CN103388006B - A kind of construction process of site-directed point mutation - Google Patents
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Abstract
The present invention discloses a kind of construction process of site-directed point mutation, to determine in rat target gene group sequence can the target sequence that identifies by artificial reconstructed CRISPR-Cas system; Can structure identify and guide CAS albumen to the guiding RNA sequence (guide of target gene target sequence? RNA, gRNA); The CAS egg white mixture of above-mentioned gRNA sequence and CAS protein-encoding nucleotide sequence or gRNA sequence and vivoexpression is imported rat embryo cell.The present invention without the need to building homologous recombination targeting vector, without the need to carrying out ES target practice cell screening, mutant ratio is high, easy and simple to handle, can realize multidigit point and knock out simultaneously, can significantly reduce experimental cost and shorten experimental period.<pb pnum="1" />
Description
Technical field
The present invention relates to gene engineering technology field, be specifically related to a kind of construction process of site-directed point mutation in rat embryo cell.
Background technology
Rat is at physiology, and pharmacology, toxicology, trophology, study of behaviour, immunology and oncology have the history more than more than 150 years as animal model.The advantages such as similar with mouse, rat has small, and the generation-inter-time is short, and a tire is given birth to more, and feeding cost is low.Meanwhile, rat compares mouse larger build, is convenient to carry out observing and operation technique; The recipe of rat and the mankind is comparatively close, is convenient to the research carrying out trophology aspect; Rat is comparatively responsive to toxicant, is suitable for drug toxicology experiment.Each medicine all needs to adopt rat to carry out toxicological experiment before listing at present.Due to inherent genetic cause, large mouse disease model can reproduce the symptom of some human diseasess more really.Rat to environment have stronger probe into for and the nervous activity of comparatively sophisticated, be suitable for nerve, cognitive and praxiology research.In the animal model of the human diseases of including at PubMed, rat is adopted to occupy first as the quantity of model.
But, along with 1981, mouse embryo stem cell vitro culture dryness maintained and in mouse extracorporeal culturing embryo stem cell, carries out the realization of the gene targeting based on homologous recombination, make to carry out personalized pointed decoration to murine genes and become routine operation, scientific circles transfer the function of more employing mice study homology Human genome and build the model of human diseases.Until within 2008, just there is the separation and ientification of Embryonic Stem Cell truly, and carry out the integration of mosaic rat reproductive tract and the reported success gone down to posterity, although there is people successfully to carry out homologous recombination gene targeting afterwards in Embryonic Stem Cell, but because the complexity of its culture condition is with harsh, and the impact that resistance screening maintains dryness, in Embryonic Stem Cell, carry out gene targeting still there is difficulty at present.With similar in mouse, the gene targeting carried out in Embryonic Stem Cell based on homologous recombination must be limited by following limiting factor: 1, the targeting vector building process cycle is long and difficulty is large.2, because homologous recombination incidence spontaneous in zooblast only has 10
-5-10
-6, after targeting vector electricity is proceeded to Embryonic Stem Cell, spend plenty of time and manpower and materials screening that the embryonic stem cell of object homologous recombination occurs.3, ES cell requires in culturing process harsh in vitro to culture condition, careless slightlyly will cause its irreversible differentiation, and makes it lose reproductive tract transfer ability, can not produce muton generation in subsequent operations.4, the ES cell of microinjection only has certain probability Successful integration to become the germ cell that can form gamete.5, require that ES cell is in good growth and undifferentiated state.If the ES cell that 6 existing ready-made goal gene are targeted, but different from the rat strains for studying, then will require a great deal of time and backcross, to dilute the original genetic background of ES cell.
Artificial Zinc finger nuclease (Zinc Finger Nucleases, the ZFN) technology that development in recent years is got up to some extent solves the problems referred to above.ZFN is a kind of recombinant protein be made up of with the FokI endonuclease cutting structure territory with nonspecific action the zinc finger protein of target specific dna sequence.A pair engineer's, identify that ZFN and the target dna sequence of specific DNA sequence dna (are generally adjacent two sections of each 18-24bp, interval 4-7bp) combine after, their FokI endonuclease will form dimer, thus cutting DNA double-strand, form double-strand break (double strand break, DSB).This damage is repaired mainly through the non-homologous end joining (Non-homologous end joining, NHEJ) of fallibility and the homologous recombination (homologous recombination, HR) of hi-fi.Wherein non-homologous end joining is by causing the frameshit of goal gene, can be used to carry out gene knockout to goal gene.By DNA or the corresponding mRNA of the ZFN that encodes in the microinjection of zygote stage, realize rat, mouse, the gene knockout of zebra fish isotype biology.
Although ZFN technology has wide prospect in fundamental research and Application Areas, but build as required and specific dna sequence had to the ZFN workload of high degree of specificity and avidity is large, the cycle is long, success ratio is low, become the bottleneck hindering this ZFN large-scale application.The DNA recognition structure territory of ZFN is made up of several zinc finger print blocks, and each zinc refers to specific 3 the continuous bases of Module recognition, continuous print 9 ~ 18bp base on 3 ~ 6 therefore adjacent zinc finger print block identifiable design DNA.This three disjunctor recognition modes mean can select handiness less for the DNA sequence dna of target.Although existing hundreds of zinc finger print block, does not contain all possible base permutation and combination.Meanwhile, the interaction due to adjacent zinc finger print interblock can affect the specificity (context dependence) of its base identification, the specific binding that ZFN could will realize goal gene through a large amount of optimization in building process.In addition, the DNA binding specificity that ZFN is relatively low, makes ZFN can cause higher cytotoxicity because of effect of missing the target (Off Target Effect) at intracellular process LAN.
