CN103233028A - Specie limitation-free eucaryote gene targeting method having no bio-safety influence and helical-structure DNA sequence - Google Patents

Abstract

The invention discloses a eukaryotic gene targeting method and a helical structure DNA sequence without species limitation and without biological safety problems, belonging to the field of genetic engineering. The steps of the method are: (1) Design and construction of CRISPR/Cas9 and chimeric RNA; (2) Cas9 mRNA is translated in vivo to form a Cas9 nuclease that binds to the chimeric RNA to achieve fixed-point shearing and generate DNA double strands after shearing Fragmentation, by inducing the cell's own natural DNA repair process "non-homologous end joining" to introduce foreign DNA, thereby modifying the cell's endogenous genes. The method has simple steps, flexible recognition sites and low consumption.

Description

A kind of eukaryotic gene target practice method and spirane structure dna sequence dna that does not have species restriction lifeless matter safety issue

Technical field

The invention belongs to the genetically engineered field, more particularly, relate to a kind of eukaryotic gene target practice method and spirane structure dna sequence dna that does not have species restriction lifeless matter safety issue.

Background technology

Gene targeting (gene targeting) technology is a kind of experimental technique of directed change organism genetic information.Along with development of biology, Zinc finger nuclease (ZFN) technology and activating transcription factor sample effector nuclease (TALEN) technology are applied in the gene targeting in recent years, and these two technology have improved the specificity of directed modification greatly.ZFN is made of a DNA differential threshold and a non-specific endonuclease.The DNA differential threshold is to be composed in series (general 3～4) by a series of Cys2-His2 zinc finger proteins (zinc-fingers), each zinc finger protein identification and in conjunction with a special triplet base.A plurality of zinc finger proteins can be together in series and form one section special base sequence of a zinc finger protein group identification, and the non-specific endonuclease that links to each other with the zinc finger protein group is to shear the territory from the DNA that 96 amino-acid residues of the C end of FokI are formed.FokI is a kind of restriction enzyme from Flavobacterium okeanokoites, only when the dimer state, just there is enzyme to cut activity, each FokI monomer links to each other with a zinc finger protein group and constitutes a ZFN, identify specific site, when two recognition sites at a distance of appropriate apart from the time (6～8bp), two monomer ZFN interact and produce enzymes and cut function.Thereby reach the purpose that the DNA fixed point is sheared.Shear the back and produce the dna double splitting of chain, introduce foreign DNA by the natural DNA repair process of inducing cell self " with the source orientation reparation " or " non-homogeneous terminal connection ", thereby revise the cell native gene.

Zinc finger nuclease technological breakthrough the restrictive factor-target practice efficiency of gene targeting, the efficient of gene targeting is increased to 10-1～10-2 by original 10-6～10-7.But Zinc finger nuclease can produce the phenomenon (off-target) of missing the target in carrying out the genetic modification process, cause cell dosage dependency toxicity.Gupta etc. carry out targeting modification research by different Zinc finger nucleases to zebra fish kdrl locus, utilize the Illumina order-checking to assess the potential site of missing the target, 141 places in the zebra fish, find to have only the site of missing the target of minority to cause the cell accumulated damage.Still need for this reason Zinc finger nuclease-gene targeting is furtherd investigate.

The TALEN technology is the albumen that utilizes phytopathogen Xanthomonas campestris (Xanthomonas) to secrete naturally---be incitant sample effector (TAL effectors, TALEs) function that can identification specificity DNA base pair, design a string suitable TALEs and identify and be attached to any particular sequence, behind TALEs, add a non-specific endonuclease FokI again, construct TALEN, thereby realized cutting off the dna double chain at specific site, by the natural DNA repair process of inducing cell self, in cellular genome, introduce new genetic material.Relative Zinc finger nuclease technology, TALEN can the longer gene order of target, and easier structure.

But up to the present, no matter be ZFN or TALEN, its recognition sequence there are a lot of requirements, can't realize each possible dna sequence dna of target, and the same period that is not easy to carry out two or more sites practice shooting, and assembling ZFN or TALEN albumen also are consuming time, a consumption power and the very high process of cost, and people do not have method cheaply a kind of and that openly can obtain to produce a large amount of ZFNs or TALENs rapidly always.

Summary of the invention

1. invent the technical problem that will solve

At realizing that the limitation that the transformation of specific gene group exists is big, take time and effort and problem that cost is high by ZFNs or TALENs in the prior art, the invention provides a kind of eukaryotic gene target practice method and spirane structure dna sequence dna that does not have species restriction lifeless matter safety issue, this method steps is simple, recognition site flexible and consume low.

