显著的提高转化效率以及提高基因编辑效率的技术效果。
所述的里氏木霉为子囊菌门木霉属真菌，作为工业菌株，被应用于分解植物材料的酶，包括纤维素酶、半纤维素酶、蛋白酶、淀粉酶等。所述的蛹虫草隶属于子囊菌 门虫草属，具有重要的药用价值。所述的米曲霉为子囊菌门曲霉属真菌，作为一种常用于食品酿造的食品安全级菌株，具有多核的特征，这使它能很好的保持遗传稳定性，但同时也使基因组编辑变得更加困难，主要体现在得到纯合的转化子。
ura5是生物体编码乳清酸磷酸核糖转移酶的基因，参与尿嘧啶的合成代谢。因它具有双向选择(ura5不能在含5-FOA的条件下生长；ura5 -不能在基本培养基上生长，需另外添加尿苷或尿嘧啶)的性质，常作为遗传操作过程中的筛选标记。wA基因是聚酮合酶基因，参与米曲霉分生孢子色素的合成。argB基因参与精氨酸生物合成，在本发明中，作为营养缺陷型筛选标记，用于敲除wA基因。其中，里氏木霉ura5(Trura5)序列具有SEQ ID NO:1所示的核苷酸序列；蛹虫草ura5(Cmura5)序列具有SEQ ID NO:2所示的核苷酸序列；米曲霉wA(AowA)序列具有SEQ ID NO:3所示的核苷酸序列；米曲霉argB序列具有SEQ ID NO:4所示的核苷酸序列。本发明的技术方案可针对上述工业学、药学或食品学上有用的菌株进行改造，体现了其良好的应用价值。
根据本发明具体实施结果，本发明还提供了针对里氏木霉的ura5基因的优选的靶基因识别位点，其具有SEQ ID NO:1所示的核苷酸序列；针对蛹虫草的ura5基因的优选的靶基因识别位点，其具有SEQ ID NO:1所示的核苷酸序列；以及针对米曲霉的AowA基因的优选的靶基因识别位点，其具有SEQ ID NO:4所示的核苷酸序列。针对这些靶基因识别位点设计的sgRNA，其与Cas酶复合后，呈现了理想的基因编辑效果。
本发明的转化及基因编辑方法，可以一次性获得较多的转化子且基因编辑效率非常高，节约了大量时间，并可能降低了脱靶概率，为真菌的基因组编辑、基因功能研究与分子育种带来了极大的便利。
本发明的主要优点在于：
(1)sgRNA-Cas酶复合物为核酸与蛋白的混合体(为双体外CRISPR/Cas9系统)，在进行转化中存在效率低、基因编辑效果不理想的问题，而本发明通过添加适当的化合物和/或优化sgRNA-Cas9浓度，有效提高了双体外基因组编辑效率，能使基因组编辑效率达到100％。
(2)通过在转化体系中添加TritonX-100或其类似物提高原生质体转化效率，通过在培养时添加抑制有丝分裂的抑菌剂或肌醇类化合物提高真菌如米曲霉纯合转化子效率。
(3)首次在蛹虫草和米曲霉中建立双体外基因组编辑技术、并实现对基因组进行高效编辑。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发 明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、sgRNA体外转录与纯化
针对靶基因，设计sgRNA识别位点，合成如下用于形成sgRNA的引物：sgRNA-F：TAATACGACTCACTATA-sgRNA site-GTTTTAGAGCTAGAAAT AGCAA。
针对里氏木霉RutC30乳清酸磷酸核糖转移酶ura5(SEQ ID NO:1)所设计的sgRNA-F序列为：
sgRNA-Trura5-F：TAATACGACTCACTATA GGCGAGGGCGGCAACAT CGTGTTTTAGAGCTAGAAATAGCAA(SEQ ID NO:5)。
针对蛹虫草Cm01的ura5(SEQ ID NO:2)所设计的sgRNA-F序列为：
sgRNA-Cmura5-F：TAATACGACTCACTATA GGACCGGATGGAGAAG CTTCGTTTTAGAGCTAGAAATAGCAA(SEQ ID NO:6)。
针对米曲霉NSAR1的聚酮合酶wA所设计的sgRNA-F序列为：
sgRNA-wA-F：TAATACGACTCACTATA GGGAAGATCTTCCGTTCAGAGTTTTAGAGCTAGAAATAGCAA(SEQ ID NO:7)。
