CN107794272A - A kind of CRISPR genome editor's systems of high specific - Google Patents
A kind of CRISPR genome editor's systems of high specific Download PDFInfo
- Publication number
- CN107794272A CN107794272A CN201610805040.2A CN201610805040A CN107794272A CN 107794272 A CN107794272 A CN 107794272A CN 201610805040 A CN201610805040 A CN 201610805040A CN 107794272 A CN107794272 A CN 107794272A
- Authority
- CN
- China
- Prior art keywords
- crispr
- editing system
- genome
- cas9
- grna
- 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.)
- Granted
Links
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
- 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
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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
- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
Abstract
The invention provides a kind of CRISPR genome editor's systems of high specific, by the gRNA that targeting Cas9 genes are introduced in CRISPR/Cas9 genome editing systems, while target cell genome editor is carried out (or afterwards), the gRNA of targeting Cas9 genes can mediate the cutting to Cas9 genes, so as to significantly reduce the expression time of Cas9 genes, then reduce due to effect of being missed the target caused by Cas9 long-term expressions in CRISPR systems, increase targeting specific.
Description
Technical field
The invention belongs to biological technical field, and specifically, the present invention relates to a kind of CRISPR genomes of high specific
Editor's system.
Background technology
Genome editing technique (Genome Editing), it is that one kind can carry out fixed point transformation to target gene group, from
And genes with unknown function is studied and the technology of gene therapy.The genome editing technique mesh of artificial endonucleases' mediation
Preceding application is most universal, and its advantage is that to break away from dependence and use range of the conventional art for embryonic stem cell wide, should in clinic
Use very big potentiality.The genome editing technique of artificial endonucleases' mediation mainly includes Zinc finger nuclease technology
(ZFNs), class activating transcription factor nucleic acid zymotechnic (TALENs) and CRISPR/Cas technologies.
ZFNs is the universal genetic group editing technique developed earliest, can be used to implement fixed point knockout and fixed point knocks in variation,
But the development of ZFNs technologies is limited to build the shortcomings of difficulty is big, cost is high.TALENs technologies develop on the basis of ZFNs,
For ZFNs technologies, TALENs technologies possess the advantages such as structure flexibility ratio is high, cost is low.Different from ZFNs and TALENs skills
Art, CRISPR/Cas technologies have unique DNA target to mechanism, and use is more convenient and flexible, therefore based on developing into
The genome edit tool wanted.At present, CRISPR/Cas systems are successfully tested in a variety of species, for example, mouse, zebra fish,
Drosophila, nematode and silkworm.
But effect of being missed the target present in genome editor hampers answering for genome editing technique wider scope always
With, therefore, this area genome editing technique stronger there is an urgent need to develop targeting specific.
The content of the invention
It is an object of the invention to provide a kind of genome editor system of high specific and its application.
The first aspect of the present invention, there is provided a kind of CRISPR genome editing systems of high specific, wherein, it is described
CRISPR genome editing systems include：First gRNA, the 2nd gRNA and Cas9 albumen；Wherein, the first gRNA targetings are treated
The genome of the target cell of editor, the 2nd gRNA target the encoding gene of the Cas9 albumen.
In another preference, the 2nd gRNA targets the gene order being selected from the group：SEQ ID NO.3 and SEQ
ID NO.4。
In another preference, CRISPR genomes editing system includes the DNA constructions shown in lower formula (I)：
P1-R1-P2-R2-P3-C, (I)
In formula (I), R1 is the first gRNA encoding genes, R2 is the 2nd gRNA encoding genes, C is Cas9 encoding histone bases
Cause, P1, P2, P3 are optionally promoter sequence, and "-" represents catenation sequence optionally.
The second aspect of the present invention, there is provided a kind of DNA constructions for CRISPR genome editors, the DNA structures
Building thing has structure shown in lower formula (I)：
P1-R1-P2-R2-P3-C, (I)
In formula (I), R1 is the first gRNA encoding genes, R2 is the 2nd gRNA encoding genes, C is Cas9 encoding histone bases
Cause, P1, P2, P3 are optionally promoter sequence, and "-" represents catenation sequence optionally.
In another preference, the 2nd gRNA targets the gene order being selected from the group：SEQ ID NO.3 and SEQ
ID NO.4。
The third aspect of the present invention, there is provided a kind of expression vector, the expression vector expression first aspect present invention institute
The CRISPR genome editing systems stated；Or the expression vector contains the DNA constructions described in second aspect of the present invention.
The fourth aspect of the present invention, there is provided a kind of genetically engineered host cell, it contains third aspect present invention
The DNA constructions described in second aspect of the present invention are integrated with described expression vector, or its genome.
In another preference, the host cell is eukaryotic, preferably zooblast.
The fifth aspect of the present invention, there is provided a kind of genome edit methods, methods described include step：
(1) the first genome editing system is provided, and base is carried out to target gene using the first genome editing system
Because of a group editor；
(2) after the genome editor occurs, the activity of the first genome editing system is reduced.