Class transcription activator nuclease (Transcription Activator-Like Effector Nuclease, TALEN) is the another kind of genome fixed point editing technique occurred after ZFN.Similar with ZFN, the TAL effectors of target specific dna sequence and FokI nuclease are formed fusion rotein by this technology.A pair engineer's, identify that TALEN and the target dna sequence of specific DNA sequence dna (are generally adjacent two sections of each 15-20bp, interval 15-20bp) combine after, their FokI endonuclease will form dimer, thus cutting DNA double-strand, form double-strand break (double strand break, DSB).This damage is repaired mainly through the non-homologous end joining (Non-homologous end joining, NHEJ) of fallibility and the homologous recombination (homologous recombination, HR) of hi-fi.Wherein non-homologous end joining is by causing the frameshit of goal gene, can be used to carry out gene knockout to goal gene.By DNA or the corresponding mRNA of the ZFN that encodes in the microinjection of zygote stage, realize rat, rat, the gene knockout of zebra fish isotype biology.
Be made up of the N-end of connect " module " and the both sides thereof of 12 or more specific recognition DNA and C-end sequence unlike TALE (TAL effectors) with ZFN.The DNA identification module of TAL effectors is made up of 34 amino acid, the specificity that the DNA identification module of each TAL effectors is combined with DNA is determined by the 12nd and the 13rd amino acids residue, is referred to as and repeats variable di-residues (RVDs) site.RVDs and A, G, C, T tetra-kinds of bases have constant corresponding relation, and namely NI identifies that A, NG identify that T, HD identify that C, NN identify G.Therefore, TAL effectors identified and in conjunction with a certain specific nucleic acid sequence, the DNA identification module of TAL effectors need only be cloned according to target dna sequence series connection.In actually operating, generally in target gene, select the target sequence (general tens bases) at two places adjacent (15 ~ 20, interval base) to carry out the structure of TAL effectors identification module respectively.
Compare ZFN, DNA identification module and the single base of TALEN have clear and definite one-to-one relationship, the DNA that the permutation and combination method of module does not affect each other identifies and binding ability, thus successfully solve conventional ZFN method and can not identify arbitrary target DNA sequence dna, and evident characteristics is often by the problem that upstream and downstream sequence affects, to make to eliminate in the building process of the series connection DNA identification module of TALEN in ZFN building process the corresponding complicated and optimal screening process of costliness, be convenient to the TALEN that Routine Test Lab oneself builds target order gene.
But in the building process of TALEN, needing the size small pieces segment DNA that the height of about 20 repeats being assembled into about 2kb, its process is still comparatively time-consuming and loaded down with trivial details, and has certain labile factor to exist.In addition because TALEN requires that target spot 5 ' holds close sites, and target spot itself 3 ' holds base to be necessary for T, add the requirement (15-20bp) of TALEN target spot itself and interval region (Spacer) suitable length, actually operating middle ideal target spot to there is frequency lower, limit the handiness that target site is selected to a certain extent.
In nearest bacterium and archeobacteria, a kind of acquired immunity mechanism being used for resisting the invasion of the exogenous dna fragment such as phage and plasmid obtains explaination.This system is by Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genomic constitution.The immune interference process of CRISPR system mainly comprises 3 stages: adapt to, express and interference.In the laundering period, the DNA short-movie section from phage or plasmid can be incorporated between leader sequence and first paragraph tumor-necrosis factor glycoproteins by CRISPR system, integrates copying all along with tumor-necrosis factor glycoproteins each time, and then repetition-intervening sequence unit that formation one is new.At expression phase, CRISPR locus can be transcribed into one section of CRISPR RNA (crRNA) precursor (pre-crRNA), and this precursor can be further processed into little crRNA at tumor-necrosis factor glycoproteins place under the existence of Cas albumen and tracrRNA.Ripe crRNA and Cas albumen forms Cas/crRNA complex body.In the interference stage, crRNA finds target spot by the regional guidance Cas/crRNA complex body of itself and target complement sequence, and causes the double-strand DNA cleavage of target position at target position by the nuclease of Cas albumen, thus makes target DNA lose original function.Wherein hold with target spot 3 ' 3 bases be close to must be the form of 5 '-NGG-3 ', thus form PAM (the protospacer adjacent motif) structure needed for Cas/crRNA complex body identification target spot.
Clustered regularly interspaced short palindromic repeats (CRISPR) system is divided into I, II, type III three families, wherein II type system only needs Cas9 albumen pre-crRNA can be processed into the ripe crRNA be combined with tracrRNA under the assistance of trans coding tiny RNA (trans-encodedsmall RNA, tracrRNA).Thus obtain the concern that researcher is more, and obtain and study the most fully.It is found that single-chain chimeric body guiding RNA (guide RNA) by artificial constructed simulation crRNA:tracrRNA complex body, effectively can mediate Cas9 albumen to the identification of target spot and cutting, thus provide wide prospect for utilizing CRISPR system to modify target dna in target species.
Summary of the invention
The invention provides a kind of construction process of site-directed point mutation, comprise the following steps:
(1) in rat target gene group sequence, determine the target sequence of CRISPR-Cas system institute target;
(2) design, structure can identify and guide CAS albumen to the gRNA nucleotide sequence of target gene target sequence;
(3) by the nucleotide sequence of described gRNA nucleotide sequence and coding CAS albumen or protein, import in described rat embryo cell.
