CN109082427A - The zebra fish msi1 gene mutation body and its construction method constructed using CRISPR-Cas9 - Google Patents
The zebra fish msi1 gene mutation body and its construction method constructed using CRISPR-Cas9 Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, and in particular to a kind of zebra fish msi1 gene mutation body for constructing zebra fish msi1 gene mutation body using CRISPR-Cas9 and being constructed using this method.Which includes the design of Cas9-gRNA target site on msi1 locus, the synthesis of Cas9mRNA and msi1-gRNA, and microinjection in vivo and subsequent genetic screening.The sequence of the target site is located on second exon of msi1 gene；A pair of primer comprising target site is designed simultaneously, passes through the verifying that PCR carries out target position point mutation.The verification mode is equally applicable to subsequent genetic screening.Msi1 zebra fish mutant of the present invention using the building of CRISPR-Cas9 technology shows high mutation rate, and mutation causes the termination in advance of msi1 protein translation.It is just obtained in addition, msi1 Gene Mutated Zebrafish of the present invention only passes through continuous two generations screening.Present invention obtains the msi1 mutant zebra fish of 2 kinds of mutation types systems.
Description
Technical field
The present invention relates to molecular biology fields, in particular to a kind of to construct zebra fish msi1 base using CRISPR-Cas9
Method because of mutant and the zebra fish msi1 gene mutation body using this method building.
Background technique
1 type of Musashi rna binding protein (Musashi RNA binding protein 1) is one kind by Msi1 gene
The albumen of coding.The albumen includes that two conservative series connection RNA identify motif, and participate in turning for gene as rna binding protein
Regulate and control after record.So that stem cell is kept undifferentiated state by adjusting the translation process after transcribing, passes through and adjust Notch, Wnt/
The signal paths such as β-catenin influence the proliferation of stem cell and the decision of cell fate and the formation of tumour, are a kind of control
The stem cell labeling object balanced between self-renewing and terminal differentiation.MSI1 is highly expressed in neural progenitor cell, for big
The normal development of brain is particularly significant.The mutation of the gene leads to autosomal recessive primary microcephaly.In addition, the gene
Being overexpressed also has relationship with the grade malignancy and proliferation activity of glioma and melanoma.And the expression for reducing Msi1 gene can
Effective induced cancer Apoptosis, prevents mitosis and cell cycle progression, inhibits tumor cell proliferation and tumour is caused to disappear
It moves back.Study Msi1 function to clinical tumor disease deeply probe into and diagnoses and treatment may provide new approach.
Zebra fish is a kind of for studying the model organism of vertebrate embryology and phenogenetics, is quickly changed based on it
The advantage in generation can perform well in the research of cancer.The zebra fish of research in to(for) msi1 gene function at present
It is still limited, this patent establishes the mutant of zebra fish msi1 by CRSPR-Cas9, for the function of further research msi1
Material foundation can be provided.
CRISPR/Cas9 is a kind of adaptive immunity defence that bacterium and archeobacteria are formed during long-term evolution, can
For fighting the virus and exogenous DNA of invasion.CRISPR refers to the short palindrome repetitive sequence (Clustered in the interval of regular cluster
Regularly interspaced short palindromic repeat, CRISPR), it is by Japanese Scientists earliest
Ishino has found in Escherichia coli.Cas then refers to CRISPR GAP-associated protein GAP, is generally located at CRISPR location proximate, is
One there are many protein families of isoform polypeptide, and Cas9 is a hypotype being most widely used.In January, 2013, Woong Y
The technology is applied in zebra fish gene editing by Hwang etc. for the first time.By external source import target gene group gRNA and
DNA double chain near Cas9mRNA, gRNA, that is, bootable Cas9 Protein cleavage genomic targets, and then the mispairing for causing cell is repaired
It is multiple, to cause gene mutation.
Summary of the invention
Present invention aims at construct the mutant of zebra fish msi1 gene.