In another preference, in the step (2), there is provided the second genome editing system, second genome are compiled
Target the encoding gene of the first genome editing system with collecting systemic characteristic；Pass through the second genome editing system
Gene editing is carried out to the encoding gene of the first genome editing system, the first genome editor is reduced so as to realize
The activity of system.
In another preference, the genome is compiled as the genome editor of cell interior；It is preferably endonuclear
Genome editor.
In another preference, the first genome editing system is the genome editor of artificial endonucleases' mediation
System；Further, the second genome editing system specifically targets the artificial of the first genome editing system
The encoding gene of endonuclease.
In another preference, the cell is selected from：Zooblast, plant cell and microbial cell.
In another preference, the first genome editing system and/or described second select genome editing system from
The following group：CRISPR-Cas genomes editing system, NgAgo-gDNA genomes editing system, Zinc finger nuclease genome editor system
System (ZFNs), class activating transcription factor nuclease gene group editing system (TALENs).
In another preference, the CRISPR-Cas genomes editing system is for CRISPR-Cas9 genomes editor
System.
In another preference, the first genome editing system is CRISPR-Cas9 genome editing systems；It is described
In step (2), there is provided the gRNA of the Cas9 genes of the CRISPR-Cas9 genomes editing system is targetted, in the CRISPR-
In the presence of the gRNA of the Cas9 albumen of Cas9 genome editing systems and the targeting Cas9 genes, the CRISPR-Cas9
The Cas9 genes of genome editing system are cut, so as to reduce the activity of the genome editing system.
In another preference, the genome editing system is the high specific described in first aspect present invention
CRISPR genome editing systems.
The sixth aspect of the present invention, there is provided CRISPR genomes editing system as described in the first aspect of the invention, sheet
The one or the four aspect institute of expression vector or the present invention described in DNA constructions, third aspect present invention described in invention second aspect
Purposes of the host cell stated in medicine or genome editor's reagent is prepared.
In another preference, the medicine is used to carry out genome editor to the genome in target cell.
The seventh aspect of the present invention, there is provided CRISPR genomes editing system as described in the first aspect of the invention, sheet
Described in expression vector described in DNA constructions, third aspect present invention or fourth aspect present invention described in invention second aspect
Host cell in transgenic animals are built purposes.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, so as to form new or preferable technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description of the drawings
Fig. 1 shows self-contr ol type CRISPR genome editor's system action principles, is compiled in common CRISPR genomes
In the system of collecting (left side), a guide RNA (guide RNA, gRNA) for target gene is opened under people's U6 transcripting promoters
Dynamic expression.Cas9 genes are activated expression under EFS promoters, after translate into Cas9 albumen.GRNA and Cas9 albumen is made simultaneously
With being cut to target gene；In self-contr ol type CRISPR genome editor systems (self-restricted CRISPR
System, hereinafter simply referred to as SeT CRISPR systems) in (right side), a gRNA for target gene is in people's U6 transcripting promoters
Under be activated expression；Another gRNA for CRISPR systems Cas9 genes itself quilts simultaneously under mouse U6 transcripting promoters
Start expression.Cas9 genes are activated expression under EFS promoters, after translate into Cas9 albumen.Two gRNA are respectively and Cas9
Albumen acts on, while cuts Cas9 sequences in targeting cellular genome and CRISPR systems, significantly reduces Cas9 genes in system
Expression time, then reduce due to effect of being missed the target caused by Cas9 long-term expressions in CRISPR systems, increase targeting specific.
Fig. 2 left figures show T7E1 testing results, and display guide-2 and guide-3 has notable cleavage activity；Right figure is
Immunoblot results, after being shown in slow-virus infection 96h, Cas9-gRNA-2 or Cas9-gRNA-3 experiment is expressed at the same time
The expression of Cas9 albumen has notable downward compared to control group (only expression lentiCRISPR v2 carriers) in group.
Fig. 3 left figures show the T7E1 testing results for cell P53 gene targets site, show that common CRISPR is sick slowly
Malicious system (being labeled as " P53gRNA ") and SeT CRISPR slow virus system (being labeled as " P53gRNA+Cas9gRNA ") have phase
As cleavage activity；Right figure shows the T7E1 testing results for Cas9 gene targets site in viral system, is shown in SeT
Cas9 is cut simultaneously in CRISPR slow virus systems.
Fig. 4 immunoblot results are shown in SeT CRISPR systems, and Cas9 protein expression levels compare common CRISPR
System has notable downward, and there is notable shortening expression time.
Fig. 5 is shown in common lentiCRISPR systems, there is one between target gene group sequence and gRNA sequences
In the case of unpaired bases, when being expressed in multiple gRNA (unpaired bases of carrying are in diverse location) within 4 days and 20 days
Cleavage activity (efficiency of missing the target) can be detected, especially at 20 days, because Cas9 expression times are longer, misses the target efficiency more
Substantially.And in SeTlentiCRISPR systems, cleavage activity caused by most of gRNA expression compares common lentiCRISPR
System significantly reduces.