将这些sgRNA-F引物分别与sgRNA-R引物PCR扩增，回收得到sgRNA模板；所述sgRNA-R引物序列如下：
sgRNA-R：AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAA(SEQ ID NO:8)。
对这些短链的gRNA模板进行纯化，等体积酚：氯仿：异戊醇＝25：24：1抽提之后，水相再用等体积氯仿抽提，得到的水相用3倍体积的无水乙醇和1/10体积的3M醋酸钠在-20℃沉淀1h，4℃离心后75％乙醇的洗涤沉淀3次，待干燥后用5ul Nuclease-free water得到纯化后的sgRNA模板，用Nano-300微量分光光度计测定浓度。
以纯化得到的DNA为模板，用体外转录试剂盒(HiScribe TM Quick T7High Yield RNA Synthesis Kit，NEB#E2050S，具体方法参考试剂盒说明书)在37℃过夜反应后得到sgRNA粗产物，经过DNA凝胶电泳验证后(图1)，用与纯化sgRNA模板相同的方法纯化这些sgRNA，将干燥后sgRNA用5ul Nuclease-free water溶解，-80℃保存。
实施例2、sgRNA-Cas9复合物体外组装
将纯化好的sgRNA和商品化的Cas9(novoprotein E365-01A)以70:1(摩尔数)的比例加入到反应缓冲液中，反应缓冲液为2×原生质体溶液2(2M山梨醇，100mM CaCl 2， 2M Tris-HCl，pH 7.5)和10×active buffer(200mM HEPES free acid，1.5M KCl，5mM DTT，1mM EDTA，100mM MgCl 2·6H 2O，pH 7.5)，混匀后37℃孵育15min。
里氏木霉ura5基因的sgRNA-Cas9组装方法：将纯化好的sgRNA-Trura5与Cas9按70：1的比例加入反应体系中，37℃孵育15min。并且通过控制sgRNA和Cas9的加入量，分别制备20nM、60nM、100nM、180nM的sgRNA-Cas9复合物。
蛹虫草ura5基因的sgRNA-Cas9组装方法：将纯化好的sgRNA-Cmura5与Cas9按70：1的比例(Cas9为6ug)加入反应体系中，37℃孵育15min。
米曲霉wA基因的sgRNA-Cas9组装方法：将纯化好的sgRNA-AowA与Cas9按70：1的比例(Cas9为6ug)加入反应体系中，将反应缓冲液中的2×原生质体溶液2替换成2×solution 1(1.6M NaCl，20mM CaCl 2，20mM Tris-HCl，pH 8.0)，37℃孵育15min。
实施例3、添加TritonX-100提高转化及基因编辑的效率
以里氏木霉Rut C30为底盘细胞，利用双体外CRISPR/Cas9技术对乳清酸磷酸核糖转移酶ura5基因(SEQ ID NO:1；其中第355～374位为sgRNA靶序列)进行突变，利用sgRNA-Cas9复合物(sgRNA模板由sgRNA-Trura5-F与sgRNA-R经PCR扩增所形成)进行转化，研究提高基因转化所获得的转化子数量及提高基因编辑的效率的方法。
原生质体制备根据文献(Rantasalo，A.et al.A universal gene expression system for fungi.Nucleic Acids Res，2018 46，e111)进行修改，制备原生质体的菌丝通过在铺有玻璃纸的PDA中28℃培养15h获得，裂解酶(sigma)的浓度为5mg/ml。
转化体系：过滤离心收集原生质体，4ml原生质体经清洗后，200ul原生质体溶液2(1M山梨醇，50mM CaCl 2，1M Tris-HCl，pH 7.5)重悬，加入sgRNA-Cas9复合物，74μl原生质体溶液3(25％PEG6000，50mM CaCl 2，1M Tris-HCl，pH 7.5)，混匀后冰上静置20min，加入2ml原生质体溶液3后混匀室温静置15min；加入4ml原生质体溶液2后混匀，快速加入到50ml上层培养基(山梨醇182.17g/l，MgSO 4·7H 2O 1g/l，KH 2PO 4 10g/l，(NH 4) 2SO 4 6g/l，柠檬酸钠·2H 2O 3g/l，葡萄糖·H 2O 10g/l，FeSO 4·7H 2O 5mg/l，MnSO 4·H 2O 1.6mg/l，ZnSO 4·7H 2O 1.4mg/l，CoCl 2 1.2mg/l，琼脂糖1.2％)中，混匀后，平铺到2％琼脂平板上，28℃培养3～5天。
