CN105543196A - Plant Cas9 variant protein VRER as well as encoding gene and application thereof - Google Patents

Plant Cas9 variant protein VRER as well as encoding gene and application thereof Download PDF

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CN105543196A
CN105543196A CN201610115904.8A CN201610115904A CN105543196A CN 105543196 A CN105543196 A CN 105543196A CN 201610115904 A CN201610115904 A CN 201610115904A CN 105543196 A CN105543196 A CN 105543196A
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vrer
gene
plant
cas9
sequence
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王克剑
胡熙璕
王春
付亚萍
刘庆
焦晓真
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China National Rice Research Institute
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China National Rice Research Institute
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    • C12N9/14—Hydrolases (3)
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/102—Mutagenizing nucleic acids
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety

Abstract

The invention discloses plant Cas9 variant protein VRER. An amino acid sequence of the protein is shown as SEQ ID No: 2. The invention also discloses a plant Cas9 variant gene VRER. The gene encodes the plant Cas9 variant protein VRER, and an amino acid sequence of the gene is shown as SEQ ID No: 1. The invention further discloses a recombinant expression vector containing the gene. The plant Cas9 variant protein VRER has the application that a genome editing function is realized under the condition that the PAM (protospacer adjacent motif) region is 5'-NGCG-3'.

