CN107828824A - The method for obtaining the homozygous mutation of seamless modification - Google Patents
The method for obtaining the homozygous mutation of seamless modification Download PDFInfo
- Publication number
- CN107828824A CN107828824A CN201711007052.1A CN201711007052A CN107828824A CN 107828824 A CN107828824 A CN 107828824A CN 201711007052 A CN201711007052 A CN 201711007052A CN 107828824 A CN107828824 A CN 107828824A
- Authority
- CN
- China
- Prior art keywords
- transposons
- cell
- resistance
- gene
- mutation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000035772 mutation Effects 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000012986 modification Methods 0.000 title claims abstract description 16
- 230000004048 modification Effects 0.000 title claims abstract description 16
- 238000012216 screening Methods 0.000 claims abstract description 30
- 108700028369 Alleles Proteins 0.000 claims abstract description 15
- 238000010362 genome editing Methods 0.000 claims abstract description 15
- 230000006801 homologous recombination Effects 0.000 claims abstract description 12
- 238000002744 homologous recombination Methods 0.000 claims abstract description 12
- 230000001413 cellular effect Effects 0.000 claims abstract description 7
- 230000005782 double-strand break Effects 0.000 claims abstract description 6
- 231100000518 lethal Toxicity 0.000 claims abstract description 6
- 230000001665 lethal effect Effects 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims abstract description 6
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 73
- 108091033409 CRISPR Proteins 0.000 claims description 42
- 238000010354 CRISPR gene editing Methods 0.000 claims description 17
- 201000010099 disease Diseases 0.000 claims description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 9
- IPVFGAYTKQKGBM-BYPJNBLXSA-N 1-[(2r,3s,4r,5r)-3-fluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-iodopyrimidine-2,4-dione Chemical compound F[C@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 IPVFGAYTKQKGBM-BYPJNBLXSA-N 0.000 claims description 7
- 230000003321 amplification Effects 0.000 claims description 5
- 230000000869 mutational effect Effects 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 238000010443 CRISPR/Cpf1 gene editing Methods 0.000 claims description 4
- 238000010459 TALEN Methods 0.000 claims description 4
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 claims description 4
- 102000008579 Transposases Human genes 0.000 claims description 4
- 108010020764 Transposases Proteins 0.000 claims description 4
- 238000002703 mutagenesis Methods 0.000 claims description 4
- 231100000350 mutagenesis Toxicity 0.000 claims description 4
- 210000000130 stem cell Anatomy 0.000 claims description 3
- -1 ZFN Proteins 0.000 claims description 2
- 238000003198 gene knock in Methods 0.000 claims description 2
- 238000003209 gene knockout Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 description 22
- 206010064571 Gene mutation Diseases 0.000 description 16
- 101000617536 Homo sapiens Presenilin-1 Proteins 0.000 description 15
- 102100022033 Presenilin-1 Human genes 0.000 description 15
- 239000013612 plasmid Substances 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 10
- 230000008859 change Effects 0.000 description 9
- 208000024827 Alzheimer disease Diseases 0.000 description 6
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 210000002569 neuron Anatomy 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 108010085895 Laminin Proteins 0.000 description 4
- 210000003618 cortical neuron Anatomy 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 108010082117 matrigel Proteins 0.000 description 4
- 108010055896 polyornithine Proteins 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 208000026350 Inborn Genetic disease Diseases 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 208000016361 genetic disease Diseases 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 229950010131 puromycin Drugs 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 2
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- 108700024394 Exon Proteins 0.000 description 2
- 102000034615 Glial cell line-derived neurotrophic factor Human genes 0.000 description 2
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 description 2
- 108020005004 Guide RNA Proteins 0.000 description 2
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 2
- 108010064397 amyloid beta-protein (1-40) Proteins 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 229920002714 polyornithine Polymers 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011435 rock Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 239000011701 zinc Substances 0.000 description 2
- 229910052725 zinc Inorganic materials 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- NEEVCWPRIZJJRJ-LWRDCAMISA-N 5-(benzylideneamino)-6-[(e)-benzylideneamino]-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound C=1C=CC=CC=1C=NC=1C(=O)NC(=S)NC=1\N=C\C1=CC=CC=C1 NEEVCWPRIZJJRJ-LWRDCAMISA-N 0.000 description 1
- 102000005869 Activating Transcription Factors Human genes 0.000 description 1
- 108010005254 Activating Transcription Factors Proteins 0.000 description 1
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 1
- 108010043324 Amyloid Precursor Protein Secretases Proteins 0.000 description 1
- 102000002659 Amyloid Precursor Protein Secretases Human genes 0.000 description 1
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 1
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108091027544 Subgenomic mRNA Proteins 0.000 description 1
- 108010076089 accutase Proteins 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 108010064539 amyloid beta-protein (1-42) Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000011559 double-strand break repair via nonhomologous end joining Effects 0.000 description 1
- 208000025688 early-onset autosomal dominant Alzheimer disease Diseases 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 208000015756 familial Alzheimer disease Diseases 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000004754 hybrid cell Anatomy 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 238000011022 operating instruction Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Transplantation (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A kind of method for the homozygous mutation for obtaining seamless modification, including：(1) cutting is carried out to the site for needing to be mutated in cellular genome with gene editing system and produces double-strand break；(2) two kinds of transposons containing purposeful mutant nucleotide sequence and different resistances are integrated into a pair of alleles of cellular genome respectively by homologous recombination；(3) screening allele is all integrated with the cell clone of transposons, then cuts off transposons；(4) it is the cell clone for remaining transposons is lethal, obtain the cell clone of the homozygous mutation containing seamless modification.Methods described can significantly improve the probability for obtaining homozygous mutation, use manpower and material resources sparingly and the time, and applied widely.
Description
Technical field
The invention belongs to biology field, more particularly to a kind of method for the homozygous mutation for obtaining seamless modification.