For the purpose for realizing above-mentioned building zebra fish msi1 gene mutation body, present invention provide the technical scheme that
1. designing Cas9 target site according to gene order；
2. confirming specificity of the target site in zebra fish genome；
3. micro- co-injection msi1-gRNA and Cas9mRNA verifies mutation efficiency to zebrafish embryo；
4. raising F0 embryo to sexal maturity and wild type diplomacy, F1 embryo mutation type is detected；
5. raising F1 embryo to 1 monthly age, identifies mutation type, raised in identical mutation set of types；
6. identical mutation type F1 adult fish is selfed, F2 embryo is obtained, grows into 1 monthly age identification homozygote；
7. obtaining the homozygous F2 adult fish of 2 kinds of mutation types, selfing obtains the Mutant progeny for stablizing heredity.
By the present invention, at least can achieve it is following the utility model has the advantages that
1. obtaining msi1 homozygous mutation body；
2. providing material for research msi1 gene function；
3. the gene mutation body can be used for inhibiting tumour cells research；
4. the gene mutation body to clinical tumor disease deeply probe into and diagnoses and treatment may provide new approach.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, with presently preferred embodiments of the present invention and cooperate symbol figure below detailed description are as follows.
Detailed description of the invention
Fig. 1 is msi1 mutation construction schematic diagram.Illustrate msi1 mutation construction schematic diagram, it is shown that msi1 locus 21
A exon, design Cas9 target site are located on the 2nd exon, and green vertical line indicates translation initiation codon ATG, and blue is perpendicular
Line indicates terminator codon TGA；Centre shows target site mutation type, respectively lacks several bases and missing base；Under
Figure shows that the amino acid length of prediction, wild-type protein amino acid are 297, and the msi1 protein translation of mutant is all whole in advance
Only, lacking 7 bases and 4 base mutation type orresponding amino acids is respectively 152 and 112.
Fig. 2 is the electrophoretic band result figure of msi1-gRNA.The electrophoretic band of msi1-gRNA is illustrated as a result, band is single,
And meet expected size, about 130bp.
Fig. 3 is second exon sequence of zebra fish msi1 gene.The present invention constructs Cas9 target position designed by mutant
Furthermore point, which is located at, to be shown on son.
Fig. 4 is msi1 gene C as9 target sequence.It illustrates this present invention and constructs Cas9 target site sequence designed by mutant.
Fig. 5 is that the mutational site msi1 PCR identifies sequence.PCR designed by diagram present invention building mutant identifies sequence,
Sequence length is 130bp.
Fig. 6 is msi1 identified for genes primer P1 sequence.Draw PCR identification upstream designed by diagram present invention building mutant
Object.
Fig. 7 is msi1 identified for genes primer P2 sequence.Draw in PCR identification downstream designed by diagram present invention building mutant
Object.
Fig. 8 is msi1 gene gRNA synthetic primer P3 sequence.In gRNA synthesis designed by diagram present invention building mutant
Swim primer.
Fig. 9 is msi1 gene order.The gene order of diagram present invention building mutant, wherein blue sequence is albumen volume
Code sequence, is located on exon；Gray system is intron sequences；Brown sequence is exon non-translational region.
Specific embodiment
With reference to the accompanying drawings and examples, the specific embodiment of the invention is described in further detail.Implement below
Example is not used in and limits the scope of the invention for illustrating the present invention.
Embodiment 1
A preferred embodiment of the present invention provides a kind of side using CRISPR-Cas9 building zebra fish msi1 gene mutation body
Method, specifically includes the following steps:
1. firstly, design msi1 gene C as9 target site, the method is as follows:
A. the albumen coded sequence (CDS) of msi1 is obtained from the website Ensemble, as shown in Fig. 9；
B. it useshttp://zifit.partners.org/ZiFiT/CSquare9Nuclease.aspxWebsite design target
Website is opened in site, inputs msi1 albumen coded sequence；
C. target site length is set as 20 bases, and 3 bases that target spot 3 ' is held constitute the area PAM, and sequence is that (N is to appoint to NGG
Meaning base), export target site sequence；
The Cas9 target site sequence of D.msi1 gene are as follows:
5 '-GGCCCAGACCAGACATGAGCTGG-3 ' are positive-sense strand target spot, are located at the 2nd exon, such as attached drawing 3-4
It is shown.