转化子数量为按上述操作获得所有转化子的数量，即转化1*10 5个原生质体所获转化子的数量，编辑效率＝阳性转化子数/所有转化子数。
转化子验证：挑取转化子在SDB培养基中培养，利用Fastprep仪破碎细胞并用酚：氯仿：异戊醇＝25：24：1抽提基因组，PCR扩增(△Trura5-F：ATGGCTACCACCTCCCAGC(SEQ ID NO:9)，△Trura5-R：TCAGTCAGTCG CCTTGTAC(SEQ ID NO:10))后测序验证突变菌株。
经过针对诸多受试样品的试验后，本发明人发现在转化体系中同时添加TritonX-100有利于提高转化及基因编辑的效率。以里氏木霉Trura5序列中的5’-GGCGAGGGCGGCAACATCGT cgg-3’(SEQ ID NO:11)为靶标序列(其中下划线为PAM序列)，利用sgRNA-Cas9复合物(sgRNA模板由sgRNA-Trura5-F与sgRNA-R PCR扩增所形成)对Trura5基因进行基因编辑，获得转化子后抽提基因组，用△Trura5-F/△Trura5-R引物扩增后进行测序分析。共测试了12个经前述转化操作的原生质体转化子样品(编号为1～12)，以野生型Trura5作为对照。
结果见图2，所有的转化子在靶标处都发生了突变。从图2可以看出，添加TritonX-100(0.006％)后转化子数量有显著的增加(sgRNA-Cas9复合物浓度180nM，12个转化子)，且基因组编辑效率保持较高，说明TritonX-100有利于提高原生质体转化效率。
实施例4、高浓度sgRNA-Cas9复合物提高基因组编辑效率
以里氏木霉Rut C30为底盘细胞，利用双体外CRISPR/Cas9技术对乳清酸磷酸核糖转移酶ura5基因(SEQ ID NO:1；其中第355～374位为sgRNA靶序列)进行突变，利用sgRNA-Cas9复合物(sgRNA模板由sgRNA-Trura5-F与sgRNA-R PCR扩增所形成)进行转化，检测sgRNA-Cas9浓度对于基因编辑效率的影响。
原生质体制备、转化以及转化体系、转化子验证方法同前述实施例3。
不同sgRNA-Cas9复合物浓度下里氏木霉ura5基因突变效率如表1，可见基因编辑效率随着sgRNA-Cas9复合物浓度的升高而升高，在100nM和180nM时达到100％。说明相对高浓度的sgRNA-Cas9复合物有利于提高双体外CRISPR/Cas9技术的基因编辑效率。
表1
Figure PCTCN2021070906-appb-000001
实施例5、化合物TritonX-100提高双体外CRISPR/Cas9基因编辑效率
以里氏木霉Rut C30和蛹虫草Cm01为底盘细胞，利用双体外CRISPR/Cas9技术 对ura5基因(SEQ ID NO:1(Trura5，其中第355～374位为sgRNA靶序列)、SEQ ID NO:2(Cmura5，其中第492～511位为sgRNA靶序列)进行突变，分别利用sgRNA-Cas9复合物1(sgRNA模板由sgRNA-Trura5-F与sgRNA-R PCR扩增所形成)、sgRNA-Cas9复合物2(sgRNA模板由sgRNA-Cmura5-F与sgRNA-R PCR扩增所形成)进行转化，通过添加TritonX-100提高基因编辑效率。
原生质体制备与转化与实施例3相同，但蛹虫草的原生质体通过酶解在SDB培养基中培养3天的芽生孢子获得。
转化子验证：抽取转化子基因组，里氏木霉ura突变转化子验证方法与实施例3相同；蛹虫草的ura5突变转化子基因组经PCR扩增(△Cmura5-F：ATGTCGGATCTCGCTCAGTAC(SEQ ID NO:12)，△Cmura5-R：CTAATCTGCATCTTTCT CC(SEQ ID NO:13))，所有的PCR产物送到公司测序。