Embodiment
The present inventor and in-depth study, obtains a kind of CRISPR genome editor's systems of high specific by extensive,
By introducing the gRNA of targeting Cas9 genes in CRISPR/Cas9 genome editing systems, it is editing into target cell genome
While row (or afterwards), the gRNA of targeting Cas9 genes can mediate the cutting to Cas9 genes, so as to significantly reduce Cas9
The expression time of gene, then reduce because effect of being missed the target caused by Cas9 long-term expressions in CRISPR systems, increase targeting are special
The opposite sex.
Before describing the present invention, it should be understood that the invention is not restricted to described specific method and experiment condition, because this
Class method and condition can change.It should also be understood that its purpose of term used herein is only that description specific embodiment, and
And it is not intended to be restricted, the scope of the present invention will be limited only by the claims which follow.
Unless otherwise defined, otherwise whole technologies used herein are respectively provided with such as art of the present invention with scientific terminology
The identical meanings that are generally understood that of those of ordinary skill.As used herein, in use, term in the numerical value specifically enumerated is mentioned
" about " mean that the value can change from the value enumerated and be not more than 1%.For example, as used herein, " about 100 " include 99 Hes for statement
101 and between whole values (for example, 99.1,99.2,99.3,99.4 etc.).
Although it can be used and heretofore described similar or of equal value any method in the implementation or test of the present invention
And material, herein place enumerate preferable method and material.
CRISPR/Cas systems
The system is to have now been found that to be present in most of bacteriums and a kind of acquired immune system in all ancient bacterium, to disappear
Go out external plastid or bacteriophage, and leave alien gene fragment in autogene group and be used as " memory ".It is complete it is entitled often between
Palindrome repetitive sequence gathers together/often between palindrome repetitive sequence gather together related protein system (clustered regularly
interspaced short palindromic repeats/CRISPR-associated proteins)。
Have now been found that three kinds of different types of CRISPR/Cas systems, be present in about 40% and 90% be sequenced it is thin
In bacterium and ancient bacterium.The composition of wherein Second-Type is relatively simple, is forming for core with Cas9 albumen and guide RNA (gRNA),
Because it disturbs DNA the characteristic of (DNAi), it is actively used at present in genetic engineering, as genosome editing instrument,
Engaged (NHEJ) also with nonhomologous end with Zinc finger nuclease (ZFN) and class transcriptional activators nuclease (TALEN)
Mechanism, in genosome produce nuclifort bifilar fracture with sharp editing.Two type CRISPR/Cas and via heredity
The restoration and reuse of engineering is in the genosome editing of cells of mamma animals and zebra fish.It designs the simple and easy characteristic of operation
Biggest advantage.Future will can be applicable among a variety of model organisms.
Referred to as CRISPR genome repeats to gather together, i.e. repetitive sequence of growing thickly in prokaryotes nucleoid DNA, 1987
On being described first in E.coli a research report.2000, similar repetitive sequence was thin in other eubacterias and Gu
It is found in bacterium and is named as short interval repetitive sequence (Short Regularly Spaced Repeats, SRSR).2002
Year, SRSR was renamed into CRISPR.The albumen of a portion gene code is nuclease and unwindase.These related proteins
(CAS, CRISPR-associated proteins) and CRISPR constitute CRISPR/CAS systems.
CRISPR/Cas technologies
" CRISPR/Cas technologies ", " CRISPR/Cas genomes editor " alleged by the present invention, " CRISPR/Cas genomes
Editing technique ", " CRISPR/Cas genomes edit methods " refer both to enter target gene using the principle of CRISPR/Cas systems
The genome editing technique of row transformation.
Cas9 albumen
CRISPR/Cas core is exactly Cas9 albumen and guide RNA (gRNA).It can be utilized in different plant species
CRISPR/Cas systems carry out the core technology of genome editor, and a primary step i.e. heterogenous expression in the species has DNA shearings
The Cas9 albumen of enzymatic activity, second step are then to obtain gRNA and target spot homologous sequence to guide Cas9 to target spot progress DNA shearings.
Wherein in second step, specific operating method is well-known to those skilled in the art.
Cas9 albumen from Streptococcus pyogenes is a kind of multi-functional Cas albumen of Multidomain,
Its N-terminal has the domain of class RuvC nucleases, and its middle part has HNH nuclease domains.Cas9 albumen and gRNA combination energy
The cutting DNA at specific site is enough realized, from Streptococcus pyogenes CRISPR/Cas system identification sequences
23bp is classified as, and 20bp can be targetted, 3 NGG sequences of its recognition site most end are referred to as PAM (protospacer adjacent
Motif) sequence, it cuts extremely important for DNA.Most of eucaryotes (including silkworm, arabidopsis, yeast, nematode at present
Deng) CRISPR/Cas systems be initially both derived from Streptococcus pyogenes, Cas9 albumen is then by humanization
Transformation.
It is preferred that Cas9 provided by the present invention derives from streptococcus pyogenes (Streptococcus pyogenes).