Background technology
Compared with animal model, a series of advantage is had based on cell model derived from people iPS, can such as be purified as certain
One specific cell type is simultaneously studied, and facilitates high flux screening compound and gene target, can be obtained several genes and be dashed forward
Become cell and cell phenotype and pathogenesis are studied with this, there is no the difference on species with people.Cell with patient is through overweight
Programming obtains iPS, can study a variety of genetic diseases.Certainly, the acquisition of patient's cell, pathogenic mutation gene is really
Recognize, the research of the common analyses of different genes background cells all to genetic disease causes obstacle.But these obstacles, it can pass through
The gene mutation related to disease is introduced in normal iPS cells, the iPS cells for obtaining series of identical genetic background are solved
Certainly.
Disease related mutation is seamlessly accurately introduced in normal iPS cells, method the most frequently used at present is CRISPR/
Cas9 combinations single strand dna oligonucleotide (ssDONs) and CRISPR/Cas9 combination piggyBac transposons.As guiding RNA
(gRNA) after matching one section of special DNA sequence dna of identification by Watson-Crick, Cas9 can make double-strand produce fracture.Double-strand
Fracture is that certainly, this can equally stimulate another more conventional endogenous dna necessary to homologous recombination obtains gene mutation
Reparation approach-non-homologous end joining (NHEJ), this will cause obstacle to accurate seamless genetic modification.Though synthesize ssODNs
Advantageously it can so be ordered from company, but it lacks screening system so that the accurate seamless modification efficiency of gene is at a fairly low.
PiggyBac transposon may build plasmid can relatively cumbersome point, but it can carry screening system, and it is seamless miscellaneous to obtain purpose
Mutation is closed relative to be easier.Certainly, for homozygous mutation, CRISPR/Cas9 combinations piggyBac method is used
It is very low to obtain efficiency, so as to cause manpower and materials and temporal huge waste.Therefore, the pure of seamless modification is efficiently obtained
It is very important for closing research of the method for mutation to current genetic disease.
The content of the invention
In order to solve problems of the prior art, the present invention proposes a kind of homozygous mutation for obtaining seamless modification
Method, the acquisition efficiency of homozygous mutation can be significantly improved.
On the one hand, the invention provides a kind of method for the homozygous mutation for obtaining seamless modification, including：(1) compiled with gene
The system of collecting carries out cutting to the site for needing to be mutated in cellular genome and produces double-strand break；(2) purposeful mutant nucleotide sequence will be contained
It is integrated into respectively in a pair of alleles of cellular genome by homologous recombination with two kinds of transposons of different resistances；(3)
Screening allele is all integrated with the cell clone of transposons, then cuts off transposons；(4) cell of transposons will be remained
Clone lethal, the cell clone of homozygous mutation of the acquisition containing seamless modification.
Preferably, methods described is carried out in inductivity versatile stem cell iPS.
Preferably, the gene editing system is CRISPR/Cas9, ZFN, TALEN or CRISPR/Cpf1.
Preferably, step (2) includes：Pass through homologous recombination side with the transposons containing the first resistance and purpose mutant nucleotide sequence
Formula is incorporated into genome, then carries out the first resistance screening；Above-mentioned mutational site is cut again with gene editing system
Cut generation double-strand break；Gene is incorporated into by homologous recombination mode with the transposons containing the second resistance and purpose mutant nucleotide sequence
In group, the second resistance screening is then carried out.
Preferably, the cell after the first resistance screening and the second resistance screening is expanded.
Preferably, two in puro, hygR or NeoR of first resistance and the second resistance.
Preferably, the transposons is piggyBac transposon.
Preferably, transposons is cut off using transposase in step (3), transposons will be remained with FIAU in step (4)
Cell clone it is lethal.
Methods described can be used for pinpointing reparation, rite-directed mutagenesis, gene knockout or gene knock-in.
Methods described can be also used for establishing disease model.
Relative to prior art, the invention has the advantages that：
(1) present invention combines the general of the transposons acquisition homozygous mutation of the different resistance screenings of carrying using gene editing system
Rate significantly rises, and has saved manpower and materials and time；
(2) the method for the invention is applied widely, suitable for CRISPR/Cas9, ZFN (zinc finger ribalgilase),
TALEN (transcription activator-like (TAL) effector nucleases), CRISPR/Cpf1 etc. are all
Gene editing system, Double range of choice is also than wide；
(3) Double screening combines different gene editing systems, is applicable not only to fixed point reparation and rite-directed mutagenesis, also
Go for knocking out (Knock out) and knock in (knock in) to gene；
(4) disease model can easily be established by introducing gene mutation by the method for the invention.
Brief description of the drawings
Figure 1A is the flow chart that seamless heterozygous mutant is obtained in embodiment 1；
Figure 1B is the schematic diagram of the piggyBac plasmids and PCR checkings in embodiment 1；
Fig. 1 C are PSEN1F105C and PSEN1 resulting after the sequence of PSEN1 in embodiment 1 and gene site-directed editor
Δ I83M84 sequences；
Fig. 1 D are the PCR testing results of puro genetic fragments in embodiment 1；
Fig. 1 E are the PCR testing results of gene after being mutated in embodiment 1 under Scr7 and NU7026 effects；
Fig. 2A is the flow chart that seamless homozygous mutation is obtained in embodiment 2；
Fig. 2 B are the schematic diagram of the piggyBac plasmids and PCR checkings in embodiment 2；
Fig. 2 C do not have the cell gram of " WT " band for preliminary screening after the screening in embodiment 2 by puro and HygR resistances
It is grand；
Fig. 2 D are the PCR testing results of puro and HygR genetic fragments in embodiment 2；
Fig. 2 E are the sequencing mirror of each iPS cell line of PSEN1F105C and PSEN1 Δ I83M84 gene mutations in embodiment 2
Determine result；
Fig. 3 is that the cell that PSEN1F105C and PSEN1 Δ I83M84 gene editings are carried in embodiment 3 obtains gene mutation
The disease-associated phenotypic situation of change of dependence：Wherein A is the cell-derived god of PSEN1F105C and PSEN1 Δ I83M84 iPS
On APP expressions it is in gene mutation dependent change through member；B is PSEN1F105C iPS cells and neuron in A β 42:
It is in gene mutation dependent change on 40 ratios；C is PSEN1 Δs I83M84 iPS cells and neuron on the ratios of A β 42: 40
In gene mutation dependent change, * * P ＜ 0.05and***P ＜ 0.001, one-way ANOVA.