TritonX-100(0.006％(w/v))对里氏木霉ura5基因突变效率的影响的测定结果如表2，可以看出添加TritonX-100，里氏木霉ura5突变转化子个数增加2～4倍，且每个转化子都在sgRNA识别位点处发生了突变。
表2
Figure PCTCN2021070906-appb-000002
TritonX-100对蛹虫草ura5基因突变效率的影响如表3，表明在蛹虫草原生质体转化过程中不添加TritonX-100，没有得到转化子，而在添加TritonX-100后，得到了12个转化子，每个转化子都在sgRNA识别位点处发生了突变，如图3。图中，Cmura5是野生型，对12个样品测序(编号为1-12)，结果发现所有的转化子在靶标处都发生了突变，靶标序列为5’-GGACCGGATG GAGAAGCTTC cgg-3’(其中下划线为PAM序列)(SEQ ID NO:18)。
表3
Figure PCTCN2021070906-appb-000003
实施例6、肌醇或苯菌灵有效增加多核丝状真菌米曲霉纯合转化子概率
以多核丝状真菌米曲霉NSAR1为底盘细胞，利用双体外CRISPR/Cas9技术对聚酮合酶wA(SEQ ID NO:3；其中第160～179位为sgRNA靶序列)基因进行突变，利用sgRNA-Cas9复合物(sgRNA模板由sgRNA-wA-F与sgRNA-R PCR扩增所形成)进行转 化。通过添加肌醇或苯菌灵提高纯合转化子概率，从而达到提高基因编辑效率的目的。
准备marker(donor-DNA)：以米曲霉RIB40的基因组，以argB-wA-F(GATGCTTTCACGCTTTGCGCCAAGAGATCGCG AGGCTTTCACCCTGTATCCTGGCCTGA(SEQ ID NO:14))/argB-wA-R(GTCAACGCACTCTCCAGAG CCGGATTAGGGTCTGACTTCGAGATGTTAATGGAGCTAGG(SEQ ID NO:15))扩增argB基因作为筛选标记，PCR扩增回收，通过真空浓缩至10ul。
原生质体制备：将在CD培养基((NH 4) 2SO 4 4.66g/l，KCl 0.52g/l，KH 2PO 4 1.52g/l，MgSO 4·7H 2O 1mol/l，葡萄糖10g/l，微量元素2 1/1000，pH 6.5，并添加0.1％(w/v)肌醇或1μg/ml苯菌灵)上产生的孢子用0.85％NaCl-0.1％Tween 80洗下，取1ml孢子接到100ml CD液体培养基(含0.1％(w/v)肌醇或1μg/ml苯菌灵)中，30℃培养。待孢子萌发后过滤收集菌丝。用20ml含有20mg/ml裂解酶(sigma)的原生质体化溶液(0.8M NaCl，10mM KH 2PO 4，pH 6.0)在30℃酶解2h。过滤收集原生质体。用4ml原生质体清洗液(0.8M NaCl)清洗原生质体2次。
原生质体转化：200ul Solution1(0.8M NaCl，10mM CaCl 2，10mM Tris-HCl，pH 8.0)重悬原生质体，加入sgRNA-Cas9复合物(终浓度为180nM)、donor-DNA与20％体积的Solution2(40％PEG4000，50mM CaCl 2，50mM Tris-HCl，pH 8.0。)，冰上静置30min。加1ml Solution2，混匀后，室温放置30min，加入9ml的原生Solution1，混匀后离心收集原生质体。200ul Solution1重悬后加入到50ml CD上层培养基(含0.8M NaCl的CD培养基，并加入肌醇(312.5μl 160g/l肌醇，终浓度为0.1％(w/v))或苯菌灵(10μl 5mg/ml苯菌灵，终浓度为1μg/ml)中，轻轻混匀，倒在下层培养基(2％琼脂)上，30℃培养3天左右可观察到菌丝长出(米曲霉原生质体转化全过程见图5)。
转化子验证：挑取转化子在SDB中培养，抽提基因组，PCR扩增(△wA-F：AGACCAGACAAGTGATTTCG(SEQ ID NO:16)，△wA-R：CGTTTCCAAGGTCTCCGTAG(SEQ ID NO:17))后DNA琼脂糖凝胶电泳检测(图4)，并测序验证。