2. further, the method for the confirmation msi1-Cas9 target site is as follows:
A. target site sequence is compared on the website Ensemble, comparison result shows that target spot is the single position of msi1 gene
Point；
B. in target site upstream and downstream design primer,
5 ' end primers are 5 '-TAATTTTGATCTCTCTGTTGTC-3 ', as shown in Fig. 6,
3 ' end primers are 5 '-TTAATATGATTAAAACTCAC-3 ', and as shown in Fig. 7, primer size is 123 bases
It is right, this section of sequence of PCR amplification from wild-type zebrafish genome, wherein should include target sequence, PCR program are as follows: 95 DEG C are pre-
It is denaturalized 5min, 95 DEG C of denaturation 30sec, 60 DEG C of annealing 30sec, 72 DEG C of extension 10sec, 40 recycle.2% agar of pcr amplification product
Sugared gel electrophoresis, size meet expection；
C. PCR product is sequenced, sequencing result is consistent with prediction target sequence.
3. it is further, prepare msi1-gRNA, the specific steps are as follows:
A. the upstream primer of template is transcribed in vitro according to target position point design gRNA, is named as P3, sequence is 5 '-GATCAC
TAATACGACTCACTATAGGCCCAGACCAGACATGAGCGTTTTAGAGCTAGAAAT-3 ', as shown in Fig. 8, downstream draw
Object is named as P4 using the universal primer on pT7-gRNA plasmid.
B. high fidelity enzyme PCR amplification is used so that carrier pT7-gRNA plasmid is transcribed in vitro as template with P3 and P4 primer pair
It obtains msi1-gRNA and template, PCR system is transcribed in vitro are as follows: 1 microlitre of template, 25 microlitres of high-fidelity buffer, dNTPs12 microlitres,
1 microlitre of high fidelity enzyme, each 1 microlitre of primer, distilled water polishing is to 50 microlitres.PCR condition are as follows: 98 DEG C of initial denaturation 3min, 98 DEG C of changes
Property 30sec, 60 DEG C of annealing 30sec, 68 DEG C of extension 30sec, 40 circulation.1 microlitre of PCR product electrophoresis is taken, is as a result confirmed as single
Band (130bp) recycles PCR product, obtains transcription templates after purification with phenol-chloroform method.
C. msi1-gRNA, reaction system is transcribed in vitro with T7 transcript reagent box are as follows: 1 microgram of template, NTP4 microlitres, buffering
2 microlitres of liquid, 1 microlitre of T7 enzyme, DEPC water is mended to 20 microlitres.Reaction condition is 37 DEG C, 1h；Then 1 microlitre of DNA enzymatic (TURBO is added
DNase), 37 DEG C, 15min is to remove DNA profiling.Take 1 microlitre of transcription product electrophoresis, primer size about 100bp or so, such as attached drawing
Shown in 2, meet expection, obtains gRNA with phenol-chloroform method purification and recovery.
D. phenol-chloroform method purification process are as follows: by recovery product with DEPC water polishing to 200 microlitres, 1:1 is added by volume
Phenol-chloroform, 12,000g is centrifuged 10min after oscillation mixes, and supernatant is taken to go in 1.5 milliliters of centrifuge tubes of RNA enzyme in new, then plus
The isopropanol for entering 2.5 times of volumes is mixed by inversion and is placed on -80 DEG C, takes out after standing 30min or more, 12,000g centrifugation 15min,
Remove supernatant, leaving white precipitate is gRNA, and 200 microlitre 75% of ethyl alcohol is added and cleans gRNA, then removes ethyl alcohol, will
Centrifuge tube, which is uncapped, to be placed in several minutes of superclean bench, and 30 microlitres of DEPC water dissolutions are added after drying gRNA, and obtained gRNA is molten
Liquid preservation set -80 DEG C it is spare.
4. it is further, prepare Cas9mRNA, the specific steps are as follows:
A. pSP6-spCas9 carrier is linearized with restriction enzyme, takes 0.5 microlitre of digestion products electrophoresis, confirmation is linear
Recycling after changing completely, and carry out purification process.
B. SP6 in-vitro transcription kit transcription linear carrier is used, system is transcribed are as follows: 10 microlitres of 2x NTP/CAP, 2 microlitres
10x buffer, 2 microlitres of SP6 transcriptases, 1 microgram carrier, DEPC water polishing is to 20 microlitres.Reaction condition is 37 DEG C, 2h, immediately
1 microlitre of DNA enzymatic is added, removes DNA profiling.Purification and recovery obtains Cas9mRNA, takes 0.5 microlitre of product electrophoresis, and 1% agarose is solidifying
Glue, primer size about 2,000bp.
C.Cas9mRNA adds ployA sequence, system are as follows: the mRNA of recycling, 5 microlitres of 10mM ATP, 5 microlitres of 10x bufferings
Liquid, 1 microlitre of E.coli Poly (A) polymerase, DEPC water are mended to 50 microlitres.Reaction condition is 37 DEG C, 1h.Purification and recovery obtains
Cas9mRNA-ployA takes 0.5 microlitre of product electrophoresis, 1% Ago-Gel, primer size about 2,000bp.
5. further, in the preparation RNA experiment, each section tests consumptive material and reagent used and all goes RNA enzyme,
It is not known in experiment and shows temperature, product and reagent should be maintained at 4 DEG C hereinafter, avoiding RNA degradation.
6. further, the preparation F0 zebra fish simultaneously detects msi1 gene target mutation efficiency, passes through microinjection
It is completed with PCR sequencing, the specific method is as follows:
A. it is pre-configured with injection system, including Cas9mRNA and msi1-gRNA, 1/10 volume of addition is phenol red, as instruction
Agent is injected into one cell stage, injects about 200 pieces of embryos, and injection dosage is Cas9mRNA 300pg, gRNA 50-
100pg。
B. the 2nd day 10 pieces of normotrophic embryo after injecting is taken, embryo alkaline lysis method of extracting genomic DNA: is placed in PCR
100 microlitres of 50mM NaOH solutions are added in Guan Zhong, and PCR instrument is heated to 95 DEG C, and 30min cracks embryo, and shaken well after taking-up adds
Enter in the Tris-HCl of 1/10 volume pH8.0 and NaOH.
C. primer P1 and P2 amplifying genom DNA is used, PCR product is connected in carrier T by TA clone, sequencing detection
Mutation type, there are two types of mutation types as the result is shown, respectively lack 7 bases and 4 bases, as shown in Fig. 1.It will mutation
The higher F0 embryonic feeder of efficiency gets up, and screens heritable mutation for subsequent detection.
7. further, the method for the detection F0 zebra fish reproduction heredity is as follows:
A. sexually matured F0 adult fish and wild type adult fish diplomacy are taken, F1 embryo is obtained, selects 5-10 embryo's mixing at random
Extract genomic DNA.
B. PCR amplification target site segment is used, PCR product determines its mutation type by TA cloning and sequencing.Show F0 zebra
Fish msi1 gene mutation can be hereditary, then the F1 of the heritable mutation of raising, in case of subsequent screening.
8. further, described confirmation F1 zebra fish msi1 gene mutation type, the method is as follows:
A.F1 grows to the 1-2 monthly age, and clip tail fin extracts genomic DNA.
B. by PCR analysis and TA cloning and sequencing, F1 zebra fish msi1 gene mutation type is further confirmed that, screening obtains
The F1 zebra fish of 2 kinds of mutation types, is named as MT-1 and MT-2, and the F1 of identical mutation type can mix raising.
9. further, described screening F2 zebra fish msi1 homozygous mutation body, specific method is: by identical mutation
F1 adult fish be selfed, obtain F2 embryo, raising to 1-2 monthly age mentions genome, PCR, sequencing confirmation F2 by cutting tail fin
Zebra fish homozygous mutation.After obtaining the homozygote of identical mutation, a large amount of homozygous mutation offsprings can be obtained by selfing, that is, establish
The msi1 gene mutation body that can stablize heredity is played.
Embodiment 2
A preferred embodiment of the present invention provides a kind of zebra fish msi1 gene mutation body constructed using CRISPR-Cas9,
It is typically characterized by:
1. msi1 gene mutation body described in includes two strains, has different mutation types；
2. msi1 gene mutation body described in, mutation type are respectively to lack 7 bases and 4 bases；
3. msi1 gene mutation body described in, mutation missing base are located near target site；
4. msi1 gene mutation body described in, mutant nucleotide sequence are as shown in Fig. 1；
5. msi1 gene mutation body described in, protein translation terminate in advance, and wild type msi1 albumen has 896 amino
Acid, and in mutant, it lacks 7 bases and 4 bases has respectively corresponded 426 amino acid and 258 amino acid.
6. msi1 gene mutation body described in, the function for further research msi1 provide material foundation, facilitate
The prognosis for the treatment of of cancer and functional dependency etc. play a role.
The above is only preferred implementation method of the invention, is not intended to restrict the invention, it is noted that for this hair
For the those of ordinary skill in bright field, without departing from the technical principles of the invention, can also make it is several improvement and
Deformation, those modifications and variations should also be regarded as the protection scope of the present invention.
The present invention is by state natural sciences fund (young Projects --- 81502500), Jiangsu Province's natural science
Fund (young Projects --- BK20150293) and scientific and technological (the health care applied basic research item of Suzhou City's people's livelihood
Mesh --- SYS2018074) subsidy is provided.
Claims (9)
1. a kind of method using CRISPR-Cas9 technology building zebra fish msi1 gene mutation body, which is characterized in that described prominent
Variant building includes the following steps:
(1) Cas9 target site is selected on msi1 gene；
(2) it is analyzed to identify msi1-Cas9 target site；
(3) Cas9 mRNA and msi1-gRNA are prepared；
(4) it prepares F0 zebra fish and detects msi1 gene target mutation efficiency；
(5) reproduction heredity of F0 zebra fish is detected；
(6) confirm F1 zebra fish msi1 gene mutation type；
(7) F2 zebra fish msi1 homozygous mutation body is screened.
2. the method according to claim 1 using CRISPR-Cas9 technology building zebra fish msi1 gene mutation body,
It is characterized in that, the msi1 gene C as9 target position design method of points is as follows:
(1) albumen coded sequence (CDS) of msi1 is obtained from the website Ensemble；
(2) 4.2 website design target site of ZiFiT Targeter Version is used, website is opened, inputs msi1 encoding histone
Sequence；
(3) target site length is set as 20 bases, and 3 bases that target spot 3 ' is held constitute the area PAM, and sequence is that (N is any to NGG
Base), export target site sequence；
(4) the Cas9 target site sequence of msi1 gene are as follows:
5 '-GGCCCAGACCAGACATGAGCTGG-3 ' are positive-sense strand target spot, are located at the 2nd exon.
3. the method according to claim 1 using CRISPR-Cas9 building zebra fish msi1 gene mutation body, feature
It is, the method for the confirmation msi1-Cas9 target site is as follows:
(1) target site sequence is compared on the website Ensemble, comparison result shows that target spot is the single site of msi1 gene；
(2) in target site upstream and downstream design primer
5 ' end primers are that 5 '-TAATTTTGATCTCTCTGTTGTC-3 ' are named as P1,
3 ' end primers are that 5 '-TTAATATGATTAAAACTCAC-3 ' are named as P2,
Primer size is 130 base-pairs, this section of sequence of PCR amplification from wild-type zebrafish genome, wherein should include target
Point sequence, amplified production electrophoresis size meet expection；
(3) PCR product is sequenced, sequencing result is consistent with prediction target sequence.
4. the method according to claim 1 using CRISPR-Cas9 technology building zebra fish msi1 gene mutation body,
It is characterized in that, the method for preparing msi1-gRNA and Cas9 mRNA is as follows:
(1) msi1-gRNA is prepared, the specific steps are as follows:
A. according to the upstream primer of target position point design gRNA transcription templates, it is named as P3, sequence is 5 '-GATCACTAATACGA
CTCACTATAGGCCCAGACCAGACATGAGCGTTTTAGAGCTAGAAAT-3 ', downstream primer use on pT7-gRNA plasmid
Universal primer, be named as P4；
B. it is obtained so that carrier pT7-gRNA plasmid is transcribed in vitro as template using high fidelity enzyme PCR amplification with P3 and P4 primer pair
Template, PCR system is transcribed in vitro in msi1-gRNA are as follows: and 1 microlitre of template, 25 microlitres of high-fidelity buffer, dNTPs12 microlitres, Gao Bao
1 microlitre of true enzyme, each 1 microlitre of primer, distilled water polishing is to 50 microlitres；PCR condition are as follows: 98 DEG C of initial denaturation 3min, 98 DEG C of denaturation
30sec, 60 DEG C of annealing 30sec, 68 DEG C of extension 30sec, 40 recycle；1 microlitre of PCR product electrophoresis is taken, single item is as a result confirmed as
Band (123bp) recycles PCR product, obtains transcription templates after purification with phenol-chloroform method；
C. msi1-gRNA, reaction system are as follows: 1 microgram of template, NTP4 microlitres, buffer 2 is micro- is transcribed in vitro with T7 transcript reagent box
It rises, 1 microlitre of T7 enzyme, DEPC water is mended to 20 microlitres；Reaction condition is 37 DEG C, 1h；Then 1 microlitre of DNA enzymatic (TURBO is added
DNase), 37 DEG C, 15min is to remove DNA profiling；1 microlitre of transcription product electrophoresis is taken, primer size about 100bp or so meets pre-
Phase obtains gRNA with phenol-chloroform method purification and recovery；
D. phenol-chloroform method purification process are as follows: by recovery product with DEPC water polishing to 200 microlitres, phenol-is added in 1:1 by volume
Chloroform, 12,000g is centrifuged 10min after oscillation mixes, and takes supernatant to go in 1.5 milliliters of centrifuge tubes of RNA enzyme in new, adds
The isopropanol of 2.5 times of volumes is mixed by inversion and is placed on -80 DEG C, takes out after standing 30min or more, and 12,000g centrifugation 15min are gone
Fall supernatant, leaving white precipitate is gRNA, and 200 microlitre 75% of ethyl alcohol is added and cleans gRNA, then removes ethyl alcohol, will be from
Heart pipe, which is uncapped, to be placed in several minutes of superclean bench, and 30 microlitres of DEPC water dissolutions, the gRNA solution that will be obtained is added after drying gRNA
Preservation set -80 DEG C it is spare；
(2) Cas9 mRNA is prepared, the specific steps are as follows:
A. pSP6-spCas9 carrier is linearized with restriction enzyme, takes 0.5 microlitre of digestion products electrophoresis, confirmation has linearized
Recycling after complete, and carry out purification process；
B. SP6 in-vitro transcription kit transcription linear carrier is used, system is transcribed are as follows: 10 microlitres of 2xNTP/CAP, 2 microlitres of 10x are slow
Fliud flushing, 2 microlitres of SP6 transcriptases, 1 microgram carrier, DEPC water polishing is to 20 microlitres；Reaction condition is 37 DEG C, and 2h is added 1 immediately
Microlitre DNA enzymatic removes DNA profiling；Purification and recovery obtains Cas9 mRNA, takes 0.5 microlitre of product electrophoresis, 1% Ago-Gel,
Primer size about 2,000bp；
C.Cas9 mRNA adds ployA sequence, system are as follows: the mRNA of recycling, 5 microlitres of 10mM ATP, 5 microlitres of 10x buffers, and 1
Microlitre E.coli Poly (A) polymerase, DEPC water are mended to 50 microlitres；Reaction condition is 37 DEG C, 1h；Purification and recovery obtains Cas9
MRNA-ployA takes 0.5 microlitre of product electrophoresis, 1% Ago-Gel, primer size about 2,000bp；
(3) each section tests consumptive material and reagent used and all goes RNA enzyme, is not known in experiment and shows temperature, product and examination
Agent should be maintained at 4 DEG C hereinafter, avoiding RNA degradation.