Description

Plant Cas9 misfolded proteins VRER and encoding gene thereof and application
Technical field
The present invention relates to biological technical field, be specially the application of Cas9 protein variant VRER and the encoding gene thereof that plant gene is edited.
Background technology
In recent years, the technical characterstic that CRISPR-Cas9 is efficient with it, easy, becomes rapidly of paramount importance genome edit in plant.CRISPR-Cas was the immunity system be present in prokaryotic organism that extensively distributes originally, the short palindrome in CRISPRs (Clusteredregularlyinterspacedshortpalindromicrepeats) i.e. rule cluster interval repeats, and Cas albumen is then a kind of nuclease guided by RNA.
The CRISPR-Cas9 system used at present is the genome editing technique developed based on II type CRISPR-Cas.Cas albumen involved by this technology only has Cas9 mono-kind, guiding RNA (being optimised for sgRNA afterwards) by tracrRNA and crRNA composition guided, and the HNH structural domain of Cas9 albumen and RuvC structural domain carry out double-strand cutting at the 3-8bp place of PAMs (protospaceradjacentmotifs) Sequences upstream and cause double-strand break.When double-strand break occurs, cell just can start two cover repair mechanisms, i.e. nonhomologous end link (Non-homologousending-joining, NHEJ) and homologous recombination (Homologousrecombination, HR).Nonhomologous end link causes double-strand break place to produce disappearance, the insertion of single or multiple base usually, and the individuality repaired in like fashion usually produces the rite-directed mutagenesis of certain gene.Homologous recombination is then using homologous sequence as template, carries out high-fidelity reparation.
In CRISPR/Cas9 system, front contiguous motif (protospaceradjacentmotif, the PAM) sequence of region sequence determines the distribution of target sequence in whole genome.PAM is the one section of sequence be positioned near target dna, has the function identifying target DNA, only has when correct combination PAM sequence, could activate the Liang Ge endonuclease activity district of Cas9.The trinucleotide region PAM of target sequence end (5 '-NGG-3 ') be Cas9 recognition site, be the key realizing shearing function.But this can not meet the demand of people for plant gene editor scope completely.Selection due to CRISPR-Cas9 system target site is subject to the restriction of PAM sequence NGG, therefore finds and transform to identify that the Cas9 variant of new PAM becomes very important in plant.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of novel plant Cas9 misfolded proteins VRER and encoding gene thereof and application.
In order to solve the problems of the technologies described above, the invention provides a kind of plant Cas9 misfolded proteins VRER, the aminoacid sequence of this protein is as shown in SEQIDNo:2.
Improvement as plant Cas9 misfolded proteins VRER of the present invention: protein is also included in the aminoacid sequence shown in SEQIDNO:2 the derivative adding, replace, insert and lack one or more amino acid and generate.
The present invention also provides a kind of plant Cas9 variant gene VRER simultaneously, and this genes encoding plant Cas9 misfolded proteins VRER, the nucleotide sequence of described gene is as shown in SEQIDNO:1.
Improvement as plant Cas9 variant gene VRER of the present invention: described gene is also included in the nucleotide sequence shown in SEQIDNO:1 the mutant, allelotrope and the derivative that add, replace, insert and lack one or more Nucleotide and generate.
The present invention also provides a kind of recombinant expression vector simultaneously, and it comprises said gene.
The present invention also provides the purposes of above-mentioned plant Cas9 misfolded proteins VRER simultaneously: can realize genome editting function when PAM district is 5 '-NGCG-3 '.N represents A, any one Nucleotide of T, C, G.
The solution of the present invention is specific as follows:
Albumen provided by the invention is following (a) or (b):
A protein that () is made up of the aminoacid sequence shown in sequence in sequence table 2;
B aminoacid sequence shown in sequence in sequence table 2 is had through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation the protein that identical active function derives by sequence 2 by ().
In above-mentioned albumen, the replacement of one or several amino-acid residue and/or disappearance and/or interpolation refer to the replacement of no more than ten amino-acid residues and/or disappearance and/or interpolation.
The gene of above-mentioned albumen of encoding also is the scope of protection of the invention.
Said gene is arbitrary described DNA molecular in following (1)-(3):
1) DNA sequence dna of sequence 1 in sequence table;
2) polynucleotide of sequence 2 protein sequence in polynucleotide;
3) DNA sequence dna limited with sequence 1 in sequence table has more than 90% homology, and coding identical function protein DNA sequence;
Recombinant vectors containing above arbitrary described gene also belongs to protection scope of the present invention, as recombinant expression vector.
Available existing plant expression vector construction contains the recombinant expression vector of described gene.
Described plant expression vector comprises double base agrobacterium vector (as pBI121, pBin19, pCAMBIA2301, pCAMBIA3301, pCAMBIA1301-UbiN, pCAMBIA1300 etc.) and can be used for the carrier etc. of plant micropellet bombardment.
The application in editor's Plant Genome of above-mentioned albumen, said gene or above-mentioned recombinant vectors, expression cassette, transgenic cell line or recombinant bacterium is also the scope of protection of the invention.
The present invention is through suddenling change to the gene of coding Cas9, and produce the gene of a kind of encoding novel Cas9 misfolded proteins VRER, the PAM of identification is NGCG.Be PAM sequence screening target site in paddy rice with NGCG and edit in the experiment of GL1-1 gene, through transgenic approach, successfully obtaining the mutant plants of GL1-1 afunction, '-NGCG is as PAM sequence and edit target site to show can to identify 5 by this albumen.The present invention has broad application prospects in expansion plant gene editor scope.