由于米曲霉是一个多细胞的菌株，为了能提高纯合转化子的效率，分别在分生孢子形成、孢子萌发阶段与原生质体再生的培养基中添加肌醇或苯菌灵(图5)，从而提高单核原生质体的概率，更利于基因组编辑。
肌醇/苯菌灵对米曲霉wA基因编辑效率的影响如表4所示，可以看出，在不添加肌醇或苯菌灵情况下，虽然可以获得阳性转化子，但是无法直接获得纯合阳性转化子，还需要大量的时间进行传代和筛选才能获得纯合阳性转化子。而在添加肌醇或苯菌灵的情况下，可以获得大量的纯合阳性转化子，且占转化子的比例分别达到46.67％和76.19％。因此，本方法可以极大地加快多核丝状真菌基因编辑的速度。
表4
Figure PCTCN2021070906-appb-000004
Figure PCTCN2021070906-appb-000005
实施例7、其他有丝分裂抑制剂有效增加多核丝状真菌米曲霉纯合转化子效率
以多核丝状真菌米曲霉NSAR1为底盘细胞，利用双体外CRISPR/Cas9技术对聚酮合酶wA(SEQ ID NO:3；其中第160～179位为sgRNA靶序列)基因进行突变，利用sgRNA-Cas9复合物(sgRNA模板由sgRNA-wA-F与sgRNA-R PCR扩增所形成；终浓度为180nM)进行转化。通过添加其他有丝分裂抑制剂如多菌灵和甲基硫菌灵提高纯合转化子概率，从而达到提高基因组编辑效率的目的。
原生质体制备、转化以及转化体系、转化子验证方法同前述实施例6。不同的是将苯菌灵替换成0.5ug/ml多菌灵。
多菌灵对米曲霉wA基因编辑效率的影响如表5所示，在添加多菌灵的条件下，可以获得纯合阳性转化子，占转化子的比例达到60％。
表5
Figure PCTCN2021070906-appb-000006
除此之外，2ug/ml的甲基硫菌灵也能显著促进米曲霉产生更多的单核原生质体。因此，其他有丝分裂抑制剂也可以提高多核丝状真菌的基因组编辑的纯合阳性转化子，加快多核丝状真菌基因编辑的速度。
实施例8、其他表面活性剂对双体外CRISPR/Cas9基因编辑效率的影响
以里氏木霉Rut C30为底盘细胞，利用双体外CRISPR/Cas9技术对乳清酸磷酸核糖转移酶ura5基因(SEQ ID NO:1；其中第355～374位为sgRNA靶序列)进行突变，利用sgRNA-Cas9复合物(sgRNA模板由sgRNA-Trura5-F与sgRNA-R经PCR扩增所形成；终浓度为180nM)进行转化，研究其他表面活性剂(如吐温-80、NP-40、PEG4000)对双体外CRISPR/Cas9基因编辑效率的影响。
原生质体制备、转化以及转化体系、转化子验证方法同前述实施例3。不同的是将原生质体转化时加入的TritonX-100替换成0.006％NP-40或0.1％吐温-80。
这些表面活性剂对里氏木霉ura5基因编辑效率的影响如表6，在添加0.006％NP-40或0.1％吐温-80的条件下，转化子的数量相对于不添加任何表面活性剂的条件下并无明显差异，只有在添加Triton X-100的条件下转化子数量明显增多，是不添加 表面活性剂的2倍。因此，Triton X-100能提高基因编辑的转化子数量，但Tween80和NP-40在该浓度下对基因编辑转化子数量并无提高作用。
表6
Figure PCTCN2021070906-appb-000007
序列信息：
里氏木霉Trura5序列(下划线表示sgRNA靶序列)(SEQ ID NO:1)
Figure PCTCN2021070906-appb-000008
蛹虫草Cmura5序列(下划线表示sgRNA靶序列)(SEQ ID NO:2)
Figure PCTCN2021070906-appb-000009
米曲霉AowA序列(下划线表示sgRNA靶序列)(SEQ ID NO:3)
Figure PCTCN2021070906-appb-000010
Figure PCTCN2021070906-appb-000011
米曲霉argB序列(SEQ ID NO:4)
Figure PCTCN2021070906-appb-000012
Figure PCTCN2021070906-appb-000013
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (16)