Particularly, in above-mentioned steps (1), obtain target dna sequence from NCBI, the range intervals of selected target sequence; In this interval sequence meeting target condition of to search by " GGNNNNNNNNNNNNNNNNNNNGG " (front 20 bases are used for and the noncoding strand complementary pairing of target spot DNA) in non-template chain; Also " CCNNNNNNNNNNNNNNNNNNNCC " can be searched find potential target spot in the reverse complementary sequence of candidate's target area.
In above-mentioned steps (2), described gRNA nucleotide sequence can with described CAS protein-specific combination and by its target target sequence.That is, RNA in-vitro transcription template from 5 ' to 3 ' is guided to hold successively by T7 promoter sequence, target spot specific sequence, and gRNA skeleton part.RNA is guided accordingly according to the design of target sequence information.
In above-mentioned steps (3), the CAS protein nucleic acid sequence of described gRNA nucleotide sequence and described coding is transcribed into RNA and purifying in vitro respectively, or, mix with the CAS albumen of vivoexpression after described gRNA nucleotide sequence is transcribed into RNA in vitro.That is, gRNA and CAS protein-encoding nucleotide sequence is transcribed into RNA in vitro, both carry out microinjection after mixing by a certain percentage; Or gRNA is transcribed into RNA, and the CAS albumen with vivoexpression and after purifying mixes, by a certain percentage then for microinjection.
In the present invention, described CRISPR-Cas system refers to and is applicable to by engineered CRISPR-Cas system, the nuclease system coming from archeobacteria II type (CRISPR)-CRISPR-associated protein (Cas) system, as compared to ZFN with TALEN, this system is simpler, more convenient operation.
In the present invention, described rat target gene group sequence comprises the genome sequence of encoding gene, lowly repetitive sequence, moderately repetitive sequence, highly repetitive sequence, low copy sequence, intergenic transcriptionally active sequence or non-transcribed sequences.
In the present invention, described rat embryo cell comprises the unicellular zygote of rat or many cells rat embryo.Such as, the many cells rat embryo of the unicellular zygote of the rat of vitro culture or vitro culture.
In the present invention, described step (3) is that described guiding RNA is imported rat embryo cell by microinjection.Described guiding RNA can pass through synthetic, and in-vitro transcription contains the methods such as the plasmid of target sequence or PCR primer and obtains.
In the present invention, in described step (3), the DNA break site that cutting target gene group is formed is repaired by non-homologous end joining or passes through homologous recombination repair.
In the present invention, described target gene sudden change comprises base insertion, disappearance, change, phase shift mutation or knocks out.
The present invention overcomes prior art to build the cycle existed in knockout rat technology long, workload is large, difficulty is high, efficiency is low waits deficiency, eliminate the process of structure and the combination of optimal screening ZFN and TALEN series connection base identification module simultaneously, and the present invention can have the higher frequency of occurrences for target sequence relative to ZFN or TALEN, thus provides more more options for accurate genome editor.The construction process rapidly and efficiently of site-directed point mutation of the present invention, by guiding RNA (guide RNA) nucleotide sequence of rat zygote microinjection in-vitro transcription and CAS protein-encoding nucleotide sequence, realizes rapid build knockout rat.The nuclease that the inventive method utilizes RNA to guide carries out rapid gene editor, and quick target also changes rat genomic dna rat, realizes site-directed point mutation rapidly and efficiently.The present invention without the need to building homologous recombination targeting vector, without the need to carrying out ES target practice cell screening, mutant ratio is high, easy and simple to handle, can realize multidigit point and knock out simultaneously, can significantly reduce experimental cost and shorten experimental period.
In the present invention, rat embryo cell refers to the rat embryo cell of vitro culture.The rat embryo cell that the present invention obtains is used for vitro culture or direct implant carrier mouse, CAS albumen target cut target gene group under the guiding of gRNA, genome after cutting is repaired under non-homogeneous recombinational repair mechanism, can sudden change be introduced in the process, thus the rat of screening target gene sudden change.The rat embryo cell that the present invention obtains is used for vitro culture or direct implant carrier mouse, expresses and guides RNA and CAS albumen also to cut target gene group, thus the rat of screening target gene sudden change.Compared with traditional method, the inventive method eliminates external process of carrying out ES cell screening, whole operating process only needs directly to import in embryo by nucleic acid, do not need ES cell to introduce embryo, avoid because of when ES cell is cultivated in vitro because of culture condition undesirable time the irreversible differentiation that causes.As compared to ZFN with TALEN, the present invention eliminates the process of structure and the combination of optimal screening ZFN and TALEN series connection base identification module, and the present invention can have the higher frequency of occurrences for target sequence relative to ZFN or TALEN, thus provides more choices for accurate genome editor.The transgenation rat that the rat embryo cell utilizing the present invention to obtain obtains further can be used for the preclinical study etc. of life science fundamental research and new drug development.
Wherein, the rat of described target gene sudden change refers to by choosing rat that target gene undergos mutation and wild-type rats is hybridized, the brood heterozygote rat choosing above-mentioned hybridization gained again carries out selfing, from above-mentioned gained neonate rat, then screen the homozygote rat of target gene sudden change.
The present invention also provides a kind of rat embryo cell prepared by the construction process of site-directed point mutation of the present invention.