Embodiment
For the object, technical solutions and advantages of the present invention are more clearly understood, below in conjunction with specific embodiment, and reference
Accompanying drawing, the present invention is described in further detail.
In an embodiment of the present invention, in order to obtain accurate seamless homozygous mutation, CRISPR/Cas9, which is combined, carries two
PiggyBac transposon containing puro/TK resistance screenings and hygR/TK respectively, Cas9 contain after particular sequence cutting
The piggyBac transposon of puro/TK resistances is first incorporated into genome by homologous recombination mode, and 3-5 is acted on puromycin
After it, CRISPR/Cas9 and the piggyBac transposon containing hygR/TK are transfected again after remaining cell amplification, is passed through
HygR resistance screenings spread single cell clone after 3-5 days, select allele all to integrate the clone of piggyBac transposon, utilize
Transposase cuts off piggyBac transposon, and reusing FIAU makes the cell clone for remaining piggyBac transposon lethal, from
And obtain the seamless modification clone containing homozygous mutation.Double use wherein for enrichment is not limited only to
Puro, hygR or NeoR combination of two.In the following embodiments, by taking normal iPS cells as an example, CRISPR/Cas9 is utilized
Familial Alzheimer disease (AD) related mutation PSEN1F105C and PSEN1 Δ is introduced with reference to piggyBac transposon
I83M84, and they are divided into neuron, its disease phenotype obtains the change of gene mutation dependence.
Embodiment 1CRISPR/Cas9 efficiently obtains miscellaneous with reference to the piggyBac transposon for carrying puro/TK resistance screenings
Close mutation
Experimental method
1st, cell culture and transfection
IPS cells kind on pre-coated Matrigel (BD Biosciences) Tissue Culture Plate, and use mTeSRTM1
(Stemcell Technologies) in 37 DEG C, 5%CO2Cell culture incubator in cultivate.Before Lipofectamine transfections,
IPS cells are pressed per hole 4 × 105~5 × 105Density is passed on coating Matrigel six orifice plates, with containing 10 μM of ROCK
Inhibitor (Sigma) mTeSRTM1 overnight incubation.Second day after passage, with 6 μ g pCas9_NeoR, 3 μ g sgRNA
Plasmid and 50nMssODN (90bpssODN) corotation iPS cells, independent turn of 2 μ g pmaxGFP of a hole cell, which are used as, in addition turns
The control of efficiency is contaminated, transfection protocol is in strict accordance with LipofectamineTM3000 official's operating instruction.In all transfection experiments
In, the wild type control cells of gene editing are used as by the use of normal iPS cells.
2nd, iPS single cell clones culture
Culture is cloned for the iPS of single cell source, cell is passed in Matrigel bags with the density of 0.5 cell per well
In 96 orifice plates of quilt, passed on after being cultivated eight days containing the mTeSR1TM containing 10 μM of ROCK inhibitor.
3rd, Cas9 and PB-based plasmids are built
Because PiggyBac modules can be cut off by PBase between two IRT sequences, and leave a TTAA sequence, institute
So that 20bp gRNAs is designed in Select gene group between TTAA and mutational site.According to the design principle 5 ' having been reported-
GN20NGG-3 ', the gRNAs being designed are cloned into the gRNA expression vectors after optimization.
For the structure of PB-based carriers, the homology arm on piggyBac plasmids is substituted for, homology arm is made with iPS cells
For template amplification.Finally, using two restriction enzymes of EcoRI and BamHI, puromaycin is substituted for HygR genes.
Primer sequence is listed in the table below：
The primer sequence table of table 1
| Primer | Sequence (5 ' -3 ') |
| PS1-EXON4-F(P1)(SEQ ID No：1) | GGAGCACAACGACAGACGG |
| PS1-EXON4-R(P2)(SEQ ID No：2) | AATGGCCCTGAGGTGGAAA |
| 183-junc-F(P3)(SEQ ID No：3) | GTCCAAACGCCTCTGACACAAGG |
| PB-junc-R(P4)(SEQ ID No：4) | CTCACGCGGTCGTTATAGTTC |
| PB-junc-F(P5)(SEQ ID No：5) | GTTCTTCTGATCTAGACCAATC |
| F105C-junc-R(P6)(SEQ ID No：6) | GTGTCAGGGATAAGAACC |
4th, heterozygous mutant is obtained
Below by taking PSEN1 Δs 183M84 as an example, illustrate seamless miscellaneous using CRISPR/Cas9 and piggyBac transposon acquisition
The flow of mutation is closed, as shown in Figure 1A.
(a) first, it is determined that the site for needing to be mutated is on PSEN1 4 exons；
(b) carry out cutting in target site using Cas9 and produce double-strand break DSB；
(c) structure carries piggyBac transposon (the SEQ ID No of puro/TK resistances：7), the piggyBac swivel bases
Son is in addition to purpose mutational site, and both sides are respectively comprising 500bp and normal cell identical sequence；
(d) piggyBac transposon is integrated into iPS cellular genomes by homologous recombination；
(e) (acted on 3-5 days with puromycin) after puro screening, using in swivel base cleavage genome
PiggyBac transposon, then FIAU processing makes the cell-lethal of residual piggyBac transposon, so as to obtain seamless modification
Heterozygous mutant cell.
Experimental result
In this way, can efficiently obtain F105C and Δ I83M84 heterozygous mutant, its efficiency 15% or so,
But in respectively being cloned for 153 and 104, again without purpose homozygous mutation (table 2).This also illustrates that piggybac is more
It is to be incorporated into the form of Figure 1B in genome, it has only been incorporated near one of allele, another equipotential base
Because neighbouring site has no any change.Because piggybac plasmids are possible to be transferred to genome other positions, in order that gained
To aim cell in do not contain the residuals of any external source piggybac plasmids, preliminary examination is done to single cell clone first, because
Piggybac contains puro genes, so only taking do not contain puro genes to send to sequencing, in this manner it is possible to obtain no external source base
Because of the hybrid cell of residual.As shown in figure iD.