One of the present invention is preferably carried out in mode, and the amino acid sequence of the Cas9 albumen is as follows：
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKN
RICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI
YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPG
EKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV
NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG
TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF
AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAF
LSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI
IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK
TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN
RLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL
SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAH
DAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL
IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS
AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSA
YNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
(SEQ ID NO.1)
Expressed in host for the ease of Cas9 genes, can also be in construction of expression vector, by the Cas9 gene structures of optimization
It build strong constitutive promoter in (such as：Pdc promoters (Li et al.Microbial Cell Factories 2012,11:
84), but not limited to this), and strong inducible promoter is (such as：Cbh1 promoters (Zou et al.Microbial Cel l
Factories2012,11:21), but not limited to this) downstream.
The DNA constructions of the present invention include cDNA, genomic DNA or artificial synthesized DNA.DNA can be it is single-stranded or
It is double-strand.DNA can be coding strand or noncoding strand.
The invention further relates to the variant of above-mentioned DNA constructions, and it is encoded has identical amino acid sequence with the present invention
The fragment of polypeptide or polypeptide, analogs and derivatives.The variant of this DNA construction can be the allelic variant naturally occurred
Or the variant that non-natural occurs.These nucleotide variants include substitution variants, Deletion variants and insert variation.Such as
Known in the art, allelic variant is the alternative forms of a polynucleotides, and it is probably taking for one or more nucleotides
Generation, missing or insertion, but not from substantially change its coding polypeptide function.
The nucleic acid fragment being related in DNA constructions of the present invention, it can generally use PCR TRAPs, recombination method or artificial synthesized
Method obtain.Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method.This is typically
Be cloned into carrier, then be transferred to cell, then by conventional method from the host cell after propagation isolated relevant sequence
Row.In addition, relevant sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.Generally, by first closing
Into multiple small fragments, the very long fragment of sequence can be obtained by being then attached again.
The present invention also relates to the carrier of the DNA constructions comprising the present invention, and the vector construction gene work with the present invention
Journey host cell.
The DNA constructions of the present invention can be plugged into recombinant expression carrier.It is ripe that term " recombinant expression carrier " refers to this area
The bacterial plasmid known, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus
Or other carriers.As long as it can be replicated in host and stably, any plasmid and carrier can be used.One weight of expression vector
It is characterized in usually containing replication orgin, promoter, marker gene and translation control element.
Method well-known to those having ordinary skill in the art can be used to build the DNA constructions of the present invention and suitably transcribe/turn over
Translate the expression vector of control signal.These methods include recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc..
Described DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to synthesize.Used in the present invention
The representative example of promoter have：Lac the or trp promoters of Escherichia coli；Bacteriophage lambda PL promoters；Eukaryotic promoter bag
Include CMV immediate early promoters, HSV thymidine kinase promoters, early and late SV40 promoters, retroviruse LTRs and
The promoter that some other known controllable gene is expressed in protokaryon or eukaryotic or its virus.Expression vector also includes
The ribosome bind site and transcription terminator of translation initiation.
In addition, expression vector preferably includes one or more selected markers, it is used to select conversion to provide
The phenotypic character of host cell, such as the dihyrofolate reductase of eukaryotic culture, neomycin resistance and green fluorescence egg
(GFP) in vain, or tetracycline or amicillin resistance for Escherichia coli.Comprising above-mentioned appropriate DNA sequence dna and suitably
The carrier of promoter or control sequence, it can be used for converting appropriate host cell, allow it to marking protein.
Host cell can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast cells；It is or high
Deng eukaryotic, such as mammalian cell.Representative example has：Escherichia coli, streptomyces；The bacterium of salmonella typhimurium
Cell；Fungal cell's such as yeast；Plant cell；Drosophila S2 or Sf9 insect cell；CHO, COS, 293 cells or Bowes are black
Zooblast of plain oncocyte etc..
When the DNA constructions of the present invention are expressed in higher eucaryotic cells, if insert enhancer sequence in the carrier
Transcription will be strengthened.Enhancer is DNA cis-acting factors, generally about has to 300 base-pairs, acts on and open
Mover is to strengthen the transcription of gene.Can illustrated example be included in 100 to 270 base-pairs of replication origin late period side
SV40 enhancers, in the polyoma enhancer of replication origin late period side and adenovirus cancers etc..
Persons skilled in the art are aware that how to select appropriate carrier, promoter, enhancer and host cell.
It can be carried out with recombinant DNA conversion host cell with routine techniques well known to those skilled in the art.When host is original
When core biology is such as Escherichia coli, can absorb DNA competent cell can harvest after exponential phase of growth, use CaCl2Method processing, institute
With the step of it is generally well-known in the art.Another method is to use MgCl2.If desired, conversion can also use the side of electroporation
Method is carried out.When host is eucaryote, following DNA transfection methods can be selected：Calcium phosphate precipitation, conventional mechanical methods are such as
Microinjection, electroporation, liposome packaging etc..