  1. 一种提高真菌的原生质体转化效率或基因编辑效率的方法，包括：将sgRNA与Cas酶混合获得sgRNA-Cas酶复合物，转化原生质体；其中，在进行原生质体转化时，添加：TritonX-100或其类似物，抑制有丝分裂的抑菌剂，或肌醇或其类似物。
  2. 如权利要求1所述的方法，其特征在于，所述的抑制有丝分裂的抑菌剂包括选自以下的试剂：苯菌灵、多菌灵、甲基硫菌灵，或其类似物；或
    所述的肌醇类似物包括：磷酸酰肌醇。
  3. 如权利要求1所述的方法，其特征在于，进行原生质体转化前，还包括：在真菌的孢子形成、孢子萌发阶段和/或原生质体再生阶段，加入所述抑制有丝分裂的抑菌剂，或所述肌醇或其类似物。
  4. 如权利要求1～3任一所述的方法，其特征在于，所述的TritonX-100或其类似物在转化体系中的浓度为0.00001～0.1％；较佳地为0.0005～0.05％；更佳地为0.001～0.02％；
    所述的抑制有丝分裂的抑菌剂在转化体系中的浓度为：0.008～10ug/ml，较佳地0.01～5ug/ml；或
    所述的肌醇或其类似物在转化体系中的浓度为：0.006％～0.8％，较佳地0.01％～0.5％。
  5. 如权利要求1所述的方法，其特征在于，所述sgRNA-Cas酶复合物在转化体系中的浓度为80～300nM；较佳地90～250nM。
  6. 如权利要求5所述的方法，其特征在于，sgRNA与Cas酶以摩尔比1:1～120:1混合。
  7. 如权利要求5所述的方法，其特征在于，所述的Cas酶为Cas9酶。
  8. 权利要求1所述的方法，其特征在于，转化体系中，原生质体的量为1×10 4个/ml～1×10 9个/ml；较佳地1×10 5个/ml～1×10 8个/ml。
  9. TritonX-100或其类似物，抑制有丝分裂的抑菌剂，或肌醇或其类似物的用途， 用于：
    提高真菌的原生质体转化效率或基因编辑效率；或
    制备提高真菌的原生质体转化效率或基因编辑效率的组合物。
  10. 一种用于真菌的原生质体转化的试剂盒，其中包括：
    靶向真菌基因组的sgRNA，Cas酶，或sgRNA-Cas酶复合物；
    化合物，选自：TritonX-100或其类似物，抑制有丝分裂的抑菌剂，或肌醇或其类似物，或它们的组合。
  11. 如权利要求10所述的试剂盒，其特征在于，其中还包括选自下组的试剂：真菌培养基；原生质体溶液；原生质体清洗液；微量元素；基因组抽提试剂。
  12. 如权利要求10所述的试剂盒，其特征在于，所述sgRNA-Cas酶复合物的浓度为80～300nM；或
    所述TritonX-100或其类似物的浓度为0.00001～0.1％；较佳地为0.0005～0.05％；更佳地为0.001～0.02％；
    所述肌醇或其类似物的浓度为0.006％～0.8％，较佳地0.01％～0.5％；或
    所述抑制有丝分裂的抑菌剂的浓度为0.008～10ug/ml，较佳地0.01～5ug/ml。
  13. 一种用于转化的组合物，包括：sgRNA-Cas酶复合物和有效量的TritonX-100或其类似物，抑制有丝分裂的抑菌剂，或肌醇或其类似物；较佳地，
    所述sgRNA-Cas酶复合物的浓度为80～300nM；或
    所述TritonX-100或其类似物的浓度为0.00001～0.1％；较佳地为0.0005～0.05％；更佳地为0.001～0.02％；
    所述肌醇或其类似物的浓度为0.006％～0.8％，较佳地0.01％～0.5％；或
    所述抑制有丝分裂的抑菌剂的浓度为0.008～10ug/ml，较佳地0.01～5ug/ml。
  14. 权利要求10～12所述的试剂盒或权利要求13所述的用于转化的组合物的用途，用于：
    提高真菌的原生质体转化效率或基因编辑效率；或
    进行真菌的原生质体转化或基因编辑。
  15. 如权利要求1、9、10、13或14任一所述，其特征在于，所述真菌为多核真菌或丝状真菌；较佳地，所述真菌为子囊菌门的真菌；较佳地，所述真菌包括：木霉 属真菌，虫草属真菌，曲霉属真菌；更佳地，所述真菌包括：里氏木霉(Trichoderma reesei)、蛹虫草(Cordyceps militaris)、米曲霉(Aspergillus oryzae)、嗜热毁丝菌(Myceliophthora thermophila)、黑曲霉(A.niger)、产黄青霉(Penicillium chrysogenum)、灵芝(Ganoderma lucidum)、香菇(Lentinus edodes)、平菇(Pleurotus ostreatus)、双孢蘑菇(Agaricus bisporus)、金针菇(Flammulina velutipes)、真姬菇(Hypsizygus marmoreus)。
  16. 如权利要求15所述，其特征在于，所述的sgRNA靶向于：
    里氏木霉的ura5基因；较佳地，靶基因识别位点具有SEQ ID NO:1中第355～374位所示的核苷酸序列；
    蛹虫草的ura5基因；较佳地，靶基因识别位点具有SEQ ID NO:2中第492～511位所示的核苷酸序列；
    米曲霉的AowA基因；较佳地，靶基因识别位点具有SEQ ID NO:3中第160～179位所示的核苷酸序列。
PCT/CN2021/070906 2020-01-09 2021-01-08 一种优化的真菌双体外基因组编辑方法 WO2021139779A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010022027 2020-01-09
CN202010022027.6 2020-01-09

