CN110511962A - A method of it is cut by double site and realizes that pig Gjb2 gene coded sequence is precisely edited - Google Patents
A method of it is cut by double site and realizes that pig Gjb2 gene coded sequence is precisely edited Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention belongs to Animal genome editing technique fields, and in particular to cut the method for realizing that pig Gjb2 gene coded sequence is precisely edited by double site.The technical problem to be solved in the present invention is to provide a kind of pig model preparation methods that Gjb2 gene coded sequence editor precisely edits.The technical solution that the present invention solves above-mentioned technical problem, which is to provide, cuts the method for realizing that pig Gjb2 gene coded sequence is precisely edited by double site.Method includes the following steps: a, sgRNA pairs of preparation；B, mixture of the sgRNA to, donor dna, mRNA the and/or spCas9 purification of recombinant proteins for encoding spCas9 is imported in pig body early embryo cytoplasm；C, pig body early embryo is moved in receptor sow fallopian tubal, is allowed to become pregnant；D, piglet is given birth to after full-term, obtains Gjb2 gene precisely edited pig model.The method of the present invention efficiently can accurately realize the accurate mutation of pig Gjb2 gene coded sequence, such mutant animals model has a good application prospect.
Description
Technical field
The invention belongs to Animal genome editing technique fields, and in particular to a kind of cut by double site realizes pig Gjb2
The method that gene coded sequence is precisely edited and the kit for this method.
Background technique
The Cx26 albumen of Gjb2 (Gap Junction Protein Beta 2) gene coding belongs to inserted by connexin base
Because of family, albumen is connect with the seam of flanking cell and forms a complete Gap junctions, these channels in Information Conduction and
It plays an important role in mass exchange, is the important channel for completing the iuntercellular conversion of electrolyte, second messenger and metabolite.
Gjb2 gene is highly conserved between species, is expressed in the positions such as inner ear, esophagus, small intestine, cerebral cortex, is important deaf phase
Correlation gene.Deafness caused by Gjb2 gene mutation is fallen ill before showing as language mostly, and leads to middle severe or pole severe deafness, is showed
For congenital deafness or Delayed onset hearing impairment.Gjb2 gene exon containing there are two, wherein First Exon is the 5 ' areas-UTR,
And albumen coded sequence is then completely contained in Second Exon.
Conservative and important biological function of the Gjb2 gene due to its height, after diallelic system knocks out
Animal often has lethal phenotype.In hereditary hearing impairment patient, Gjb2 gene mutation mode is complicated and changeable, has 111 at present
Kind mutational formats are reported, wherein 9 kinds of dominant mutation.The 235th bit base C of Gjb2 gene coded sequence missing
(Gjb2c.235delC) mutation is the most common hereditary hearing impairment mutation of Chinese population, and crowd is mutated carrying rate and is up to 1.88-
1.925%.C.235delC homozygous mutation leads to autosomal recessive non-syndrome hereditary hearing impairment to Gjb2, and patient is mostly ears
Weight depth is deaf (ears are greater than 90dB), and not only homozygous mutation can be so that deaf, and Gjb2c.235delC and other pathogenic mutations are located at
When forming double heterozygous mutation on two different chromosomes, deafness can also be caused.Except Gjb2c.235delC be mutated with
Outside, the 37th valine mutation of Gjb2 gene coded protein is that isoleucine (GJB2 p.V37I) is mutated in Chinese population
There is very high disease incidence, and the high correlation with hearing is presented.But it is possible to precisely simulate " personalizing " of human mutant mode
Gjb2 mutant animals model lacks, and leads to that its pathogenic mechanism is unclear, magnetic target therapy scheme lacks.Therefore, Gjb2 is precisely constructed
Gene mutant animals model causes deaf mechanism, research and development more accurately based on the targeting of mechanism further investigation Gjb2 gene mutation
Therapeutic strategy etc. has great importance.
Pig and the mankind have similitude in terms of anatomy, physiology and biochemistry, and pig model is considered raw
Transformation model with bright prospects in object medical research and exploitation.Pig is the ideal material for formulating deaf animal model: pig with
People has high similitude in the form and configuration aspects of hearing organ；Pig is high to the sensibility and its threshold value of sound frequency and people
Degree is consistent；Pig cochlea size, form, structure and the mankind are closely similar；As people, in pig birth, anatomy of internal ear but is basic
It reaches maturity, and has normal good hearing, and the rodents such as mouse are the late-maturing animal of auditory system: without hearing when birth, out
Inner ear, which need to continue to develop, after life just has normal good hearing；Further, since cochlea bone wall thickness, intensity and people are more closely, pig ear
Snail has been widely used in the research and development of the surgical technics such as the fine Tomography of cochlea windowing, cochlea, cochlear implant implantation,
It is the important model animal of otology translational medicine research.
Although pig (especially weight and organ size and the similar miniature pig of people's height) has been used for the great disease of a variety of mankind
The basis of disease is studied with translational medicine, such as angiocarpy, metabolic syndrome, digestion (stomach) and skeletal diseases, diabetes, heart
Disease, skin disease and acute and chronic intestinal inflammatory etc. still there is no Gjb2 gene mutation pig and other big animal models at present
Report.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of preparations of pig model that Gjb2 gene coded sequence is precisely edited
Method.The technical solution that the present invention solves above-mentioned technical problem cuts realization pig Gjb2 gene by double site there is provided a kind of
The method that coded sequence is precisely edited.
Method includes the following steps:
A, prepare a pair of sgRNA, wherein the cleavage site of a sgRNA identification Gjb2 gene coded sequence upstream, another
The cleavage site in sgRNA identification gene coded sequence downstream；
The sgRNA of the identification Gjb2 gene coded sequence downstream cleavage site can identify the PAM on the Gjb2 gene of pig
Site is CGG；
The sgRNA of the identification Gjb2 gene coded sequence upstream cleavage site can identify the PAM on the Gjb2 gene of pig
Site is GGG.
B, by a pair of of sgRNA described in step a, the donor dna with expected mutant nucleotide sequence, the mRNA for encoding spCas9
And/or after spCas9 purification of recombinant proteins is sufficiently mixed, mixture is imported to the cytoplasm of pig body early embryo by microinjection
In；
C, by step a, treated that pig body early embryo migrates in the fallopian tubal of receptor sow, and receptor sow is made to become pregnant；
D, the receptor sow become pregnant gives birth to piglet after full-term, obtains Gjb2 gene precisely edited pig model.
Further, in sgRNA described in the above method:
The boot sequence for identifying the sgRNA of pig Gjb2 gene coded sequence downstream cleavage site is 5 '-
GAAUCUGUAUCUGGGAGACG-3 ' or its complementary series；
The boot sequence for identifying the sgRNA of pig Gjb2 gene coded sequence upstream cleavage site is 5 '-
GACCAAGCGACACGGCACAG-3 ' or its complementary series.
Further, donor dna described in the above method is by 37 valine (V37) codons of pig Gjb2 gene
Sport the donor dna of isoleucine (I) codon.
Preferably, the nucleotides sequence of donor dna described in the above method is classified as shown in SEQ ID No.1 or is that it is complementary
Sequence.
Further, sgRNA described in upper method and step b to, donor dna and encode spCas9 mRNA and/or
Proportion between spCas9 purification of recombinant proteins is by 10~15ng/uL of final concentration sgRNAs, spCas9 in the mixture
5~10ng/uL of 20~25ng/uL of mRNA and/or spCas9 purification of recombinant proteins and donor dna.
Wherein, the embryo of pig body early embryo described in the above method for 4 cell stages and before.
Further, further include having detection verification step in above method step c: using primer pair amplifies piglet genome
DNA sample is sequenced according to amplification and determines whether containing expected gene editing genotype Gjb2c.109-111GTG > ATC, if
Show to model successfully containing the genotype.
Wherein, the detection primer is to for Gjb2-CDS-F/Gjb2-CDS-R primer pair, sequence are as follows:
Gjb2-CDS-F:5-TCACCTTTGTGGGTGAGGTTGCGTAAGAA-3；
Gjb2-CDS-R:5-GCTTGACGACGGGAATCTGTATCTGG-3.
The present invention also provides a kind of kits for preparing Gjb2 gene mutation pig model.The kit includes boot sequence
As follows sgRNA pairs:
5′-GAAUCUGUAUCUGGGAGACG-3′；
5′-GACCAAGCGACACGGCACAG-3′。
Further, it is different bright that mentioned reagent box, which further includes by 37 valine (V) codon mutations of pig Gjb2 gene,
The donor dna of propylhomoserin (I) codon.Preferably, the nucleotides sequence of the donor dna is classified as shown in SEQ ID No.1 or for it mutually
Complementary series.
Further, mentioned reagent box further includes mRNA the and/or spCas9 recombinant protein for encoding spCas9 albumen.
Key of the invention first is that using special donor dna, it is close by the 37th in pig Gjb2 gene coded sequence
Numeral sports ATC (c.109-111GTG > ATC) by GTG, and the amino acid for encoding it sports different bright ammonia by valine (V)
Sour (I).Donor dna is inserted into one section of heterologous sequence containing general introne shearing receptor sequence in the upstream of coding sequence of mutation
It arranges (heterology), it is non-in the flank that mutant coding sequences downstream introduces one section of people's Gjb2 gene translation terminator codon 3 ' end
Coded sequence (3 ' UTR) sequence is using as heterologous sequence；The two sides of heterologous sequence in upstream and downstream, accurately according to upstream and downstream
Cleavage site of the sgRNA in pig Gjb2 gene respectively introduces one section of pig Gjb2 genome sequence using as homology arm.Cooperate specific
SgRNA pairs, utilize crispr/cas9 system, realize pig Gjb2 gene accurate efficient editor.
The beneficial effects of the present invention are: it may be implemented by double site cutting to pig Gjb2 gene coded sequence high efficiency
The accurate editor based on " homologous sequence guidance repair " (homology-directed repair, HDR) mechanism；This method one
Aspect can expand sgRNA range of choice, solve the problems, such as may not have the efficient site sgRNA in target editing area；It is another
Aspect is template using long chain DNA, is able to achieve the Gjb2 gene coded sequence different experiments such as from single base mutation to sequence substitutions
Purpose；In addition, avoiding the direct cutting to Gjb2 gene coded sequence, can theoretically reduce to the potential of animal growth
It influences, is more advantageous to the acquisition of mutant animals；As application case, sgRNA pairs that efficient double site cutting can be achieved is devised,
With matched achievable Gjb2 c.109-111GTG > the long-chain DNA profiling precisely edited of ATC；Based on above-mentioned discovery,
The Gjb2 gene mutation model of pig has been obtained, expected precisely mutation is realized.The personnel of this research field can be cut using above-mentioned
Site is cut, using the DNA profiling for containing different mutation, the mutation of any required pig Gjb2 gene coded sequence is realized or sets
It changes, has a good application prospect.
Detailed description of the invention
The long single-stranded DNA templates structure chart that Fig. 1, the present invention design.
Fig. 2, the method for the present invention mechanism of action schematic diagram.
Fig. 3, pig Gjb2 gene structure and sgRNA pairs of cleavage site location diagram.E1, E2 indicate exon,
Box in E2 indicates that the coded sequence of pig Gjb2 gene, ATG indicate initiation codon, and TAA indicates terminator codon；White edge arrow
Head indicates that transcriptional orientation, pGjb2-5fs-sgRNA and pGjb2-3fs-sgRNA respectively indicate the cleavage site of upstream and downstream sgRNA
Corresponding position.
The design diagram for the long single-stranded DNA templates that Fig. 4 is accurately mutated for pig Gjb2 gene coded sequence humanization.Side
Frame: c.109-111GTG > ATC mutation (i.e. p.V37I mutation) pig Gjb2 gene coded sequence has occurred；Light grey hachure:
Upstream and downstream heterologous sequence；Heavy black line item: upstream and downstream homologous recombination arm.
Fig. 5 is cut by double site, and cell realizes displacement and the mutational site of pig Gjb2 coded sequence by HDR mechanism
It is accurate to import.Left Cas9/sgRNA: left side (upstream) sgRNA cleavage site；Right Cas9/sgRNA: right side (downstream)
SgRNA cleavage site；Pig Gjb2 CDS: pig Gjb2 gene coded sequence；Pig genomic locus: pig genomic locus；
Right arm: right side (upstream) homology arm；Left arm: left side (downstream) homology arm；LssODN: long single donor DNA；
PGJB2p.V37I CDS: the coded sequence of coding pGJB2 p.V37I mutain；Mutant locus: it is expected that mutational site.
C.109- Fig. 6 realizes pig Gjb2 gene coded sequence by the unit point cutting to pig Gjb2 gene coded sequence
The importing of 111GTG > ATC (p.V37I) mutation.A: white edge arrow indicates genetic transcription direction；The first of E1 pig Gjb2 genes
Exon；E2 pig Gjb2 gene Second Exons；Boxed area in Second Exon indicates the code sequence of pig Gjb2 gene
Column；The bond area of black arrow expression pGjb2-V37-sgRNA；The area of coverage of grey lines expression pGjb2-V37I-ssODN
Domain；B: white edge arrow indicates the bond area pGjb2-V37-sgRNA；The gtg of black overstriking indicates the target site of quasi- mutation；Black
The atc of overstriking indicates targeted mutagenesis base
Fig. 7 head builds the genetic analysis of pig individual otic tissues.PGjb2 genomic locus: pig Gjb2 gene order；
Founderpiglet1,2 indicates that the head that expected mutation occurs builds pig individual；Sequencing chromatography:PCR is produced
Peak figure is sequenced in object.
Fig. 8 GJB2 p.V37I is mutated pig passage analysis.PGjb2 genomic sequence: pig Gjb2 gene order；F1
Piglet#1-6:6 F1 generation piglet；Sequencing chromatography: the sequencing peak figure in mutational site.
Specific embodiment
Deafness caused by GJB2 mutation meeting is fallen ill before showing as language mostly, and leads to middle severe or pole severe deafness.Research
The screening of its fall ill specific mechanism and treatment means all be unable to do without suitable animal model.
Existing clinical research shows that the 37th valine (V) of people GJB2 molecule sports the GJB2 of isoleucine (I)
P.V37I (Gjb2 c.109-111GTG > ATC) mutation is highly relevant with deafness.GJB2 molecule is highly conserved between species, people
The 37th valine (V) of GJB2 molecule corresponds to the valine of pig GJB2 molecule corresponding position.In order to prepare and human inheritance
The consistent pig model of Catastrophe Model height, early-stage study of the present invention utilize CRISPR system, attempt by directly to pig Gjb2 base
Codon site because encoding the 37th valine in coded sequence is cut, to prepare the pig mould of GJB2 p.V37I mutation
Type.However, it was found that using the method for directly carrying out unit point cutting to Gjb2 gene coded sequence, it is difficult to obtain containing expection
The pig model of Gjb2 gene mutation.
In view of the above-mentioned problems, the present invention respectively devises 1 sgRNA in pig Gjb2 gene coded sequence upstream and downstream, make its work
Cut the non-coding region of pig Gjb2 gene coded sequence upstream and downstream simultaneously for a pair of of sgRNA；Meanwhile devise one with it is upper
State sgRNA to it is mating, carry Gjb2 c.109-111GTG > ATC mutation long chain DNA as donor dna, then by " together
The DNA of source sequence guidance is repaired " (HDR) mechanism, achieve the purpose that gene editing.Based on sgRNA pairs provided by the invention, ability
Any accurate mutation or sequence substitutions required for domain researcher can carry out pig Gjb2 gene coded sequence.
Specifically, in one embodiment provided by the invention, by the 37th password in pig Gjb2 gene coded sequence
Son sports ATC (c.109-111GTG > ATC) by GTG, and the amino acid for encoding it sports isoleucine by valine (V)
(I)；One section of heterologous sequence containing general introne shearing receptor sequence is inserted into the upstream of coding sequence of mutation
(heterology), the non-volume of flank at one section of people's Gjb2 gene translation terminator codon 3 ' end is introduced in mutant coding sequences downstream
Code sequence (3 ' UTR) sequence is using as heterologous sequence；The two sides of heterologous sequence in upstream and downstream, accurately according to upstream and downstream sgRNA
Cleavage site in pig Gjb2 gene respectively introduces one section of pig Gjb2 genome sequence using as homology arm, specific structure such as Fig. 4
It is shown.The homologous sequence is that its sequence refers to above-mentioned sgRNA to the area between the cleavage site in pig Gjb2 gene order
The upstream and downstream flanking sequence in domain is identical；
Devise carry Gjb2 c.109-111GTG > ATC mutation long chain DNA after, carry out the principle of gene editing
Are as follows:
1, by the RNA in in-vitro transcription, reverse transcription and RNaseH digestion RNA-DNA heteroduplex, above-mentioned length is obtained
Single-stranded (long single stranded DNA, long the single-stranded DNA, lssDNA) of chain donor dna；
2, in the non-coding region of gene coded sequence two sides, the CRISPR cutting that two activity are strong, specificity is high is chosen
Site and its corresponding sgRNAs；
3, the sgRNA of two cleavage sites will be directed to, the recombination spCas9 albumen of spCas9 mRNA or purifying and as same
The long single stranded DNA of source recombination template is imported intracellular by approach such as cell transfecting or microinjections；
4, identify that the sgRNA of two sides cleavage site in coded sequence two sides cutting DNA, is formed double-strand break (DSB)；
5, cell is circumscribed to the progress 5 ' -3 ' of DNA break end at DSB by exonuclease, and formation contains 3 ' ends
Local single stranded DNA；
6, cell is to draw with single stranded DNA that can be complementary with the homologous sequence of long single-stranded DNA templates in the DSB of wherein side
Object synthesizes the complementary strand (synthesizing first chain) of long single-stranded template by DNA replication dna using long single stranded DNA as template；
7, cell is using the single stranded DNA in the DSB of the other side as primer, using the complementary strand of newly synthesized long single stranded DNA as template,
Article 2 chain is synthesized by DNA replication dna；
8, by the duplication of two chains, sequence of the target gene between two cleavage sites is dissociated, and duplexed
Long single-stranded DNA templates sequence is connected with the upstream and downstream flanking sequence of two cleavage sites；
9, in the design of long-chain DNA profiling, first is that the position of two sides homologous sequence is accurately corresponded to target exon two sides
Cleavage site, the efficiency of homologous recombination is improved, second is that introducing one section of heterologous sequence in the inside of two sides homologous sequence, promoting
Cell must start the duplication of DNA using the single stranded DNA of opposite side DSB as primer, and cannot be to draw with the single stranded DNA of the same side DSB
Object starting duplication, it is ensured that accurate displacement of the mutant nucleotide sequence to target sequence improves gene editing efficiency.
In an example of the present invention, using the long single stranded DNA that designs of the present invention as donor dna and two sides
On the basis of sgRNA pairs, those skilled in the art can be by the genome editing system of CRISPR/Cas9, will be above-mentioned
After sgRNA is sufficiently mixed mRNA the and/or spCas9 purification of recombinant proteins of, donor dna and coding spCas9, mixture is led to
Microinjection is crossed to import in the cytoplasm of pig body early embryo；Treated pig body early embryo is migrated into receptor sow again
In fallopian tubal, receptor sow is made to become pregnant；The receptor sow become pregnant gives birth to piglet after full-term, obtains Gjb2 mutation pig mould
Type.
Wherein, above-mentioned sgRNA to, donor dna and encodes mRNA the and/or spCas9 purification of recombinant proteins of spCas9
Between be final concentration of by the mixture with ratio: sgRNA10~15ng/uL, spCas9 mRNA and/or spCas9
5~10ng/uL of 20~25ng/uL of purification of recombinant proteins and donor dna.
Further, the embryo of pig body early embryo described in the above method for 4 cell stages and before.It is above-mentioned in order to detect
Whether Method Modeling succeeds, and can also use primer amplification piglet genome DNA sample, is determined whether according to amplification sequencing
Containing expected gene editing genotype, if showing to model successfully containing the genotype.Present invention provides effective detections
Use primer.
In an example of the present invention, the sgRNA being transcribed in vitro accordingly is made to external using the method for the present invention
Standby long single-stranded DNA templates and spCas9 mRNA are imported in the fertilized eggs of 1~4 cell stage of pig simultaneously by microinjection, and
By the zygote transplation after injection in the adult sow fallopian tubal being in heat, pig Gjb2c.109- is efficiently obtained
111GTG > ATC is mutated pig, and the accurate mutation rate that head builds pig is 50%；The breeding of the mating between pig is further built by head, thereafter
Homozygous mutation rate for piggy is 100%.And use the control group of routine CRISPR-Cas9 method, then it can not obtain prominent
Become pig.
Below by way of to embodiment, the present invention will be described in more detail.
Accurate mutation one, experimental program of one pig Gjb2 gene coded sequence of embodiment:
Experiment purpose is precisely to be mutated to the coded sequence of the Gjb2 gene of pig, keeps its 37th valine (V) prominent
Become isoleucine (I).
1, the preparation of long single stranded DNA
1) double-stranded DNA for containing long single-stranded DNA templates sequence is obtained by gene chemical synthesis, and places it in T7 promoter sequence
The downstream of column obtains long single-stranded DNA templates and plasmid is transcribed in vitro；
2) in above-mentioned in-vitro transcription plasmid long single-stranded DNA templates sequence downstream, select suitable restriction enzyme site to cut
Plasmid, realize plasmid it is abundant linearisation (restriction enzyme site of selection cannot cut long single-stranded DNA templates sequence or T7 promoter sequence
Column).
3) it carries out the purifying of linear plasmid DNA and removes RNA enzyme pollution；
4) DNA precipitating is sufficiently washed with 70% ethyl alcohol of no RNA enzyme, it is heavy with the deionized water dissolving DNA of no RNA enzyme later
It forms sediment；
5) the template plasmid DNA that 1ug is linear is taken, with in-vitro transcription kit MEGAshortscriptTM T7
Transciption Kit (invitrogen, AM1354) is transcribed in vitro, and obtains the complementary RNA of long single-stranded template (in detail
Experimental procedure is referring to kit specification)；
6) the complementary RNA for taking 1ug long single-stranded template, using with the primer of 3 ' the end sequences complementation of complementary RNA, with complementation
RNA is reverse transcribing template, utilizes Reverse Transcriptase kit PrimeScriptTM II 1st Strand cDNA Synthesis Kit
(TAKARA, 6210A) synthesizes complementary DNA by reverse transcription, and obtaining RNA-DNA heteroduplex product, (operation, which is detailed in kit, to be said
Bright book)；
7) in the reaction product of step 6), RNAaseH is added, in specific digestion RNA-DNA heteroduplex
RNA obtains long single stranded DNA；
8) long single stranded DNA is recycled with QIAquick PCR Purification Kit, is washed with the deionization of no RNase
Long single stranded DNA is taken off, dispenses and saves backup after survey concentration.
2, the preparation of sgRNA
1) synthesize two it is complementary containing sgRNA identification sequence oligonucleotide single stranded DNA (oligo DNA) (note:
Two complementary single-stranded dna renaturation is after double-strand, the subcloning sites that end will be transcribed in vitro after the digestion on plasmid with sgRNA are mutual
It mends；It is pUC57kan-T7-gRNA (Addgene#115520, for Asia that plasmid, which is transcribed in vitro, in sgRNA used in the present embodiment
The restriction enzyme site of clone is BsaI)；
2) the single-stranded oligo DNA of every synthesis takes 1OD, is dissolved in deionized water with the concentration of 0.2ug/uL, and with
It is saved backup after the packing of 20uL/ pipe；
3) the single-stranded oligo DNA solution for respectively taking the complementation of 1 pipe after mixing and mix well in equal volume, is containing 1L originally
In the water-bath of water, in 95 degrees Celsius of incubation 10min, so that two complementary single-stranded oligo DNA are sufficiently denaturalized as single-stranded shape
State；
4) turn off water-bath power supply, make its natural cooling, complementary single-stranded oligo DNA renaturation is allowed to be double-stranded state；
5) vector plasmid pUC57Kan-T7-gRNA is transcribed in vitro with the abundant digestion sgRNA of BsaI, recycles linear plasmid piece
Section；
6) the linear plasmid segment that the double-stranded DNA and step 5) obtained step 4) obtains, is connected with T4DNA ligase,
Obtain recombinant plasmid；
7) the connection product transformed competence colibacillus bacterium of step 6), picking kalamycin resistance bacterium colony, and amplification cultivation obtain
Resistance bacterium solution；
8) recombinant plasmid, and sequence verification are extracted using resistance bacterium solution；
9) plasmid obtained with the correct step 8) of the abundant digestion sequence verification of restriction endonuclease DraI realizes that recombinant plasmid is complete
Entirely, it adequately linearizes；
10) pass through protease K digesting, phenol: chloroform recovery, sodium acetate neutralization, ice ethanol precipitation, ice bath and refrigerated centrifuge
And etc., DNA precipitating is obtained, the purifying of linear plasmid DNA is realized and removes RNA enzyme pollution；
11) DNA precipitating is sufficiently washed with 70% ethyl alcohol of no RNA enzyme, later with the deionized water dissolving DNA of no RNA enzyme
Precipitating；
12) linear plasmid for the purifying for taking 1ug step 11) to obtain is template, uses in-vitro transcription kit
MEGAshortscriptTMT7 Transciption Kit (invitrogen, AM1354) is transcribed in vitro, and sgRNA is obtained
(operating procedure is detailed in kit specification)；
13) MEGAClear Transcription Clean-Up Kit (invitrogen, AM1908), purifying step are utilized
The rapid in-vitro transcription product 12) obtained, is packed as 5-10uL/ pipe according to concentration and saves backup that (operating procedure is detailed in reagent
Box specification).
3, the preparation of Cas9 mRNA
1) preparation of transcription templates:
With AgeI restriction endonuclease sufficiently (overnight) digested plasmid pST1374-N-NLS-flag-linker-Cas9 (Addgene#
44758), the preparation of template is transcribed in vitro with sgRNA for remaining step.
2) it is transcribed in vitro:
Cas9 mRNA is transcribed using mMESSAGEmMACHINE T7 Ultra kit, experimental procedure is detailed in kit explanation
Book.1ul Turbo DNase, 37 DEG C of incubation 15min, to remove remaining template DNA are added into system after completing for transcription.
3) tailing:
(1) reagent in table 1 is sequentially added into T7 Ultra Reaction system:
Table 1
| Ingredient | Dosage |
| T7 Ultra Reaction | 20ul |
| 5xE-PAP Buffer | 20ul |
| MnCl2 | 10ul |
| ATP | 10ul |
| Water | 36ul |
(2) after mixing, 2.5ul mix is drawn as control；4ul E-PAP enzyme is added, 37 DEG C of incubations after mixing
45min。
4) mRNA purification and recovery
Utilize the RNAeasy kit of Qiagen company, purification and recovery mRNA:
(1) template is adjusted to 100ul with NFW water；
(2) the RLT Buffer that 350ul is added is sufficiently mixed uniformly；
(3) dehydrated alcohol of 250ul is added, mixes；
(4) it is transferred in the included pillar of kit, >=8000g is centrifuged 15s；
(5) waste liquid in collecting pipe is abandoned, the Buffer RPE of 500ul, >=8000g is added, is centrifuged 15s；
(6) it repeats the above steps
(7) pillar is transferred to new 2ml collecting pipe, dally 1min；
(8) pillar is put in 1.5ml centrifuge tube, the RFW of 40ul is added, >=8000g, 1min；
(9) it takes 2ul to survey concentration, dispenses mRNA solution to the EP of no RNA enzyme according to the volume of 1-5uL/ pipe according to concentration
MRNA solution after packing is placed in -80 degrees Celsius of refrigerators and frozen by Guan Zhong.
(10) 1 pipe mRNA electrophoresis is taken, is control with the mRNA before tailing, detects the quality and tailing effect (tailing of mRNA
The pillar location of mRNA afterwards should slightly lag behind the mRNA before tailing).Cas9 mRNA is diluted to 20ng/ul using preceding.
4, the acquisition of pig body early embryo, the microinjection of CRISR reagent and transplanting
1) acquisition of pig body early embryo
(1) the 3-5 healthy sexal maturity sows being in heat are chosen, are used as embryo after breeding with healthy sexal maturity boar
Donor sow, and an another standby healthy sexal maturity sow being in heat is as receptor sow；
(2) 24-36h after donor insemination of sows, the Nembutal sodium solution for being 3% by auricular vein implantation quality score
10-15mL or the general anesthesia for realizing donor pig by ventilator using isoflurane, and sow is bound on V-arrangement operating table；
(3) last and third is to nipple in sow along abdomen median line after conventional cleaning, disinfection sow abdomen
Between, hara kiri skin, fascia, muscle and peritonaeum, size incision 5-8cm；
(4) sow ovary, fallopian tubal and part uterus are taken out, visible sow Ovarian surface ovulation point, shows sow at this time
It has ovulated；
(5) it is the grass tube of the both ends 4-6mm passivation by internal diameter, passes through fimbriae tubae portion and be inserted into fallopian tubal；
(6) embryo washing water (PBS+1% fetal calf serum) the about 20mL sufficiently incubated in 38 DEG C of water-baths is extracted with syringe, led to
It crosses intravenous infusion needle and the syringe containing embryo washing water is connected to (its one end containing syringe needle passes through fallopian tubal and uterus with fallopian tube lumen
Fallopian tube lumen is inserted into engaging portion, and the other end is connected with syringe)；
(7) embryo washing water is injected by fallopian tubal by syringe, and with the collection of the 50mL centrifuge tube of sterilizing flow through fallopian tubal, from
It is inserted into the embryo washing water flowed out in the grass tube in fimbriae tubae portion；
(8) embryo washing water of collection is transferred in the sterile petri dish that diameter is 9cm, picks embryo under stereomicroscope,
The embryo picked at this time is generally 1-cell the or 2-cell phase；
(9) Pig embryos picked are placed in the culture solution drop for covering paraffin oil, sufficiently having incubated balance in the incubator
Middle culture is spare, and (Pig embryos culture solution used in present case is PZM-3, and formula is shown in document Biology of
Reproduction,2002,66:112)；
2) microinjection of pig body early embryo:
By the sgRNA of the above-mentioned preparation of step, spCas9 mRNA and homologous recombination template DNA (donor dna), respectively with end
Concentration sgRNA 10ng/uL, spCas9 mRNA 20ng/uL and donor dna 10ng/uL are sufficiently mixed, later by mixed solution
The cytoplasm of pig body early embryo is injected by microinjection.Hogan et al.Manipulating Mouse is shown in operation in detail
Embryo Manipulation Manual,Cold Spring Harbor Laboratory Press,1994,Second
Edition。
3) Pig embryos are transplanted
(1) it anaesthetizes and Baoding receptor sow, cuts abdominal cavity, takes out ovary, defeated ovum according to obtaining identical mode with embryo
Pipe and part uterus；
(2) embryo suction pipe (U.S., Agtech company) will be picked up to be connected with 1mL syringe, and inhaled in advance in picking up embryo suction pipe
Enter one section of air；
(3) under stereomicroscope, embryo's sucking after 20-30 pieces of injection in culture solution is picked up by the syringe of connection
(note in embryo suction pipe: sucking one section of air before liquid section where embryo, and suck one section of liquid again before air, with antifouling
Dye)；
(4) the embryo suction pipe of picking up equipped with embryo is inserted into receptor pig fallopian tubal by umbrella portion, syringe is pushed to import embryo
In fallopian tubal；
(5) receptor pig uterus, fallopian tubal and ovary are put back in abdomen, successively peritoneal suture, muscle, fascia and skin, and
Routine disinfection processing is done to wound；The receptor sow become pregnant can give birth to piglet after full-term.
Two, experimental result:
1, the long single stranded DNA of pig Gjb2 gene mutation
Our early-stage study shows that pig Gjb2 gene contains 2 exons, and wherein it is complete to contain it for Second Exon
Coded sequence.The present invention has selected two in the position of the coded sequence upstream and downstream of pig Gjb2 gene by largely studying
(title of sgRNA is respectively pGjb2-5fs-sgRNA and pGjb2-3fs-sgRNA, recognition site sequence to sgRNA recognition site
Column are shown in Table 5,6) boot sequence of identification target site is shown in Table, and test cuts mediation as template, by double site using long single stranded DNA
The validity of gene editing.The structure of pig Gjb2 gene and the selection in the site sgRNA are referring to Fig. 3.
It designs according to the structure of Fig. 3 long single-stranded DNA templates, is started by artificial synthesized obtain of overall length double-stranded DNA containing T7
The double chain DNA fragment of sub and long single-stranded DNA templates sequence, and be subcloned to cloning vector plasmids (such as pUC19), thus to obtain length
The in-vitro transcription vector plasmid of single-stranded DNA templates.
Carry out above-mentioned overall length double chain DNA fragment synthesis when, by the 37th codon in pig Gjb2 gene coded sequence by
GTG sports ATC (c.109-111GTG > ATC)；The upstream of coding sequence of mutation be inserted into one section of general introne shearing by
Body sequence introduces the flank non-coding at one section of people's Gjb2 gene translation terminator codon 3 ' end in mutant coding sequences downstream respectively
Sequence (3 ' UTR) sequence, using as heterologous sequence (heterology)；The two sides of heterologous sequence in upstream and downstream, accurately according to
Cleavage site of the upstream and downstream sgRNA (Gjb2-5fs-sgRNA/Gjb2-3fs-sgRNA) in pig Gjb2 gene, respectively introduces one
Section pig Gjb2 genome sequence is using as homology arm.(long single donor DNA template sequence is SEQ to specific structure as shown in Figure 4
Shown in ID NO.1).
The base sequence of the non-overstriking of black is upstream and downstream homology arm；The base sequence of scribing line is respectively to import Gjb2 base
Because coded sequence upstream and downstream heterologous sequence (upstream heterologous sequence include general introne shear receptor sequence, splice
acceptor；Downstream heterologous sequence is originated from the 3 ' areas UTR of people Gjb2 gene)；Italicized bases sequence be pGjb2 c.109-
111GTG > ATC (pGJB2 p.V37I) mutant coding sequences；Black overstriking base is T7 promoter complementary series.
It is that template is transcribed in vitro that plasmid, which is transcribed in vitro, with the long single-stranded DNA templates of above-mentioned building, aforementioned according to the present embodiment
Long single stranded DNA prepare experimental procedure, respectively by being transcribed in vitro, reverse transcription and RNaseH enzymic digestion, obtain long single stranded DNA mould
Plate.
It using the long single stranded DNA of above-mentioned preparation as template, is cut by double site, cell is expected to according to machine shown in Fig. 2
System realizes the precise permutation to pig Gjb2 gene coded sequence by " DNA of homologous sequence guidance is repaired " (HDR), introduces pig
The coded sequence (pGJB2 p.V37I CDS, referring to Fig. 5) of Gjb2 gene V37I mutation.
2, control experiment designs
The present embodiment with it is currently used with single strain oligonucleotide (ssODN) be template, it is straight by being carried out to target site
The unit point cutting connect realizes that the scheme of gene editing is control experiment group.In the 109-111 of pig Gjb2 gene coded sequence
At bit base GTG (c.109-111GTG), with three base sequences, the 5 '-TGG-3 ' apart from one, site base for PAM structure,
Choose the sgRNA recognition site (name are as follows: pGjb2-V37-sgRNA for covering c.109-111GTG site；Shearing site position
It sets and sees Fig. 6,6) boot sequence that shearing site sequence is shown in Table 5, sgRNA identification target site is shown in Table.The sgRNA recognition site is located at
Positive-sense strand (coding strand).When designing short single-stranded HDR template DNA (pGjb2-V37I-ssODN), Gjb2 gene coded sequence is taken
109-111 bit base GTG upstream and downstream about 60bp sequence is homology arm and includes c.109-111GTG > ATC mutation justice
Chain-ordering is template sequence (structure is as shown in Figure 6).It is theoretical in Cas9 after pGjb2-V37-sgRNA recognition site cutting DNA
Upper cell can realize c.109-111GTG > ATC mutation importing (ginseng by HDR mechanism using pGjb2-V37I-ssODN as template
See Fig. 6).
The sequence of pGjb2-V37I-ssODN is (SEQ ID NO.2):
Wherein the base sequence of the non-overstriking of black is upstream and downstream homology arm；The base ATC of scribing line overstriking is targeted mutagenesis alkali
Base；The sequence that black overstriking base sequence is covered by pGjb2-V37-sgRNA.
3, gene editing efficiency analysis
3.1 head build the genetic analysis of pig individual
Experimental group 4 head that are born altogether build pig.The upstream and downstream of code area respectively choose one in pig Gjb2 genome sequence respectively
(2) Gjb2-CDS-F/Gjb2-CDS-R, sequence are shown in Table, amplification head builds the gene coding region Gjb2 of pig, are expanded for upstream and downstream primer
Increasing primer size is 931bp.Clip head, which builds pig otic tissues and extracts head, builds pig genomic DNA, and building pig genomic DNA with head is
Template obtains the pcr amplification product of above-mentioned primer.First the direct sample presentation of pcr amplification product is sequenced, it, should if covering peak without sequencing
Individual is homozygote genotype；If there is sequencing set peak, illustrate that the individual is heterozygosis or mosaic gene type, then further by PCR
Product carries out TA clone, and every head builds 15-20 transformed bacteria clone's sample presentation of pig random picking and carries out Sanger sequencing.
2 head of table builds pig genetic analysis the primer
| Primer | Primer sequence |
| Gjb2-CDS-F (SEQ ID NO.3): | 5-TCACCTTTGTGGGTGAGGTTGCGTAAGAA-3 |
| Gjb2-CDS-R (SEQ ID NO.4): | 5-GCTTGACGACGGGAATCTGTATCTGG-3 |
The result shows that being built in pig in 4 head, found by PCR product direct Sequencing, have in 4 individuals 2 have occurred it is pre-
Phase mutation, it is 50% that head, which builds the accurate mutation rate of pig,；PCR product sequencing illustrates that the genotype of its otic tissues is pure without set peak
Genotype is closed, and is all expected precisely mutation.The Genetic Detection result that head builds pig is shown in that Fig. 7, head build pig genotype statistics and be shown in Table 3.
3 head of table builds animal genotypes statistics
| Number | Gender | Genotype |
| G00101 | Female | Gjb2 c.109-111GTG>ATC |
| G00102 | Male | Gjb2 c.109-111GTG>ATC |
| G00103 | Male | WT |
| G00104 | Male | WT |
The gene editing efficiency analysis of 3.2 control groups
According to the above-mentioned site sgRNA and homologous recombination template pGjb2-V37I-ssODN, trial carries out passing through unit point
Cut exon realize pGjb2 c.109-111GTG > ATC mutation.By mode identical with experimental group by pGjb2-V37-
It is early that sgRNA, pGjb2-V37I-ssODN and Cas9 mRNA (concentration is respectively 20ng/uL, 10ng/uL and 10ng/uL) import pig
Phase embryonic cell is simultaneously transplanted in receptor sow；After receptor is become pregnant, early stage fetus is obtained to assess the validity of gene editing.As a result
It was found that: there is no gene editings for target site in 4 fetuses of acquisition.Illustrate that unit point cutting exon is difficult to realize
PGjb2 c.109-111GTG > ATC (pGJB2 p.V37I) mutation.Spy traces it to its cause or may be directly against pig Gjb2 gene
Exon is cut with certain embryonic lethality, and the fetus edited is caused to fail to develop, and Successful development is not
Carry out the fetus of gene editing；Certainly, also having certain may be that selected sgRNA site activity is not strong enough, not be able to achieve
Gene editing.Experimental group and the gene editing efficiency of control group, which summarize, is shown in Table 4.
4 experimental group of table and control group gene editing efficiency
The acquisition of 3.3GJB2 p.V37I homozygous mutation offspring pig
It is bred by the way that the GJB2 p.V37I mutation head of above-mentioned acquisition is built the breeding between pig, obtains 6 offspring (F1
Generation) pig.Mutation target site is expanded using above-mentioned Genetic Detection primer (Gjb2-CDS-F/Gjb2-CDS-R), and directly PCR is produced
Object carries out Sanger sequencing, and discovery: the PCR product sequencing result of all individuals is unimodal, and there is expected Gjb2
C.109-111GTG > ATC mutation, show 5 F1 generation individuals obtained be GJB2 p.V37I homozygous mutation individual (referring to
Fig. 8).The above results prompt head build in pig obtain GJB2 p.V37I mutated-genotype can stablize pass on and can efficiently obtain pure
Close mutated individual.
Recognition site sequence of the table 5sgRNA on pig Gjb2 gene
| Recognition site title | Recognition site sequence |
| Gjb2 gene coded sequence upstream shearing site (SEQ ID NO.5) | 5′-GAATCTGTATCTGGGAGACG-CGG-3′ |
| Gjb2 gene coded sequence downstream shearing site (SEQ ID NO.6) | 5′-GACCAAGCGACACGGCACAG-GGG-3′ |
| Gjb2 gene coded sequence V37 shearing site (SEQ ID NO.7) | 5′-CCGCGTCATGATCCTGATCG-TGG-3′ |
The boot sequence of table 6sgRNA identification target site
| SgRNA title | Boot sequence |
| pGjb2-3fs-sgRNA(SEQ ID NO.8) | 5′-GAAUCUGUAUCUGGGAGACG-3′ |
| pGjb2-5fs-sgRNA(SEQ ID NO.9) | 5′-GACCAAGCGACACGGCACAG-3′ |
| pGjb2-V37-sgRNA(SEQ ID NO.10) | 5′-CCGCGUCAUGAUCCUGAUCG-3′ |
Present invention discover that using long single stranded DNA as template, by double site cutting may be implemented higher efficiency based on HDR's
Animal genome is precisely edited；Based on above-mentioned discovery, the Gjb2 gene mutation model of pig is established, and realizes expected essence
Quasi- mutation.And since cleavage site is in introne, theoretically reduce the influence to animal individual development；And the utilization compareed
Conventional is template, by unit point cutting with single strain oligonucleotide (ssODN), and gene does not occur in identical gene loci
Editor's event, it was demonstrated that the validity and feasibility of used technology of the invention.
Sequence table
<110>army medical university, ground force, the Chinese People's Liberation Army
<120>a kind of that the method for realizing that pig Gjb2 gene coded sequence is precisely edited is cut by double site
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1058
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tttaaaggtt ttgtcctcac ctttgtgggt gaggttgcgt aagaatcagt gtttgcccag 60
gaagaggttt cacgctgcct gctccccctg gtcctgtccc gagcggtcac ccctgctgac 120
atccactttg cctttctctc cacaggaccg tccggaggag aagatggact ggggcgcgct 180
gcagaccatc ctgggcggcg tgaacaagca ctccaccagc atcggcaaga tctggctcac 240
ggtgctcttc atcttccgcg tcatgatcct gatcgtggcg gccaaggagg tgtggggcga 300
cgagcaggcc gacttcatct gcaacaccct gcagcccggc tgcaagaacg tgtgctacga 360
ccactacttc cccatctcgc acatccggct ctgggcgctg cagctcatct tcgtgtccac 420
gcccgccctg ctggtggcca tgcacgtggc ctaccggcga cacgagaaga aaaggaagtt 480
catcaagggc gagatgaaga gcgagttcaa ggacatcgaa gagatcaaga cccagaaggt 540
ccgcatcgag ggcgccctct ggtggaccta caccggcagc atcttcttcc gcatcctctt 600
cgaggccgcc ttcatgtacg tgttctacgt catgtacagc gggttctcca tgcagcgcct 660
ggtcaagtgc aacgcgtggc cttgtcccaa cacggtggac tgcttcgtgt ccaggcccac 720
ggagaagacg gtctttacgg ttttcatggt cgtggtgtcg gggatctgca ttctgctcaa 780
cgtcaccgaa ctgtgctatt tgctcatcag atattgttcc ggaaagtcca agaagccagt 840
gtaacgcatt gcccagttgt tagattaaga aatagacagc atgagaggga tgaggcaacc 900
cgtgctcagc tgtcaaggct ctcccagata cagattcccg tcgtcaagct cccccccacc 960
cgcgaccctc ctgtggccca cgtgggactc cagacgcctc cggctgcggc cccgcggtcg 1020
gccccgcggt ccacaggcgc ctatagtgag tcgtatta 1058
<210> 2
<211> 123
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tccaccagca tcggcaagat ctggctcacg gtgctcttca tcttccgcgt catgatcctg 60
atcgtggcgg ccaaggaggt gtggggcgac gagcaggccg acttcatctg caacaccctg 120
cag 123
<210> 3
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tcacctttgt gggtgaggtt gcgtaagaa 29
<210> 4
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gcttgacgac gggaatctgt atctgg 26
<210> 5
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gaatctgtat ctgggagacg cgg 23
<210> 6
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gaccaagcga cacggcacag ggg 23
<210> 7
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
ccgcgtcatg atcctgatcg tgg 23
<210> 8
<211> 20
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gaaucuguau cugggagacg 20
<210> 9
<211> 20
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gaccaagcga cacggcacag 20
<210> 10
<211> 20
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ccgcgucaug auccugaucg 20
Claims (10)
1. cutting the method precisely edited to pig Gjb2 gene coded sequence by double site, it is characterised in that including following
Step:
A, prepare a pair of sgRNA, wherein the cleavage site of a sgRNA identification Gjb2 gene coded sequence upstream, another
The cleavage site in sgRNA identification gene coded sequence downstream；
The sgRNA of the identification Gjb2 gene coded sequence downstream cleavage site can identify the site PAM on the Gjb2 gene of pig
For CGG；
The sgRNA of the identification Gjb2 gene coded sequence upstream cleavage site can identify the site PAM on the Gjb2 gene of pig
For GGG.
B, by a pair of of sgRNA described in step a, the donor dna with expected mutant nucleotide sequence, the mRNA for encoding spCas9 and/or
After spCas9 purification of recombinant proteins is sufficiently mixed, mixture is imported by microinjection in the cytoplasm of pig body early embryo；
C, by step b, treated that pig body early embryo migrates in the fallopian tubal of receptor sow, and receptor sow is made to become pregnant；
D, the receptor sow become pregnant gives birth to piglet after full-term, obtains Gjb2 gene precisely edited pig model.
2. according to claim 1 cut the side precisely edited to pig Gjb2 gene coded sequence by double site
Method, it is characterised in that the sgRNA centering:
The boot sequence for identifying the sgRNA of pig Gjb2 gene coded sequence downstream cleavage site is 5 '-
GAAUCUGUAUCUGGGAGACG-3 ' (SEQ ID NO.8) or its complementary series；
The boot sequence for identifying the sgRNA of pig Gjb2 gene coded sequence upstream cleavage site is 5 '-
GACCAAGCGACACGGCACAG-3 ' (SEQ ID NO.9) or its complementary series.
3. according to claim 1 cut the side precisely edited to pig Gjb2 gene coded sequence by double site
Method, it is characterised in that: it is isoleucine that the donor dna, which is by 37 valine (V) codon mutations of pig Gjb2 gene,
(I) donor dna of codon.
4. according to claim 1 cut the side precisely edited to pig Gjb2 gene coded sequence by double site
Method, it is characterised in that: the nucleotides sequence of the donor dna is classified as shown in SEQ ID No.1 or is its complementary series.
5. according to claim 1 cut the side precisely edited to pig Gjb2 gene coded sequence by double site
Method, it is characterised in that: the sgRNA is to, donor dna and encodes mRNA and/or spCas9 the purifying recombination egg of spCas9
Proportion between white is pure by 10~15ng/uL of final concentration sgRNAs, spCas9 mRNA and/or spCas9 in the mixture
Change 20~25ng/uL of recombinant protein and 5~10ng/uL of donor dna.
6. the method according to claim 1, it is characterised in that: embryo of the pig body early embryo for 4 cell stages and before.
7. according to method for claim 9, it is characterised in that further include having detection verification step in the step c: using primer
To amplification piglet genome DNA sample, determined whether according to amplification sequencing containing expected gene editing genotype
Gjb2c.109-111GTG > ATC, if showing to model successfully containing the genotype.
8. preparing the kit of Gjb2 gene mutation pig model, it is characterised in that including as follows sgRNA pairs of boot sequence:
5′-GAAUCUGUAUCUGGGAGACG-3′；
5′-GACCAAGCGACACGGCACAG-3′。
9. the kit according to claim 8 for preparing Gjb2 gene mutation pig model, it is characterised in that further include by pig
37 valine (V) codon mutations of Gjb2 gene are the donor dna of isoleucine (I) codon, and nucleotides sequence is classified as
It shown in SEQ ID No.1 or is its complementary series.
10. the kit for preparing Gjb2 gene mutation pig model according to claim 8 or claim 9, it is characterised in that: further include
Encode mRNA the and/or spCas9 recombinant protein of spCas9 albumen.
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| CN110272900B (en) * | 2019-04-19 | 2024-03-26 | 中国人民解放军陆军军医大学 | sgRNA for preparing skeletal dysplasia pig model and application thereof |
| RU2780677C1 (en) * | 2021-12-28 | 2022-09-29 | Федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр акушерства, гинекологии и перинатологии имени академика В.И. Кулакова" Министерства здравоохранения Российской Федерации | METHOD FOR EDITING THE GJB2 GENE TO CORRECT THE PATHOGENIC VARIANT OF c.del35G IN HUMAN CELLS CULTURED IN VITRO |
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