CN106282228A - A kind of method that point mutation is repaired - Google Patents
A kind of method that point mutation is repaired Download PDFInfo
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- CN106282228A CN106282228A CN201610692418.2A CN201610692418A CN106282228A CN 106282228 A CN106282228 A CN 106282228A CN 201610692418 A CN201610692418 A CN 201610692418A CN 106282228 A CN106282228 A CN 106282228A
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Abstract
The present invention relates to the gene editing method that the most accurate a kind of point mutation is repaired, when utilizing nuclease to combine from different length sgRNA, genomic DNA is pinpointed the different of cutting power, it is aided with targeting transcription regulatory factor, significantly improve homologous recombination reaction efficiency, accurately repairing or being imported in genes of interest group by exogenous gene of gene mutation can be carried out accurately and efficiently.The invention also discloses the coupling molecule of the compositions of a kind of gene editing, a kind of RNA aptamers sequence and ssgRNA, and the purposes for gene editing.
Description
Technical field
The present invention relates to molecular biology gene editing field.Specifically, the present invention is for by gene editing method
Gene mutation carries out in mammalian cell fixed point reparation knock in exogenous gene.
Background technology
The ultimate principle of genome fixed point editor/modification is to utilize or the DNA double chain of induction spontaneous in target site district at present
Breach (double-strain breaks, DSBs), the DNA repair mechanism in active cell is carried out changing of genome by DSBs
Making, the end in the most non-homogeneous district connects (Non-homologous end joint, NHEJ) or homologous recombination
(Homologousrecombination, HR) (accompanying drawing 1).
In mammalian cell, the probability of the spontaneous generation of DSB is less than about 1/104If adopted by gene engineering method
With nuclease induction DSBs such as spCas9 and SaCas9, efficiency can improve to more than 10%, and has locus specificity, therefore side
Next step gene repair process that endogenous gene target site is carried out be smoothed out.DNA in DSBs active cell repairs
After path, having a reparation of two kinds of different repair mechanisms emulative participation DSBs, a kind of is that the end of non-homology region connects
NHEJ, one is homologous recombination HR.The accurate editor of genome target site to be realized, needs to rely on the homologous recombination repair of intracellular
Mechanism.The most directly improve the frequency of homologous recombination generation in DSBs repair process or suppress the end in non-homogeneous district to connect
(NHEJ) both contribute to improve the efficiency of genome fixed point editor/modification.
The end in non-homogeneous district connects the generation of (NHEJ) and homologous recombination (HR) has multiple protein to participate, wherein
LIG4 albumen is the important factor of NHEJ, and Rad51, Rad52 and Brca1 are then the important participation eggs of homologous recombination repair process
In vain.Therefore if able to the expression of the expression of suppression LIG4 albumen and/or promotion Rad51, Rad52 and Brca1 will assist in and carries
The efficiency of high gene group fixed point editor/modification.
The nucleases such as Cas9 need the guiding of sgRNA to the cutting of genome, as a length of 20-21bp of sgRNA,
Cas9 can identify and cut target site, thus leaves DSBs breach at target site；As a length of 14-15bp of sgRNA,
Cas9 can identify and combine target site, but cannot cut this site.This characteristic can be transformed for genetic transcription
Suppression/activation.By designing the sgRNA of different length, we can also realize activation/suppression and the target position of target gene simultaneously
The cutting of point and homology reparation.
Summary of the invention
The invention provides a kind of gene editing method based on homologous recombination, including using nuclease, target gene is entered
Row cutting, it is characterised in that: also including ssgRNA and targeting transcription regulatory factor in described method, described ssgRNA can draw
Lead the code nucleic acid of nuclease target gene editor's associated protein but will not cut, described targeting transcription regulatory factor energy
Transcribing of enough controlling gene editor's associated protein.
Wherein, described nuclease target gene is carried out homologous recombination include use sgRNA, described sgRNA can draw
Lead nuclease targeting target gene to cut；The most described sgRNA includes the adjacent motif in conservative interval of 3' end, preferably described
Motif be NGG；The most described sgRNA is 18-23bp, preferably 20bp.
In some specific embodiments of the present invention, described nuclease preferably selects selected from Cas9 and CPF1, described Cas9
From SaCas9, SpCas9.
In some specific embodiments of the present invention, described method also includes homologous complementary recovery template, preferably same
The integration site of source restructuring deletes termination codon.