Main advantages of the present invention are：
(1) effect of missing the target can be significantly reduced using the gene editing system of the present invention, increases targeting specific；
(2) gene editing system of the invention is easy to use, without other gene expressions in addition to Cas9 expression systems,
Use without small molecule induction agent；
(3) it can completely retain the high of viral system and import efficiency and cell/tissue importing specificity.
With reference to specific embodiment, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted detailed conditions in the following example, generally according to conventional strip
Part such as U.S. Sambrook.J etc. writes《Molecular Cloning: A Laboratory room guide》(Huang Peitang etc. is translated, Beijing：Science Press, 2002)
Described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and number be by weight
Calculate.Experiment material and reagent used can obtain from commercially available channel unless otherwise instructed in following examples.
Material and method
1. experiment material
1) reagent
DMEM, hyclone, 0.25%Trypsin-EDTA are purchased from Thermo Fisher Scientific companies；Mould
Element-streptomysin (100X) solution is purchased from green skies company；T7 endonucleases I is purchased from NEB companies；Extracting genome DNA reagent
Box and the quick QIAquick Gel Extraction Kit of DNA product are purchased from Tiangeng company；ANTI-FLAG antibody is purchased from SIGMA companies
2) cell line
293T cells and Huh7 cells are purchased from Shanghai life science institute cell bank, add 10% hyclone and
Adhere-wall culture in the DMEM culture mediums of 1% penicillin-streptomysin (100X).
3) carrier
PlentiCRISRP v2 (Plasmid#52961) plasmids and slow virus packaging plasmid：pMDLg/pRRE
(Addgene#12251), pRSV-Rev (Addgene#12253), pMD2.G (Addgene#12259) are purchased from Addgene companies.
2. method
2.1 slow virus are packed
293T cells are transfected with polyethyleneimine (polyethylenime, PEI) method.Cell is in the day before transfection with appropriate
Density divides disk into 10cm culture dishes, and cell growth is changed culture medium after DMEM is carried out with the transfection of PEI methods into when about 80%.
PEI transfection slow virus packaging plasmids are pMDLg/pRRE 6.53ug, pRSV-Rev 2.53ug, pMD2.G 3.51ug, core matter
Grain lentiCRISRP v2 10ug.4-6 hours are changed to complete medium culture after transfection.48h and 96h collects supernatant and obtains disease
Venom.
2.2T7E1 detection
T7E1 detects a kind of method of detection CRISPR shear actives.T7E1 endonucleases can identify and cut gene
The site of incomplete pairing in group.Postgenome is extracted, the band with CRISPR target sites is amplified with PCR.Purifying recovery
95 DEG C are heated 5min and are slowly cooled to room temperature afterwards, make DNA fragmentation random incorporation.With 1ul T7EI endonucleases, 37 is Celsius
Degree cutting 500ng DNA fragmentation 60min.Digestion result is tested and analyzed with 2% agarose gel electrophoresis.By comparing cutting
Open with the electrophoretic band brightness do not cut open to judge CRISPR shear actives.
Embodiment 1 designs and screened the high activity gRNA for targeting Cas9
Experimental procedure：
1) by sequence alignment, high activity gRNA, the gRNA sequence for designing five targeting Cas9 sequences is shown in Table 1.
2) five gRNA are cloned into lentiCRISPR v2 carriers (addgene#52961) respectively
3) slow virus postoperative infection 293T cells are packed respectively
4) 48h and 96h receives cell respectively after infecting, and carrying out Cas9 genes to cell by T7E1 detection kits respectively cuts
The situation of cutting is analyzed；And immunoblotting reaction is analyzed Cas9 protein expression situations
Table 1 is for Cas9 design targeting gRNA sequences, and the primer sequence information for T7E1 detections
Experimental result is as shown in Fig. 2 left figure shows T7E1 testing results, and band does not have to cut in negative control group
Cut, obvious cutting, display guide-2 (target stripe 243bp occur for band in Cas9guide-2 and Cas9guide-3 groups
+ 423bp) and guide-3 (target stripe 298bp+368bp) there is notable cleavage activity；Right figure is immunoblot results, is shown
Show after slow-virus infection 96h, express Cas9 albumen in Cas9-gRNA-2 or Cas9-gRNA-3 experimental group at the same time
Expression has notable downward compared to control group (only expression lentiCRISPR v2 carriers).As a result Cas9guide-2 and Cas9 are shown
Guide-3 has higher gene editing activity.