2. technical scheme

Inventive principle: utilize procaryotic rule cluster to lack the palindrome at interval and repeat the orientation identification of (CRISPR/Cas) system and shear function, method of the present invention is appraised and decided the position by increasing this system in eukaryote, realization is to the shearing of the specific site of target DNA in the eukaryote, can introduce the sudden change of target dna sequence dna in the repair process after shearing, thereby realize the purpose of gene targeting.

The present invention is achieved through the following technical solutions above-mentioned purpose:

A kind of eukaryotic gene target practice method of not having species restriction lifeless matter safety issue the steps include:

(1) design of CRISPR/Cas9 and chimeric RNA and structure

1) codon of optimization coding Cas9

2) Cas9 nuclease vector construction and Cas9 nuclease mRNA are synthetic

Sequence after optimizing according to step 1), the dna sequence dna of customization composite coding Cas9 nuclease, and it is inserted into respectively in the following expression vector forms: pST1374-Cas9, pST1374-NLS-Flag-Cas9;

Utilize Cas9C-NLS Cla For and two primers of Cas9C-NLS Apa Rev, by dna fragmentation of chain polymerization enzyme reaction (PCR) amplification, use dna fragmentation and the pST1374-NLS-Flag-Cas9 of two digestion with restriction enzyme amplifications of ClaI and ApaI, the dna fragmentation of amplification is inserted into pST1374-NLS-Flag-Cas9, makes up the pST1374-NLS-Flag-Cas9-NLS carrier;

The sequence of three nucleus signal for locating NLS of encoding utilizes Cas9-N-3NLS For and Cas9-N-3NLS Rev primer to come out for the template pcr amplification with the oligonucleotide, the PCR product is behind NheI and two digestion with restriction enzyme of NotI, be inserted in the pST1374-NLS-Flag-Cas9 carrier by same restriction enzyme site again, make up pST1374-3XNLS-Flag-Cas9;

Spirane structure (Helix) dna sequence dna is inserted into after synthetic in the middle of the sequence and coding Cas9 nuclease sequence of code tag albumen Flag through customizing, and forms the pST1374-NLS-Flag-Helix-Cas9 carrier;

The Cas9 system derives from prokaryotic cell prokaryocyte, is difficult to shift to nucleus in eukaryotic cell, thereby can't guarantees that this system realizes its shearing function in eukaryotic cell.The present invention has solved this problem by adding the expression of spirane structure dna sequence dna increase Cas9 in eukaryotic cells nuclear between cell nucleus signal for locating sequence and Cas9 encoding sequence, sees Fig. 2.In this process, used human 293T.

Cas9 nuclease serial carrier obtains Cas9 nuclease mRNA after utilizing T7Ultra Kit in-vitro transcription after the AgeI linearizing, be directly used in gene targeting; Because the unstable of mRNA, can the long-term existence organism in, can not produce further influence to environment, thereby not have the biological safety problem.

3) chimeric RNA's is synthetic

T7 promoter sequence on the chimeric RNA template, be to be that template produces by PCR with synthetic oligonucleotide, utilize T7Shortscript Kit in-vitro transcription to go out chimeric RNA, it comprises crRNA and tracrRNA structure, can be directed in conjunction with the site on the target DNA;

(2) translation back formation Cas9 nuclease is combined with chimeric RNA in the Cas9mRNA body, realize the fixed point shearing, shear the back and produce the dna double splitting of chain, introduce foreign DNA by the natural DNA repair process of inducing cell self " non-homogeneous terminal the connection ", thereby revise the cell native gene.

A kind of spirane structure dna sequence dna, the ability that the consideration convey of this spirane structure dna sequence dna enhancing albumen under nuclear localization signal instructs moves ability and is combined with nucleic acid, its gene order is:

ctggaacccggcgagaagccatacaaatgcccagagtgtggtaaatctttctctcagtctggtgctctgaccagacaccagcggacccacactaga。

3. beneficial effect

Than prior art, the invention has the advantages that:

(1) accuracy is higher.Adopt method of the present invention, as long as there is the difference of any base pair between RNA target sequence and the genome sequence, Cas9 not can in conjunction with, thereby can't realize shearing to DNA;

(2) genome multidigit point is practiced shooting.Adopt method of the present invention, can carry out a plurality of sites on the target gene simultaneously and shear, realize the purpose of genome manipulation;

(3) use is more convenient, and expense is lower.Be the recognition sequence that ZFN or TALEN need change the nucleases front at different target spots, the synthetic or assembling of these recognition sequences take time and effort and expense very high.In the method for the present invention, the CRISPR/Cas system only need change the specific recognition that very short RNA sequence just can realize different loci.In the foundation of animal genetic engineering model, if use ZFN or TALEN technology, also ZFN or TALEN plasmid to be transcribed into mRNA, this has increased the difficulty of expense and experiment again, adopt the CRISPR/Cas system only need transcribe a Cas nuclease and just can finish repeatedly experiment, greatly reduced cost;

(4) lifeless matter safety issue.The present invention takes mRNA and RNA as the gene targeting starting material, uses resistant gene without any screening, thereby does not have the biological safety problem;

(5) adopt method of the present invention, no species restriction.