In some specific embodiments of the present invention, described ssgRNA includes the adjacent motif in conservative interval of 3' end, excellent
Motif described in choosing is NGG；Preferably described ssgRNA is 14-16bp, more preferably 14bp, 15bp or 16bp.
The present invention utilizes DNA sequence specific chromosome double-strand DSBs generation technique, by transforming sgRNA and designing not
With the sgRNA of length, it is aided with targeting transcription regulatory factor to realize genome efficient, accurate fixed point transformation.In the present invention
Some specific embodiments in, described targeting transcription regulatory factor is transcribed selected from targeting activating transcription factor or targeting
At least one of inhibitive factor, the most described targeting activating transcription factor is selected from VP64, the most described targeting Transcription inhibition
The factor is selected from KRAB.
When the present invention utilizes Cas9 to combine from different length sgRNA, activity is different, it is achieved the difference to different target sites
Regulation and control, repair the expression of key enzyme, improve homologous recombination efficiency thus reality by activating the expression/suppression NHEJ of homologous recombination enzyme
The most precisely repairing of existing endogenous gene sudden change.In some specific embodiments of the present invention, described gene editing is correlated with
Albumen connects at least one of associated protein and homologous recombination associated protein, the most described non-homogeneous district selected from non-homogeneous district end
End connects associated protein and is selected from Rad51, Rad52 and Brca1 or its group selected from LIG4, the most described homologous recombination associated protein
Close.
The present invention utilizes sequence-specific nuclease (sequence-specific endonuclease) at target site
Place or one DSBs of neighbouring manufacture；On this basis, the present invention utilizes sgRNA that length is different and targeting transcriptional activation/press down
The factor processed, activates homologous recombination correlative protein expression, and the end of suppression non-homology connects correlative protein expression, it is achieved target site
Locate efficient specific homologous recombination repair (see accompanying drawing 2, schematic diagram).
In some specific embodiments of the present invention, the nuclease in described method is formed with targeting transcription regulatory factor
Fusion protein, the most described fusion protein comprises flexible linker.
In some specific embodiments of the present invention, described ssgRNA also coupling RNA aptamers sequence, described RNA
Aptamers sequence can specific be combined with related protein, described targeting transcription regulatory factor and described related protein shape
Become fusion protein.
In some specific embodiments of the present invention, the nuclease in described method and the first targeting transcription regulatory factor
Forming fusion protein, the most described fusion protein comprises flexible linker, described ssgRNA also coupling RNA aptamers sequence
Row, described RNA aptamers sequence can specific be combined with related protein, and the second targeting transcription regulatory factor is with described
Related protein forms fusion protein.
In the present invention, the strategy introducing transcription regulatory factor can be, but not limited to, strategy 1: nuclease is transcribed with targeting
Regulatory factor forms fusion protein；Or strategy 2: targeting transcription regulatory factor and described related protein (such as MS2 associated proteins
Or PCP associated proteins) form fusion protein, then the ssgRNA of RNA aptamers sequence (such as MS2 or PP7) is combined with coupling；
Or strategy 3: in a method, there is above two strategy simultaneously.
In the gene editing method of homologous recombination, the strategy introducing transcription regulatory factor also includes those skilled in the art
According to prior art or routine techniques, transcription regulatory factor (such as VP64 or KRAB) is introduced described homologous recombination associated protein
Other of (being selected from Rad51, Rad52 and Brca1 or a combination thereof) promoter region may be tactful, such as by fusion protein
Expressing, transcription regulatory factor is expressed on nuclease-ssgRNA complex, can activate as long as described transcription regulatory factor has
Or suppress the effect transcribed of described homologous recombination associated protein.
It addition, applicant finds in an experiment, strategy 1 is similar to the result of strategy 2 with 3, and these gene editing methods are equal
Efficiency that in mammalian cell homologous recombination occur can be greatly improved.Wherein, strategy 3 compare strategy 1 and 2 can be more efficient
Activate or suppress transcribing of target gene.According to this principle, those skilled in the art design homologous recombination associated protein described in targeting
Transcription regulatory factor (such as) is also drawn by the ssgRNA of (being selected from Rad51, Rad52 and Brca1 or a combination thereof) promoter region
Enter any possibility of described homologous recombination associated protein (being selected from Rad51, Rad52 and Brca1 or a combination thereof) promoter region
Strategy falls within the summary of the invention of the present invention.