Embodiment 2 builds SeT CRISPR slow virus carriers
Experimental procedure：
1) (synthesized in synthetic plasmid pmU6-gRNA by Jin Weizhi companies, its sequence is as shown in SEQ ID NO.11), wherein
Include the guide RNA of targeting target cell genome frame sequence and mouse U6 promoters, and in mouse U6 promoters 5 ' and
GRNA frame sequences 3 ' are with EcoRI limitation cleavage sites；
The plasmid pmU6-gRNA of synthesis sequence is following (SEQ ID NO.11)：
GAATTCAGATAGATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGA
AGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAA
AGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATAC
TAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAA
ATATCCCTTGGAGAAAAGCCCACCTTGTCTTCGAGAAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC
TAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTACTGAATTC
2) gRNA-2 for targetting Cas9 is cloned into pmU6-gRNA, obtains pm6-Cas9-gRNA
3) by EcoRI digestions, m6-Cas9-gRNA fragments are accessed into EcoRI digestions in plenti-CRISPRv2 carriers
At site (particular location is shown in Fig. 1)
Embodiment 3 verifies SeT CRISPR slow virus systems and the gene target efficiency in common CRISPR slow virus system
With the presence or absence of difference
Experimental procedure：
1) by taking the P53 genes for targetting people as an example, common CRISPR slow virus system (being labeled as " P53gRNA ") is built respectively
With SeT CRISPR slow virus system (being labeled as " P53gRNA+Cas9gRNA ")
2) slow virus postoperative infection human hepatoma cell strain Huh7 is packed respectively
3) 4 days and 8 days receipts cells, the cutting situation to P53 and Cas9 target sites respectively by T7E1 kits are infected
Detected
Table 2. is directed to the targeting gRNA sequences of P53 designs, and the primer sequence information for T7E1 detections
Experimental result is not as shown in figure 3, negative control lentiCRISPR v2 group bands are sent out in P53T7E1 testing results
Raw cutting, and all there occurs obvious cutting (target stripe 245bp+ for P53gRNA and P53gRNA+Cas9gRNA groups band
285bp).In Cas9T7E1 testing results, all without band cutting occurs as P53gRNA with lentiCRISPR v2, only
Having P53gRNA+Cas9gRNA groups band, there occurs cutting (target stripe 243bp+423bp).As a result the SeT of structure is shown
CRISPR slow virus carriers have an efficient gene editing ability as common CRISPR slow virus carriers, and SeTCRISPR
Slow virus carrier can successfully cut and be transferred to intracellular Cas9 genes simultaneously.
Embodiment 4. verifies whether the expression time of the Cas9 in SeT CRISPR systems significantly shortens
Experimental procedure：
1) by taking the P53 genes for targetting people as an example, common CRISPR slow virus system (being labeled as " P53gRNA ") is built respectively
With SeT CRISPR slow virus system (being labeled as " P53gRNA+Cas9gRNA ")
2) slow virus postoperative infection human hepatoma cell strain Huh7 is packed respectively
3) infect 2 days, 4 days, 6 days and 8 days and receive cell, the expression of Cas9 albumen in cell is detected by immunoblotting reaction
It is horizontal
Experimental result is as shown in Figure 4, the results showed that P53gRNA groups are as lentiCRISPR v2 groups, Cas9 eggs after 2 days
Significant change does not occur for white content.And just there occurs bright after 2 days for the Cas9 protein contents of P53gRNA+Cas9gRNA groups
It is aobvious to reduce.Illustrate that the expression time of the Cas9 in SeT CRISPR systems significantly shortens.
Embodiment 5. verifies whether miss rate is remarkably decreased in SeT CRISPR systems
1) the targeting sequence of P53 genes is directed to, designs a plurality of guideRNA, a non-matching core is introduced in different positions
Thuja acid, CRISPR systems cuts when a non-matching nucleotides be present with sequence on system research gRNA and target gene group sequence
Cut efficiency.Specific gRNA sequences are shown in Table lattice three.
2) slow virus postoperative infection human hepatoma cell strain Huh7 is packed respectively
3) cell is received after infecting 4 days and 20 days respectively, is carried out by T7E1 kits for P53 target sites in difference
In CRISPR systems with the cutting situation in the case of the different gRNA of expression
Table 3. carries the gRNA sequence informations of a non-matching nucleotides
Experimental result is as shown in figure 5, common CRISPR groups mispairing group from the 4th day is imitated it is seen that significantly editing
Rate, and the editorial efficiency to the 20th day mispairing group is then more obvious.And SeT CRISPR groups mispairing group from the 4th day can be observed
To certain editorial efficiency, the editorial efficiency to the 20th day mispairing group also has a slow rising, but more common CRISPR its mispairing
The editorial efficiency of group will be reduced substantially.As a result show that SeT CRISPR plasmids substantially reduce compared to common CRISPR miss rates.
All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (10)
- A kind of 1. CRISPR genome editing systems of high specific, it is characterised in that the CRISPR genomes editing system Including：First gRNA, the 2nd gRNA and Cas9 albumen；Wherein, the first gRNA targets the gene of target cell to be edited Group, the 2nd gRNA target the encoding gene of the Cas9 albumen；Preferably, CRISPR genomes editing system includes the DNA constructions shown in lower formula (I)：P1-R1-P2-R2-P3-C, (I)In formula (I), R1 is the first gRNA encoding genes, R2 is the 2nd gRNA encoding genes, C is Cas9 protein coding genes, P1, P2, P3 are optionally promoter sequence, and "-" represents catenation sequence optionally.
- 2. a kind of DNA constructions for CRISPR genome editors, the DNA constructions have the structure shown in lower formula (I)：P1-R1-P2-R2-P3-C, (I)In formula (I), R1 is the first gRNA encoding genes, R2 is the 2nd gRNA encoding genes, C is Cas9 protein coding genes, P1, P2, P3 are optionally promoter sequence, and "-" represents catenation sequence optionally.
