CN109321548A - A kind of Cas9 albumen, CRISPR/Cas9 system, the method and application of mushroom gene editing - Google Patents
A kind of Cas9 albumen, CRISPR/Cas9 system, the method and application of mushroom gene editing Download PDFInfo
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Abstract
The invention discloses Cas9 albumen, CRISPR/Cas9 system, the methods and application of mushroom gene editing.The codon of source of people Cas9 albumen, which is optimized, in the Preference that the present invention is used according to Pleurotus eryngii genome codon obtains the nucleotide sequence of coding Cas9, and the promoter that 4 obtained as transcription sgRNA are capable of high expression U6snRNA gene is screened, it establishes endogenous gene mutation and resistance is caused to change the genetic conversion system alternatively marked.It is of the invention the experimental results showed that, system established by the present invention, which can succeed that efficiently Pleurotus eryngii gene pyrG is lacked, is inserted into and knocked out etc., edits.Gene editing technology of the invention can be applied and popularized to other produced mushrooms in addition to Pleurotus eryngii, be applied in produced mushroom improvement and metabolic pathway regulation.
Description
Technical field
The present invention relates to a kind of gene editing technologies, in particular to a kind of Cas9 albumen, CRISPR/ in gene editing technology
Cas9 system, the method and application of mushroom gene editing.
Background technique
Edible mushroom be can fructification edible, with large-scale meat or sclerotium tissue higher fungus etc. it is total
Claim.Edible mushroom has huge biological activity, can generate a variety of different secondary metabolites, outstanding in terms of drug research
It finds that new active material has great potential quality.In addition, extracting natural products tool from edible mushroom compared with chemicals
There is the characteristics of hypotoxicity and low side effect, therefore edible mushroom is a kind of potential natural pharmaceutical resources.Pleurotus eryngii (Pleurotus
Eryngii) belong to Basidiomycotina, Hymenomycetes, basidiomycetes subclass, Agaricales, Pleurotaceae, Pleurotus, it has very high medicinal
Value.With deepening continuously for basic theory and application technical research, a set of efficient gene editing is developed in Pleurotus eryngii
Technology just seems very urgent.
With the raising of human living standard, people are this for edible mushroom while having natural drug and health-care efficacy
The demand of resource be huge.Exploitation is used for the new technology of Pleurotus eryngii genetic modification, important medicinal for disclosing Pleurotus eryngii
The genetic improvement of the mechanism of action and character is of great significance.Gene editing technology, in genome in-situ accomplishes to the essence of gene
Really, it pinpoints, heritable modification, is the desirable route of Pleurotus eryngii genetic improvement and MOLECULE DESIGN.
The third generation artificial nucleic acid zymotechnic emerging as one, by RNA mediate CRISPR-Cas9 system addresses its be
System building is simple and convenient, target practice precision is high, construction cost is low, can carry out each of fixed point editor to multiple sites of design simultaneously
Item advantage, becomes hot technology studied both at home and abroad at present, has been successfully applied to plant, bacterium, yeast, fish and lactation
In zooblast.This research and probe implements the technology of fixed point editor using CRISPR-Cas9 system to Pleurotus eryngii genome, takes
The technology platform that the mushrooms genoid group such as Pleurotus eryngii efficiently pinpoints editor is built.
Short palindrome repetitive sequence (the clustered regularly interspaced short of the regular intervals of cluster
Palindromic repeat, CRISPR) it is that acquired self is immune for one kind that bacterium and archeobacteria are formed in evolution process
Defense system.CRISPR-Cas9 system be by choose one section of invasion sequence as newly between region sequence be inserted into CRISPR-
In the locus of Cas9, there are prototype intervening sequences to adjoin motif (protospacer-associated for region sequence between this section
Motif, PAM), utilize CRISPR RNA (CRISPR-derived RNA, crRNA) and transacting RNA (trans-
Activating RNA, tracrRNA) specifically identify target sequence, and using Cas9 albumen the of PAM Sequences upstream
Shearing forms flat end at 3-4bp, occurs nonhomologous end reparation (Non-homologous end joining, NHEJ) and same
Repair (homology-mediated end joining, HR) in source end.
Presently, there are the technical issues of include following two points: (1) lack effective selection markers, common external source hygromycin
Resistant gene (hph) is unable to stable integration into Pleurotus eryngii genome, and transformant hph segment after passage goes out active, from
And lead to not obtain the transformant after gene editing.(2) Pleurotus eryngii is amphiploid basidiomycetes, with the side of traditional homologous recombination
Method generally can not obtain homozygote to gene knockout, therefore the prior art brings very big difficulty to gene knockout.(3) current state
The gene site-directed editing technique of the inside and outside Pleurotus eryngii for not having foundation also.
Summary of the invention
Goal of the invention: the present invention provides a kind of Cas9 albumen, which can be used for CRISPR/Cas9 system.This hair
It is bright to additionally provide the CRISPR/Cas9 system editor's target gene group formed using the Cas9 albumen.The present invention has been also set up pair
The method of mushroom gene editing and the mushroom cell strain that can carry out gene editing, the cell strain are to lose orotidine -5'- phosphoric acid
The Pleurotus eryngii uracil auxotrophy bacterial strain of decarboxylase function.
Technical solution: in order to overcome the drawbacks of the prior art, first aspect present invention provides a kind of Cas9 albumen, is applicable in
In CRISPR/Cas9 system, the coded sequence of the Cas9 albumen is as shown in SEQ ID NO.3.Cas9 used in the present invention
Albumen is source of people Cas9 albumen of the optimization with Codon degeneracy feature according to mushroom genome codon Preference.
Second aspect of the present invention provides the polynucleotides for encoding above-mentioned Cas9 albumen, and sequence is as shown in SEQ ID NO.3.
Third aspect present invention provides expression vector, which contains the polynucleotides of coding Cas9 albumen.
Fourth aspect present invention provides a kind of edit tool of DNA genomic fragment, is CRISPR/Cas9 system, institute
Stating CRISPR/Cas9 system includes above-mentioned Cas9 albumen, polynucleotides, the multicore containing Cas9 albumen for encoding Cas9 albumen
The expression vector of thuja acid；In order to realize the editor to DNA genomic fragment, the CRISPR/Cas9 system also includes for mesh
Mark one or more sgRNA of DNA fragmentation.
Fifth aspect present invention provides a kind of CRISPR/Cas9 carrier for mushroom gene editing, the CRISPR/
Cas9 carrier is that the nucleotide sequence of Cas9 albumen, U6 promoter, sgRNA are loaded on plasmid, is obtained for editing mushroom
The recombinant vector of gene；The nucleotide sequence of the U6 promoter is selected from SEQ ID NO.4, SEQ ID NO.5, SEQ ID
NO.6 or SEQ ID NO.7.
In order to improve the target cell strain identification work after transcriptional efficiency and gene editing, further include in recombinant vector
The left and right homology arm nucleotide sequence of GFP nucleotide sequence and target gene.
Sixth aspect present invention provides a kind of U6 promoter using CRISPR/Cas9 carrier, the U6 promoter
Nucleotide sequence is selected from SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7.The present invention identifies 4
The Pleurotus eryngii U6snRNA promoter of a transcription sgRNA, can efficiently start the transcription of sgRNA.
Seventh aspect present invention provides a kind of edit methods of mushroom cell strain genomic fragment, by above-mentioned CRISPR/
Cas9 carrier transfects mushroom cell strain, edits to the target gene of mushroom cell strain.
In order to improve mushroom cell strain genomic fragment editor's success rate and gene editing tool of the invention it is pervasive
Property, resistance screening is carried out to target cell strain, the mushroom cell strain is CbxRResisting cell strain.
The CbxRResisting cell strain is to compile in coding succinate dehydrogenase-iron-sulfur protein subunit sdhB gene order
The 240th hyte amino acid of code has carried out rite-directed mutagenesis, and the sdhB gene order after mutation is as shown in SEQ ID NO.2.
Eighth aspect present invention provides a kind of mushroom cell strain that can be used for gene editing, and the mushroom cell strain is
CbxRResisting cell strain, wild type succinate dehydrogenase-iron-sulfur protein subunit sdhB gene are right as shown in SEQ ID NO.1
It is prominent to have carried out fixed point for the 240th hyte amino acid of coding in coding succinate dehydrogenase-iron-sulfur protein subunit sdhB gene order
Become (His to Leu, CAC to CTC), the sdhB gene order after mutation is as shown in SEQ ID NO.2.
By the tool of genome editor of the invention, the pyrG gene of Pleurotus eryngii is edited.