Description of drawings

Fig. 1 is combined with chimeric RNA for Cas9 and is realized that the fixed point shearing causes dna double chain-breaking process synoptic diagram;

Fig. 2 is that the Laser Scanning Confocal Microscope of each carrier of Cas9 nuclease is taken fluorescence photo.

Embodiment

Below in conjunction with accompanying drawing and specific embodiment technical scheme of the present invention is done further introduction.

Embodiment 1

A kind of eukaryotic gene target practice method of not having species restriction lifeless matter safety issue of present embodiment the steps include:

(1) design of CRISPR/Cas9 and chimeric RNA and structure

1) codon of optimization coding Cas9

Cas9 belongs to the second class CRISPR/Cas system, to derive from prokaryotic cell prokaryocyte, in order can in eukaryotic cell, expressing better, at first Cas9 encoding sequence process to be optimized, do not changing under the amino acid whose prerequisite, using the codon of coded amino acid in the eukaryotic cell;

2) Cas9 nuclease vector construction and Cas9 nuclease mRNA are synthetic

According to the sequence after the step 1) optimization, the dna sequence dna of customization composite coding Cas9 nuclease, and it is inserted into respectively in the following expression vector forms: except containing carrier pST1374-Cas9 all sequences, also have the sequence NLS of a Codocyte nuclear localization signal and the sequence of a code tag albumen Flag on this carrier of pST1374-Cas9, pST1374-NLS-Flag-Cas9();

Utilize Cas9C-NLS Cla For and two primers of Cas9C-NLS Apa Rev (primer sequence sees Table 1), by dna fragmentation of chain polymerization enzyme reaction (PCR) amplification, use dna fragmentation and the pST1374-NLS-Flag-Cas9 of two digestion with restriction enzyme amplifications of ClaI and ApaI, the dna fragmentation of amplification is inserted into pST1374-NLS-Flag-Cas9, make up the pST1374-NLS-Flag-Cas9-NLS carrier, except containing carrier pST1374-Cas9 all sequences, also have the sequence NLS of former and later two Codocyte nuclear localization signals and the sequence of a code tag albumen Flag on this carrier;

It is that template (template sequence sees Table 1) pcr amplification comes out with the oligonucleotide that the sequence of three nucleus signal for locating NLS of encoding is utilized Cas9-N-3NLS For and Cas9-N-3NLS Rev primer (primer sequence sees Table 1), the PCR product is behind NheI and two digestion with restriction enzyme of NotI, be inserted in the pST1374-NLS-Flag-Cas9 carrier by same restriction enzyme site again, make up pST1374-3XNLS-Flag-Cas9, except containing carrier pST1374-Cas9 all sequences, also have the sequence NLS of three Codocyte nuclear localization signals and the sequence of a code tag albumen Flag on this carrier;

Spirane structure (Helix) dna sequence dna (seeing Table 2) is inserted into after synthetic in the middle of the sequence and coding Cas9 nuclease sequence of code tag albumen Flag through customizing, form the pST1374-NLS-Flag-Helix-Cas9 carrier, on this carrier except containing carrier pST1374-Cas9 all sequences, the sequence NLS that also has a Codocyte nuclear localization signal, the sequence of a code tag albumen Flag and a spirane structure dna sequence dna;

The Cas9 system derives from prokaryotic cell prokaryocyte, is difficult to shift to nucleus in eukaryotic cell, thereby can't guarantees that this system realizes its shearing function in eukaryotic cell.The present invention has solved this problem by adding the expression of spirane structure dna sequence dna increase Cas9 in eukaryotic cells nuclear between cell nucleus signal for locating sequence and Cas9 encoding sequence, sees Fig. 2.In this process, used human 293T.