Purposes of the present invention is: for carrying out gene editing to the specific target site of Plant Genome, namely PAM district is the target site of 5 '-NGCG-3 '.As a member in gene editing technology CRISPR-Cas9 system, can only edit compared to traditional C as9 albumen the restriction that PAM district is the target site of 5 '-NGG-3 ', expand this system editable scope in Plant Genome.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is restructuring carrier part element and mutational site schematic diagram.
Fig. 2 is that GL1-1 target site enzyme cuts detected result; M is marker; 1,5,6,7,11,12,15,16,17 for knocking out heterozygote plant; 14 for knocking out homozygote plant; Latter two swimming lane is respectively the wild-type that enzyme is cut and cut without enzyme; All the other swimming lanes are then for knocking out negative plant, and enzyme is cut the wild-type that result and enzyme cut and is as good as.
Fig. 3 is GL1-1 target site sequencing result; What draw down wavy line sign is target site; What single underscore indicated is PAM sequence; What collimation mark was shown cuts recognition site for enzyme; Disappearance represents with hyphen thick stick, inserts and represents with lowercase.
Fig. 4 is the phenotype spending 11 and mutant GL1-1 in wild-type.
Fig. 5 is LG1 target site sequencing result; What draw down wavy line sign is target site; What single underscore indicated is PAM sequence; What collimation mark was shown cuts recognition site for enzyme; Disappearance represents with hyphen thick stick, inserts and represents with lowercase.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The acquisition of embodiment 1, Cas9 variant VRER
By overlap extension pcr by the specific site in sudden change introducing Cas9 encoding gene.Need by complete Cas9 with the amino acid of 1135,1218 amino acids and 1336 amino acids places for boundary, be divided into 4 sections, then by PCR, its encoding sequence expanded.The primer used is as shown in table 1; KODFXDNA polysaccharase (spinning bio tech ltd purchased from Japan) pcr amplification object band, reaction conditions is as follows:
Remarks illustrate: pC1300-Cas9 can from application number 201510485573.2.
Response procedures: 94 DEG C, sex change 2 minutes; Then 98 DEG C of sex change 10 seconds, 58 DEG C of annealing 30 seconds, 68 DEG C of extensions (1000bp/min), 35 circulations of increasing; Finally 68 DEG C of downward-extensions 5 minutes.
1,2,2,3 and 3, comprise 20bp overlapping region between 4 sections; First paragraph and the 4th section comprise the region with carrier upstream and downstream homology respectively.At the beginning of design primer, specific sudden change is included in the lap of two fragments.The lap of the reverse primer of fragment 1 and the forward primer of fragment 2 comprises the codon of amino acid " V ", replaces the amino acid " D " of original 1135.The lap of the reverse primer of fragment 2 and the forward primer of fragment 3 comprises the codon of amino acid " R ", replaces the amino acid " G " of original 1218.The lap of the reverse primer of fragment 3 and the forward primer of fragment 4 comprises the codon of amino acid " Q " and " R ", replaces amino acid " R " and 1337 amino acids " T " of original 1335 respectively.The forward primer of fragment 1 and the reverse primer of fragment 3 then comprise the sequence with carrier upstream and downstream homology respectively.Subsequently by the recombinant technology of multiple clips, several amplified fragments lap splices is become complete Cas9 variant VRER encoding sequence.Multiple clips recombination kit is purchased from Nanjing Vazyme Biotechnology Co., Ltd., and carrier pC1300-Cas9 is through AatII and SpeI (purchased from NEB company) double digestion linearizing, and double digestion reaction system is as follows:
37 DEG C of enzymes cut 3 hours, reclaim test kit (Biomed, DR0103) carry out purifying by product description with Biomed glue; Obtain fragment pC1300-Cas9/AatII+SpeI.
Recombining reaction system is as follows:
Be placed in 37 DEG C of reaction 30min.After question response completes, immediately reaction tubes is placed in ice-water bath and cools 5min.Product conversion competent escherichia coli cell DH5 α obtains recombinant plasmid pC1300-VRER, and order-checking recombinant plasmid determines that sudden change is introduced correct.That is, the gene as shown in SEQIDNO:1 is comprised in this recombinant plasmid pC1300-VRER.
Remarks illustrate:
SEQIDNO:1 is VRERDNA sequence; Underscore is nuclear localization signal region (NLS); Be written as terminator codon greatly; Rest part is VRER albumen coded sequence, and transformation site counting is from VRER albumen coded sequence.
SEQIDNO:2 is corresponding VRER protein sequence; Underscore is nuclear localization signal region (NLS); Rest part is VRER protein sequence, and transformation site counting is from VRER albumen coded sequence.
The amplimer of each fragment of table 1
Fragment Forward primer Reverse primer
Fragment 1 cgcctctcggattacgacgtcgatca caccggtgtgagggaactagttttga
Fragment 2 gttggtgacacgaaacctccatactt tggagctcgcgtgcggaagccaacat
Fragment 3 cttccgcacgcgagctccagaagggt ttcgtagagcggtactccttccgatcaatcgt
Fragment 4 atcggaaggagtaccgctctacgaaggaggtg caccggtgtgagggaactagttttga
The design of embodiment 2, GL1-1 gene sgRNA and construction of recombinant plasmid
Select GL1-1 gene order " 5 '-ACGTTGCAGTGGC cCATGGcGCG " be target sequence, " CGCG " is PAM sequence, and underscore is that NcoI enzyme cuts recognition site.Synthesize positive and negative two oligonucleotide chains, as shown in table 2.G++ and the g--primer of 100 μMs (namely, Oligonucleolide primers in table 2 (5 '-3 ')) each 20 μ L mix, 100 DEG C of placements are placed on room temperature in 5 minutes, cool gradually, sex change is annealed, form the fragment (that is, g++/g--primer annealing product) with sticky end.
SK-gRNA is carried out AarI enzyme to cut (enzyme and system agents useful for same are all purchased from Ferment company), form the carrier with sticky end, endonuclease reaction system is as follows:
Remarks illustrate: SK-gRNA can from application number 201510485573.2.
37 DEG C of enzymes cut 3 hours, reclaim test kit (Biomed, DR0103) carry out purifying by product description with Biomed glue; Obtain linear carrier SK-gRNA/AarI.
Carrier and fragment are carried out T4 enzyme (purchased from NEB company) to be connected, react as follows:
Room temperature reaction 1 hour.Connect product 5 μ L transformation of E. coli competent cell DH5 α to obtain connecting plasmid SK-gRNA gL1-1.Use primer T7:5 '-TAATACGACTCACTATAGG-3 ', it is correct that order-checking determines that clone builds.That is, containing VRER and target site correlated series.