Screen the DNA sequence dna with section of DNA sequence base complementrity special on the exon of pyrG gene N-terminal, feature
Meet 5 '-GGN (17) GG, 5 '-GGN (17) CGG or 5 '-G (17) GGNGG put in order, gene as a purpose
SgRNA, above-mentioned pyrG-sgRNA target sequence are located on exon, and sequence is unique, nucleotide sequence such as SEQ
Shown in ID NO.8.
The present invention utilizes above-mentioned sgRNA preparation and reorganization plasmid, and the recombinant plasmid obtains by the following method: by Cas9 egg
(nucleotide sequence is selected from SEQ ID NO.4, SEQ ID NO.5, SEQ for white (sequence is as shown in SEQ ID NO.3), U6 promoter
ID NO.6 or SEQ ID NO.7), sgRNA, pyrG Donor DNA (GFP segment), pyrG or so homology arm sequence be loaded intoOn-Blunt Zero Cloning Kit plasmid, recombinant plasmid is obtained, by Transfected Recombinant Plasmid Pleurotus eryngii cell strain,
To the pyrG gene editing of Pleurotus eryngii cell strain
Above-mentioned Pleurotus eryngii cell strain is by following processing: Pleurotus eryngii mycelia is collected, with the ddH of sterilizing2O is washed twice.Configuration
The lywallzyme enzymolysis liquid for hydrolyzing Pleurotus eryngii cell wall, digests Pleurotus eryngii mycelia, and the protoplast for obtaining Pleurotus eryngii carries out
Transfection.
Further, clone has obtained coding Pleurotus eryngii succinate dehydrogenase-iron-sulfur protein subunit gene order, to amber
The group amino acid that amber acidohydrogenase-iron-sulfur protein subunit coding region is the 240th has carried out rite-directed mutagenesis, is connected into-
In Blunt Zero Cloning Kit plasmid, recombinant plasmid is obtained, strain of Pleurotus eryngii is transfected, obtains CbxRResistant strain,
Utilize CbxRThe Pleurotus eryngii cell strain of resistance carries out pyrG gene editing.
The utility model has the advantages that (1), the present invention provides a kind of Cas9 albumen of optimization, which has Pleurotus eryngii genome password
Sub- Preference, the genome editor for mushroom cell strain；It (2), can the present invention provides the U6 promoter that 4 are transcribed sgRNA
Efficiently to start the transcription of sgRNA；(3) use endogenous gene mutation that resistance is caused to change as apricot Bao the present invention provides a kind of
Mushroom selected marker completes the system of screening, overcomes common external source hygromycin gene (hph) and is unable to stable integration and arrives
The defect for the transformant that acquisition gene editing is crossed cannot be stablized in Pleurotus eryngii genome；(4) it is provided in the present invention
It is a kind of can be with the sgRNA of specific localization pyrG gene, can targeting knockout pyrG gene.
Detailed description of the invention
Fig. 1 is Pleurotus eryngii succinate dehydrogenase-iron-sulfur protein subunit (SdhB) rite-directed mutagenesis sequencing signal in the present invention
Figure；
Fig. 2 is Cbx in the present inventionRThe point bacterium schematic diagram of resistant strain, the results showed that, it encodes iron-sulfur protein subunit (SdhB)
Site-directed point mutation after, can be grown on the culture medium containing carboxin bacteriostatic agent；
Fig. 3 is the fragment structure schematic diagram of U6 promoter transcription sgRNA in the present invention；
Fig. 4 is to be constructed in the present invention for knocking out the Cas9 encoding histone base of the sgRNA of Pleurotus eryngii pyrG gene, optimization
The recombinant plasmid figure of cause, the external source GFP segment for needing to be inserted into；
Fig. 5 is that the point bacterium schematic diagram of Pleurotus eryngii pyrG gene function is successfully destroyed in the present invention, and display transformant cannot be
Growth, illustrates pyrG gene lacks functionality in basal medium (MM)；And WT and pyrG transformant is in addition UU (pyrimidine nucleoside
Acid) culture medium in be can be with normal growth.
Specific embodiment
Embodiment 1: with the structure for the Pleurotus eryngii genetic system that endogenous gene mutation causes resistance change alternatively to mark
It builds
1.1 clone's coding Pleurotus eryngii succinate dehydrogenase-iron-sulfur protein subunit (SdhB) gene orders (including promoter
And terminator, 3015bp) be connected into plasmid (- Blunt Zero Cloning Kit), as shown in SEQ ID NO.1.
The primer of a pair of reverse complementals comprising mutational site of 1.2 designs, primer sequence is as follows, and wherein primer sequence is such as
Under, wherein Mutant-F and Mutant-R respectively represent forward and reverse primer:
Mutant-F:GTTCCGCTGCCTCACCATCT
Mutant-R:AGATGGTGAGGCAGCGGAAC
1.3PCR system are as follows: 25 μ l of high fidelity enzyme；Each 2 μ l of primer；18 μ l of water；3 μ l of plasmid template.
1.4PCR reaction condition is 95 DEG C of initial denaturation, 5min；95 DEG C of denaturation, 15s；56 DEG C of annealing, 15s；Extend 72 DEG C,
2min；Totally 32 circulations；Finally extend 5min.
After 1.5PCR reaction, 10 μ l PCR reaction solutions are taken；DpnI 1μl；10xT Buffer 2μl；7 μ l of water.It is anti-in 20 μ l
It answers in liquid, is reacted at a temperature of 37 DEG C 1 hour, 70 DEG C of heat treatment 15min, can be such that enzyme inactivates later.
The reaction solution of previous step is directly converted competent escherichia coli cell, plated overnight by 1.6；Picking recombination is correct
Single colonie shakes bacterium 12-15h, extracts plasmid, and sequencing obtains CbxRR plasmid- Blunt-Zero-sdhB, sequencing knot
Fruit sees SEQ ID NO.2.
1.7 sequencings and result prove to occur really in 240 histidine conserved positions of iron-sulfur protein subunit (SdhB)
Point mutation, by codon CAC is mutated into CTC, Pleurotus eryngii succinate dehydrogenase-iron-sulfur protein subunit (SdhB) rite-directed mutagenesis
Schematic diagram is shown in Fig. 1.
1.8 will expressionThe method that the plasmid of-Blunt-Zero-sdhB carries out the protoplast transfection of PEG mediation
It is transfected, the screening of transformant is carried out with carboxin bacteriostatic agent.
1.9 picking transformants extract its DNA, sequencing discovery transfection- Blunt-Zero-sdhB plasmid is successful
Transformant bacterial strain can grow out on resistance carboxin plate really, as a result see Fig. 2, and be sequenced correct.It proves above-mentioned
The endogenous transformation system of the Pleurotus eryngii established is the important selection markers to Pleurotus eryngii gene editing.
1.10 generate the feature construction of resistance with iron-sulfur protein subunit (SdhB) 240 hyte propylhomoserin point mutation to carboxin
Endogenous transformation system be for later stage compilation Pleurotus eryngii related gene it is vital, can use this endogenous transformation system as
Edit the basis of Pleurotus eryngii related gene.
Embodiment 2: transcription sgRNA and the plasmid construction for expressing Cas9 albumen
The optimization of 2.1 source of people Cas9 albumen codon preferences
The Preference that 64 kinds of codons encode corresponding amino acid is analyzed according to the coded sequence of Pleurotus eryngii genome, to people
Source Cas9 albumen carries out rearranging for codon, and final optimization pass goes out the sequence that can stablize the Cas9 albumen of expression in Pleurotus eryngii
Column information, as shown in SEQ ID NO.3.
The clone of 2.2 Pleurotus eryngii High-expression promoter Pgpd promoters
Primer are as follows:
Pgpd-R:AGTCACAAGGGATGGGTGGT
Pgpd-F:GCGAACACTCAAAGCAAAG
PCR system are as follows: 25 μ l of high fidelity enzyme；Each 2 μ l of primer；18 μ l of water；3 μ l of WT template.
PCR reaction condition are as follows: 95 DEG C of initial denaturation, 5min；95 DEG C of denaturation, 15s；56 DEG C of annealing, 15s；Extend 72 DEG C,
2min；Totally 32 circulations；Finally extend 5min.
The clone of 2.3 aspergillus nidulans trpC terminators
Primer are as follows:
trpC-F:AAAGCCTTCGAGCGTCCCA
trpC-R:TCCTGCCCGTCACCGAGAT
PCR system are as follows: 25 μ l of high fidelity enzyme；Each 2 μ l of primer；18 μ l of water；3 μ l of plasmid template.
PCR reaction condition are as follows: 95 DEG C of initial denaturation, 5min；95 DEG C of denaturation, 15s；56 DEG C of annealing, 15s；Extend 72 DEG C,
2min；Totally 32 circulations；Finally extend 5min.
2.4 fusion DNA vaccine
By Pgpd promoter, the source of people Cas9 albumen of the Pleurotus eryngii Preference optimized, aspergillus nidulans trpC terminator three
Duan Ronghe is connected intoIn-Blunt Zero Cloning Kit plasmid, recombinant plasmid is formed-Blunt
Zero-Pgpd-Cas9-trpC。
2.5 sgRNA target spots are selected
SgRNA is located on exon of the pyrG gene close to N-terminal in the target sequence on pyrG gene, such as SEQ ID NO.8
It is shown.
The identification of 2.6 sgRNA promoter Pol III RNA polymerase class promoters
4 U6snRNA promoters (promoter), sequence such as SEQ ID are identified from the genome of Pleurotus eryngii itself
NO.4, SEQ ID NO.5, shown in SEQ ID NO.6 and SEQ ID NO.7.
The clone of 2.7 sgRNA splice segments (scaffold).
SgRNA scaffold is expanded from PX330 plasmid (plasmid).
SgRNA scaffold-F:GTTTTAGAGCTAGAAATAGC
SgRNA scaffold-R:AAAAAAGCACCGACTCGGTGCC
2.8 concentration measured according to two U6 promoter, sgRNA scaffold segments, by U6 promoter, sgRNA
The mixing of scaffold equal proportion, two segments are connected to composition U6-sgRNA-scaffold segment together by fusion DNA vaccine, such as Fig. 3 institute
Show, forms NotI-U6-sgRNA-scaffold-NotI segment plus NotI restriction enzyme site at fusion segment both ends.
2.9The single endonuclease digestion in the site NotI of-Blunt Zero-Pgpd-Cas9-trpC.
2.10 willThe single endonuclease digestion segment in the site NotI of-Blunt Zero-Pgpd-Cas9-trpC and 2.9 institutes
The NotI-U6-sgRNA-scaffold-NotI segment stated carries out recombination connection.
2.11 recombination systems are as follows:
4 μ l single endonuclease digestion carriers-Blunt Zero-Pgpd-Cas9-trpC
1 μ l NotI-U6-sgRNA-scaffold-NotI segment
2μl 5xCE II Buffer
1μlRecombinase
Moisturizing directly converts competent escherichia coli cell, plated overnight to 10 μ l, 37 DEG C of incubation 30min；Picking recombination
Successful single colonie shakes bacterium 12-15h, extracts plasmid-Blunt Zero-Pgpd-Cas9-trpC-U6-sgRNA-
Scaffold。
2.12 clones need to edit left and right homology arm (Donor) DNA fragmentation in the site gene (pyrG), left homology arm sequence
As shown in SEQ ID NO.17, it is same to need to edit the access of gene loci both ends as shown in SEQ ID NO.18 for right homology arm sequence
Source arm sequence.
The left homology arm of pyrG, GFP segment, three sections of the right homology arm of pyrG fusions, sequence are with pyrG by 2.13 fusion DNA vaccines
The GFP segment that Donor DNA (GFP segment) is inserted into as template reparation, as a result as shown in figure SEQ ID NO.19.Left and right arms point
SpeI connector is not taken forms speI-pyrG left arm-GFP-pyrG right arm-speI.
2.14 plasmidThe site speI of-Blunt Zero-Pgpd-Cas9-trpC-U6-sgRNA-Scaffold
Single endonuclease digestion.
2.15The list in the site speI of-Blunt Zero-Pgpd-Cas9-trpC-U6-sgRNA-Scaffold
PyrG left arm-GFP-pyrG right arm in above-mentioned the 2.13 of endonuclease bamhi and speI connector) recombination.
2.16 recombination systems are as follows:
4 μ l single endonuclease digestion carriers-Blunt Zero-Pgpd-Cas9-trpC-U6-gRNA-Scaffold
1 μ l long homology arm pyrG Donor DNA (GFP segment)
2μl 5xCE II Buffer
1μlRecombinase
Moisturizing directly converts competent escherichia coli cell, plated overnight to 10 μ l, 37 DEG C of incubation 30min；Picking recombination
Successful single colonie shakes bacterium 12-15h, extracts plasmid-Blunt Zero-Pgpd-Cas9-trpC-U6-sgRNA-
Scaffold-pyrG-Donor-DNA (GFP segment), the recombinant plasmid figure of building are shown in Fig. 4.
Embodiment 3: the acquisition of the vegetative mutant bacterial strain of Pleurotus eryngii pyrG gene knockout
In embodiment 2-Blunt-Zero-Pgpd-Cas9-trpC-U6-gRNA-scaffold-pyrG-
The method that the plasmid of Donor-DNA (GFP segment) carries out the protoplast transfection of PEG mediation is transfected.
The method for the protoplast transfection that 3.1 PEG are mediated is as follows:
3.1.1 Pleurotus eryngii mycelia is collected, with the ddH of sterilizing2O is washed twice.
3.1.2 the lywallzyme enzymolysis liquid of configuration hydrolysis Pleurotus eryngii cell wall, and filtration sterilization, the mycelia of collection is transferred to
In enzymolysis liquid, with 28 DEG C in shaking table, 80rpm revolving speed digests 3.5h or so.
3.1.3 after digesting, product is transferred in 50ml centrifuge tube, 10ml Trapping Buffer is added,
5000rpm, 4 DEG C of centrifugation 15min.
3.1.4 by the protoplast layer of white after being centrifuged, it is gently drawn in another 50ml centrifuge tube with liquid-transfering gun,
Buffer, 5000rpm, 4 DEG C of centrifugation 10min of STC of equal volume are added.
3.1.5 supernatant is removed, 1ml STC Buffer is added and mixes protoplast, it is spare to be put into refrigerator.
It 3.1.6 is the plasmid after the protoplast and vacuum concentration of 100 μ l by volume with liquid-transfering gun-Blunt-
Zero-Pgpd-Cas9-trpC-U6-gRNA-scaffold-pyrG-Donor-DNA (GFP segment) is mixed gently, and is put on ice
50min is set, 1.25ml PEG solution is added, is placed at room temperature for 25min, 10ml is added and converts upper layer culture medium, it is good in advance to pour into
Lower layer's culture medium, 28 DEG C cultivate 15 days.
3.2 select pyrG transformant to be edited, and (MM) carries out a bacterium on basal medium, and point bacterium result is shown in Fig. 5 institute
Show, shows that transformant cannot be grown in basal medium (MM), illustrate pyrG gene lacks functionality；And WT and pyrG is edited
Transformant addition UU (pyrimidine nucleotide) culture medium in can be with normal growth.
3.3 pairs of transformants extract DNA sequencing analysis, analyze the editing sites of target gene, analyze result
It is shown in Table 1.
The sequencing result of the auxotrophic strain of nonhomologous end reparation occurs for the fixed point editor's Pleurotus eryngii pyrG gene of table 1
It is found according to the sequencing result of table 1: the orotidine -5'- phosphate decarboxylase (OMPDC) of Pleurotus eryngii pyrG gene coding
The orotic acid contained in culture medium can be changed into pyrimidine nucleotide, so the Pleurotus eryngii of damage pyrG gene function can not
It is normally grown on basal medium (MM).30 transformants are selected altogether, and transformant sequencing result demonstrates pyrG gene
Really the different types of editor such as insertion and missing is completed in expected editor's target spot, as shown in SEQ ID NO.9-16,
The cleavage site CGCTGTA of Cas9 albumen detects 25 muton (mutation of nonhomologous end reparation for causing frameshift mutation
Son has 10, and the muton that insertion GFP sequence is repaired in homologous end has 15), mutation rate 83.3%, part of transformant
Sequencing result it is as shown in table 1, show target gene pyrG the sequence location that sgRNA is targeted occur be inserted into and lack, cause
Frameshift mutation has occurred in pyrG gene, illustrates that gene knockout is successful.
The DNA sequence dna that targets identification sequence complementary pairing is directed in the present invention is SEQ ID NO.8, is thus recruited
Cas9 protein sequence is SEQ ID NO.3, and the Cas9 albumen optimized according to Pleurotus eryngii genome codon Preference is designing
Target site carry out pinpointing accurate editor.Illustrate that the U6-Cas9 system of optimization can efficiently edit a system such as Pleurotus eryngii pyrG
Column gene, and editorial efficiency is very high.
Sequence table
<110>Nanjing Normal University
<120>a kind of method and application of Cas9 albumen, CRISPR/Cas9 system, mushroom gene editing
<160> 19
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3015
<212> DNA
<213>Pleurotus eryngii sdhB gene (Pleurotus eryngii-sdhB gene)
<400> 1
tggctccctt agtcacatac tgtcaaggct cttttagaga acttttctca agcagcgtga 60
aagcctagtt ggtatcgacg aaagcggaaa gataccaaga tatcacggca ttcgtagctt 120
cgccgaggga atgacatagg cgggtataac ctcctttgag ttgagacgaa agtgtggctt 180
caaactcacg tcctcaataa gcgctcgcga catcgcggat tcacatcaca ccactctcca 240
agaatctctc gttcaaaggt atcctgtatt tgtaagtcaa gccaacgttt attccatgat 300
acaaggctgg ccacattcat ggtattctct ccgtaaatag ccatttacca cccatgacac 360
gacatattcg gattcctgcc gcttgtgaac caaccgttca actgtgcaaa cgctaataca 420
tggcttctga gactcccaac gttgtttgga gaaaaattct caaaaacggc tgccgaagac 480
ctaagccagt gttgctccta cattttctca acactcgata gaagctgcaa ctaggtcaac 540
aaagttggca ttccatcgcc tttgactaca aattgattcg gggggttcga cgatggacac 600
catgagcgag ctttgtccgt tacgacgaga acatccgaag tcccatcctc tcctgaactc 660
ttcaagacca ccattcaagg gaatttcgag gtctcggagc gccgttgaac ccaggttttc 720
gggtgacacc tcaattgctt tccccctttt ctgaccagag tccccgaggt acagcccggg 780
tacggcacat attctacctt gtaaacctca ctcataacgt tccgaggaga gtggcatcac 840
acagcgcgct ggcctaatag tggtgtcaag ttggaaggga agcgagtcgg ggtcattggg 900
actggggcca gcgccgagtt cagatcatcc aagaagcagg caaattggca caagggctcg 960
tggtattcca gcgtacaccg aatctgacat tccgcttgcg acaacccacg caagccaaag 1020
tgtcagaagt tgtcgaaatg caatggaatc atgttcacga agaggccttg ggtagtgacc 1080
ctatcaggta aacaagtatc gtcgatttct tgacacttct agctgctccg tccacgtgta 1140
taccttcgac tcaccattga agagcatgat gggcgcaaag attacgttct atcgctcttc 1200
tctctttact ttgataatcg aaagctgcac tactcagcgc cgcaattata catgtttttc 1260
taaaaatcta ccgactcacc gctgacgcgc tgacggccgg taagcttcaa gtaacctgca 1320
gttccaatcg ggcaatttca aagtgtgccg gcatttaccg aagagtaaca tgcgccctct 1380
gattggttcg gatcgcgagg gctcctttcc cgctccgaat tcacgcgacg ggatcccgcc 1440
gcatctctcc tcttcaccgt cgtcgttgac gtccccgaaa cacagaaatc accgaatcat 1500
gcaggcgctc acctccaggt cgttggctcg ctcaccccgc tcgattcgtt ctttctccac 1560
ttcgtgtgga aggtggcagg ctgagctcct ccagaagccc gttctccaga aagaattcaa 1620
gatctaccgt tgggtgagct gagacgcttg tgtatccaga tgtgttgctc acattgtggc 1680
acagaatccg gatgaaccag ccaagaagcc tcatctccaa tcgtacacca ttgacttgaa 1740
ccagacgggc cccatggtac gtacaattcc aaggcgattg tctctatgct cacgggctcg 1800
tagattctgg acgctcttat caagatcaag aacgaaatcg atcctacgct cacattccgt 1860
cgttcgtgta gagaagggat ttgcggctcg tgcgcgatga atattgacgg acagaacacg 1920
ttggcttgcc tgtgccgaat tgaccgtaac gccagcaagg acagcaagat ctaccctttg 1980
ccgcacagta tgacatgtct tttggttcct aatagcattg actgacggtc agttacagtg 2040
tacatcgtga aagacctcgt acccgacctc acacttttct acaagcagta caagtccatc 2100
aagccttacc tgcagaacga caatgtcccc gagagggagc acctccagtc gccagaggac 2160
cgcaagaagc tggacgggat gtatgaatgc atcctgtgcg cgtgctgcag cacatcgtgt 2220
cccagctatt ggtggaacca agatgagtat ttggaccggc tgctttgatg gccgcgtata 2280
ggtggattgc ggactcacga gtgcgttatg ttgccgtcga tacatgctct cattcatagt 2340
ctaacatttc gcaggatacg tatggcgcac aacgcaagga acatttccag aatgagatga 2400
gtttgttccg ctgcctcacc atcttcaatt gtacgtcgct ttcgccttga tatggattgg 2460
ctgttaatca tatcccttct caaggctctc gcacttgtcc aaagggcctc aaccctgcga 2520
aagccattgc agagatcaag ctcgcgctcg ctactgagta aaccctagtc aacagccacg 2580
gatcaaaagc attaagtcag aggcagattt ctttcctgta gcagttcgca gttcttttca 2640
cttcatcgta tggtgtccat tgcagacatc taatcatatt cattctatca catccacttg 2700
ttcttgagcc actcttaagg taggcatacc ctcggtctca cttgcggtgc tgtacgaaac 2760
aagaacggat acatattcta tattctatgg gacatctaac gactcaggca attcatagtc 2820
tactaggctc aacatcgaga gcgaagaagg gatacgacgt gtagccgtcg gagtccacgc 2880
tgccaggtct gtagtaatcc tttctatcac ctttttccac ttgccaatca ttcttcagtc 2940
gcagcggcaa atcgggctat cgtccagcac tcgaactcag ccaacacctc tcctcaggac 3000
gaaggaaggc ttacg 3015
<210> 2
<211> 3015
<212> DNA
<213>Pleurotus eryngii sdhB gene (Pleurotus eryngii-sdhB gene)
<400> 2
tggctccctt agtcacatac tgtcaaggct cttttagaga acttttctca agcagcgtga 60
aagcctagtt ggtatcgacg aaagcggaaa gataccaaga tatcacggca ttcgtagctt 120
cgccgaggga atgacatagg cgggtataac ctcctttgag ttgagacgaa agtgtggctt 180
caaactcacg tcctcaataa gcgctcgcga catcgcggat tcacatcaca ccactctcca 240
agaatctctc gttcaaaggt atcctgtatt tgtaagtcaa gccaacgttt attccatgat 300
acaaggctgg ccacattcat ggtattctct ccgtaaatag ccatttacca cccatgacac 360
gacatattcg gattcctgcc gcttgtgaac caaccgttca actgtgcaaa cgctaataca 420
tggcttctga gactcccaac gttgtttgga gaaaaattct caaaaacggc tgccgaagac 480
ctaagccagt gttgctccta cattttctca acactcgata gaagctgcaa ctaggtcaac 540
aaagttggca ttccatcgcc tttgactaca aattgattcg gggggttcga cgatggacac 600
catgagcgag ctttgtccgt tacgacgaga acatccgaag tcccatcctc tcctgaactc 660
ttcaagacca ccattcaagg gaatttcgag gtctcggagc gccgttgaac ccaggttttc 720
gggtgacacc tcaattgctt tccccctttt ctgaccagag tccccgaggt acagcccggg 780
tacggcacat attctacctt gtaaacctca ctcataacgt tccgaggaga gtggcatcac 840
acagcgcgct ggcctaatag tggtgtcaag ttggaaggga agcgagtcgg ggtcattggg 900
actggggcca gcgccgagtt cagatcatcc aagaagcagg caaattggca caagggctcg 960
tggtattcca gcgtacaccg aatctgacat tccgcttgcg acaacccacg caagccaaag 1020
tgtcagaagt tgtcgaaatg caatggaatc atgttcacga agaggccttg ggtagtgacc 1080
ctatcaggta aacaagtatc gtcgatttct tgacacttct agctgctccg tccacgtgta 1140
taccttcgac tcaccattga agagcatgat gggcgcaaag attacgttct atcgctcttc 1200
tctctttact ttgataatcg aaagctgcac tactcagcgc cgcaattata catgtttttc 1260
taaaaatcta ccgactcacc gctgacgcgc tgacggccgg taagcttcaa gtaacctgca 1320
gttccaatcg ggcaatttca aagtgtgccg gcatttaccg aagagtaaca tgcgccctct 1380
gattggttcg gatcgcgagg gctcctttcc cgctccgaat tcacgcgacg ggatcccgcc 1440
gcatctctcc tcttcaccgt cgtcgttgac gtccccgaaa cacagaaatc accgaatcat 1500
gcaggcgctc acctccaggt cgttggctcg ctcaccccgc tcgattcgtt ctttctccac 1560
ttcgtgtgga aggtggcagg ctgagctcct ccagaagccc gttctccaga aagaattcaa 1620
gatctaccgt tgggtgagct gagacgcttg tgtatccaga tgtgttgctc acattgtggc 1680
acagaatccg gatgaaccag ccaagaagcc tcatctccaa tcgtacacca ttgacttgaa 1740
ccagacgggc cccatggtac gtacaattcc aaggcgattg tctctatgct cacgggctcg 1800
tagattctgg acgctcttat caagatcaag aacgaaatcg atcctacgct cacattccgt 1860
cgttcgtgta gagaagggat ttgcggctcg tgcgcgatga atattgacgg acagaacacg 1920
ttggcttgcc tgtgccgaat tgaccgtaac gccagcaagg acagcaagat ctaccctttg 1980
ccgcacagta tgacatgtct tttggttcct aatagcattg actgacggtc agttacagtg 2040
tacatcgtga aagacctcgt acccgacctc acacttttct acaagcagta caagtccatc 2100
aagccttacc tgcagaacga caatgtcccc gagagggagc acctccagtc gccagaggac 2160
cgcaagaagc tggacgggat gtatgaatgc atcctgtgcg cgtgctgcag cacatcgtgt 2220
cccagctatt ggtggaacca agatgagtat ttggaccggc tgctttgatg gccgcgtata 2280
ggtggattgc ggactcacga gtgcgttatg ttgccgtcga tacatgctct cattcatagt 2340
ctaacatttc gcaggatacg tatggcgcac aacgcaagga acatttccag aatgagatga 2400
gtttgttccg ctgcctcacc atcttcaatt gtacgtcgct ttcgccttga tatggattgg 2460
ctgttaatca tatcccttct caaggctctc gcacttgtcc aaagggcctc aaccctgcga 2520
aagccattgc agagatcaag ctcgcgctcg ctactgagta aaccctagtc aacagccacg 2580
gatcaaaagc attaagtcag aggcagattt ctttcctgta gcagttcgca gttcttttca 2640
cttcatcgta tggtgtccat tgcagacatc taatcatatt cattctatca catccacttg 2700
ttcttgagcc actcttaagg taggcatacc ctcggtctca cttgcggtgc tgtacgaaac 2760
aagaacggat acatattcta tattctatgg gacatctaac gactcaggca attcatagtc 2820
tactaggctc aacatcgaga gcgaagaagg gatacgacgt gtagccgtcg gagtccacgc 2880
tgccaggtct gtagtaatcc tttctatcac ctttttccac ttgccaatca ttcttcagtc 2940
gcagcggcaa atcgggctat cgtccagcac tcgaactcag ccaacacctc tcctcaggac 3000
gaaggaaggc ttacg 3015
<210> 3
<211> 4272
<212> DNA
<213>gene order (Pleurotus eryngii Cas9 protein gene) of Pleurotus eryngii Cas9 albumen
<400> 3
atggactaca aggaccacga cggcgactac aaggatcacg acatcgacta caaagacgac 60
gacgacaaga tggcgcccaa gaagaagcgc aaggtcggca tccacggcgt ccccgcagct 120
gacaagaaat actccatcgg cctggacatc ggcaccaaca gcgtcggatg ggctgttatc 180
accgacgaat acaaagtccc atccaagaag ttcaaggtcc tcggtaacac cgacagacac 240
tccatcaaga agaacctcat cggagctctc ctcttcgact ccggagaaac cgctgaagcc 300
accagactca aaagaaccgc ccgccgccgc tacacccgca gaaaaaaccg catctgctac 360
ctccaagaaa tcttctccaa cgaaatggct aaggtcgacg actccttctt ccaccgcctc 420
gaagaatcct tcctcgtcga agaagacaag aagcacgaac gccaccctat cttcggcaac 480
atcgtcgacg aagtcgctta ccacgaaaag taccctacca tctaccacct ccgcaagaag 540
ctcgtcgact ccaccgacaa ggctgacctc cgcctcatct acctcgctct cgctcacatg 600
atcaagttcc gcggccactt cctcatcgaa ggcgacctca accctgacaa ctccgacgtc 660
gacaagctct tcatccaact cgtccaaacc tacaaccaac tcttcgaaga aaaccctatc 720
aacgcttccg gcgtcgacgc taaggctatc ctctccgctc gcctctccaa gtcccgccgc 780
ctcgaaaacc tcatcgctca actccctggc gaaaagaaga acggcctctt cggcaacctc 840
atcgctctct ccctcggcct cacccctaac ttcaagtcca acttcgacct cgctgaagac 900
gctaagctcc aactctccaa ggacacctac gacgacgacc tcgacaacct cctcgctcaa 960
atcggcgacc aatacgctga cctcttcctc gctgctaaga acctctccga cgctatcctc 1020
ctctccgaca tcctccgcgt caacaccgaa atcaccaagg ctcctctctc cgcttccatg 1080
atcaagcgct acgacgaaca ccaccaagac ctcaccctcc tcaaggctct cgtccgccaa 1140
caactccctg aaaagtacaa ggaaatcttc ttcgaccaat ccaagaacgg ctacgctggc 1200
tacatcgacg gcggcgcttc ccaagaagaa ttctacaagt tcatcaagcc tatcctcgaa 1260
aagatggacg gcaccgaaga actcctcgtc aagctcaacc gcgaagacct cctccgcaag 1320
caacgcacct tcgacaacgg ctccatccct caccaaatcc acctcggcga actccacgct 1380
atcctccgcc gccaagaaga cttctaccct ttcctcaagg acaaccgcga aaagatcgaa 1440
aagatcctca ccttccgcat cccttactac gtcggccctc tcgctcgcgg caactcccgc 1500
ttcgcttgga tgacccgcaa gtccgaagaa accatcaccc cttggaactt cgaagaagtc 1560
gtcgacaagg gcgcttccgc tcaatccttc atcgaacgca tgaccaactt cgacaagaac 1620
ctccctaacg aaaaggtcct ccctaagcac tccctcctct acgaatactt caccgtctac 1680
aacgaactca ccaaggtcaa gtacgtcacc gaaggcatgc gcaagcctgc tttcctctcc 1740
ggcgaacaaa agaaggctat cgtcgacctc ctcttcaaga ccaaccgcaa ggtcaccgtc 1800
aagcaactca aggaagacta cttcaagaag atcgaatgct tcgactccgt cgaaatctcc 1860
ggcgtcgaag accgcttcaa cgcttccctc ggcacctacc acgacctcct caagatcatc 1920
aaggacaagg acttcctcga caacgaagaa aacgaagaca tcctcgaaga catcgtcctc 1980
accctcaccc tcttcgaaga ccgcgaaatg atcgaagaac gcctcaagac ctacgctcac 2040
ctcttcgacg acaaggtcat gaagcaactc aagcgccgcc gctacaccgg ctggggccgc 2100
ctctcccgca agctcatcaa cggcatccgc gacaagcaat ccggcaagac catcctcgac 2160
ttcctcaagt ccgacggctt cgctaaccgc aacttcatgc aactcatcca cgacgactcc 2220
ctcaccttca aggaagacat ccaaaaggct caagtctccg gccaaggcga ctccctccac 2280
gaacacatcg ctaacctcgc tggctcccct gctatcaaga agggcatcct ccaaaccgtc 2340
aaggtcgtcg acgaactcgt caaggtcatg ggccgccaca agcctgaaaa catcgtcatc 2400
gaaatggctc gcgaaaacca aaccacccaa aagggccaaa agaactcccg cgaacgcatg 2460
aagcgcatcg aagaaggcat caaggaactc ggctcccaaa tcctcaagga acaccctgtc 2520
gaaaacaccc aactccaaaa cgaaaagctc tacctctact acctccaaaa cggccgcgac 2580
atgtacgtcg accaagaact cgacatcaac cgcctctccg actacgacgt cgaccacatc 2640
gtccctcaat ccttcctcaa ggacgactcc atcgacaaca aggtcctcac ccgctccgac 2700
aagaaccgcg gcaagtccga caacgtccct tccgaagaag tcgtcaagaa gatgaagaac 2760
tactggcgcc aactcctcaa cgctaagctc atcacccaac gcaagttcga caacctcacc 2820
aaggctgaac gcggcggcct ctccgaactc gacaaggctg gcttcatcaa gcgccaactc 2880
gtcgaaaccc gccaaatcac caagcacgtc gctcaaatcc tcgactcccg catgaacacc 2940
aagtacgacg aaaacgacaa gctcatccgc gaagtcaagg tcatcaccct caagtccaag 3000
ctcgtctccg acttccgcaa ggacttccaa ttctacaagg tccgcgaaat caacaactac 3060
caccacgctc acgacgctta cctcaacgct gtcgtcggca ccgctctcat caagaagtac 3120
cctaagctcg aatccgaatt cgtctacggc gactacaagg tctacgacgt ccgcaagatg 3180
atcgctaagt ccgaacaaga aatcggcaag gctaccgcta agtacttctt ctactccaac 3240
atcatgaact tcttcaagac cgaaatcacc ctcgctaacg gcgaaatccg caagcgccct 3300
ctcatcgaaa ccaacggcga aaccggcgaa atcgtctggg acaagggccg cgacttcgct 3360
accgtccgca aggtcctctc catgcctcaa gtcaacatcg tcaagaagac cgaagtccaa 3420
accggcggct tctccaagga atccatcctc cctaagcgca actccgacaa gctcatcgct 3480
cgcaagaagg actgggaccc taagaagtac ggcggcttcg actcccctac cgtcgcttac 3540
tccgtcctcg tcgtcgctaa ggtcgaaaag ggcaagtcca agaagctcaa gtccgtcaag 3600
gaactcctcg gcatcaccat catggaacgc tcctccttcg aaaagaaccc tatcgacttc 3660
ctcgaagcta agggctacaa ggaagtcaag aaggacctca tcatcaagct ccctaagtac 3720
tccctcttcg aactcgaaaa cggccgcaag cgcatgctcg cttccgctgg cgaactccaa 3780
aagggcaacg aactcgctct cccttccaag tacgtcaact tcctctacct cgcttcccac 3840
tacgaaaagc tcaagggctc ccctgaagac aacgaacaaa agcaactctt cgtcgaacaa 3900
cacaagcact acctcgacga aatcatcgaa caaatctccg aattctccaa gcgcgtcatc 3960
ctcgctgacg ctaacctcga caaggtcctc tccgcttaca acaagcaccg cgacaagcct 4020
atccgcgaac aagctgaaaa catcatccac ctcttcaccc tcaccaacct cggcgctcct 4080
gctgctttca agtacttcga caccaccatc gaccgcaagc gctacacctc caccaaggaa 4140
gtcctcgacg ctaccctcat ccaccaatcc atcaccggcc tctacgaaac ccgcatcgac 4200
ctctcccaac tcggcggcga caagcgccct gctgctacca agaaggctgg ccaagctaag 4260
aagaagaagt aa 4272
<210> 4
<211> 695
<212> DNA
<213>Pleurotus eryngii U6 promoter 1 (Pleurotus eryngii U6 Promoter1)
<400> 4
cccatatgag catcttgatc aaccaacgaa ggccaagtgg ggcacaggtc ttctaccgtc 60
ctgggtgtgt caggttgcgc cggctacaaa tcatcctgct ccgatgtaga ttacgaccac 120
ggggatggaa gaatgtggac tggagacgtc cgcgtggtat acccgtaccg gctgggaggg 180
agcgagagac agagatggtg gcggaagcag acactaggga ttgcccaacg gcaatggaca 240
taccttgctt agaattgtgt ttgcaatgag actttactac gaaggacacc ccgtcgctca 300
acttgcggtc gaacttactc aatggttgag gagaacgaga actttgaaaa ttcagaataa 360
gtggattctt gtgcctacag ttggcgcagt gcgggctacc gaaaatctgt gccatttgtc 420
attcttcgaa ccactatcgc aatgtttttg gtagtaagtt caaggtggag tggtgtgtcg 480
acatcttttg tcaacctcgt cgaacttcac gcaaagcgtc ggagcggaga tactgccatt 540
caacaagagt acggtgcgca agtacggtga gagagtcaag actgcgcgcg attcgagccc 600
acgtgctacc aggcactgcc actgtgatcg aacccagtga gctggacaaa acacagcact 660
cagtggacgc cgtagcaaca gtactaacaa agact 695
<210> 5
<211> 453
<212> DNA
<213>Pleurotus eryngii U6 promoter 2 (Pleurotus eryngii U6 Promoter2)
<400> 5
gacctccgcc aactatacat ctaagcacac gccttggaaa atccaagtct ggagaggcga 60
ggacgggttt gttaagagaa tggtgaatgt tgatagtctt gttgtgtcgc ccagtcctgt 120
tgaaaagtta ctccaccccc gttctgaata ttgcagtcaa cctaacacac aggtacgcgt 180
aaagtattta cgccttcgtg acctatcgaa tgttccacgg ccccaaactt ccatgctcta 240
tcaggtacag acacaatcaa acttccgtga tatacgtgcc attcttgaca agtttcctca 300
ggaaatagat caagtcgaaa atacccctac acaaggacag acaataggta tcgcaatgag 360
cccctttcag ttggcgggta cccctgggaa gctcgcgcgc tgaacaaaac gcacaagtca 420
tgttgtatcc agtcaacacg agcaacaact att 453
<210> 6
<211> 707
<212> DNA
<213>Pleurotus eryngii U6 promoter 3 (Pleurotus eryngii U6 Promoter3)
<400> 6
agaaagccca gagttaccgt cagtcattgt cagcaccact gcttgtcaat gtcaatcgct 60
tcttgttgtt cctatatata tactagggat cgcccggcgg cgcttcaaaa tataccttgc 120
gtgggttggc atctcggttt cagtcctgct gcctccgttg ttttgcacac ccatctgtgt 180
cgaacgtcct gctttccaga atcccgcaca gcctcggagc atttcccaac gtctttctgg 240
gttgttatgg taggatacca ttcatttgcc gcgtccatac agaacgttgc tgttgacacg 300
gcacccgagt gaggatctct attccgaaga tgcaaggttg gcggcgctca acaaagatac 360
tgcgcagaag ttcggcaagc agtagggggg actcagagat tcagtgaagg gaacagtaca 420
ttgcgagcga cagtgcaggg agcattcctg tatacaagat taatattatt gtatgaatgt 480
aaattaggtg tgacttctga gggagcgtgg tagcgttgac gaccaccagc gcttcaaacg 540
tgggaatgtg cttgttgaga atcccgacga attgggcgag ttccagggag ggggacctag 600
ctttccaagc aaggtaatat atatataggg gtacacgtga tggacttcgc gaactgaaca 660
aaatacgaag ctcaggtcca gggctccttc ttcatacatt aattaat 707
<210> 7
<211> 793
<212> DNA
<213>Pleurotus eryngii U6 promoter 4 (Pleurotus eryngii U6 Promoter4)
<400> 7
gcgtgtcatc cttgcgcagg ggccatgcta atcttctctg tatcgttcca attttttcgt 60
atgtcacccc gaaggggaca atagttgttg ctcgtgttga ctggatacaa catgacttgt 120
gcgttttgtt cagcgcgcga gcttcccagg ggtacccgcc aactgaaagg ggctcattgc 180
gattacctat tgtctgtcct tgtgtagggg tattttcgac ttgatctatt tcctgaggaa 240
acttgtcaag aatggcacgt atatcacgga agtttgattg tgtctgtacc tgatagagca 300
tggaagtttg gggccgtgga acattcgata ggtcacgaag gcgtaaatac tttacgcgta 360
cctgtgtgtt agttgactgc aatattcaga acgggggtgg agtaactttt caacaggact 420
gggcgacaca acaagactat caacattcac cattctctaa caaacccgtc ctcgcctctc 480
cagactggat ttttccaagg cgtgtgctta gatgtatagt tggcggaggt ctcgcggacg 540
agctgttcgt tgatttctat accgattcca ggacctgcga aacaaggcgt cagatataaa 600
gtcttagaca acgaagatgc agttgacttg ccatggagca acccaagatg gccttccttg 660
atctcaaaca ccgatgggtg cgaaaggtag gtgtacaagt cagcctcttg atcactttca 720
ggagatacat tgtagtgtat ctatgcaatt acaatatgtt actgcgttaa gcaacgggag 780
tgtgtgataa cgc 793
<210> 8
<211> 19
<212> DNA
<213>target site (Pleurotus eryngii pyrG sgRNA) of Pleurotus eryngii pyrG gene
<400> 8
ggcaatcatc gacgctgta 19
<210> 9
<211> 920
<212> DNA
<213>WT type Pleurotus eryngii pyrG gene (wild type Pleurotus eryngii pyrG)
<400> 9
atgagctcaa agggagtcca ggcattgtca tatgtccaaa gggctgacaa ctacaccaat 60
cctgctgcga aggaactgct tctcaccatg gaacgcaaga agtccaacct ttccgttagc 120
gtggatgtga cgaaatcaag agatttcctg gcaatcatcg acgctgtagg gccatatgcc 180
tgtttaatca aggtaaaccg acctcatttg ttgcacaata ctcgggatct gatgcccaga 240
tctgcgaaag actcatgttg atatacttga agattttgac tttacgttga ttgaaagctt 300
gcaagctttg agcaaaaaac atgacttcat gatcttcgag gacagaaagt ttgcagacat 360
aggtgccact cttcgtagct caacttgaat cgccgttcac agcgcttgta ggaaacaccg 420
ttgcgttaca gtattcaagt ggcgtgcatc gcatcgcgag ttggtcgcag atcacgaatg 480
cccactcagt ccctggtcca tccatcgtcg cagggctttc ttcagtaggc ttacccctcg 540
gacggggtct cctcctcttg gcagaaatga gcacggcggg aaaccttgct gtgggccaat 600
acacagaaga gacttatcag atggctcgcg atcaccggga cttcgtaatc gggttcattg 660
gacaaagacg cccatctggc gagggagatg aggatttcct agtcctgaca cccggagtag 720
gattggatgt gaaggcagat ggtatggggc agcagtacag aacgcctcgc gaagtcatct 780
tggaatcagg ttgcgatgtg attattgtag gacgagggat atatgggaaa gattacagct 840
tgactgaagc catcgctcag caagcggaga ggtaccggga atcggggtgg agcgcatact 900
tagaaaggtg taaatcgtaa 920
<210> 10
<211> 54
<212> DNA
<213>(the Pleurotus eryngii pyrG gene segment of Pleurotus eryngii pyrG genetic fragment transformant 1