Present embodiment has also made up the expression vector of a series of Cas9 of containing, and these carrier transfections afterwards just can be showed that by the immunostaining reaction Cas9 is in intracellular location situation in cell.Before the cell transfecting, with cell culture on the cover plate of poly-lysine bag quilt, utilize the Lipofectamine2000 transfection reagent that Cas9 series plasmid DNA transfection is gone in the 293T cell.In immunostaining experiment, at room temperature with cell fixation 15 minutes, PBS washed twice to the Paraformaldehyde 96 with 4% with damping fluid, added 0.2% Triton X-100 immersion 5 minutes with PBS again, washed twice with PBS again.With sealing under the normal lowlenthal serum room temperature 1 hour, will resist the antibody of label protein FLAG to add in the confining liquid incubated at room 2 hours more afterwards.After the PBS cleaning, again with two anti-and Hoechst incubated at room cells of the goat anti-mouse that is dissolved in the cy3 combination among the PBS 1 hour, after the PBS cleaning, cell is cultivated fluid-tight with Vectashield close, take the fluorescence photo (see figure 2) with Olympus Flueview1000 Laser Scanning Confocal Microscope then.Only Cas9 is expressed adding the spirane structure dna sequence dna between cell nucleus signal for locating sequence and Cas9 encoding sequence in eukaryotic cells nuclear, ensure that further it exercises the shearing function in gene targeting.

Cas9 nuclease serial carrier obtains Cas9 nuclease mRNA after utilizing T7Ultra Kit in-vitro transcription after the AgeI linearizing, can be directly used in gene targeting; Because the unstable of mRNA, can the long-term existence organism in, can not produce further influence to environment, thereby not have the biological safety problem.

3) chimeric RNA's is synthetic

The section of having T7 promoter sequence on the chimeric RNA template, this is to be (template sequence sees Table 1) that template produces by PCR with synthetic oligonucleotide, utilize T7Shortscript Kit in-vitro transcription to go out chimeric RNA, it comprises crRNA and tracrRNA structure, can be directed in conjunction with the site on the target DNA;

(2) translation back formation Cas9 nuclease is combined with chimeric RNA in the Cas9mRNA body, realize the fixed point shearing, shear the back and produce the dna double splitting of chain, introduce foreign DNA by the natural DNA repair process of inducing cell self " non-homogeneous terminal the connection ", thereby revise the cell native gene; See Fig. 1.

Table 1

Italic, binding sequence; Black matrix, the T7 promotor.

Table 2

Claims (2)

1. an eukaryotic gene target practice method of not having species restriction lifeless matter safety issue the steps include:

(1) design of CRISPR/Cas9 and chimeric RNA and structure

1) codon of optimization coding Cas9

2) Cas9 nuclease vector construction and Cas9 nuclease mRNA are synthetic

Sequence after optimizing according to step 1), the dna sequence dna of customization composite coding Cas9 nuclease, and it is inserted into respectively in the following expression vector forms: pST1374-Cas9, pST1374-NLS-Flag-Cas9;

Utilize Cas9 C-NLS Cla For and two primers of Cas9 C-NLS Apa Rev, by dna fragmentation of chain polymerization enzyme reaction amplification, use dna fragmentation and the pST1374-NLS-Flag-Cas9 of two digestion with restriction enzyme amplifications of ClaI and ApaI, the dna fragmentation of amplification is inserted into pST1374-NLS-Flag-Cas9, makes up the pST1374-NLS-Flag-Cas9-NLS carrier;

The sequence of three nucleus signal for locating NLS of encoding utilizes Cas9-N-3NLS For and Cas9-N-3NLS Rev primer to come out for the template pcr amplification with the oligonucleotide, the PCR product is behind NheI and two digestion with restriction enzyme of NotI, be inserted in the pST1374-NLS-Flag-Cas9 carrier by same restriction enzyme site again, make up pST1374-3XNLS-Flag-Cas9;

The spirane structure dna sequence dna is inserted into after synthetic in the middle of the sequence and coding Cas9 nuclease sequence of code tag albumen Flag through customizing, and forms the pST1374-NLS-Flag-Helix-Cas9 carrier;

Cas9 nuclease serial carrier obtains Cas9 nuclease mRNA after utilizing T7 Ultra Kit in-vitro transcription after the AgeI linearizing, be directly used in gene targeting;

3) chimeric RNA's is synthetic

T7 promoter sequence on the chimeric RNA template is to be that template is passed through the chain polymerization enzyme reaction and produced with synthetic oligonucleotide, utilizes T7 Shortscript Kit in-vitro transcription to go out chimeric RNA;

(2) translation back formation Cas9 nuclease is combined with chimeric RNA in the Cas9mRNA body, realize the fixed point shearing, shear the back and produce the dna double splitting of chain, introduce foreign DNA by the natural DNA repair process of inducing cell self " non-homogeneous terminal the connection ", thereby revise the cell native gene.

2. a spirane structure dna sequence dna is characterized in that, the ability that the consideration convey of this spirane structure dna sequence dna enhancing albumen under nuclear localization signal instructs moves ability and is combined with nucleic acid, and its gene order is:

ctggaacccggcgagaagccatacaaatgcccagagtgtggtaaatctttctctcagtctggtgctctgaccagacaccagcggacccacactaga。

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