In some specific embodiments of the present invention, described RNA aptamers is hairpin structure；The most described hairpin structure selects
From MS2 or PP7, described related protein is selected from MS2 associated proteins or PCP associated proteins.The such as present invention utilizes the sgRNA of transformation
(carrying the special hairpin structures such as MS2/PP7, can be by specific recognition such as MS2 associated proteins/PCP albumen), is aided with targeting
Property transcriptional activation/inhibitive factor (MS2/PCP-VP64/KRAB), can activate/suppress transcribing of specific gene.
On the other hand, present invention also offers a kind of compositions for gene editing, turn including ssgRNA and targeting
Record regulatory factor, described ssgRNA can guide the code nucleic acid of nuclease target gene editor's associated protein but will not cut
Cutting, described targeting transcription regulatory factor can the transcribing of controlling gene editor's associated protein.
Wherein, described compositions also includes nuclease, sgRNA, homologous complementary recovery template or a combination thereof.
In some specific embodiments of the present invention, the invention provides a kind of compositions for gene editing, including
Fusion protein, sgRNA, ssgRNA and homologous complementary that nuclease, nuclease are formed with targeting transcription regulatory factor repair mould
Plate, described ssgRNA can guide the code nucleic acid of nuclease target gene editor's associated protein but will not cut, described
Targeting transcription regulatory factor can the transcribing of controlling gene editor's associated protein.
In some specific embodiments of the present invention, the invention provides a kind of compositions for gene editing, including
Nuclease, the coupling molecule of sgRNA, RNA aptamers sequence and ssgRNA, homologous complementary recovery template, targeting is provided to transcribe
Regulatory factor and the fusion protein of RNA aptamers related protein, described ssgRNA can guide nuclease target gene editor's phase
Closing the code nucleic acid of albumen but will not cut, described targeting transcription regulatory factor can controlling gene editor's associated protein
Transcribe.
In some specific embodiments of the present invention, the invention provides a kind of compositions for gene editing, including
Fusion protein, sgRNA, RNA aptamers sequence and ssgRNA that nuclease and the first targeting transcription regulatory factor are formed are provided
The fusion of coupling molecule, homologous complementary recovery template and the second targeting transcription regulatory factor and RNA aptamers related protein
Albumen, described ssgRNA can guide the code nucleic acid of nuclease target gene editor's associated protein but will not cut, institute
Stating targeting transcription regulatory factor can the transcribing of controlling gene editor's associated protein.
In some specific embodiments of the present invention, described targeting transcription regulatory factor is selected from targeting transcriptional activation
At least one of the factor or targeting transcription inhibitory factor, the most described targeting activating transcription factor is selected from VP64, preferably institute
State targeting transcription inhibitory factor selected from KRAB.
In some specific embodiments of the present invention, described gene editing associated protein connects selected from non-homogeneous district end
At least one of associated protein and homologous recombination associated protein, the most described non-homogeneous district end connects associated protein and is selected from
LIG4, the most described homologous recombination associated protein is selected from Rad51, Rad52 and Brca1 or a combination thereof.
On the other hand, present invention also offers the coupling molecule of a kind of RNA aptamers sequence and ssgRNA, described
SsgRNA part can guide the code nucleic acid of nuclease target gene editor's associated protein but will not cut, described RNA
Aptamers Sequence can specific be combined with related protein；Preferably described ssgRNA part include 3' end conservative between
Every adjacent motif, preferably described motif is NGG；Preferably described ssgRNA part is 14-16bp, preferably 14bp, 15bp
Or 16bp.
In some specific embodiments of the present invention, described RNA aptamers is hairpin structure；The most described hairpin structure selects
From MS2 or PP7, described related protein is selected from MS2 associated proteins or PCP associated proteins.
On the other hand, present invention also offers a kind of test kit, including any one heretofore described described combination
Thing or the coupling molecule that any one is described.
On the other hand, present invention also offers in the present invention any one described gene editing method or any one is described
Compositions or the coupling molecule that any one is described are used for the purposes of gene editing.
In the present invention, described gene comes from microorganism, plant, animal, cell, mammal or people.
Beneficial effects of the present invention
The present invention is greatly improved the efficiency that in mammalian cell, homologous recombination occurs；
The present invention improves homologous recombination efficiency in the case of not importing exogenous gene, it is possible to avoid existing raising same
Source recombinant technique such as micromolecular compound etc. is prone to produce the shortcomings such as chromosome set unstability.
The invention provides the method carrying out gene editing accurate, fine in mammal.
The invention provides and multiple genes can be carried out in mammal the method that sudden change is repaired simultaneously.