transformant 1)
<400> 10
aatcaagaga tttcctggca atcatcgacc gcttgtaggg ccatatgcct gttt 54
<210> 11
<211> 51
<212> DNA
<213>(the Pleurotus eryngii pyrG gene segment of Pleurotus eryngii pyrG genetic fragment transformant 2
transformant 2)
<400> 11
aatcaagaga tttcctggca atcatcgacg gtagggccat atgcctgttt a 51
<210> 12
<211> 43
<212> DNA
<213>(the Pleurotus eryngii pyrG gene segment of Pleurotus eryngii pyrG genetic fragment transformant 3
transformant 3)
<400> 12
aatcaagaga tttcctggca atgtagggcc atatgcctgt tta 43
<210> 13
<211> 46
<212> DNA
<213>(the Pleurotus eryngii pyrG gene segment of Pleurotus eryngii pyrG genetic fragment transformant 4
transformant 4)
<400> 13
aatcaagaga tttcctggca atcattgtag ggccatatgc ctgttt 46
<210> 14
<211> 46
<212> DNA
<213>(the Pleurotus eryngii pyrG gene segment of Pleurotus eryngii pyrG genetic fragment transformant 5
transformant 5)
<400> 14
aatcaagaga tttcctggca atcatgtagg gccatatgcc tgttta 46
<210> 15
<211> 120
<212> DNA
<213>(the Pleurotus eryngii pyrG gene segment of Pleurotus eryngii pyrG genetic fragment transformant 6
transformant 6)
<400> 15
aatcaagaga tttcctggca atcatcgacg ctaataatat taatcttgta tacaggaatg 60
ctccctgcac tgtcgctcgc aatgtactgt tcccttcacg tagggccata tgcctgttta 120
<210> 16
<211> 173
<212> DNA
<213>(the Pleurotus eryngii pyrG gene segment of Pleurotus eryngii pyrG genetic fragment transformant 7
transformant 7)
<400> 16
aatcaagaga tttcctggca atcatcgacg ctaaagctcg aggaacggac agtaactctg 60
cccgatccca aaccgaagct gaaactagca cacaaaccta tcattgagac tataggtgat 120
actaacgcgg aaaagtcgag cttgaattcg aagtagggcc atatgcctgt tta 173
<210> 17
<211> 923
<212> DNA
<213>the left homology arm sequence of Pleurotus eryngii pyrG gene (Pleurotus eryngii pyrG Upstream
homologous sequence)
<400> 17
gaatcaaggc tcacctctgc tttataatcc cctcgatctg ggagactccg cgaccgacgt 60
attctgatat ccctacatca gtctctctga acttgtaccc agagccgtct tcagccttct 120
caggtcgagg tacgcctagg agaacgtggc ttgggggtag aatgtcgtcg gcatgcccat 180
ctttgggcgg ggcagtatct gtcgtgactt cgggatcgga ttcctcggaa ggggacgtgg 240
gttctagaga atcctctaat ctagcacgtt ttgaggcgcg ttcaagactc tccgcctcgg 300
cttcgtgttt gagagacatg atcgaagtta ttttgcgaga tggcaagatc acgcgccaga 360
ttgaactcga accatcacca tctcgaggtt tcccacggtt gactcccagt tctcgaggct 420
catcgtcagc cacgcacagg cttccaatgc ctctcttgca ctacgacgac gaggaaaggc 480
agcatggtct cctgccaggg ggtgcttctt ctttaaggag cttaaatgaa gttcacgccc 540
tctgagtcct ggaagggcta tagacttcct ggtgtcggtg ctcccttggt aaaggagctt 600
aaactgggta tatccacgcc tgagtcctgg aatgatgaaa cttccttcgc tatgcctcta 660
tgtccaacac taccttacca accattatca tgcccaccgt ccttgcctgc agcgttaatt 720
cgttgtactc aacccgtacc gcgtcttagt agctttagca tacctgctgc ggtatataaa 780
tgcagacatt aatgaaaata aaaataaaag gcatgatcac gcgcttagta gatgttagat 840
ttcacagccc ctgtctttca ccaaaacgtc ggagccatgt cgaaggttca tccacttccc 900
gaaggcacct actttcaacc atc 923
<210> 18
<211> 996
<212> DNA
<213>the right homology arm sequence of Pleurotus eryngii pyrG gene (Pleurotus eryngii pyrG downstream
homologous sequence)
<400> 18
ccgacctcat ttgttgcaca atactcggga tctgatgccc agatctgcga aagactcatg 60
ttgatatact tgaagatttt gactttacgt tgattgaaag cttgcaagct ttgagcaaaa 120
aacatgactt catgatcttc gaggacagaa agtttgcaga cataggtgcc actcttcgta 180
gctcaacttg aatcgccgtt cacagcgctt gtaggaaaca ccgttgcgtt acagtattca 240
agtggcgtgc atcgcatcgc gagttggtcg cagatcacga atgcccactc agtccctggt 300
ccatccatcg tcgcagggct ttcttcagta ggcttacccc tcggacgggg tctcctcctc 360
ttggcagaaa tgagcacggc gggaaacctt gctgtgggcc aatacacaga agagacttat 420
cagatggctc gcgatcaccg ggacttcgta atcgggttca ttggacaaag acgcccatct 480
ggcgagggag atgaggattt cctagtcctg acacccggag taggattgga tgtgaaggca 540
gatggtatgg ggcagcagta cagaacgcct cgcgaagtca tcttggaatc aggttgcgat 600
gtgattattg taggacgagg gatatatggg aaagattaca gcttgactga agccatcgct 660
cagcaagcgg agaggtaccg ggaatcgggg tggagcgcat acttagaaag gtgtaaatcg 720
taaaaaggtg taaatcgtaa ttgatccatt acacccttgc aaactagaaa agcgtatatc 780
tgcacagaca gtctatcggc cacttcaggc ttttgtcgta gaagaaagga aaataacgta 840
caaagagtgg tttccgctta acccgcccta ttgatttggt tcgtattgta cacatggcgc 900
agctcaatca tcgatatcga agacatcttc attatcttgt ttcgaccgtg aagcggacga 960
aaccttgtaa agctgatcta tctggtttgt tggcgc 996
<210> 19
<211> 2636
<212> DNA
<213>Pleurotus eryngii pyrG gene or so homology arm is inserted into GFP sequence (Pleurotus eryngii pyrG gene
homologous sequence and GFP)
<400> 19
gaatcaaggc tcacctctgc tttataatcc cctcgatctg ggagactccg cgaccgacgt 60
attctgatat ccctacatca gtctctctga acttgtaccc agagccgtct tcagccttct 120
caggtcgagg tacgcctagg agaacgtggc ttgggggtag aatgtcgtcg gcatgcccat 180
ctttgggcgg ggcagtatct gtcgtgactt cgggatcgga ttcctcggaa ggggacgtgg 240
gttctagaga atcctctaat ctagcacgtt ttgaggcgcg ttcaagactc tccgcctcgg 300
cttcgtgttt gagagacatg atcgaagtta ttttgcgaga tggcaagatc acgcgccaga 360
ttgaactcga accatcacca tctcgaggtt tcccacggtt gactcccagt tctcgaggct 420
catcgtcagc cacgcacagg cttccaatgc ctctcttgca ctacgacgac gaggaaaggc 480
agcatggtct cctgccaggg ggtgcttctt ctttaaggag cttaaatgaa gttcacgccc 540
tctgagtcct ggaagggcta tagacttcct ggtgtcggtg ctcccttggt aaaggagctt 600
aaactgggta tatccacgcc tgagtcctgg aatgatgaaa cttccttcgc tatgcctcta 660
tgtccaacac taccttacca accattatca tgcccaccgt ccttgcctgc agcgttaatt 720
cgttgtactc aacccgtacc gcgtcttagt agctttagca tacctgctgc ggtatataaa 780
tgcagacatt aatgaaaata aaaataaaag gcatgatcac gcgcttagta gatgttagat 840
ttcacagccc ctgtctttca ccaaaacgtc ggagccatgt cgaaggttca tccacttccc 900
gaaggcacct actttcaacc atcatgagta aaggagaaga acttttcact ggagttgtcc 960
caattcttgt tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg 1020
gtgaaggtga tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac 1080
tacctgttcc atggccaaca cttgtcacta ctttctctta tggtgttcaa tgcttttcaa 1140
gatacccaga tcatatgaag cggcacgact tcttcaagag cgccatgcct gagggatacg 1200
tgcaggagag gaccatcttc ttcaaggacg acgggaacta caagacacgt gctgaagtca 1260
agtttgaggg agacaccctc gtcaacagga tcgagcttaa gggaatcgat ttcaaggagg 1320