The present invention also provides a kind of homozygote rat, is the rat embryo cell that the construction process containing site-directed point mutation of the present invention prepares.That is, the rat embryo cell prepared by the inventive method is used for vitro culture or direct implant carrier mouse, express TALEN and also cut target gene group, screening obtains the rat of target gene sudden change; Again itself and wild-type rats are hybridized, the more brood heterozygote rat choosing above-mentioned hybridization gained carries out selfing, from above-mentioned gained neonate rat, then screen the homozygote rat of target gene sudden change.
The endonuclease (RNA-guided endonucleases, RGENs) that the present invention adopts RNA to guide realizes cutting the specificity of target gene sequence.RGENs is made up of the guiding RNA of mosaic type and Cas9 albumen, wherein the CRISPR RNAs (crRNAs) in naturally occurring II type CRISPR-Cas system and trans-activating crRNA (tracrRNA) are fused into a strand to guide RNA (gRNA) by the former, thus guide the latter to carry out specific cutting to target spot DNA sequence dna with Cas9 protein binding.Cutting will form double-strand break (double strand break, DSB), this damage is by non-homologous end joining (the Non-homologous end joining of fallibility, NHEJ) after repairing, by the frameshit having the probability of 2/3 to cause goal gene, thus realize knocking out goal gene.In the present invention, such as, in rat embryo, implement gene knockout method, comprise step as follows:
(1) in target rat gene N end encoding sequence, suitable target sequence is found.
(2) template DNA needed for in-vitro transcription gRNA is obtained according to above-mentioned target sequence by the method for overlapping primers PCR.
(3) above-mentioned template DNA in-vitro transcription is become RNA, and carry out purifying.
(4) plasmid linearization of coding Cas9 albumen is carried out in-vitro transcription simultaneously.
(5) above-mentioned two kinds of RNA are mixed also microinjection and enter the rat zygote of artificial insemination formation.
Further, after stating step on the invention, above-mentioned zygote is implanted in the female mouse uterine tube of false pregnancy; The genomic dna extracting above-mentioned steps gained Offspring rat carries out genotype identification; The rat of target gene generation phase shift mutation is hybridized with wild-type rats respectively; Heterozygote rat brood for above-mentioned hybridization gained is carried out selfing; Identify the genotype of above-mentioned selfing gained rat, the homozygote rat of screening target gene phase shift mutation.
Advantage of the present invention comprises: need in the gene Knockout of ZFN or TALEN mediation the enzyme of repetition cut the method such as connection build reach hundreds of or on the expression plasmid of kilobase, gRNA vector construction process of the present invention very letter time in contrast, directly can synthesize the complementary oligonucleotide containing target spot of two about 20-bp, after annealing, be connected into gRNA expression plasmid.Can in the selection of target sequence, ZFN generally needs to be optimized screening from multiple candidate targets, and it must be all base T that the target spot of TALEN is selected also to be subject to left and right target spot 0, and target spot last be the restriction of base T.And the restriction that CRISPR target spot is selected only comes from and hold with target spot 3 ' 3 bases be close to must be the form of 5 '-NGG-3 ', thus formation is by PAM (the protospacer adjacent motif) structure of Cas9 identification itself.Thus for carry out on genome more accurate and flexibly edit provide possibility.Through the optimization to microinjection RNA concentration used, the F0 that the present invention obtains can reach 78% for the mutation rate of rat, is significantly higher than the mutation rate adopting ZFN or TALEN to reach.By more than one gRNA of a shot, the present invention can realize once editing the multiple genes in rat zygote simultaneously.By the gRNA of shot target two adjacent target spots, the present invention can realize deleting the genomic dna compared with large fragment.With traditional compared with being knocked out target gene by homologous recombination in Embryonic Stem Cell or changing, the present invention avoids the cycle long and the targeting vector building process that difficulty is large.Secondly, by the guiding RNA nucleotide sequence of microinjection in-vitro transcription and CAS protein-encoding nucleotide sequence gained Offspring rat in the present invention, as described embodiments, the probability of a wherein allelotrope generation base deletion is up to 86%.And prior art and document show, by homologous recombination in Embryonic Stem Cell and follow-up screening, the probability occurring to fix a point to knock out is no more than 15-65%.The ES cell filtered out also needs through embryo's microinjection and produces mosaic rat.If mosaic rat generation reproductive tract transfer (germline transmission) just likely obtains the heterozygote rat that specific gene knocks out.And there is irreversible differentiation due to the change of ES cell culture condition in screening process, the daughter cell of the ES cytodifferentiation of probably suddenling change can not enter reproductive tract and can not get knocking out rat.By the embryo of injection one cell stage, there is not the problem of ES cytodifferentiation in the present invention, is therefore the rat strains that generally can obtain gene knockout by repeating to test.The present invention passes through realized high mutation efficiency, in the input of saving experimental period and manpower and materials, have very large meaning.And, this high mutation efficiency also by contribute to this technology lay eggs less, growth cycle is longer and application in the economic costly macrofauna of single animal.Again, the present invention directly to the guiding RNA nucleotide sequence of microinjection in-vitro transcription in the rat zygote of artificial insemination and CAS protein mRNA, the process that the cycle that avoids is long, labor capacity is large, object homologous recombination Embryonic Stem Cell occurs in screening.In addition, gRNA plasmid is built and the link of carrying out in-vitro transcription only needs less than 1 time-of-week in the present invention, only need about 1 month from microinjection to the heterozygote rat identifying target gene generation frameshit, and traditional cycle of passing through to carry out homologous recombination construction knockout rat in Embryonic Stem Cell needs at least year usually.The inventive method greatly shortens experimental period, is conducive to the efficient propelling studied.