Accompanying drawing explanation
Fig. 1 is illustrated that the figure of the genetic modification method that DSB mediates
Fig. 2 is illustrated that the figure that system is illustrated.
A., as a length of 14bp of ssgRNA, Cas9 does not possess nuclease.14bp can be realized by three kinds of strategies
The ssgRNA transcriptional control to specific gene: strategy one, builds the fusion protein of Cas9-VP64/KRAB, is aided with 14bp
SsgRNA can activate/suppress transcribing of target gene；Strategy two, uses wild type Cas9, adds MS2/ in ssgRNA sequence
The RNA aptamers sequences such as PCP, then it is aided with MS2/PCP-VP64/KRAB fusion protein, can activate/suppress transcribing of target gene；
Strategy three, builds the fusion protein of Cas9-VP64/KRAB, adds the RNA aptamers sequences such as MS2/PCP in ssgRNA sequence,
It is aided with MS2/PCP-VP64/KRAB fusion protein again, transcribing of activation/suppression target gene that can be more efficient.According to this principle,
The ssgRNA of design targeting LIG4/RAD51/RAD52/BRCA1, can regulate and control the expression of serial genes.
B., in being embodied as, the method for the present invention is applied to the reparation (referring to experiment flow) of GFP reporter gene, outward
In the GFP expression vector in source there is termination codon and cannot translate generation green fluorescent protein in GFP coding region.The most accurate
Fixed point is repaired, and its is possible to realize intended GFP and expresses, and by the ratio of fluidic cell sorting detection GFP positive cell, can
The efficiency repaired with quantitative fixed point.This method is also apply to repairing of source TP53 gene mutation in U251 human glioma cells system
Multiple, by cell transfecting, cultivate, extract cellular genome and PCR order-checking by the way of, effect gene mutation fixed point can repaired
Rate carries out detecting (referring to experiment flow).
Fig. 3 is illustrated that the figure knocking in vector construction
Fig. 4 is illustrated that the figure of gene editing efficiency comparison
Fig. 5 is illustrated that in BFP stably transfected cell line that gene mutation remediation efficiency compares
Fig. 6 is illustrated that source TP53 gene mutation remediation efficiency compares
Detailed description of the invention
Below by detailed description of the invention and experimental data, the present invention is further illustrated.Although for clearly mesh
, proprietary term used below, but these terms are not meant to definition or limit the scope of the present invention.
In the present invention, term " target site " i.e. in target gene group any one section be intended to the DNA that transformed or repair
Sequence.DNA sequence near target site, it is allowed to exogenous array integration at target site, includes but not limited to gene knock-in
(knock-in).In a specific embodiment, target dna sequence is the DNA sequence of double-strand, includes, but not limited to the dye of cell
DNA sequence in colour solid genome, the DNA sequence outside cell chromosome genome (such as mitochondrial genome), plasmid, virus
Deng DNA sequence.
In the present invention, term " fixed point restructuring " refers to, is incorporated into specifically by nonrandom mode by exogenous array
At target site, including being incorporated into the 5 ' upstreams in certain particular target site, between 3 ' downstreams or target site.
In the present invention, term " exogenous DNA array " refers to, it is desirable to by the DNA sequence at fixed point restructuring to target site.Outward
Source DNA sequence can be the sequence not existing at target site or being changed.
In the present invention, " sgRNA " refers to that guide RNA, bootable nuclease targeting target gene cut；Described
SgRNA can include the adjacent motif in conservative interval of 3' end, and such as motif is NGG；The length of described sgRNA can be 18-
Between 23bp.
In the present invention, " ssgRNA " refers to the guide RNA of a length of 14-16bp, and under the guiding of ssgRNA, Cas9 can
To identify and to combine target site, but target site cannot be cut.
In the present invention, " aptamers ", " fit ", " in conjunction with fit (aptamer) " have identical meanings, refer to through
A kind of in-vitro screening technology (systematic evolution of ligands by exponential enrichment,
SELEX), obtain from random single chain oligonucleotide library can Specific binding proteins or the strand widow of other small-molecule substances
Polynucleotide, it is also possible to be RNA, it is also possible to be DNA, length is generally 25～60 nucleotide.
Specific embodiment
Embodiment 1GFP reporter gene is transformed, and recovers the expression of GFP green fluorescent protein
1) transformation ssgRNA, it is thus achieved that with the ssgRNA of specific RNA hairpin structure
Specific site at ssgRNA (SpCas9 and SaCas9) accesses unique RNA hair clip such as MS2/PP7, this kind of hair clip
Structure can be by MS2 associated proteins/PCP albumen institute specific recognition.
The ssgRNA total order having PP7 hairpin structure is classified as:
ccggtgagaccgagagagggtctcagttttagagctagcgagggagcagacgatatggcgtcgctccctcgttagca
agttaaaataaggctagtccgttatcaacttgcgagggagcagacgatatggcgtcgctccctcgtaagtggcaccg
agtcggtgctttttgaattc
SsgRNA total order with MS2 hairpin structure is classified as:
ccggtgagaccgagagagggtctcagttttagagctaggccaacatgaggatcacccatgtctgcagggcctagcaa
gttaaaataaggctagtccgttatcaacttggccaacatgaggatcacccatgtctgcagggccaagtggcaccgag
tcggtgcttttttt
Above ssgRNA complete sequence all synthesizes in Nanjing Genscript Biotechnology Co., Ltd..
2) potential at GFP gene target site Cas9 recognition site is analyzed
In this experiment, the target of GFP reporter gene transformation is to replace termination codon, recovers GFP green fluorescent protein
Express.The operation of corresponding transgenic is to introduce DNA double chain interruption (DSBs) near the termination codon of GFP coding region, afterwards
Repaired by homology recovery template.Therefore it is first necessary to determine near the termination codon of i.e. GFP coding region, target site district
Integration site.In the present invention program, i.e. selecting Cas9 recognition site, integration site can be selected in the middle of recognition site, to reach
Destroy recognition site, it is ensured that integration sequence is not continued cutting.
As mentioned above, specific dna sequence effectively produces DSBs and can have multiple strategy, including genetic engineering modified mistake
I-sceI, I-AniI, FoxI, Cas9 and some synthetic polyribonucleotidess, such as LNA, PNA etc..This experiment use spCas9 and
SaCas9, with the supporting ssgRNAs transformed combination.According to sequence analysis, find have near the termination codon of GFP coding region
Potential spCas9 recognition site (sequence is as follows):
Sequence chart near GFP termination codon (ATG), marks recognition site
ATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGA
CGGCtagGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGC
ATCGACTTCAAGGAGGACGGCAACATCCTGGGGC
Lower case tag is termination codon
Tilted letter regionFor spCas9 recognition site, draw horizontal line region "agG”For
PAM sequence
3) design homologous complementary recovery template (donor DNA template)
Homologous complementary recovery template design such as Fig. 3, the upstream and downstream at ssgRNA target site is respectively arranged with the homology sequence of 100-200bp
Row (upstream and downstream homology arm).Termination codon subsequence is deleted at integration site.Entirety is connected into carrier T and extracts plasmid through amplification
Become foreign donor.
Foreign donor construct:
Homologous recombination arm 100bp downstream, 100bp upstream homologous recombination arm
Particular sequence is:
gacgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggcta
cgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgaca
ccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtac
aactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgtttttgagctcccaacg
cg
4) MS2/PCP-VP64/KRAB fusion protein expression vector is built
Repair relevant gene loci for being accurately positioned by transcription regulatory factor to DSBs, must be connected into specific in ssgRNA
Hairpin structure such as MS2 or PP7, build the expressing fusion protein of MS2/PCP and transcription regulatory factor such as VP64 or KRAB afterwards
Carrier, MS2 albumen can recognize that the MS2 hairpin structure in ssgRNA herein, and PCP then can identify PP7 hairpin structure.Build for this
MS2/PCP-VP64/KRAB fusion protein expression vector, merges with VP64 and KRAB respectively by the coded sequence of MS2/PCP, even
It is connected in the expression vector with EF1alpha promoter, it is ensured that reading frame is consistent readable logical.
5) rotaring redyeing 293 cell system determines the remediation efficiency of DNA profiling
Experimental group uses the GFP expression vector with termination codon sudden change, and spCas9 expression vector, GFP is specific
SgRNA (20bp), with ssgRNA (14bp), the MS2-KRAB of MS2 hairpin structure targeting LIG4/RAD52/RAD51/BRCA1
(corresponding LIG4)/VP64 (corresponding RAD52/RAD51/BRCA1) fusion protein is thin by Lipofectamine 3000 corotation 293
Born of the same parents are, after 37 DEG C of incubators are cultivated 48 hours, are accounted for by fluidic cell sorting (flow cytometry) detection GFP positive cell
The ratio of total cell.