- A kind of 3. expression vector, it is characterised in that the CRISPR genome editors described in the expression vector expression claim 1 System；Or the expression vector contains the DNA constructions described in claim 2.
- 4. a kind of genetically engineered host cell, it is characterised in that it contains the expression vector described in claim 3, or its The DNA constructions described in claim 2 are integrated with genome.
- 5. a kind of genome edit methods, it is characterised in that methods described includes step：(1) the first genome editing system is provided, and genome is carried out to target gene using the first genome editing system Editor；(2) after the genome editor occurs, the activity of the first genome editing system is reduced；Preferably, in the step (2), there is provided the second genome editing system, the second genome editing system specificity Ground targets the encoding gene of the first genome editing system；By the second genome editing system to first base Because the encoding gene of group editing system carries out gene editing, so as to realize the activity for reducing the first genome editing system.
- 6. method as claimed in claim 5, it is characterised in that the genome editing system mediates for artificial endonucleases Genome editing system；Preferably, the second genome editing system specifically targets the first genome editor The encoding gene of the artificial endonucleases of system.
- 7. method as claimed in claim 5, it is characterised in that the first genome editing system is CRISPR-Cas9 bases Because of a group editing system；In the step (2), there is provided target the Cas9 genes of the CRISPR-Cas9 genomes editing system GRNA, the CRISPR-Cas9 genomes editing system Cas9 albumen and the targeting Cas9 genes gRNA effect Under, the encoding gene of the Cas9 albumen of the CRISPR-Cas9 genomes editing system is cut, so as to reduce the genome The activity of editing system.
- 8. in another preference, the genome editing system is the CRISPR genes of the high specific described in claim 1 Group editing system.
- 9. DNA constructions, claim described in CRISPR genomes editing system as claimed in claim 1, claim 2 Purposes of the host cell described in expression vector or claim 4 in medicine or genome editor's reagent is prepared described in 3.
- 10. the DNA constructions, right described in CRISPR genomes editing system as claimed in claim 1, claim 2 will Seek the purposes in transgenic animals are built of the host cell described in expression vector or the claim 4 described in 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610805040.2A CN107794272B (en) | 2016-09-06 | 2016-09-06 | High-specificity CRISPR genome editing system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610805040.2A CN107794272B (en) | 2016-09-06 | 2016-09-06 | High-specificity CRISPR genome editing system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107794272A true CN107794272A (en) | 2018-03-13 |
| CN107794272B CN107794272B (en) | 2021-10-12 |
Family
ID=61530582
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610805040.2A Active CN107794272B (en) | 2016-09-06 | 2016-09-06 | High-specificity CRISPR genome editing system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107794272B (en) |
Cited By (26)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| CN110592141A (en) * | 2018-06-13 | 2019-12-20 | 中国科学院上海有机化学研究所 | Compound for regulating gene editing efficiency and application thereof |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| CN112266418A (en) * | 2019-07-08 | 2021-01-26 | 中国科学院微生物研究所 | Improved genome editing system and application thereof |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| CN114958920A (en) * | 2021-02-25 | 2022-08-30 | 北京中因科技有限公司 | Novel CRISPR-Cas9 system vector and preparation method and application thereof |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140357523A1 (en) * | 2013-05-29 | 2014-12-04 | Agilent Technologies, Inc. | Method for fragmenting genomic dna using cas9 |
| CN104450785A (en) * | 2014-12-08 | 2015-03-25 | 复旦大学 | Genome editing method using attachment carrier for encoding targeted endonuclease and kit |
| CN104513814A (en) * | 2014-11-18 | 2015-04-15 | 李云英 | I-TevIN201-Cas9null fusion protein and application thereof |
| WO2015070083A1 (en) * | 2013-11-07 | 2015-05-14 | Editas Medicine,Inc. | CRISPR-RELATED METHODS AND COMPOSITIONS WITH GOVERNING gRNAS |
| WO2016061481A1 (en) * | 2014-10-17 | 2016-04-21 | The Penn State Research Foundation | Methods and compositions for multiplex rna guided genome editing and other rna technologies |
| CN105647922A (en) * | 2016-01-11 | 2016-06-08 | 中国人民解放军疾病预防控制所 | Application of CRISPR-Cas9 system based on new gRNA (guide ribonucleic acid) sequence in preparing drugs for treating hepatitis B |
-
2016
- 2016-09-06 CN CN201610805040.2A patent/CN107794272B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140357523A1 (en) * | 2013-05-29 | 2014-12-04 | Agilent Technologies, Inc. | Method for fragmenting genomic dna using cas9 |