By SK-gRNA correct for order-checking gL1-1plasmid KpnI and BglII double digestion, reclaim test kit (Biomed, DR0103) with Biomed glue and carry out purifying by product description, obtain linear fragment.Between BamHI and the KpnI recognition site this linear fragment being connected into pC1300-VRER binary vector (that is, the recombinant plasmid pC1300-VRER of embodiment 1 gained), finally knocked out paddy rice binary expression vector pC1300-VRER-gRNA gL1-1.
KpnI, BglII, BamHI and system other reagent interior are all purchased from TAKARA company, and double digestion system is as follows:
37 DEG C of enzymes cut 3 hours, reclaim test kit (Biomed, DR0103) carry out purifying by product description with Biomed glue; Obtain linear fragment gRNA gL1-1/ KpnI+BglII.
37 DEG C of enzymes cut 3 hours, reclaim test kit (Biomed, DR0103) carry out purifying by product description with Biomed glue; Obtain linear carrier pC1300-VRER/KpnI+BamHI.
Ligation is as follows:
Room temperature reaction 1 hour.Get and connect product 5 μ L for transformation of E. coli competent cell DH5, obtain connecting plasmid.Use primer pC1300-F:5 '-ACACTTTATGCTTCCGGCTC-3 ', it is correct that order-checking determines that clone builds.When comparison is consistent for sequencing result and implementation sequence (comprising target site sequence), judge to build correctly; Otherwise, then incorrect for building.
Target site, oligonucleotide, the primer of table 2GL1-1
The acquisition of embodiment 3, transfer-gen plant
Paddy rice binary expression vector pC1300-VRER-gRNA is finally knocked out by above-mentioned gL1-1proceeded in Agrobacterium (Agrobacterium) strain EHA105 by the method for electric shock, utilize agrobacterium-mediated transformation this binary expression vector to be proceeded to the fine mature embryo callus of paddy rice Japan.The concrete grammar transformed cuts out after the mature embryo sterilizing of fine for Japan seed, is inoculated in the substratum of evoked callus.After cultivating 1 week, select growth vigorous, color is pale yellow, more open embryo callus, is used as the acceptor transformed.With containing pC1300-VRER-gRNA gL1-1the EHA105 bacterial strain of plasmid infects Rice Callus, after 25 DEG C, dark place cultivates 3 days, the Selective agar medium containing 50mg/L Totomycin screens resistant calli and transfer-gen plant.Select the transfer-gen plant of normal growth on Hygromycin selection media, identify.
The qualification of embodiment 4, transfer-gen plant
1. enzyme cuts qualification
Molecular biology method is utilized to identify goal gene catastrophe.CTAB method individual plant extracts the transgenic plant genomic dna of wild type control and embodiment 3 gained, and use primer and KODFXDNA polysaccharase (spinning bio tech ltd purchased from Japan) pcr amplification object band described in table 2, reaction conditions is as follows:
Response procedures: 94 DEG C, sex change 2 minutes; Then 98 DEG C of sex change 10 seconds, 58 DEG C of annealing 30 seconds, 68 DEG C extend 40 seconds, 34 circulations of increasing; Finally 68 DEG C of downward-extensions 5 minutes.
The PCR primer obtained, about 688bp, use NcoI (purchased from NEB company) to carry out enzyme and cut detection, system is as follows:
37 DEG C of enzymes cut 3 hours, agarose gel electrophoresis (145V) 25min of 2%, and result as shown in Figure 2, the sample be completely degraded as 428bp and 260bp two less bands is wild-type and knocks out feminine gender (as wt and 2 ~ 4,8 ~ 10,13,18 ~ 20); The sample be not degraded completely is for knocking out homozygote (as 14); Sample containing two kinds of banding patterns is for knocking out heterozygote (as 1,5,6,7,11,12,15,16,17).
2. check order
Above-mentioned PCR primer is sent order-checking company (Hangzhou Qing Ke Zi Xi Bioisystech Co., Ltd), with F in table 2 for primer checks order.Acquired results and wild-type sequence are compared.Sequencing result is bimodal, with degenerate codon analysis of strategies (http://dsdecode.scgene.com/ carries out peak map analysis), can directly obtain heterozygous mutant information.Result as shown in Figure 3, all have in various degree knock out homozygote, heterozygote detects.
3. phenotypic evaluation
As shown in Figure 4, by Crystal drops were dripping down to blade surface, if water can maintain water droplet proterties, can illustrate that rice leaf has wax synthesis, be wild-type.Otherwise (namely can not maintain drops), then illustrate that waxy gene is destroyed, for knocking out positive plant.
Conclusion:
The original Cas9 albumen expressed by Cas9 gene can only realize genome editting function when PAM district is 5 '-NGG-3 '.And compared to original Cas9 albumen, VRER (variant Cas9 albumen) can realize genome editting function when PAM district is 5 '-NGCG-3 ', common Cas9 albumen then cannot realize editor in this case.Remarks illustrate: N is represented as A, any one Nucleotide of T, C, G, and in present case (Fig. 3), N is then G.
Embodiment 5, the gene GL1-1 in embodiment 2 ~ 4 made into LG1 (comprise GL1-1 contained in subscript, all change LG1 into), accordingly, by " 5 '-ACGTTGCAGTGGC cCATGGcGCG " change " 5 '-GCCAAGAAGAG into cTGCAGgAAGCG ", " CGCG " changes into " AGCG ", and " NcoI " changes into " PstI "; The system used, method are all constant, and the Oligonucleolide primers g++/g--used becomes " g++:GGCAGCCAAGAAGAGCTGCAGGA; G--:AAACTCCTGCAGCTCTTCTTGGC "; Primers F/R becomes " F:GATCTGGCCTGAGCAACCT; R:CCCCAGTAGCTGTGTCTCG ".
Its result is as follows: CTAB method individual plant extracts the transgenic plant genomic dna of wild type control and gained, and use above-mentioned primer and KODFXDNA polysaccharase (spinning bio tech ltd purchased from Japan) pcr amplification object band, reaction conditions is as follows:
Response procedures: 94 DEG C, sex change 2 minutes; Then 98 DEG C of sex change 10 seconds, 58 DEG C of annealing 30 seconds, 68 DEG C extend 40 seconds, 34 circulations of increasing; Finally 68 DEG C of downward-extensions 5 minutes.
Obtain PCR primer, about 671bp, with amended F for primer checks order.Acquired results and wild-type sequence are compared.Sequencing result is bimodal, with degenerate codon analysis of strategies (http://dsdecode.scgene.com/ carries out peak map analysis), can directly obtain heterozygous mutant information.Result as shown in Figure 5, has sudden change in various degree to detect.
Finally, it is also to be noted that what enumerate above is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.