The mutation efficiency of table 2
| 153 single cell clones | 104 single cell clones |
| PS1WTF105C(29) | PS1WTΔIM(17) |
| PS1F105CF105C(0) | PS1ΔIMΔIM(0) |
On this basis, SCR7 and NU7026 are employed, can see promote piggybac while be incorporated into a pair of equipotential bases
Because near, schemed from Fig. 2 B, if piggybac is incorporated near a pair of alleles simultaneously, can't detect script size
For the WT bands of 527bp length, in other words, it is about 6160bp to expand the band come.Picking single cell clone is verified, is sent out
Both existing micromolecular compounds are not obviously promoted piggybac while are integrated into an equity under the conditions of its best use of
Effect (Fig. 1 E) near the gene of position.
The piggyBac that embodiment 2CRISPR/Cas9 combines respectively containing puro/TK and hygR/TK resistance screenings turns
Stand efficiently obtains homozygous mutation
Experimental method
Experimental method is the same as the method therefor of embodiment 1.
1st, cell culture and transfection
With embodiment 1.
2nd, iPS single cell clones culture
With embodiment 1.
3rd, Cas9 and PB-based plasmids are built
With embodiment 1.
4th, homozygous mutation is obtained
Below still by taking PSEN1 Δs I83M84 as an example, illustrate to utilize CRISPR/Cas9 and new piggyBac transposon system
System obtains the flow of seamless homozygous mutation, as shown in Figure 2 A：
(a) first, it is determined that the site for needing to be mutated is on PSEN1 4 exons；
(b) carry out cutting in target site using Cas9 and produce DSB；
(c) structure carries piggyBac transposon (the SEQ ID No of puro/TK and HygR/TK resistances respectively：7 Hes
SEQ ID No：8), for the piggyBac transposon in addition to purpose mutational site, both sides respectively include 500bp and normal cell phase
Same sequence；
(d) the piggyBac transposon system is all integrated into by homologous recombination a pair of iPS cellular genomes
In allele, concretely comprise the following steps：PiggyBac transposon containing puro/TK resistances is first incorporated into by homologous recombination mode
In genome, after being acted on 3-5 days with puromycin, CRISPR/Cas9 is transfected again after remaining cell amplification and is contained
HygR/TK piggyBac transposon, after hygR resistance screenings 3-5 days, single cell clone is spread, select allele all whole
Close the clone of piggyBac transposon；
(e) puro and HygR screening is passed through, using piggyBac transposon in swivel base cleavage genome, then FIAU
Processing make the cell-lethal of residual piggyBac transposon, so as to obtain the homozygous mutant cells of seamless modification.
Experimental result
Want to obtain homozygous mutation, a crucial step is that a pair of alleles nearby all incorporates piggybac plasmids (figure
2B).So in order to efficiently obtain homozygous mutation, it is necessary to take new method on this basis.I.e. for same gene site, adopt
Transfected jointly with two sets of different piggybac plasmids, often cover and contain different resistance puro and hygR respectively, using not
What same resistance enrichment was wanted：A pair of alleles nearby all integrates piggybac plasmids.Then transposase and FIAU are passed through
Effect, you can to obtain desired purpose homozygous mutation (Fig. 2A).Once a pair of alleles nearby all incorporates piggybac
Plasmid, will as shown in Figure 2 B, and cell clone at this time is by the WT bands of no script, but simultaneously, should also be in this cell gram
It is grand to detect left and right arms.PCR amplifications will be continued without WT bands, obtain expected left and right arms (Fig. 2 C).This explanation,
Near these cloning clusters allele to be mutated, piggybac plasmids are all incorporated.FIAU screening after, it is necessary to nothing appoint
The cell of what external source piggybac genes residual, because two piggybac plasmids respectively contain puro and hygR genes, so tentatively
The cell of screening should be without the presence of both genes.As shown in Figure 2 D.Utilize new piggybac system combinations CRISPR/
Cas9, the probability for obtaining homozygous mutation significantly rise, and as shown in the results, it is homozygous to obtain PSEN1F105C and PSEN1 Δs I83M84
The efficiency of mutation is respectively 13.7% and 11.9% or so (table 3).PSEN1F105C and PSEN1 Δ 183M84 gene mutations it is each
The sequencing result of iPS cell line is as shown in Figure 2 E.
The mutation efficiency of table 3
| 51 single cell clones | 42 single cell clones |
| PS1F105CF105C(7) | PS1ΔIMΔIM(5) |
Embodiment 3 carries the character mutation experimental method of the cell acquisition gene mutation dependence of AD associated gene mutations
1st, the differentiation of cortical neuron
People iPS cells break up to NSC direction, and the iPS cells of culture spread to small cloning cluster with cell, with
2.5-3.0×104Individual cell/cm2Density, with PSC Neural Induction Medium (Life Technologies)
Y23632 (sigma) cultures are added to change within every two days and once cultivate on pre-coated matrigel (BD Biosciences) 6 orifice plates
Base, continuous culture 7 days.It is digested to after 7 days with Accutase (Sigma) unicellular with 1.0-1.2 × 105Individual cell/cm2It is close
Degree carries out amplification cultivation.
Break up for cortical neuron, in pre-coated Polyornithine/laminin (poly-L-ornithine/
Laminin on Tissue Culture Plate), NSC is with 2 × 105Individual cell/cm2Density, with N2B27 culture mediums (DMEM-
F12：Neural Basal medium 1: 1,2%B27,1%N2,1% nonessential amino acid (non-essential amino
Acids), 2mM Glutamax, every milliliter of 100U penicillin, every milliliter of 0.1mg streptomysin) (it is all Life
Technologies BDNF (PeproTech, 20ng/mL), GDNF (PeproTech, 20ng/mL) and NT3) are added
(PeproTech, 10ng/mL) be digested to after cultivating 7 days, 7 days it is unicellular, then with pre-coated Polyornithine/laminin
(poly-L-ornithine/laminin) Tissue Culture Plate is with 5 × 104-10×104Individual cell/cm2Density, use N2B27
Culture medium is plus GDNF (PeproTech, 20ng/mL), BDNF (PeproTech, 20ng/mL), NT3 (PeproTech, 10ng/
ML), 0.2mM ascorbic acid (ascorbic acid) (Sigma Aldrich) and cAMP (10 μM, Sigma Aldrich) are trained again
Support 14 days.