The same amount of GFP expression vector with termination codon sudden change of same employing in matched group, spCas9 expresses load
Body, the specific sgRNA of GFP (20bp), with MS2 hairpin structure without targeting ssgRNA, MS2-KRAB (corresponding LIG4)/
VP64 (corresponding RAD52/RAD51/BRCA1) fusion protein is by Lipofectamine 3000 corotation 293 cell line, 37 DEG C of trainings
After supporting case cultivation 48 hours, accounted for the ratio of total cell by fluidic cell sorting (flow cytometry) detection GFP positive cell
Example (Fig. 4)
We utilize the BFP of point mutation to construct stably transfected cell line, can produce GFP by the reparation of BFP point mutation
The efficiency repaired is done by green fluorescent protein determines statistics of variables.We have found that suppression NHEJ path Lig4 can significantly improve homology
The efficiency (Fig. 5 is left) of recombination repair.And promote that path component Rad51/52 of homologous recombination repair also can remarkably promote homology weight
The efficiency (Fig. 5 is right) that group is repaired.Prompting is used in combination as expressed suppression Lig4 to express with promotion Rad51/52, can enter one
Step promotes gene mutation homologous recombination repair efficiency.
The experimental design that embodiment 2 people's TP53 point mutation is repaired
1) selection U-251 human glioma cells system, sequencing result shows that the TP53 gene of this cell line carries R273H
(CGT-> CAT) point mutation of isozygotying.For repairing this point mutation, design the sgRNA of selectively targeted TP53 gene at this location proximate
(SaCas9) (Fig. 6).
Its particular sequence:
agagaccggcgcacagaggaa
PAM:gagaat
2) design recovery template: template selects the mono-oligonucleotide chain of DNA, and sequence is
actgggacggaacagctttgaggtgcgtgtttgtgcctgtcctgggcgtgataggcgaacggaagaggaaaacctcc
gcaagaaaggggagcctcaccacgagctgcccccagggag.At the sgRNA recognition site of TP53 introduce samesense mutation with
The site after repairing is avoided to be continued cutting by SaCas9 and introduce insertion/deletion sudden change.
3) transfection U251 cell line checking sudden change remediation efficiency: proceed to carry TP53 in experimental group selectively targeted
The SaCas9 expression vector of sgRNA, homology recovery template, puromycin screening plasmid and reparative factor expression vector (with
SsgRNA (14bp), MS2-KRAB (corresponding the LIG4)/VP64 of MS2 hairpin structure targeting LIG4/RAD52/RAD51/BRCA1
(corresponding RAD52/RAD51/BRCA1) fusion protein), matched group proceeds to carry the selectively targeted sgRNA's of TP53
SaCas9 expression vector, homology recovery template, puromycin screening plasmid and control vector.By PEI corotation U-251 cell
System, after 37 DEG C of incubators are cultivated 48 hours, the puromycin adding 2ug/ml continues to cultivate 48 hours, extracts cellular genome,
Use TP53 specific primer to expand restoring area by PCR experiment, send sequencing analysis repairing effect (Fig. 6, factor 1:Rad51+
MS2-VP64；Factor 2:Rad52+MS2-VP64；Factor 3:Brca1+MS2-VP64；Factor 4:LIG4+MS2-KRAB).
4) interpretation
By the repair system of external source GFP mutant gene is tested, it has been found that Rad52 recombinase can effectively improve same
Source recombination efficiency is to more than 50% (seeing Fig. 4), and this result will be greatly increased the remediation efficiency of gene mutation, this research contents and
Thinking there is no any international research paper publishing, belongs to the discovery that the world is pioneering.
In the Part II sudden change reparative experiment to endogenous oncogene, we verify further, and Rad52 can effectively facilitate
The reparation of TP53 gene mutation, the experiment of this recovery test not yet sorts positive cell with fluidic cell, i.e. by the method for PCR order-checking
Obtained very uniform sudden change reparation order-checking peak figure, illustrated that remediation efficiency is the highest, can reach internal reparation mutant oncogenes with
And other are in body gene mutation reparation and the requirement of gene editing.
Above, it is illustrated based on embodiments of the present invention, but the present invention is not limited to this, those skilled in the art
Member should be understood that and can implement in the way of carrying out deforming and change in the range of the purport of the present invention, such deformation with
The mode of change, ought to belong to protection scope of the present invention.