Publications (1)

Publication Number Publication Date
WO2021139779A1 true WO2021139779A1 (zh) 2021-07-15

Family

ID=76709204

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/070906 WO2021139779A1 (zh) 2020-01-09 2021-01-08 一种优化的真菌双体外基因组编辑方法

Country Status (2)

Country Link
CN (1) CN113106126B (zh)
WO (1) WO2021139779A1 (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106676130A (zh) * 2016-12-30 2017-05-17 华智水稻生物技术有限公司 利用crispr‑cas9技术对水稻badh2基因定点突变的方法
CN107474129A (zh) * 2017-10-12 2017-12-15 洛阳轩智生物科技有限公司 特异性增强crispr‑cas系统基因编辑效率的方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE480632T1 (de) * 2004-04-02 2010-09-15 Dsm Ip Assets Bv Filamentöse pilzmutanten mit verbesserter homologer rekombinationseffizienz
CA2913234A1 (en) * 2013-05-22 2014-11-27 Northwestern University Rna-directed dna cleavage and gene editing by cas9 enzyme from neisseria meningitidis
CA2975486A1 (en) * 2017-08-04 2019-02-04 Rutgers, The State University Of New Jersey Compositions and methods comprising endophytic bacterium for application to target plants to increase plant growth, and increase resistance to abiotic and biotic stressors
WO2019046703A1 (en) * 2017-09-01 2019-03-07 Novozymes A/S METHODS OF ENHANCING GENOME EDITION IN FUNGI