2nd, Amyloid- beta determinations
The supernatant (iPS cells are persistently cultivated 2 days, and cortical neuron is persistently cultivated 4 days) of culture cell is taken to determine A β 1-
40 and A β 1-42.- 80 DEG C of preservations are put after required time point gathers supernatant nutrient solution.The A β 1-40 and A β 1-42 of cell secretion are used
Human/Rat β Amyloid (40) ELISA Kit (Wako) and Human/Rat β Amyloid (42) ELISA Kit (Wako) examinations
Agent box is measured to specifications.
Experimental result
After being divided into neuron, phenotype A β 42: 40 typical to AD is detected, and it is all in gene to find this two indexs
Mutation dependence sexually revises.Meanwhile exception will occur because carrying the cell gamma-secretase function of PS1 mutation, thus influence god
Processing through the APP in member.Compared with normal control, the neuron derived from PSEN1F105C and PSEN1 Δs I83M84 is in APP
Also occurs gene mutation dependent change (Fig. 3 A-3C) in level.Result of study proves, passes through accurate and seamless gene editing
The cell of resulting carrying early stage morbidity AD associated gene mutations, according to the change of its heterozygous mutant and homozygous mutation, thin
Disease phenotype that can be related loyal simulation AD in born of the same parents' level.
Above test result indicates that：CRISPR/Cas9 combines the piggyBac transposon for carrying puro/TK resistance screenings
Heterozygous mutant can be efficiently obtained, it can be difficult to obtaining homozygous mutation, acquisition can not be also improved even if Scr7 and NU7026 is subject to
The efficiency of homozygous mutation；And CRISPR/Cas9 combines the piggyBac respectively containing puro/TK and hygR/TK resistance screenings
Transposons, using the enrichment of different resistances, it can efficiently obtain homozygous mutation.Wherein Double selection, is also not only limited
In puro, hygR or NeoR combination of two.
It is pointed out that Double utilization, be merely not only suitable for CRISPR/Cas9 systems, it is in addition to all
For gene editing system such as ZFN (zinc finger ribalgilase), TALEN (activating transcription factor sample effector nuclease,
Transcription activator-like (TAL) effector nucleases), the gene editing such as CRISPR/Cpf1
System.Meanwhile Double screening combines different gene editing systems, fixed point reparation and rite-directed mutagenesis are applicable not only to, also
Go for knocking out (Knock out) and knock in (knock in) to gene.
The cell differentiation of the AD associated gene mutations obtained be cortical neuron after, compared with normal control cells, obtain
The character mutation of gene mutation dependence is obtained, further confirms that CRISPR/Cas9 combinations piggyBac transposon introduces gene and dashed forward
Become the convenience and necessity for establishing disease model.
Particular embodiments described above, the purpose of the present invention, technical scheme and beneficial effect are carried out further in detail
Describe in detail bright, it should be understood that the foregoing is only the present invention specific embodiment, be not intended to limit the invention, it is all
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvements done etc., the protection of the present invention should be included in
Within the scope of.
Sequence table
<110>University of Macao
<120>The method for obtaining the homozygous mutation of seamless modification
<130> IB179379
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ggagcacaac gacagacgg 19
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
aatggccctg aggtggaaa 19
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gtccaaacgc ctctgacaca agg 23
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ctcacgcggt cgttatagtt c 21
<210> 5
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
gttcttctga tctagaccaa tc 22
<210> 6
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gtgtcaggga taagaacc 18
<210> 7
<211> 5317
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
gaaatcacag aaagctgaat ttgaaaaagg tgcttggagc tgcagccagt aaacaagttt 60
tcatgcaggt gtcagtattt aaggtacatc tcaaaggata agtacaattg tgtatgttgg 120
gatgaacaga gagaatggag caagccaaga cccaggtaaa agagaggacc tgaatgcctt 180
cagtgaacaa tgatagataa tctagacttt taaactgcat acttcctgta cattgttttt 240
tcttgcttca ggtttttaga actcatagtg acgggtctgt tgttaatccc aggtctaacc 300
gttaccttga ttctgctgag aatctgattt actgaaaatg tttttcttgt gcttatagaa 360
tgacaataga gaacggcagg agcacaacga cagacggagc cttggccacc ctgagccatt 420
atctaatgga cgaccccagg gtaactcccg gcaggtggtg gagcaagatg aggaagaaga 480
tgaggagctg acattgaaat atggcgcaaa gcatgtgact agtctctttg tccctgtgac 540
tctctgcatg gtggtggtcg tggctaccat taaccctaga aagatagtct gcgtaaaatt 600
gacgcatgca ttcttgaaat attgctctct ctttctaaat agcgcgaatc cgtcgctgtg 660
catttaggac atctcagtcg ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta 720
agtgtcactg attttgaact ataacgaccg cgtgagtcaa aatgacgcat gattatcttt 780
tacgtgactt ttaagattta actcatacga taattatatt gttatttcat gttctactta 840
cgtgataact tattatatat atattttctt gttatagata tcaactagaa tgctagcatg 900
ggcccatctc gacctacttc gtatagcata cattatacga agttatgtgc gggcctcttc 960
gctattacgc cagcgcgcgt tgacattgat tattgactag ttattaatag taatcaatta 1020
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 1080
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 1140
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 1200
ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 1260
atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 1320
cttggcagta catctacgta ttagtcatcg ctattaccat gggtcgaggt gagccccacg 1380
ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt 1440
ttttaattat tttgtgcagc gatgggggcg gggggggggg gggcgcgcgc caggcggggc 1500
ggggcggggc gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag 1560
cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa 1620