The present invention by carrying out any genomic dna of target and making it change thus rapid build knockout rat method in rat embryo, identify by introducing direct in rat zygote and cut guiding RNA nucleotide sequence and the CAS protein-encoding nucleotide sequence of the microinjection in-vitro transcription of target gene, by DNA double splitting of chain (double strand break, DSB) non-homologous end joining (the Non-homologous end joining and thus caused, NHEJ) frameshit causing target gene is repaired, thus the site-directed point mutation realized in rat embryo cell rapidly and efficiently.Utilize the present invention, the F0 that further acquisition target gene knocks out is for heterozygote or homozygote rat.Instant invention overcomes by injecting artificial Zinc finger nuclease (Zinc Finger Nucleases to rat zygote, ZFN) arbitrary target DNA sequence dna can not be identified, and evident characteristics is often by the problem that upstream and downstream sequence affects, and the complicated and optimal screening process of costliness in the ZFN building process brought thus.Overcome splicing comparatively loaded down with trivial details in TALEN institute module assembled process, and TALEN target spot selects the problem of upper limited flexibility.With traditional by compared with knocking out target gene with homologous recombination targeting vector in Embryonic Stem Cell, the present invention has without the need to building homologous recombination targeting vector, without the need to carrying out ES target practice cell screening, mutant ratio is high, easy and simple to handle, experimental cost and cycle such as to greatly reduce at the advantage.
Accompanying drawing explanation
Fig. 1 is CRISPR/Cas systemic effect principle schematic.
Fig. 2 is Cas9mRNA and guiding rna expression carrier schematic diagram.
Fig. 3 is microinjection Cas9mRNA/MC4R-gRNA rat gained neonate rat corresponding PCR primer T7E1 restriction enzyme digestion and electrophoresis figure.
Fig. 4 is microinjection Cas9mRNA/MC4R-gRNA gained neonate rat base deletion situation schematic diagram.
Fig. 5 is given birth to F1 generation rat base deletion situation schematic diagram by No. 12 head build rat.
Fig. 6 is CAS nuclease prokaryotic expression carrier schematic diagram.
Fig. 7 is microinjection Cas9 albumen/MC4R-gRNA and Cas9 albumen/MC3R-gRNA gained neonate rat base deletion situation schematic diagram.
Embodiment
In conjunction with following specific embodiments and the drawings, the present invention is described in further detail, and protection content of the present invention is not limited to following examples.Under the spirit and scope not deviating from inventive concept, the change that those skilled in the art can expect and advantage are all included in the utility model, and are protection domain with appending claims.Implement process of the present invention, condition, reagent, experimental technique etc., except the following content mentioned specially, be universal knowledege and the common practise of this area, the present invention is not particularly limited content.As according to people such as Sambrook, molecular cloning, the described people of laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989), or according to the suggestion condition of manufacturer.
The construction process of the present invention's site-directed point mutation in rat embryo cell, a kind ofly carry out any genomic dna of target and the method making it change, the method comprises: introduce in rat cell the coding nucleic acid of guideRNA identifying target gene and CRISPR/Cas9 albumen and the biologically active substance such as coding nucleic acid, albumen or manually modified virus, thus target gene group DNA sequence dna is identified and is cut.Then, cell carried out vitro culture or directly proceeded in suitable female mouse uterine tube or uterus, make class transcription activator enzyme nucleic acid expression and make the target gene group DNA near cleavage site that duplex fracture occur, then this DNA break site being repaired.
Wherein, repair mode comprises: the reparation of (a) non-homologous end joining.Non-homologous end joining reparation causes transgenation (base insertion, disappearance) to be introduced in goal gene group sequence.(b) homologous recombination repair.Homologous recombination repair makes donor exogenous DNA array be incorporated in target gene group DNA sequence dna, causes the change of endogenous targets gene order.
The construction process of the present invention's site-directed point mutation in rat embryo cell, comprises the steps:
(1) NLS-hCas9-NLS in-vitro transcription carrier is built
On carrier, by the NcoI restriction endonuclease sites overlapping with NLS-hCas9-NLS encoding sequence initiator codon, introduce SP6 promotor and Kozak sequence; Synthesizing single-stranded oligonucleotide P1 and P2; With PNK Phosphoric acid esterase, 5 ' phosphate group is added to above-mentioned single stranded oligonucleotide, and anneal afterwards with the process of NcoI single endonuclease digestion and dephosphorylized carrier be connected; Choose the SP6-Cas9 plasmid that SP6 promotor direction of insertion is correct;
Wherein, P1 primer sequence: CATGGATTTAGGTGACACTATAGAAGAGGCCGCCAC;
P2 primer sequence: CATGGTGGCGGCCTCTTCTATAGTGTCACCTAAATC;
(2) Cas9 nuclease mRNA is prepared
Choose the SP6-Cas9 plasmid that direction of insertion is correct, carry out linearizing with the NotI restriction endonuclease in downstream, Cas9 coding region; In-vitro transcription is carried out with SP6mMESSAGEmMACHINE Kit test kit;
(3) Cas9 target site is determined
Determine that " GGCTGCTGCGGTTCCAGAGG " is as target sequence;
(4) preparation guides RNA in-vitro transcription template
Synthesizing single-stranded oligonucleotide P3, P4, P5, P6 carry out primer over-lap PCR;
Wherein,
P3 primer sequence: GATCACTAATACGACTCACTATAGGCTGCTGCGGTTCCAGAGGGTTTTAGAGCTAG AAAT;
P4 primer sequence: AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTA ACTTGCTATTTCTAGCTCTAAAAC;
P5 primer sequence: GATCACTAATACGACTCAC;
P6 primer sequence: AAAAAAGCACCGACTCGGTGCC;
(5) with above-mentioned PCR primer for template, carry out in-vitro transcription, and mix with the Cas9 nuclease mRNA prepared;
In-vitro transcription gained is guided RNA and Cas9mRNA mixing, with TE dilution, make guiding RNA final concentration be 25ng/ μ l, Cas9mRNA final concentration is 50ng/ μ l, is injected into the protokaryon part of rat zygote in vitro fertilization, vitro culture 1-2 hour.