Claims (23)
1. a gene editing method based on homologous recombination, including using nuclease that target gene is cut, its feature
Being: also include ssgRNA and targeting transcription regulatory factor in described method, described ssgRNA can guide nuclease targeting
The code nucleic acid of gene editing associated protein but will not cut, described targeting transcription regulatory factor can be compiled by controlling gene
Collect transcribing of associated protein.
2. the method for claim 1, described nuclease carries out homologous recombination and includes using sgRNA target gene,
Described sgRNA can guide nuclease targeting target gene to cut；The most described sgRNA includes the conservative interval phase of 3' end
Adjacent motif, preferably described motif is NGG；The most described sgRNA is 18-23bp, preferably 20bp.
3. method as claimed in claim 1 or 2, described nuclease is preferably selected from selected from Cas9 and CPF1, described Cas9
SaCas9、SpCas9。
4. the method as described in any one of claim 1-3, also includes homologous complementary recovery template, preferably same in described method
The integration site of source restructuring deletes termination codon.
5. the method as described in any one of claim 1-4, described ssgRNA includes the adjacent motif in conservative interval of 3' end, excellent
Motif described in choosing is NGG；Preferably described ssgRNA is 14-16bp, more preferably 14bp, 15bp or 16bp.
6. the method as described in any one of claim 1-5, described targeting transcription regulatory factor is transcribed sharp selected from targeting
At least one of the factor of living or targeting transcription inhibitory factor, the most described targeting activating transcription factor is selected from VP64, preferably
Described targeting transcription inhibitory factor is selected from KRAB.
7. the method as described in any one of claim 1-6, described gene editing associated protein is selected from non-homogeneous district end even
Connecing at least one of associated protein and homologous recombination associated protein, the most described non-homogeneous district end connects associated protein and is selected from
LIG4, the most described homologous recombination associated protein is selected from Rad51, Rad52 and Brca1 or a combination thereof.
8. the method as described in any one of claim 1-7, the nuclease in described method and targeting transcription regulatory factor shape
Becoming fusion protein, the most described fusion protein comprises flexible linker.
9. the method as described in any one of claim 1-7, described ssgRNA also coupling RNA aptamers sequence, described RNA
Aptamers sequence can specific be combined with related protein, described targeting transcription regulatory factor and described related protein shape
Become fusion protein.
10. the method as described in any one of claim 1-7, nuclease in described method and the first targeting transcriptional control because of
Son forms fusion protein, and the most described fusion protein comprises flexible linker, described ssgRNA also coupling RNA aptamers sequence
Row, described RNA aptamers sequence can specific be combined with related protein, and the second targeting transcription regulatory factor is with described
Related protein forms fusion protein.
11. methods as described in any one of claim 9 or 10, described RNA aptamers is hairpin structure；The most described hair clip is tied
Structure is selected from MS2 or PP7, and described related protein is selected from MS2 associated proteins or PCP associated proteins.
12. 1 kinds of compositionss for gene editing, including ssgRNA and targeting transcription regulatory factor, described ssgRNA can
Guide the code nucleic acid of nuclease target gene editor's associated protein but will not cut, described targeting transcription regulatory factor
Can the transcribing of controlling gene editor's associated protein.
13. compositionss as claimed in claim 12, described compositions also includes nuclease, sgRNA, homologous complementary recovery template
Or a combination thereof.
14. 1 kinds of compositionss for gene editing, including nuclease, nuclease and melting that targeting transcription regulatory factor is formed
Hop protein, sgRNA, ssgRNA and homologous complementary recovery template, described ssgRNA can guide nuclease target gene editor's phase
Closing the code nucleic acid of albumen but will not cut, described targeting transcription regulatory factor can controlling gene editor's associated protein
Transcribe.
15. 1 kinds of compositionss for gene editing, including providing nuclease, sgRNA, RNA aptamers sequence and ssgRNA's
Coupling molecule, homologous complementary recovery template, targeting transcription regulatory factor and the fusion protein of RNA aptamers related protein, institute
State ssgRNA to guide the code nucleic acid of nuclease target gene editor's associated protein but will not cut, described targeting
Transcription regulatory factor can the transcribing of controlling gene editor's associated protein.
16. 1 kinds of compositionss for gene editing, are formed including providing nuclease and the first targeting transcription regulatory factor
Fusion protein, sgRNA, RNA aptamers sequence turn with coupling molecule, homologous complementary recovery template and second targeting of ssgRNA
Record regulatory factor and the fusion protein of RNA aptamers related protein, described ssgRNA can guide nuclease target gene editor
The code nucleic acid of associated protein but will not cut, described targeting transcription regulatory factor can controlling gene editor be correlated with egg
White transcribes.