gcgaagcgcg cggcgggcgg gagtcgctgc gttgccttcg ccccgtgccc cgctccgcgc 1680
cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg 1740
ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc tcgtttcttt 1800
tctgtggctg cgtgaaagcc ttaaagggct ccgggagggc cctttgtgcg ggggggagcg 1860
gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc ccgcgctgcc 1920
cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgcg tgtgcgcgag 1980
gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg ctgcgagggg aacaaaggct 2040
gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcggcg gtcgggctgt 2100
aacccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg 2160
ctccgtgcgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg 2220
gtgccgggcg gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc 2280
cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat 2340
cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg gcggagccga aatctgggag 2400
gcgccgccgc accccctcta gcgggcgcgg gcgaagcggt gcggcgccgg caggaaggaa 2460
atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctcca tctccagcct 2520
cggggctgcc gcagggggac ggctgccttc gggggggacg gggcagggcg gggttcggct 2580
tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt 2640
tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat 2700
tcaccatggg gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc 2760
gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg 2820
acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg 2880
ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca 2940
cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt 3000
tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc 3060
ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg 3120
gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg 3180
ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg 3240
tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg 3300
gtgccggatc catgcccacg ctactgcggg tttatataga cggtcctcac gggatgggga 3360
aaaccaccac cacgcaactg ctggtggccc tgggttcgcg cgacgatatc gtctacgtac 3420
ccgagccgat gacttactgg caggtgctgg gggcttccga gacaatcgcg aacatctaca 3480
ccacacaaca ccgcctcgac cagggtgaga tatcggccgg ggacgcggcg gtggtaatga 3540
caagcgccca gataacaatg ggcatgcctt atgccgtgac cgacgccgtt ctggctcctc 3600
atatcggggg ggaggctggg agctcacatg ccccgccccc ggccctcacc ctcatcttcg 3660
accgccatcc catcgccgcc ctcctgtgct acccggccgc gcgatacctt atgggcagca 3720
tgacccccca ggccgtgctg gcgttcgtgg ccctcatccc gccgaccttg cccggcacaa 3780
acatcgtgtt gggggccctt ccggaggaca gacacatcga ccgcctggcc aaacgccagc 3840
gccccggcga gcggcttgac ctggctatgc tggccgcgat tcgccgcgtt tacgggctgc 3900
ttgccaatac ggtgcggtat ctgcagggcg gcgggtcgtg gcgggaggat tggggacagc 3960
tttcggggac ggccgtgccg ccccagggtg ccgagcccca gagcaacgcg ggcccacgac 4020
cccatatcgg ggacacgtta tttaccctgt ttcgggcccc cgagttgctg gcccccaacg 4080
gcgacctgta caacgtgttt gcctgggcct tggacgtctt ggccaaacgc ctccgtccca 4140
tgcacgtctt tatcctggat tacgaccaat cgcccgccgg ctgccgggac gccctgctgc 4200
aacttacctc cgggatggtc cagacccacg tcaccacccc cggctccata ccgacgatct 4260
gcgacctggc gcgcacgttt gcccgggaga tgggggaggc taactgagct ctagagctcg 4320
ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt 4380
gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat 4440
tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag 4500
caagggggag gattgggaag acaatagcag gcatgctggg gaactagtta aaagttttgt 4560
tactttatag aagaaatttt gagtttttgt ttttttttaa taaataaata aacataaata 4620
aattgtttgt tgaatttatt attagtatgt aagtgtaaat ataataaaac ttaatatcta 4680
ttcaaattaa taaataaacc tcgatataca gaccgataaa acacatgcgt caattttacg 4740
catgattatc tttaacgtac gtcacaatat gattatcttt ctagggttaa gtcagtcagc 4800
ttttataccc ggaaggatgg gcagctgtac gtatgagttt tgttttatta ttctcaaagc 4860
cagtgtggct tttctttaca gcatgtcatc atcaccttga aggcctctgc attgaagggg 4920
catgacttag ctggagagcc catcctctgt gatggtcagg agcagttgag agagcgaggg 4980
gttattactt catgttttaa gtggagaaaa ggaacactgc agaagtatgt ttcctgtatg 5040
gtattactgg atagggctga agttatgctg aattgaacac ataaattctt ttccacctca 5100
gggccattgg gcgcccattg ctcttctgcc tagaatattc tttccttttc taactttggt 5160
ggattaaatt cctgtcatcc ccctcctctt ggtgttatat ataaagtttt ggtgccgcaa 5220
aagaagtagc actcgaatat aaaattttcc ttttaattct cagcaaggca agttacttct 5280
atatagaagg gtgcaccctt acagatggaa caatggc 5317
<210> 8
<211> 5740
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
gaaatcacag aaagctgaat ttgaaaaagg tgcttggagc tgcagccagt aaacaagttt 60
tcatgcaggt gtcagtattt aaggtacatc tcaaaggata agtacaattg tgtatgttgg 120
gatgaacaga gagaatggag caagccaaga cccaggtaaa agagaggacc tgaatgcctt 180
cagtgaacaa tgatagataa tctagacttt taaactgcat acttcctgta cattgttttt 240
tcttgcttca ggtttttaga actcatagtg acgggtctgt tgttaatccc aggtctaacc 300
gttaccttga ttctgctgag aatctgattt actgaaaatg tttttcttgt gcttatagaa 360
tgacaataga gaacggcagg agcacaacga cagacggagc cttggccacc ctgagccatt 420
atctaatgga cgaccccagg gtaactcccg gcaggtggtg gagcaagatg aggaagaaga 480
tgaggagctg acattgaaat atggcgcaaa gcatgtgact agtctctttg tccctgtgac 540
tctctgcatg gtggtggtcg tggctaccat taaccctaga aagatagtct gcgtaaaatt 600
gacgcatgca ttcttgaaat attgctctct ctttctaaat agcgcgaatc cgtcgctgtg 660
catttaggac atctcagtcg ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta 720
agtgtcactg attttgaact ataacgaccg cgtgagtcaa aatgacgcat gattatcttt 780
tacgtgactt ttaagattta actcatacga taattatatt gttatttcat gttctactta 840
cgtgataact tattatatat atattttctt gttatagata tcaactagaa tgctagcatg 900
ggcccatctc gacctacttc gtatagcata cattatacga agttatgtgc gggcctcttc 960
gctattacgc cagcgcgcgt tgacattgat tattgactag ttattaatag taatcaatta 1020
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 1080
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 1140
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 1200
ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 1260
atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 1320
cttggcagta catctacgta ttagtcatcg ctattaccat gggtcgaggt gagccccacg 1380
ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt 1440