5. the method according to claim 1 using CRISPR-Cas9 technology building zebra fish msi1 gene mutation body,
Be characterized in that, the preparation F0 zebra fish simultaneously detects msi1 gene target mutation efficiency, by microinjection and PCR sequencing come
It completes, the specific method is as follows:
(1) it is pre-configured with injection system, including Cas9 mRNA and msi1-gRNA, it is phenol red to be added 1/10 volume, as indicator,
It is injected into one cell stage, injects about 200 pieces of embryos, injection dosage is Cas9 mRNA 300pg, gRNA 50-100pg；
(2) the 2nd day 10 pieces of normotrophic embryo after injecting is taken, embryo alkaline lysis method of extracting genomic DNA: is placed in PCR pipe
In, 100 microlitres of 50mM NaOH solutions are added, PCR instrument is heated to 95 DEG C, and 30min cracks embryo, and shaken well after taking-up is added
In the Tris-HCl of 1/10 volume pH8.0 and NaOH；
(3) primer P1 and P2 amplifying genom DNA is used, PCR product is connected in carrier T by TA clone, sequencing detection is prominent
Become type；The higher F0 embryonic feeder of mutation efficiency is got up, screens heritable mutation for subsequent detection.
6. the method for the zebra fish msi1 gene mutation body according to claim 1 using the building of CRISPR-Cas9 technology,
It is characterized in that, the method for the detection F0 zebra fish reproduction heredity is as follows:
(1) sexually matured F0 adult fish and wild type adult fish diplomacy are taken, F1 embryo is obtained, 5-10 embryo is selected at random and mixes extraction
Genomic DNA；
(2) PCR amplification target site segment is used, PCR product determines its mutation type by TA cloning and sequencing；Show F0 zebra fish
Msi1 gene mutation can be hereditary, then the F1 of the heritable mutation of raising, in case of subsequent screening.
7. the method according to claim 1 using CRISPR-Cas9 technology building zebra fish msi1 gene mutation body,
It is characterized in that, the confirmation F1 zebra fish msi1 gene mutation type, the method is as follows:
(1) F1 grows to the 1-2 monthly age, and clip tail fin extracts genomic DNA；
(2) by PCR analysis and TA cloning and sequencing, F1 zebra fish msi1 gene mutation type is further confirmed that, screening obtains 2 kinds
The F1 zebra fish of mutation type, is named as MT-1 and MT-2, and the F1 of identical mutation type can mix raising.
8. the method according to claim 1 using CRISPR-Cas9 technology building zebra fish msi1 gene mutation body,
Be characterized in that, the screening F2 zebra fish msi1 homozygous mutation body, specific method is, by the F1 adult fish of identical mutation into
Row selfing, obtains F2 embryo, and raising to 1-2 monthly age mentions genome, PCR, sequencing confirms that F2 zebra fish is homozygous by cutting tail fin
Mutation；After obtaining the homozygote of identical mutation, a large amount of homozygous mutation offsprings can be obtained by selfing, that is, establishing can stablize
The msi1 gene mutation body of heredity.
9. a kind of zebra fish msi1 gene mutation body, which is characterized in that the zebra fish msi1 gene mutation body is using right
It is required that the method for any one of 1-8 constructed.
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