Claims (6)

1. plant Cas9 misfolded proteins VRER, is characterized in that: the aminoacid sequence of this protein is as shown in SEQIDNo:2.
2. plant Cas9 misfolded proteins VRER, is characterized in that: described protein is also included in the aminoacid sequence shown in SEQIDNO:2 the derivative adding, replace, insert and lack one or more amino acid and generate.
3. plant Cas9 variant gene VRER, is characterized in that: this genes encoding plant Cas9 misfolded proteins VRER, the nucleotide sequence of described gene is as shown in SEQIDNO:1.
4. plant Cas9 variant gene VRER, is characterized in that: described gene is also included in the nucleotide sequence shown in SEQIDNO:1 the mutant, allelotrope and the derivative that add, replace, insert and lack one or more Nucleotide and generate.
5. recombinant expression vector, is characterized in that: comprise the gene described in claim 3 or 4.
6. the purposes of plant Cas9 misfolded proteins VRER as claimed in claim 1 or 2, is characterized in that: can realize genome editting function when PAM district is 5 '-NGCG-3 '.
CN201610115904.8A 2016-03-01 2016-03-01 Plant Cas9 variant protein VRER as well as encoding gene and application thereof Pending CN105543196A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140273234A1 (en) * 2012-12-12 2014-09-18 The Board Institute, Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
CN105112435A (en) * 2015-08-09 2015-12-02 中国水稻研究所 Establishment and application of plant multi-gene knockout vector

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140273234A1 (en) * 2012-12-12 2014-09-18 The Board Institute, Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
CN105112435A (en) * 2015-08-09 2015-12-02 中国水稻研究所 Establishment and application of plant multi-gene knockout vector

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KLEINSTIVER, B.P. ET AL.,: ""Engineered CRISPR-Cas9 nucleases with altered PAM specificities"", 《NATURE》 *
谢科等: ""基因组编辑技术在植物中的研究进展与应用前景"", 《中国生物工程杂志》 *

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