ttttaattat tttgtgcagc gatgggggcg gggggggggg gggcgcgcgc caggcggggc 1500
ggggcggggc gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag 1560
cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa 1620
gcgaagcgcg cggcgggcgg gagtcgctgc gttgccttcg ccccgtgccc cgctccgcgc 1680
cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg 1740
ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc tcgtttcttt 1800
tctgtggctg cgtgaaagcc ttaaagggct ccgggagggc cctttgtgcg ggggggagcg 1860
gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc ccgcgctgcc 1920
cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgcg tgtgcgcgag 1980
gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg ctgcgagggg aacaaaggct 2040
gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcggcg gtcgggctgt 2100
aacccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg 2160
ctccgtgcgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg 2220
gtgccgggcg gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc 2280
cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat 2340
cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg gcggagccga aatctgggag 2400
gcgccgccgc accccctcta gcgggcgcgg gcgaagcggt gcggcgccgg caggaaggaa 2460
atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctcca tctccagcct 2520
cggggctgcc gcagggggac ggctgccttc gggggggacg gggcagggcg gggttcggct 2580
tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt 2640
tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat 2700
tcaccatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt 2760
tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct 2820
tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca 2880
aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg 2940
acattgggga gttcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca 3000
cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca 3060
tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc 3120
aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg 3180
tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg 3240
atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt 3300
tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg 3360
aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt 3420
tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat 3480
cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg 3540
ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat 3600
ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg 3660
atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg 3720
caaaggaagg atccatgccc acgctactgc gggtttatat agacggtcct cacgggatgg 3780
ggaaaaccac caccacgcaa ctgctggtgg ccctgggttc gcgcgacgat atcgtctacg 3840
tacccgagcc gatgacttac tggcaggtgc tgggggcttc cgagacaatc gcgaacatct 3900
acaccacaca acaccgcctc gaccagggtg agatatcggc cggggacgcg gcggtggtaa 3960
tgacaagcgc ccagataaca atgggcatgc cttatgccgt gaccgacgcc gttctggctc 4020
ctcatatcgg gggggaggct gggagctcac atgccccgcc cccggccctc accctcatct 4080
tcgaccgcca tcccatcgcc gccctcctgt gctacccggc cgcgcgatac cttatgggca 4140
gcatgacccc ccaggccgtg ctggcgttcg tggccctcat cccgccgacc ttgcccggca 4200
caaacatcgt gttgggggcc cttccggagg acagacacat cgaccgcctg gccaaacgcc 4260
agcgccccgg cgagcggctt gacctggcta tgctggccgc gattcgccgc gtttacgggc 4320
tgcttgccaa tacggtgcgg tatctgcagg gcggcgggtc gtggcgggag gattggggac 4380
agctttcggg gacggccgtg ccgccccagg gtgccgagcc ccagagcaac gcgggcccac 4440
gaccccatat cggggacacg ttatttaccc tgtttcgggc ccccgagttg ctggccccca 4500
acggcgacct gtacaacgtg tttgcctggg ccttggacgt cttggccaaa cgcctccgtc 4560
ccatgcacgt ctttatcctg gattacgacc aatcgcccgc cggctgccgg gacgccctgc 4620
tgcaacttac ctccgggatg gtccagaccc acgtcaccac ccccggctcc ataccgacga 4680
tctgcgacct ggcgcgcacg tttgcccggg agatggggga ggctaactga gctctagagc 4740
tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc 4800
cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga 4860
aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga 4920
cagcaagggg gaggattggg aagacaatag caggcatgct ggggaactag ttaaaagttt 4980
tgttacttta tagaagaaat tttgagtttt tgtttttttt taataaataa ataaacataa 5040
ataaattgtt tgttgaattt attattagta tgtaagtgta aatataataa aacttaatat 5100
ctattcaaat taataaataa acctcgatat acagaccgat aaaacacatg cgtcaatttt 5160
acgcatgatt atctttaacg tacgtcacaa tatgattatc tttctagggt taagtcagtc 5220
agcttttata cccggaagga tgggcagctg tacgtatgag ttttgtttta ttattctcaa 5280
agccagtgtg gcttttcttt acagcatgtc atcatcacct tgaaggcctc tgcattgaag 5340
gggcatgact tagctggaga gcccatcctc tgtgatggtc aggagcagtt gagagagcga 5400
ggggttatta cttcatgttt taagtggaga aaaggaacac tgcagaagta tgtttcctgt 5460
atggtattac tggatagggc tgaagttatg ctgaattgaa cacataaatt cttttccacc 5520
tcagggccat tgggcgccca ttgctcttct gcctagaata ttctttcctt ttctaacttt 5580
ggtggattaa attcctgtca tccccctcct cttggtgtta tatataaagt tttggtgccg 5640
caaaagaagt agcactcgaa tataaaattt tccttttaat tctcagcaag gcaagttact 5700
tctatataga agggtgcacc cttacagatg gaacaatggc 5740
Claims (10)
1. a kind of method for the homozygous mutation for obtaining seamless modification, comprises the following steps：
(1) cutting is carried out to the site for needing to be mutated in cellular genome with gene editing system and produces double-strand break；
(2) two kinds of transposons containing purposeful mutant nucleotide sequence and different resistances are integrated into cell base by homologous recombination respectively
Because in a pair of alleles of group；
(3) screening allele is all integrated with the cell clone of transposons, then cuts off transposons；
(4) it is the cell clone for remaining transposons is lethal, obtain the cell clone of the homozygous mutation containing seamless modification.
2. the method as described in claim 1, it is characterised in that：Methods described is carried out in inductivity versatile stem cell iPS.
3. the method as described in claim 1, it is characterised in that：The gene editing system be CRISPR/Cas9, ZFN,
TALEN or CRISPR/Cpf1.