The construction process of the present invention's targeted mutagenesis in rat embryo cell, comprises transgenation or gene knockout, and its step comprises:
(1) in target rat gene N end encoding sequence, suitable target sequence is found.
(2) according to two single stranded oligonucleotides that above-mentioned target sequence synthesis is complementary, annealing is connected into gRNA expression vector.
(3) by above-mentioned expression vector linearizing, in-vitro transcription becomes RNA, and carries out purifying.
(4) plasmid linearization of coding Cas9 albumen is carried out in-vitro transcription simultaneously.
(5) above-mentioned two kinds of RNA are mixed also microinjection and enter the rat zygote of artificial insemination formation.
Further, above-mentioned zygote is implanted in the female mouse uterine tube of false pregnancy; The genomic dna extracting above-mentioned steps gained Offspring rat carries out genotype identification; The rat of target gene generation phase shift mutation is hybridized with wild-type rats respectively; Heterozygote rat brood for above-mentioned hybridization gained is carried out selfing; Identify the genotype of above-mentioned selfing gained rat, the homozygote rat of screening target gene phase shift mutation.
In the present invention, rat cell is rat embryo cell.Rat embryo comprises the unicellular zygote of rat or many cells rat embryo.The transcriptionally active sequence that rat target gene group sequence is the genome sequence of encoding gene, lowly repetitive sequence, moderately repetitive sequence, highly repetitive sequence, low copy sequence, gene are asked or non-transcribed sequences.
Embodiment 1 builds Mc4r knockout rat by injecting RNA in rat cell
1, the structure of NLS-hCas9-NLS in-vitro transcription carrier
As shown in Figure 2, Cas9 protein expression module is held successively by SP6 promoter sequence from 5 ' end to 3 ', N holds nuclear localization signal (Nuclear localization sequence, NLS), humanized Cas9 DNA sequences encoding, C holds nuclear localization signal, and polyadenylic acid (polyA) is formed.Concrete construction strategy is, on the basis of carrier pX260 (Addgene plasmid #42229), by the NcoI restriction endonuclease sites overlapping with NLS-hCas9-NLS encoding sequence initiator codon, introduces SP6 promotor and Kozak sequence.I.e. synthesizing single-stranded oligonucleotide P1 (SEQ ID NO.1) and P2 (SEQ ID NO.2), use PNK Phosphoric acid esterase to add 5 ' phosphate group to above-mentioned single stranded oligonucleotide, and anneal afterwards with the process of NcoI single endonuclease digestion and dephosphorylized pX260 carrier be connected.The some clones of picking, the correct plasmid of SP6 promotor direction of insertion is chosen in order-checking.
P1 primer sequence: CATGGATTTAGGTGACACTATAGAAGAGGCCGCCAC (SEQ ID NO.1)
P2 primer sequence: CATGGTGGCGGCCTCTTCTATAGTGTCACCTAAATC (SEQ ID NO.2)
2, the preparation of Cas9 nuclease mRNA
Choose the SP6-Cas9 plasmid that direction of insertion is correct, use the NotI restriction endonuclease in downstream, Cas9 coding region to carry out linearizing.Reclaim linearizing SP6-Cas9 fragment by phenol chloroform, use SP6mMESSAGEmMACHINE Kit test kit to carry out in-vitro transcription.The LiCl solution that in-vitro transcription gained mRNA test kit provides is precipitated, uses microinjection TE resuspended.
3, the prediction of Cas9 target site
Target dna sequence is obtained from NCBI, be the CDS region of first exon of rat Mc4r in the present embodiment, target sequence is copied and pastes into Word document, use " searching " function (Ctrl+F), search in content text frame input " GG GG " (front 20 bases are used for and the noncoding strand complementary pairing of target spot DNA) be used for the sequence meeting target condition of searching in non-template chain.It is that the product of transcribing due to T7 promotor could must be transcribed efficiently so that " GG " is initial that above-mentioned base is formed.II type CRISPR system then requires that target spot 3 ' holds the adjacent motif of former intervening sequence (proto-spacer-adjacent motifs, PAM) of next-door neighbour to meet the formation of " NGG " simultaneously.According to same reason, also can search in the reverse complementary sequence of candidate's target area " CC CC " find potential target spot.Final selected " GGCTGCTGCGGTTCCAGAGG " is as target sequence.