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106676130A (zh) * 2016-12-30 2017-05-17 华智水稻生物技术有限公司 利用crispr‑cas9技术对水稻badh2基因定点突变的方法
CN107474129A (zh) * 2017-10-12 2017-12-15 洛阳轩智生物科技有限公司 特异性增强crispr‑cas系统基因编辑效率的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DENG HUAXIANG; GAO RUIJIE; LIAO XIANGRU; CAI YUJIE: "CRISPR system in filamentous fungi: Current achievements and future directions", GENE, ELSEVIER AMSTERDAM, NL, vol. 627, 16 June 2017 (2017-06-16), NL, pages 212 - 221, XP085144954, ISSN: 0378-1119, DOI: 10.1016/j.gene.2017.06.019 *

Also Published As

Publication number Publication date
CN113106126A (zh) 2021-07-13
CN113106126B (zh) 2023-07-18

Similar Documents

Publication Publication Date Title
Zou et al. Efficient genome editing in filamentous fungi via an improved CRISPR‐Cas9 ribonucleoprotein method facilitated by chemical reagents
US7125982B1 (en) Microbial production of nuclease resistant DNA, RNA, and oligo mixtures
Liu et al. A dual-plasmid CRISPR/Cas system for mycotoxin elimination in polykaryotic industrial fungi
Desjardins et al. Linkage among genes responsible for fumonisin biosynthesis in Gibberella fujikuroi mating population A
CN110268057B (zh) 用于鉴定和表达基因簇的系统和方法
EP2718442B1 (en) Genetic manipulation and expression systems for pucciniomycotina and ustilaginomycotina subphyla
Dong et al. Efficient genome editing in Aspergillus niger with an improved recyclable CRISPR-HDR toolbox and its application in introducing multiple copies of heterologous genes
CN109161480B (zh) 拟茎点霉的原生质体制备方法及基因敲除方法
Zhang et al. Agrobacterium tumefaciens-mediated transformation in the entomopathogenic fungus Lecanicillium lecanii and development of benzimidazole fungicide resistant strains
EP3715462A1 (en) Method for regulating in vitro biosynthesis activity by knocking-out of nuclease system
BR112020005217A2 (pt) métodos para a engenharia genética de células hospedeiras de kluyveromyces
Králová et al. CRISPR/Cas9 genome editing in ergot fungus Claviceps purpurea
Chen et al. Improvement of the CRISPR-Cas9 mediated gene disruption and large DNA fragment deletion based on a chimeric promoter in Acremonium chrysogenum
CN111057654B (zh) 一种适用于巴戟天内生真菌A761的Cas9基因敲除载体及其构建方法和应用
Yu et al. Efficient genome editing in Claviceps purpurea using a CRISPR/Cas9 ribonucleoprotein method
WO2021139779A1 (zh) 一种优化的真菌双体外基因组编辑方法
Chai et al. Liposome-mediated mycelial transformation of filamentous fungi
US20130196374A1 (en) Cis-acting element and use thereof
EP3412765B1 (en) Method for producing mutant filamentous fungi
CN105886415B (zh) 一种生产白桦脂酸的酿酒酵母工程菌及其构建方法
Shen et al. Application of CRISPR in Filamentous Fungi and Macrofungi: From Component Function to Development Potentiality
Chen et al. An efficient genetic transformation system for Chinese medicine fungus Tolypocladium ophioglossoides
Zhang et al. Application progress of CRISPR/Cas9 genome-editing technology in edible fungi
CN113728097A (zh) 具有ruvc结构域的酶
Faugeron-Fonty et al. A comparative study of the ori sequences from the mitochondrial genomes of twenty wild-type yeast strains

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21738108

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21738108

Country of ref document: EP

Kind code of ref document: A1