4. the method as described in claim 1, it is characterised in that：Step (2) includes：With contain the first resistance and purpose mutation sequence
The transposons of row is incorporated into genome by homologous recombination mode, then carries out the first resistance screening；With gene editing system
Carry out cutting again to above-mentioned mutational site and produce double-strand break；Led to the transposons containing the second resistance and purpose mutant nucleotide sequence
Cross homologous recombination mode to be incorporated into genome, then carry out the second resistance screening.
5. method as claimed in claim 4, it is characterised in that：Cell after first resistance screening and the second resistance screening is entered
Row amplification.
6. the method as described in claim 4 or 5, it is characterised in that：First resistance and the second resistance are selected from puro, hygR
Or two in NeoR.
7. the method as described in claim 1, it is characterised in that：The transposons is piggyBac transposon.
8. the method as described in claim 1, it is characterised in that：Transposons is cut off using transposase in step (3), step
(4) it is with FIAU that the cell clone for remaining transposons is lethal in.
9. the method as described in claim 1, it is characterised in that：Methods described is used to pinpoint reparation, rite-directed mutagenesis, gene knockout
Or gene knock-in.
10. the method as described in claim 1, it is characterised in that：Methods described is used to establish disease model.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711007052.1A CN107828824A (en) | 2017-10-24 | 2017-10-24 | The method for obtaining the homozygous mutation of seamless modification |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711007052.1A CN107828824A (en) | 2017-10-24 | 2017-10-24 | The method for obtaining the homozygous mutation of seamless modification |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107828824A true CN107828824A (en) | 2018-03-23 |
Family
ID=61648994
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711007052.1A Pending CN107828824A (en) | 2017-10-24 | 2017-10-24 | The method for obtaining the homozygous mutation of seamless modification |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107828824A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109971791A (en) * | 2019-04-17 | 2019-07-05 | 杭州荣泽基因科技有限公司 | A kind of candidate stem cell progress modified method of gene of patient poor over the ground |
| CN111961686A (en) * | 2020-08-28 | 2020-11-20 | 西北农林科技大学 | System for realizing biallelic precise genome editing by using CRISPR/Cas9 and PiggyBac |
| EP4085145A4 (en) * | 2019-12-30 | 2024-02-21 | The Broad Institute Inc. | Guided excision-transposition systems |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567734A (en) * | 2016-01-18 | 2016-05-11 | 丹弥优生物技术（湖北）有限公司 | Method for precisely editing genome DNA sequence |
| CN106520829A (en) * | 2016-10-17 | 2017-03-22 | 扬州大学 | Method for terminating biallelic gene transcription |
| US20170099813A1 (en) * | 2015-10-07 | 2017-04-13 | Recombinetics, Inc. | Method of generating sterile terminal sires in livestock and animals produced thereby |
-
2017
- 2017-10-24 CN CN201711007052.1A patent/CN107828824A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170099813A1 (en) * | 2015-10-07 | 2017-04-13 | Recombinetics, Inc. | Method of generating sterile terminal sires in livestock and animals produced thereby |
| CN105567734A (en) * | 2016-01-18 | 2016-05-11 | 丹弥优生物技术（湖北）有限公司 | Method for precisely editing genome DNA sequence |
| CN106520829A (en) * | 2016-10-17 | 2017-03-22 | 扬州大学 | Method for terminating biallelic gene transcription |
Non-Patent Citations (2)
| Title |
|---|
| GANG WANG等: "Efficient, footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies", 《NATURE PROTOCOLS》 * |
| RETO EGGENSCHWILER等: "Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase", 《SCIENTIFIC REPORTS》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109971791A (en) * | 2019-04-17 | 2019-07-05 | 杭州荣泽基因科技有限公司 | A kind of candidate stem cell progress modified method of gene of patient poor over the ground |
| EP4085145A4 (en) * | 2019-12-30 | 2024-02-21 | The Broad Institute Inc. | Guided excision-transposition systems |
| CN111961686A (en) * | 2020-08-28 | 2020-11-20 | 西北农林科技大学 | System for realizing biallelic precise genome editing by using CRISPR/Cas9 and PiggyBac |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108779466B (en) | Therapeutic targets and methods of use for correction of human dystrophin genes by gene editing | |
| JP2024023294A (en) | CPF1-related methods and compositions for gene editing | |
| US8598328B2 (en) | Tol1 factor transposase and DNA introduction system using the same | |
| JP2022518329A (en) | CRISPR-Cas12j Enzymes and Systems | |
| JP2019525756A (en) | Therapeutic application of genome editing based on CPF1 | |
| US10253332B2 (en) | Protein with recombinase activity for site-specific DNA-recombination | |
| JP2003528594A (en) | Modified recombinase for genome modification | |
| CA2361191A1 (en) | Gene repair involving the induction of double-stranded dna cleavage at a chromosomal target site | |
| CN107828824A (en) | The method for obtaining the homozygous mutation of seamless modification | |
| JP2011045380A (en) | Trap vector and method for gene trapping using the same | |
| CN110760511B (en) | gRNA, expression vector and CRISPR-Cas9 system for treating duchenne muscular dystrophy | |
| CN107868798A (en) | A kind of method for building up of the positive-selecting system based on Knockout cells | |
| JP4769796B2 (en) | Hybrid recombinase for genome manipulation | |
| US20070254291A1 (en) | Gene Targeting in Eukaryotic Cells by Group II Intron Ribonucleoprotein Particles | |
| US11274317B2 (en) | Targeted RNA knockdown and knockout by type III-A Csm complexes | |
| CA3156789A1 (en) | Genome editing in bacteroides | |
| JP6956995B2 (en) | Genome editing method | |
| KR20130108317A (en) | Use of a hspc117 molecule as rna ligase | |
| US20200080991A1 (en) | Screening for agents that target the actin cytoskeleton using c. elegans exposed to heat shock | |
| EP3918058A1 (en) | Controllable genome editing system | |
| JPWO2020122195A1 (en) | How to make genome-edited cells | |
| Ma et al. | PhiC31 integrase induces efficient site-specific recombination in the Capra hircus genome | |
| JP5773403B2 (en) | Method for producing genetically modified cells | |
| EP3652310B1 (en) | Gene editing system for correcting splicing defects | |
| US20030134270A1 (en) | Rapid method of selecting cells for gene disruption by homologous recombination |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180323 |