4, the preparation of RNA in-vitro transcription template is guided
As shown in Figure 2, guide RNA in-vitro transcription template from 5 ' to 3 ' to hold successively by T7 promoter sequence, target spot specific sequence, and guide RNA skeleton part.After determining target, synthesizing single-stranded oligonucleotide P3 (SEQ ID NO.3), P4 (SEQ ID NO.4), wherein single stranded oligonucleotide P3 from 5 ' to 3 ' holds successively containing T7 promoter sequence, target spot specific sequence, and guide RNA skeleton 5 ' end portion base.Single stranded oligonucleotide P4 encodes and guides RNA skeleton part, and its 3 ' end holds complementation with 3 ' of single stranded oligonucleotide P3.Synthesizing single-stranded oligonucleotide P5, P6, hold base sequence identical with 5 ' of single stranded oligonucleotide P3, P4 respectively.Use single stranded oligonucleotide P3, P4, P5, P6 carry out primer over-lap PCR.By phenol chloroform and isopropanol precipitating purified pcr product, carry out resuspended with RNase Free ddH2O, measure concentration.
P3 primer sequence (SEQ ID NO.3): GATCACTAATACGACTCACTATAGGCTGCTGCGGTTCCAGAGGGTTTTAGAGCTAG AAAT
P4 primer sequence (SEQ ID NO.4): AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTA ACTTGCTATTTCTAGCTCTAAAAC
P5 primer sequence: GATCACTAATACGACTCAC
P6 primer sequence: AAAAAAGCACCGACTCGGTGCC
5, the preparation of Cas9 nuclease mRNA
With above-mentioned PCR primer for template, illustrate according in vitro Transcription T7Kit (Takara#6140) test kit and carry out in-vitro transcription.Phenol chloroform and isopropanol precipitating purifying transcription product, use microinjection TE resuspended.
6, the microinjection of RNA and Cas9mRNA is guided
Guide RNA and Cas9mRNA mix and use microinjection TE to dilute in-vitro transcription gained, make guiding RNA final concentration be 25ng/ μ l, Cas9mRNA final concentration is 50ng/ μ l.Use microinjection instrument above-mentioned mRNA injection of solution to be entered the protokaryon part of rat zygote in vitro fertilization, build and form specific rat embryo cell (zygote), namely complete the structure of the present invention's site-directed point mutation in rat embryo cell.
The present invention's site-directed point mutation construction process in rat embryo cell whether is successfully realized below in order to qualification.
(1) genotype identification of neonate rat
The zygote transplation of survival, after vitro culture 1-2 hour, is entered the uterine tube of the female mouse of false pregnancy by the rat embryo cell (zygote) step 6 obtained.After neonate rat is born 1 week, clip toe, extracting genomic dna.At target spot upstream design PCR primer P7 (SEQ ID NO.7), downstream design PCR primer P8 (SEQ ID NO.8), with rat genomic dna to be identified for template carries out pcr amplification, PCR reaction terminates to carry out purifying to product afterwards, sex change, slow annealing, adds identifiable design and carries out the T7Endonuclease I that cuts in non-matching site, carrying out agargel electrophoresis.Fig. 3 is PCR primer T7E1 restriction enzyme digestion and electrophoresis figure corresponding to microinjection gained neonate rat.As shown in Figure 3,13 are had at target position with sudden change in 15 neonate rats.Wherein,
P7 primer sequence: TACTGTTTAGCAGGGTGATGACG (SEQ ID NO.7)
P8 primer sequence: GAAGAGACCAACAACTCCTTTGC (SEQ ID NO.8)
T7E1 is detected positive rats PCR primer and is connected into pMD18T carrier respectively, the some clones of picking extract plasmid and check order respectively.Rat base deletion situation comparison result figure is built headed by Fig. 4.As shown in Figure 4, wherein underscore part is target spot region, former interval (protospacer) i.e., and TGG is below the adjacent motif of former intervening sequence (proto-spacer-adjacent motifs, PAM).Every section of short-term represents the base of a disappearance.
(2) head builds the reproductive tract heredity of rat Mc4r transgenation
Choose No. 12 Mc4r transgenation head and build rat, hybridize with wild-type rats, by the genotype of aforesaid method qualification neonate rat.Fig. 5 is given birth to F1 generation rat base deletion situation schematic diagram by No. 12 head build rat.As shown in Figure 5,3 mouse are had to carry sudden change in 6 newborn F1 generation rats.Wherein underscore part is target spot region, former interval (protospacer) i.e., after TGG be the adjacent motif of former intervening sequence (proto-spacer-adjacent motifs, PAM).Every section of short-term represents the base of a disappearance.Prove Mc4r transgenation energy genetic stability.
Embodiment 2 by injecting CAS albumen and gRNA structure Mc3r/Mc4r knockout rat in rat cell
1, the structure of CAS nuclease prokaryotic expression carrier PET-28a-H6-3FLAG-NLS-CAS9-NLS
As shown in Figure 6, Cas9 fusion protein prokaryotic expression carrier is held successively by T7 promoter sequence from 5 ' end to 3 ', His6Tag, 3 × Flag, N holds nuclear localization signal (Nuclear localization sequence, NLS1), humanized Cas9 DNA sequences encoding, C holds nuclear localization signal NLS2 to form.First, His6 and 3 × FLAG label and new N are introduced by the method for over-lap PCR in the basis of NLS-hCas9-NLS in-vitro transcription carrier and hold NcoI restriction enzyme site and C-terminal EcoRI restriction enzyme site.By NcoI with the EcoRI restriction enzyme site on PET-28a new H6-3FLAG-NLS-CAS9-NLS is connected and inserts wherein and by the initiator codon that includes in the NcoI restriction enzyme site translation initiation site as protein expression.