17. compositionss as described in any one of claim 12-16, described targeting transcription regulatory factor turns selected from targeting
At least one of record activity factor or targeting transcription inhibitory factor, the most described targeting activating transcription factor is selected from VP64,
The most described targeting transcription inhibitory factor is selected from.
18. compositionss as described in any one of claim 12-16, described gene editing associated protein is selected from end, non-homogeneous district
End connects at least one of associated protein and homologous recombination associated protein, and the most described non-homogeneous district end connects associated protein choosing
From LIG4, the most described homologous recombination associated protein is selected from Rad51, Rad52 and Brca1 or a combination thereof.
19. 1 kinds of RNA aptamers sequences and the coupling molecule of ssgRNA, described ssgRNA part can guide nuclease targeting
The code nucleic acid of gene editing associated protein but will not cut, described RNA aptamers Sequence can specific with
Related protein combines；Preferably described ssgRNA part includes the adjacent motif in conservative interval of 3' end, and preferably described motif is
NGG；Preferably described ssgRNA part is 14-16bp, preferably 14bp, 15bp or 16bp.
20. coupling molecules as claimed in claim 19, described RNA aptamers is hairpin structure；The most described hairpin structure selects
From MS2 or PP7, described related protein is selected from MS2 associated proteins or PCP associated proteins.
21. 1 kinds of test kits, including the compositions described in any one of claim 12-18 or any one of claim 19-20 institute
The coupling molecule stated.
Method described in 22. any one of claim 1-11 or the compositions described in any one of claim 12-18 or right are wanted
Ask the coupling molecule described in any one of 19-20 for the purposes of gene editing.
Method described in 23. any one of claim 1-11 or the purposes described in claim 22, described gene comes from micro-
Biology, plant, animal, cell, mammal or people.
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| PCT/CN2017/097797 WO2018033110A1 (en) | 2016-08-19 | 2017-08-17 | Method for repairing gene point mutation |
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Cited By (6)
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| WO2018033110A1 (en) * | 2016-08-19 | 2018-02-22 | 苏州兰希亚生物科技有限公司 | Method for repairing gene point mutation |
| WO2019041296A1 (en) * | 2017-09-01 | 2019-03-07 | 上海科技大学 | Base editing system and method |
| WO2020063178A1 (en) * | 2018-09-26 | 2020-04-02 | 中国科学技术大学 | Gene editing composition or kit used for in vivo gene therapy |
| CN111630175A (en) * | 2018-01-22 | 2020-09-04 | 庆尚大学校产学协力团 | Method for improving gene editing efficiency in plants based on homologous recombination |
| CN112980880A (en) * | 2021-03-08 | 2021-06-18 | 浙江大学 | Method for constructing Fzd6-Q152E site-directed mutagenesis mouse model based on CRISPR/Cas9 and application |
| CN114807240A (en) * | 2021-01-21 | 2022-07-29 | 深圳市第二人民医院(深圳市转化医学研究院) | Aptamer-linked template molecule and kit thereof |
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| US11597924B2 (en) | 2016-03-25 | 2023-03-07 | Editas Medicine, Inc. | Genome editing systems comprising repair-modulating enzyme molecules and methods of their use |
| WO2017180694A1 (en) | 2016-04-13 | 2017-10-19 | Editas Medicine, Inc. | Cas9 fusion molecules gene editing systems, and methods of use thereof |
| WO2019014564A1 (en) | 2017-07-14 | 2019-01-17 | Editas Medicine, Inc. | Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites |
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| WO2018033110A1 (en) * | 2016-08-19 | 2018-02-22 | 苏州兰希亚生物科技有限公司 | Method for repairing gene point mutation |
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| CN111630175A (en) * | 2018-01-22 | 2020-09-04 | 庆尚大学校产学协力团 | Method for improving gene editing efficiency in plants based on homologous recombination |
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| WO2020063178A1 (en) * | 2018-09-26 | 2020-04-02 | 中国科学技术大学 | Gene editing composition or kit used for in vivo gene therapy |
| CN114807240A (en) * | 2021-01-21 | 2022-07-29 | 深圳市第二人民医院(深圳市转化医学研究院) | Aptamer-linked template molecule and kit thereof |
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| WO2018033110A1 (en) | 2018-02-22 |
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