acggaaacat cctcggccac aagttggaat acaactacaa ctcccacaac gtatacatca 1380
tggccgacaa gcaaaagaac ggcatcaaag ccaacttcaa gacccgccac aacatcgaag 1440
acggcggcgt gcaactcgct gatcattatc aacaaaatac tccaattggc gatggccctg 1500
tccttttacc agacaaccat tacctgtcca cacaatctgc cctttcgaaa gatcccaacg 1560
aaaagagaga ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggca 1620
tggatgaact atacaaataa ccgacctcat ttgttgcaca atactcggga tctgatgccc 1680
agatctgcga aagactcatg ttgatatact tgaagatttt gactttacgt tgattgaaag 1740
cttgcaagct ttgagcaaaa aacatgactt catgatcttc gaggacagaa agtttgcaga 1800
cataggtgcc actcttcgta gctcaacttg aatcgccgtt cacagcgctt gtaggaaaca 1860
ccgttgcgtt acagtattca agtggcgtgc atcgcatcgc gagttggtcg cagatcacga 1920
atgcccactc agtccctggt ccatccatcg tcgcagggct ttcttcagta ggcttacccc 1980
tcggacgggg tctcctcctc ttggcagaaa tgagcacggc gggaaacctt gctgtgggcc 2040
aatacacaga agagacttat cagatggctc gcgatcaccg ggacttcgta atcgggttca 2100
ttggacaaag acgcccatct ggcgagggag atgaggattt cctagtcctg acacccggag 2160
taggattgga tgtgaaggca gatggtatgg ggcagcagta cagaacgcct cgcgaagtca 2220
tcttggaatc aggttgcgat gtgattattg taggacgagg gatatatggg aaagattaca 2280
gcttgactga agccatcgct cagcaagcgg agaggtaccg ggaatcgggg tggagcgcat 2340
acttagaaag gtgtaaatcg taaaaaggtg taaatcgtaa ttgatccatt acacccttgc 2400
aaactagaaa agcgtatatc tgcacagaca gtctatcggc cacttcaggc ttttgtcgta 2460
gaagaaagga aaataacgta caaagagtgg tttccgctta acccgcccta ttgatttggt 2520
tcgtattgta cacatggcgc agctcaatca tcgatatcga agacatcttc attatcttgt 2580
ttcgaccgtg aagcggacga aaccttgtaa agctgatcta tctggtttgt tggcgc 2636
Claims (11)
1. a kind of Cas9 albumen, which is characterized in that the Cas9 albumen is suitable for CRISPR-Cas9 system, the Cas9 albumen
Coded sequence as shown in SEQ ID NO.3.
2. a kind of polynucleotides for encoding Cas9 albumen as described in claim 1.
3. a kind of expression vector contains polynucleotides as claimed in claim 2.
4. a kind of edit tool of DNA genomic fragment, is CRISPR/Cas9 system, the CRISPR/Cas9 system includes such as
Cas9 albumen described in claim 1 or polynucleotides as claimed in claim 2 or expression as claimed in claim 3 carry
Body；The CRISPR/Cas9 system also includes one or more sgRNA for target DNA fragments.
5. a kind of CRISPR/Cas9 carrier for mushroom gene editing, which is characterized in that the CRISPR/Cas9 carrier is
The nucleotide sequence of Cas9 albumen, U6 promoter, sgRNA are loaded on plasmid, the recombination for editing mushroom gene is obtained
Carrier；The nucleotide sequence of the U6 promoter is selected from SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID
NO.7。
6. the CRISPR/Cas9 carrier according to claim 5 for mushroom gene editing, which is characterized in that described heavy
It further include the left and right homology arm nucleotide sequence of GFP nucleotide sequence and target gene in group carrier.
7. a kind of applied to such as claim 5 or the U6 promoter of CRISPR/Cas9 carrier as claimed in claim 6, feature
It is, the nucleotide sequence of the U6 promoter is selected from SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID
NO.7。
8. a kind of edit methods of mushroom cell strain genomic fragment, which is characterized in that will be such as claim 5 or claim 6
The CRISPR/Cas9 carrier transfects mushroom cell strain, edits to the target gene of mushroom cell strain.
9. the edit methods of mushroom cell strain genomic fragment according to claim 8, which is characterized in that the mushroom is thin
Born of the same parents' strain is CbxRResisting cell strain.
10. the edit methods of mushroom cell strain genomic fragment according to claim 8, which is characterized in that the CbxRIt is anti-
Property cell strain be to coding succinate dehydrogenase-iron-sulfur protein subunit sdhB gene order in encode the 240th hyte amino acid
Rite-directed mutagenesis is carried out, the sdhB gene order after mutation is as shown in SEQ ID NO.2.
11. a kind of mushroom cell strain that can be used for gene editing, which is characterized in that the mushroom cell strain is CbxRResisting cell
Strain, to be carried out to the 240th hyte amino acid of coding in coding succinate dehydrogenase-iron-sulfur protein subunit sdhB gene order
Rite-directed mutagenesis, the sdhB gene order after mutation is as shown in SEQ ID NO.2.
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| CN109825532A (en) * | 2019-03-04 | 2019-05-31 | 中国科学院昆明植物研究所 | Application of the CRISPR/Cas12a gene editing system in small liwan moss gene editing |
| CN111349649A (en) * | 2020-03-16 | 2020-06-30 | 三峡大学 | Method for gene editing of agaricus bisporus and application |
| CN111575291A (en) * | 2020-05-29 | 2020-08-25 | 南京师范大学 | Promoter capable of efficiently driving exogenous gene expression in pleurotus eryngii |
| CN112111507A (en) * | 2020-08-10 | 2020-12-22 | 江苏大学 | Grifola frondosa CRISPR-Cas9 gene editing system, method and application |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825532A (en) * | 2019-03-04 | 2019-05-31 | 中国科学院昆明植物研究所 | Application of the CRISPR/Cas12a gene editing system in small liwan moss gene editing |
| CN109825532B (en) * | 2019-03-04 | 2019-12-10 | 中国科学院昆明植物研究所 | Application of CRISPR/Cas12a gene editing system in physcomitrella patens gene editing |
| CN111349649A (en) * | 2020-03-16 | 2020-06-30 | 三峡大学 | Method for gene editing of agaricus bisporus and application |
| CN111575291A (en) * | 2020-05-29 | 2020-08-25 | 南京师范大学 | Promoter capable of efficiently driving exogenous gene expression in pleurotus eryngii |
| CN111575291B (en) * | 2020-05-29 | 2022-06-14 | 南京师范大学 | Promoter capable of efficiently driving exogenous gene expression in pleurotus eryngii |
| CN112111507A (en) * | 2020-08-10 | 2020-12-22 | 江苏大学 | Grifola frondosa CRISPR-Cas9 gene editing system, method and application |
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