P9 primer sequence: CCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAA GAC
P10 primer sequence: GATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGT CGG
P11 primer sequence: GAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCA TCG
P12 primer sequence: GGCAAAAAAGAAAAAGTAAGAATTCCTA
2, the expression and purification of Cas9 nuclease
Build and the PET-28a-H6-3FLAG-NLS-CAS9-NLS plasmid transfection Rosseta DE3 host competent cell identified.24 DEG C of active cells to OD values are add IPTG induction after 0.8 ~ 1.0, make IPTG final concentration be 0.1mM, 16 DEG C of inductions 17 hours.Supernatant liquor is collected and with ni-sepharose purification, for subsequent use after sonicated cells.
3, the prediction of Cas9 target site
With embodiment 1, final selected " GGCTGCTGCGGTTCCAGAGG ", as the target sequence of Mc4r, selected " GGGCTGCAGGGTGCTGGGAG " is as the target sequence of Mc4r.
4, the preparation of RNA in-vitro transcription template is guided
With embodiment 1.
5, the microinjection of RNA and Cas9 albumen is guided
Guide the Cas9 albumen of RNA and prokaryotic expression mix and use microinjection TE to dilute in-vitro transcription gained, make guiding RNA final concentration be 12.5ng/ μ l, Cas9 final concentration of protein is 5ng/ μ l.Use microinjection instrument above-mentioned mRNA injection of solution to be entered the protokaryon part of rat zygote in vitro fertilization, build and form specific rat embryo cell (zygote), namely complete the structure of the present invention's site-directed point mutation in rat embryo cell.
The present invention's site-directed point mutation construction process in rat embryo cell whether is successfully realized below in order to qualification.
(1) genotype identification of neonate rat
With embodiment 1.The zygote transplation of survival, after vitro culture 1-2 hour, is entered the uterine tube of the female mouse of false pregnancy by the rat embryo cell (zygote) step 6 obtained.Have 3 in 5 neonate rats at target position with sudden change, wherein 2 there is dual-gene simultaneous mutation.
Wherein Mc4r identifies by PCR primer and is: P7 primer sequence: TACTGTTTAGCAGGGTGATGACG
P8 primer sequence: GAAGAGACCAACAACTCCTTTGC
Mc3r identifies by PCR primer: P13 primer sequence AATCTAGACTGGACAGCATCCAC
P14 primer sequence TGATAACCACGATCATGATGGTC
T7E1 is detected positive rats PCR primer and is connected into pMD18T carrier respectively, the some clones of picking extract plasmid and check order respectively.Rat base deletion situation comparison result figure is built headed by Fig. 7.As shown in Figure 7, wherein underscore part is target spot region, former interval (protospacer) i.e., and TGG is below the adjacent motif of former intervening sequence (proto-spacer-adjacent motifs, PAM).Every section of short-term represents the base of a disappearance.
Claims (6)
1., to the construction process carrying out site-directed point mutation in rat embryo cell, it is characterized in that, comprising:
(1) in rat target gene group sequence, determine the target sequence of CRISPR-Cas system institute target;
(2) design, structure can identify and guide CAS9 recombinant protein to the gRNA nucleotide sequence of target gene target sequence;
(3), after described gRNA nucleotide sequence being mixed with CAS9 recombinant protein, import in described rat embryo nucleus by microinjection; Wherein,
In described step (1), in candidate's target area, search the target sequence in non-template chain by " GGNNNNNNNNNNNNNNNNNNNGG "; Or target sequence is searched by " CCNNNNNNNNNNNNNNNNNNNCC " in the reverse complementary sequence of candidate's target area;
In described step (2), described gRNA nucleotide sequence can with described CAS protein-specific combination and by its target target sequence;
In described step (3), in advance described gRNA nucleotide sequence is transcribed into RNA and purifying in vitro.
2. method as claimed in claim 1, wherein prepare described CAS9 recombinant protein by CAS nuclease prokaryotic expression carrier PET-28a-H6-3FLAG-NLS-CAS9-NLS, wherein said H6-3FLAG-NLS-CAS9-NLS is successively by T7 promoter sequence from 5 ' end to 3 ' end, His6Tag, 3 × Flag, N holds nuclear localization signal NLS1, humanized Cas9 DNA sequences encoding, C holds nuclear localization signal NLS2 to form, and by NcoI with the EcoRI restriction enzyme site on PET-28a this H6-3FLAG-NLS-CAS9-NLS is connected and inserts wherein and by the initiator codon that includes in the NcoI restriction enzyme site translation initiation site as protein expression, thus obtain described CAS nuclease prokaryotic expression carrier PET-28a-H6-3FLAG-NLS-CAS9-NLS.
3. method as claimed in claim 1, the gene of wherein said rite-directed mutagenesis is Mc3r or Mc4r gene.
4. the method as described in any one of claim 1-3, is characterized in that, in described step (3), the DNA break site that cutting target gene group is formed is repaired by fallibility non-homologous end joining or passes through homologous recombination repair.
5. the method as described in any one of claim 1-3, is characterized in that, described target gene sport that base is inserted, disappearance, change, phase shift mutation or knock out.
6. the method as described in any one of claim 1-3, is characterized in that, described rat embryo cell is the unicellular zygote of rat or many cells rat embryo.
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