| WO2015070083A1 (en) * | 2013-11-07 | 2015-05-14 | Editas Medicine,Inc. | CRISPR-RELATED METHODS AND COMPOSITIONS WITH GOVERNING gRNAS |
| WO2016061481A1 (en) * | 2014-10-17 | 2016-04-21 | The Penn State Research Foundation | Methods and compositions for multiplex rna guided genome editing and other rna technologies |
| CN104513814A (en) * | 2014-11-18 | 2015-04-15 | 李云英 | I-TevIN201-Cas9null fusion protein and application thereof |
| CN104450785A (en) * | 2014-12-08 | 2015-03-25 | 复旦大学 | Genome editing method using attachment carrier for encoding targeted endonuclease and kit |
| CN105647922A (en) * | 2016-01-11 | 2016-06-08 | 中国人民解放军疾病预防控制所 | Application of CRISPR-Cas9 system based on new gRNA (guide ribonucleic acid) sequence in preparing drugs for treating hepatitis B |
Non-Patent Citations (5)
| Title |
|---|
| IAN M.SLAYMAKER 等: "Rationally engineered Cas9 nucleases with improved specificity", 《SCIENCE》 * |
| KEVIN M.DAVIS 等: "Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity", 《NAT CHEM BIOL.》 * |
| YANHAO CHEN 等: "A Self-restricted CRISPR System to Reduce Off-target Effects", 《MOLECULAR THERAPY》 * |
| 蒲强 等: "CRISPR/Cas9基因组编辑技术的研究进展及其应用", 《中国生物工程杂志》 * |
| 郑武等: "CRISPR/Cas9 的应用及脱靶效应研究进展", 《遗传》 * |
Cited By (40)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11702651B2 (en) | 2016-08-03 | 2023-07-18 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| CN110592141B (en) * | 2018-06-13 | 2023-07-07 | 中国科学院上海有机化学研究所 | Compounds for modulating gene editing efficiency and uses thereof |
| CN110592141A (en) * | 2018-06-13 | 2019-12-20 | 中国科学院上海有机化学研究所 | Compound for regulating gene editing efficiency and application thereof |
| US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| CN112266418A (en) * | 2019-07-08 | 2021-01-26 | 中国科学院微生物研究所 | Improved genome editing system and application thereof |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| CN114958920A (en) * | 2021-02-25 | 2022-08-30 | 北京中因科技有限公司 | Novel CRISPR-Cas9 system vector and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107794272B (en) | 2021-10-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107794272A (en) | A kind of CRISPR genome editor's systems of high specific | |
| Thurtle‐Schmidt et al. | Molecular biology at the cutting edge: A review on CRISPR/CAS9 gene editing for undergraduates | |
| US11802277B2 (en) | Thermostable Cas9 nucleases | |
| CN106566838B (en) | A kind of miR-126 full-length gene knockout kit and its application based on CRISPR-Cas9 technology | |
| CN110312793A (en) | Safety for enhancing from the restricted circuit Cas9 (SLiCES) plasmid and its slow virus system | |
| CN104745626B (en) | A kind of fast construction method of conditional gene knockout animal model and application | |
| US11788088B2 (en) | CRISPR/Cas system and method for genome editing and modulating transcription | |
| EP3222728A1 (en) | Method for regulating gene expression using cas9 protein expressed from two vectors | |
| Zeng et al. | Expansion of CRISPR targeting sites in Bombyx mori | |
| CN109072207A (en) | Improved method for modifying target nucleic acid | |
| CN106414740A (en) | Method for specific knockout of swine SLA-3 gene using CRISPR-Cas9 specificity, and sgRNA used for specifically targeting sla-3 gene | |
| CN105899658A (en) | Delivery, use and therapeutic applications of CRISPR-CAS systems and compositions for HBV and viral diseases and disorders | |
| JP2022520428A (en) | Enzyme with RUVC domain | |
| JP2015523860A (en) | Supercoil MiniVector as a tool for DNA repair, modification and replacement | |
| CN106282228A (en) | A kind of method that point mutation is repaired | |
| CN110484538A (en) | Identify sgRNA and its coding DNA, gene editing method, kit and the application of porcine ROSA 26 gene | |
| CN107365803A (en) | Exempt from the method that drug screening quickly obtains mouse Rosa26 Gene targeting foreign genes | |
| Palazzo et al. | Transposable elements: A jump toward the future of expression vectors | |
| CN106480081A (en) | A kind of CRISPRi carrier of single pUC pUC and preparation method thereof | |
| Wong et al. | Gene targeting and genome editing | |
| CN104232676B (en) | It is a kind of obtain minicircle dna parental plasmid and its application | |
| CN108559731A (en) | A kind of human embryonic stem cell line of tetracycline-regulated gene expression and its application | |
| CN105462969B (en) | Pig specificity close friend site Pifs102 and its application | |
| EP4227409A1 (en) | Technique for modifying target nucleotide sequence using crispr-type i-d system | |
| CN109689693A (en) | Improve the method and system of gene editing efficiency |
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 | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 200031 Yueyang Road, Shanghai, No. 319, No. Applicant after: Shanghai Institute of nutrition and health, Chinese Academy of Sciences Address before: 200031 Yueyang Road, Shanghai, No. 319, No. Applicant before: SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCES |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |