CN104928321B - A kind of scale loss zebra fish pattern and method for building up by Crispr/Cas9 inductions - Google Patents
A kind of scale loss zebra fish pattern and method for building up by Crispr/Cas9 inductions Download PDFInfo
- Publication number
- CN104928321B CN104928321B CN201510071972.4A CN201510071972A CN104928321B CN 104928321 B CN104928321 B CN 104928321B CN 201510071972 A CN201510071972 A CN 201510071972A CN 104928321 B CN104928321 B CN 104928321B
- Authority
- CN
- China
- Prior art keywords
- eda
- zebra fish
- scale
- sgrna
- cas9
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108091033409 CRISPR Proteins 0.000 title claims abstract description 47
- 241000252212 Danio rerio Species 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 19
- 230000006698 induction Effects 0.000 title claims description 6
- 101150053890 EDA gene Proteins 0.000 claims abstract description 17
- 238000003780 insertion Methods 0.000 claims abstract description 12
- 230000037431 insertion Effects 0.000 claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 12
- 238000012216 screening Methods 0.000 claims abstract description 7
- 210000004209 hair Anatomy 0.000 claims abstract description 6
- 239000003814 drug Substances 0.000 claims abstract description 4
- 229940079593 drug Drugs 0.000 claims abstract description 4
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 25
- 101000880080 Homo sapiens Ectodysplasin-A Proteins 0.000 claims description 21
- 238000012408 PCR amplification Methods 0.000 claims description 21
- 238000013518 transcription Methods 0.000 claims description 20
- 230000035897 transcription Effects 0.000 claims description 20
- 238000000338 in vitro Methods 0.000 claims description 18
- 230000035772 mutation Effects 0.000 claims description 17
- 108020004999 messenger RNA Proteins 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- 238000012163 sequencing technique Methods 0.000 claims description 14
- 238000004043 dyeing Methods 0.000 claims description 8
- 239000012634 fragment Substances 0.000 claims description 8
- 108020004414 DNA Proteins 0.000 claims description 7
- 239000000969 carrier Substances 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 7
- 238000001556 precipitation Methods 0.000 claims description 7
- 238000012795 verification Methods 0.000 claims description 7
- 238000013461 design Methods 0.000 claims description 6
- 210000002257 embryonic structure Anatomy 0.000 claims description 6
- 210000004027 cell Anatomy 0.000 claims description 5
- 238000011161 development Methods 0.000 claims description 5
- 230000018109 developmental process Effects 0.000 claims description 5
- 206010064571 Gene mutation Diseases 0.000 claims description 4
- 101710137500 T7 RNA polymerase Proteins 0.000 claims description 4
- 230000007547 defect Effects 0.000 claims description 4
- 108700028369 Alleles Proteins 0.000 claims description 3
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 claims description 3
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 claims description 3
- 206010058314 Dysplasia Diseases 0.000 claims description 3
- 230000013020 embryo development Effects 0.000 claims description 3
- 238000000520 microinjection Methods 0.000 claims description 3
- 102000002322 Egg Proteins Human genes 0.000 claims description 2
- 108010000912 Egg Proteins Proteins 0.000 claims description 2
- 210000004681 ovum Anatomy 0.000 claims description 2
- 210000003981 ectoderm Anatomy 0.000 claims 1
- 208000031068 ectodermal dysplasia syndrome Diseases 0.000 abstract description 6
- 208000002169 ectodermal dysplasia Diseases 0.000 abstract description 5
- 102100037354 Ectodysplasin-A Human genes 0.000 description 16
- 239000000047 product Substances 0.000 description 16
- 210000003491 skin Anatomy 0.000 description 12
- 241000251468 Actinopterygii Species 0.000 description 5
- 235000013601 eggs Nutrition 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 102000008186 Collagen Human genes 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 229920001436 collagen Polymers 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 4
- 230000008685 targeting Effects 0.000 description 4
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 description 3
- 102000004961 Furin Human genes 0.000 description 3
- 108090001126 Furin Proteins 0.000 description 3
- 241000982642 Gasterosteus aculeatus Species 0.000 description 3
- 108020005004 Guide RNA Proteins 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 210000000106 sweat gland Anatomy 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241001580935 Aglossa pinguinalis Species 0.000 description 2
- 101150031037 EDARADD gene Proteins 0.000 description 2
- 102100030809 Ectodysplasin-A receptor-associated adapter protein Human genes 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 101000920026 Homo sapiens Tumor necrosis factor receptor superfamily member EDAR Proteins 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 102100030810 Tumor necrosis factor receptor superfamily member EDAR Human genes 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000013505 freshwater Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000003161 ribonuclease inhibitor Substances 0.000 description 2
- 210000004243 sweat Anatomy 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 241000271566 Aves Species 0.000 description 1
- 241000448255 Congiopodus peruvianus Species 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000283070 Equus zebra Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 1
- 208000008017 Hypohidrosis Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101100332755 Mus musculus Edar gene Proteins 0.000 description 1
- 241000123069 Ocyurus chrysurus Species 0.000 description 1
- 241000276569 Oryzias latipes Species 0.000 description 1
- 101100332756 Oryzias latipes edar gene Proteins 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 108010065868 RNA polymerase SP6 Proteins 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 208000021018 autosomal dominant inheritance Diseases 0.000 description 1
- 230000002902 bimodal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 210000003499 exocrine gland Anatomy 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 210000003746 feather Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 210000003780 hair follicle Anatomy 0.000 description 1
- 210000003026 hypopharynx Anatomy 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 210000004175 meibomian gland Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000032724 odontogenesis Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 230000006128 skin development Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of scale loss zebra fish pattern induced by Crispr/Cas9, which refers to the scale loss zebra fish for including the 4 site base insertion of EDA gene extrons.Meanwhile the method for building up the invention also discloses the pattern and application.The scale loss zebra fish pattern that the present invention establishes has very big application value in ectodermal dysplasia drugs such as the functional study of appendages of skin related gene, screening treatment human hair's rareness etc..
Description
Technical field
The present invention relates to gene engineering technology field more particularly to a kind of scale loss spots induced by Crispr/Cas9
Horse fish pattern and method for building up.
Background technology
The hair of the mankind and mammal, tooth, nail, sweat gland, the feather of birds, scale, hypopharynx tooth, the antenna of fish
Etc. belonging to the appendages of skin, and the development of the appendages of skin is most important for the mankind, and appendages of skin depauperation is suffered from
Person has Familial Occurrence, at present in mouse, fish etc. it has also been found that having such phenomenon, by genetics research it has proven convenient that skin is attached
The development of part is mainly regulated and controled by EDA family genes.
Eda (Eetodysplasin) and receptor Edar are associated with TNF, belong to TNF superfamily members, and EDA signals are
It is adjusted by Eetodysplasin, EDAR, EDARADD, is formed in the vertebrate appendages of skin regulation and control of fish to the mankind
One tnf ligand-receptor-aptamer family.EDA signal paths are found for the first time in the ectodermal dysplasia syndrome of the mankind.Nothing
Sweat or hypohidrosis type are ectodermal dysplasia（EDA）It is most common a kind of symptom in ectodermal dysplasia group, it is main to influence
The form of two or more appendages of skin occurs.
EDA, which mainly influences exocrine gland in hair, tooth sum aggregate, includes sweat gland, Meibomian gland and preputial gland.As a kind of something lost
It passes and learns disease, EDA is occurred in the form of X- linked recessives, autosomal dominant and recessiveness etc..Darwin is for the first time his within 1875
Book《The variations of animals and plants under domestication》In describe the chain shapes of X-
Formula has recorded the phenotype and the distinctive hereditary pattern of male of impacted Indian family's appendages of skin in detail.More than 100 years
Three kinds of various forms of variations of gene are found that in patient later, wherein X- is chainEDAGene mutation account in the case 90% with
On, remaining case is mainlyEDARWithEDARADDThe rare autosomal dominant inheritance of genetic comparison.Substantial amounts of result provesEDA、EDARWithEDARADDGene code participates in tnf ligand-receptor-aptamer family of appendages of skin development.
Eetodysplasin is one chain by X sex chromosomeEDATwo type transmembrane proteins of gene code, in extracellular space
Include a short collagenous portion（collagen）With a TNF domain.There is furin close to collagen areas（furin）
Site generates soluble EDA ligands, re-forms active ligand trimer.Have now been found that TNF, collagen,
Furin cuts the deletion mutation occurred with cross-film site in missense or reading frame, can cause EDA syndromes.TNF is
The functional domain of Eetodysplasin, however some the mutation displays found in patient, collagen areas also have critically important
Effect, which includes 19 G-X-Y and repeats, though the missing that 2 G-X-Y in reading frame are repeated or 4 G-X-Y are repeated
The function in TNF domains is not so influenced, but can equally cause EDA syndromes.Comparative genomics and genetics research finder,
Mouse, fish, Africa xenopus, chicken, ox, chimpanzee etc.EDA, EDAR, EDARADD geneIt is highly conserved during evolution.
Have now been found that similar spontaneous mutation animal model hasEDARThe medaka of mutation,EDAThe dog and ox of mutation,
Tabby, Downless/Sleek and Crinkled mouse mutant.People'sEDAGene mutation and the tabby of natural mutation
The phenotype of mouse is consistent, shows as the decline of hair follicle quantity or missing and sweat duct and the defects of tooth development.The appendages of skin
Species specificity is shown as, compared with the hair of mammal, sweat gland, fish are mainly bone plate, scale etc..Colosimo etc.
（2005）The study found that EDA also assists in the adaptive evolution of bone plate pattern in transition of the three-spined stickleback from ocean to fresh water.It is small
The EDA-A1 of mouse can make fresh water three-spined stickleback bone plate recover the consistent pattern with ocean three-spined stickleback, further point outEDAGene
For the adaptability of appendages of skin parallel evolution（Plasticity）.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of scale loss zebra fish mould induced by Crispr/Cas9
Formula.
Another technical problem to be solved by this invention is to provide the method for building up of the scale loss zebra fish pattern.
To solve the above problems, a kind of scale loss zebra fish pattern induced by Crispr/Cas9 of the present invention,
It is characterized in that：The pattern refers to the scale loss zebra fish for including the 4 site base insertion of EDA gene extrons.
A kind of method for building up of scale loss zebra fish pattern by Crispr/Cas9 inductions as described above, including with
Lower step：
(1), using Ensemble online databases, zebra fish EDA gene orders no is searched：ENSDARG00000074591 is simultaneously
It downloads, searches the PAM i.e. 5 '-GGNNNNNNNNNNNNNNNNNNNGG-3 ' of sequence in the sequence, found in EDA gene extrons
Targeting sequence is TTAGGCAAGAAAGGGCCCCC【TGG】, and design and design abrupt climatic change primers F-eda:
Ttgttttgcttctcatcagttg and abrupt climatic change primer R-eda: tttgctctgctgcttcactc；
(2) the sequence verification of the wild-type allele of target sequence：
5 pieces of pf wild-type zebrafish embryos for 24 hours are randomly selected, genomic DNA template are extracted, then with F-eda and R-eda
PCR amplification is carried out, obtains the pcr amplification product I that fragment length is 358bp, which is drawn with the abrupt climatic change
Object F-eda direct Sequencings, to show that target gene group sequence is consistent with the sequence in database, no SNP applications overlapping；
(3) sgRNA is built：
(4) eda-sgRNA templates are established：
The template of the in-vitro transcription of sgRNA is obtained with PCR methods, the template sequence is as follows：TAATACGACT CACTATA GTT AGGCAAGAAA GGGCCCCCgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt t；
(5) sgRNA in-vitro transcriptions：
It is template with the pcr amplification product I, carries out sgRNA in-vitro transcriptions with T7 RNA polymerases, obtain sgRNA；Institute
State sgRNA makes its concentration be 1000ng/ul after absolute ethyl alcohol method precipitation；
(6) Cas9 mRNA in-vitro transcriptions：
With the Cas9 coded sequences of the humanization of U7 drivings（Mali et al., 2013. Science, 339(6121):
823-6）For template, Cas9 in-vitro transcriptions are carried out with U7 RNA polymerases, simultaneously tailing of then attaching the names of pre-determined candidates obtains Cas9 mRNA；It is described
Cas9 mRNA make its concentration be 600ng/ul after absolute ethyl alcohol method precipitation；
(7) the microinjection of zebra fish fertilized egg and abrupt climatic change：
After the sgRNA and the Cas9 mRNA mixed in equal amounts, the micro- wild type zebra for being injected into 1 ~ 2 cell stage
Fish fertilized egg when embryonic development is to 24 hpf, takes 5 pieces of embryos, prepares genomic DNA template, then with the abrupt climatic change
Primers F-eda and abrupt climatic change primer the R-eda carries out PCR amplification, obtains pcr amplification product II；The PCR amplification is produced
Object II is subcloned into pEASY-T3 carriers；And transformant is expanded with T7 and Sp6 universal primers, by PCR product ten
One group totally two groups directly send sequencing, sequencing primer is T7 or Sp6 wherein one；
(8) mutant scale Observation On The Development and dyeing identification：
The scale developmental state for having the zebra fish of EDA gene mutations to detection carries out microexamination, and screening obtains EDA bases
Cause scale loss mutant because 4 target site of extron introduces insertion mutation, scale dyeing is carried out to scale dysplasia individual
Identification, to determine whether scale has developmental defect.
A kind of application of scale loss zebra fish pattern by Crisper/Cas9 inductions as described above, feature exist
In：The scale loss zebra fish pattern is applied to the verification of appendages of skin related gene function and ectodermal dysplasia drug
Screening.
The present invention has the following advantages compared with prior art：
1st, the present invention is for zebra fish EDA genes (containing 8 extrons), and design targeting is in 4 site of extron
EDA-sgRNA（See Fig. 1）, by building EDA-sgRNA and Cas9 mRNA carriers, fertilized eggs are injected after reverse transcription, are expanded through PCR
Increasing and sequence verification detect the insertion mutation in 4 12 bases of site primer of extron（See Fig. 2）.
2nd, the present invention carries out the observation of scale developmental state and dyeing to individual and the wild type individual of EDA gene insertion mutations
Identification finds that the mutant there is no that scale is developed, and wild type individual scale covering whole body（See Fig. 3）.
3rd, the scale loss zebra fish pattern that the present invention establishes is in the functional study of appendages of skin related gene, screening treatment people
Class exiguous human hair etc. is ectodermal dysplasia, and drug etc. has very big application value.
Description of the drawings
The specific embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is the zebra fish EDA genes of the present invention and Crispr/Cas9 target sites.Zebra fish EDA genes are shared outside 8
Aobvious son, the Crispr target sites at 4 site of extron have been screened by Photographing On-line.
Fig. 2 is the zebra fish of Crispr/Cas9 of the present invention inductionsEDADetection in Gene Mutation.F1 individual extractions to foundation
Genome is expanded using abrupt climatic change primers F-eda and R-eda, and carries out direct Sequencing to PCR product, is detected whether
There is mutation to introduce（A and B）；In addition the specific mutation type of TA clone identifications is carried out to the PCR product for having mutation to introduce（C and D）.
Wherein：
A figures are that wild-type zebrafish EDA genes include target site region PCR product sequencing result, are target site in frame；
B figures are that PCR product the bimodal saltant type zebra fish testing result for indicating insertion or deletion mutation occurs；
C figures are the individual PCR product T-A cloning and sequencings of B figure detections as a result, relatively there is base insertion with wild type；
D target site sequences compare, wild type individual target site sequence（WT）With there is the saltant type individual of 15 base insertions
Sequence (MT+15bp).
Fig. 3 is zebra fish individual Alizarin red staining of the present invention.To EDA processing zebra fish wild type and saltant type individual into
Row scale dyeing identification scale developmental state, wherein A is mutant zebra fish, and scale lacks substantially；B is wild-type zebrafish,
Whole body covers scale.
Specific embodiment
A kind of scale loss zebra fish pattern induced by Crispr/Cas9 of embodiment 1, the pattern refer to comprising EDA bases
Because of the scale loss zebra fish of 4 site base insertion of extron.
The method for building up of the scale loss zebra fish pattern, comprises the following steps：
(1), using Ensemble online databases, zebra fish EDA gene orders no is searched：ENSDARG00000074591 is simultaneously
It downloads, PAM is searched in the sequence（protospacer-adjacent motif ）Sequence i.e. 5 '-
GGNNNNNNNNNNNNNNNNNNNGG-3 ' is in the targeting sequence that EDA gene extrons are found
TTAGGCAAGAAAGGGCCCCC【TGG】, and design and design abrupt climatic change primers F-eda:
Ttgttttgcttctcatcagttg and abrupt climatic change primer R-eda: tttgctctgctgcttcactc.
(2) the sequence verification of the wild-type allele of target sequence：
5 pieces of pf wild-type zebrafish embryos for 24 hours are randomly selected, genomic DNA template are extracted, then with F-eda and R-eda
Carry out PCR amplification, obtain fragment length be 358bp pcr amplification product I, the pcr amplification product I with abrupt climatic change primers F-
Eda direct Sequencings, sequencing work well, and chromatogram readability is good.The result shows that the sequence in target gene group sequence and database
Row are consistent, no SNP applications overlapping.
(3) sgRNA is built：
1. carrier and Insert Fragment processing：
SgRNA carrier constructions are usedBbsL digestions pT7-gRNA, gel extraction obtain sgRNA clone's skeletons pT7-gRNA_BbsIoFollowing processing is done as Insert Fragment, the Insert Fragment in IE sites：Use ddH2The Oligo of target site is dissolved as 10 μ by O
M respectively takes the 5 isometric mixings of μ L, anneals by following procedure：95 DEG C of 5min, -1 DEG C/30sec/cycle, 4 DEG C of to.
2. linked system：
Take target site Oligo and sgRNA clone's skeleton pT7-gRNA_ after annealingBbsEach luL of I run glue, to Insert Fragment
It is carried out with carrier after quantifying, the target site Oligo after annealing is connected with gRNA carriers.Because Insert Fragment is smaller, final institute
Determining linked system is：Oligo l μ L, pT7-gRNA_Bbs2 μ L, T4 ligase l of I l μ L, 5 × T4 ligase buffer
μ L, totally 10 μ L systems, 25 DEG C of 3 ~ 4h of connection.5 μ L are taken, are transformed into Trans-Tl competent cells.
3. bacterium colony PCR is verified：
5 ~ 10 monoclonals of picking take l μ L to be used as template and carry out PCR amplification, primer is into l0 μ L LB fluid nutrient mediums
Carrier universal primer RV-M and target site specific mutagenesis detection primer R-eda, with EsayTaq DNA Polymerase, l0 μ L bodies
System is expanded.Purpose band about 130bp.Picking positive colony send sequencing, chooses sequence and correctly clones progress glycerine guarantor bacterium,
Extract plasmid.
(4) eda-sgRNA templates are established：
The template of the in-vitro transcription of sgRNA is obtained with PCR methods, the template sequence is as follows：TAATACGACT CACTATA GTT AGGCAAGAAA GGGCCCCCgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt t。
Wherein：Italicized capitals part for T7 start subdivision, underscore part be gRNA templates, lowercase sgRNA
Skeleton.
(5) sgRNA in-vitro transcriptions：
It is template with pcr amplification product I, carries out sgRNA in-vitro transcriptions with T7 RNA polymerases, obtain sgRNA；sgRNA
It is 1000ng/ul to make its concentration after absolute ethyl alcohol method precipitation.
Wherein：In-vitro transcription system refers to 5 × Transcription Buffer l0 μ L；l00Mm DTT 5μL；NTP
mix (lOmM each) l0μL；Template (>lμg) 20μL；2 μ L of T7 RNA polymerases；RNase Inhibitor
(40U) l μ L DEPC water supplies system to 50 μ L, 37 DEG C of reaction 2h,
2 μ L is taken to detect transcription effects with the agarose gel electrophoresis of 1 %.
(6) Cas9 mRNA in-vitro transcriptions：
With the Cas9 coded sequences of the humanization of U7 drivings（Mali et al., 2013. Science, 339(6121):
823-6）For template, Cas9 in-vitro transcriptions are carried out with U7 RNA polymerases, simultaneously tailing of then attaching the names of pre-determined candidates obtains Cas9 mRNA；Cas9
MRNA makes its concentration be 600ng/ul after absolute ethyl alcohol method precipitation.
Wherein：In-vitro transcription system refers to 5 × Tanscription Buffer l0 μ L, l00mMDTT 5 μ L, NTPmix
(ATP/CTP/UTP each l0mM, GTP：ImM)：L0 μ L SP6 RNA Polymerase (SOU) 2 μ L, Rnase
Inhibitor (SOU) l μ L, Template 17 μ L, CAP (lOmM) 5 μ L.37 DEG C, water-bath 1.5h.
L μ L is taken to carry out 1% Ago-Gel detection mRNA synthetic effects.
1. Cas9 mRNA templates are digested：Add in DNasel 2 μ L, 37 DEG C of digestion 15min.
2. phenol chloroform and LiCl precipitations：
Cas9 mRNA in-vitro transcription systems are supplied to 100 μ L with DEPC water, add in phenol：Chloroform：Isoamyl alcohol（25：24：
1）100 μ L, misfortune rotation 15s mixings, 12000rpm are centrifuged 30 seconds, are drawn supernatant into new centrifuge tube, add in chloroform：Isoamyl alcohol（24：
1）100 μ L, misfortune rotation 15s mix hook, and 12000rpm is centrifuged 30 seconds, again moved to supernatant in new centrifuge tube, adds in 1/10 volume
3 MNaAc (pH4.0) and the absolute ethyl alcohol of 2 times of volume precoolings, turn upside down, -80 DEG C of standing 5min.12000rpm is centrifuged
15min removes supernatant, is blotted on blotting paper, 400 μ L Ua of addition, reverse mixing, 12000rpm centrifugation 15min, in absorption
Clearly, 700 μ L 70%DEPC ethyl alcohol are added in, 12000rpm centrifugation 15min carefully outwell supernatant, add 700 μ L 70%DEPC second
Alcohol centrifuges 12000rpm15min again, removes supernatant, is dissolved in l0 μ L Rnase after being stored at room temperature the ethyl alcohol that thoroughly volatilizees for 3 ~ 5 minutes
In Free water, 0.5 μ L is taken to detect gRNA concentration for 1% agarose gel electrophoresis.
(7) the microinjection of zebra fish fertilized egg and abrupt climatic change：
After sgRNA and Cas9 mRNA mixed in equal amounts, the micro- wild-type zebrafish fertilized eggs for being injected into 1 ~ 2 cell stage,
Zebrafish embryo after injection is put into 28 DEG C of insulating boxs and is cultivated.General 1 μ L can inject 300 ovum.
When embryonic development is to 24 hpf, takes 5 pieces of embryos, prepare genomic DNA template, then with abrupt climatic change primers F-
Eda and abrupt climatic change primer R-eda carries out PCR amplification, obtains pcr amplification product II；The pcr amplification product II is subcloned
Into pEASY-T3 carriers.And transformant is expanded with T7 and Sp6 universal primers, by ten one group totally two groups of PCR product
Sequencing is directly sent, sequencing primer is T7 or Sp6 wherein one.
TA clone's used carriers are pEASY-T3 carriers.Specific steps：
Following reaction solution second is added in the EP pipes of 0.2mL and takes turns 2 μ L, T4 DNA ligase of PCR products, 1 μ L, 10
1 μ L, pGM-T vector of × T4 DNA ligation buffer 1 μ L, ddH26 μ L of O, 16 DEG C of connections overnight, turn for second day
In Trans-Tl competent cells.The random white single bacterium colony of picking 10 carries out bacterium colony PCR with T7 and SP6 universal primers, in advance
Phase size segment is 350bp, one brightness of day is sent far to be sequenced positive colony.
(8) mutant scale Observation On The Development and dyeing identification：
The scale developmental state for having the zebra fish of EDA gene mutations to detection carries out microexamination, and screening obtains EDA bases
Cause scale loss mutant because 4 target site of extron introduces insertion mutation, scale dyeing is carried out to scale dysplasia individual
Identification, to determine whether scale has developmental defect.
It is as follows：It is fixed for 24 hours with 4% formalin solution, 70% ethanol dehydration 5h, then in the madder of 1g/L
It is dyed for 24 hours in the red solution of element, it is micro- using ZESIS Discovery V13 after 1%KOH+5% glycerine carries out decoloration 48h
Mirror is imaged.
The verification applied to appendages of skin related gene function of the scale loss zebra fish pattern of embodiment 2 and outer embryo
The screening of layer depauperation drug.
PAM sequences
5’-GGNNNNNNNNNNNNNNNNNNNGG-3’
Targeting sequence
TTAGGCAAGAAAGGGCCCCC【TGG】
Abrupt climatic change primers F-eda
ttgttttgcttctcatcagttg
Abrupt climatic change primer R-eda
tttgctctgctgcttcactc
Template sequence
TAATACGACT CACTATAGTT AGGCAAGAAA GGGCCCCCgt tttagagcta gaaatagcaa
gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt t
Claims (2)
1. a kind of method for building up of scale loss zebra fish pattern by Crispr/Cas9 inductions, it is characterised in that：The pattern is
Refer to the scale loss zebra fish for including the 4 site base insertion of EDA gene extrons；
Comprise the following steps：
(1), using Ensemble online databases, zebra fish EDA gene orders no is searched：ENSDARG00000074591 and under
It carries, the PAM i.e. 5 '-GGNNNNNNNNNNNNNNNNNNNGG-3 ' of sequence is searched in the sequence, in the target that EDA gene extrons are found
It is TTAGGCAAGAAAGGGCCCCC to acting sequences【TGG】, and design abrupt climatic change primers F-eda:
Ttgttttgcttctcatcagttg and abrupt climatic change primer R-eda: tttgctctgctgcttcactc；
(2) the sequence verification of the wild-type allele of target sequence：
5 pieces of pf wild-type zebrafish embryos for 24 hours are randomly selected, genomic DNA template is extracted, is then carried out with F-eda and R-eda
PCR amplification, obtain fragment length be 358bp pcr amplification product I, the pcr amplification product I with the abrupt climatic change primers F-
Eda direct Sequencings, to show that target gene group sequence is consistent with the sequence in database, no SNP applications overlapping；
(3) sgRNA is built：
(4) eda-sgRNA templates are established：
The template of the in-vitro transcription of sgRNA is obtained with PCR methods, the template sequence is as follows：TAATACGACT CACTATA GTT AGGCAAGAAA GGGCCCCCgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
gaaaaagtgg caccgagtcg gtgctttttt t；
(5) sgRNA in-vitro transcriptions：
It is template with the pcr amplification product I, carries out sgRNA in-vitro transcriptions with T7 RNA polymerases, obtain sgRNA；It is described
SgRNA makes its concentration be 1000ng/ul after absolute ethyl alcohol method precipitation；
(6) Cas9 mRNA in-vitro transcriptions：
Using the Cas9 coded sequences of the humanization of U7 drivings as template, Cas9 in-vitro transcriptions are carried out with U7 RNA polymerases, then
It attaches the names of pre-determined candidates and tailing, obtains Cas9 mRNA；The Cas9 mRNA make its concentration be 600ng/ul after absolute ethyl alcohol method precipitation；
(7) the microinjection of zebra fish fertilized egg and abrupt climatic change：
By after the sgRNA and the Cas9 mRNA mixed in equal amounts, the micro- wild-type zebrafish for being injected into 1 ~ 2 cell stage by
Smart ovum when embryonic development is to 24 hpf, takes 5 pieces of embryos, prepares genomic DNA template, then with the abrupt climatic change primer
The F-eda and abrupt climatic change primer R-eda carries out PCR amplification, obtains pcr amplification product II；By the pcr amplification product II
It is subcloned into pEASY-T3 carriers；And transformant is expanded with T7 and Sp6 universal primers, by ten one group of PCR product
Directly send sequencing for totally two groups, sequencing primer is T7 or Sp6 wherein one；
(8) mutant scale Observation On The Development and dyeing identification：
The scale developmental state for having the zebra fish of EDA gene mutations to detection carries out microexamination, and screening is obtained outside EDA genes
4 target sites of aobvious son, which introduce insertion mutation, causes scale loss mutant, and scale dyeing identification is carried out to scale dysplasia individual,
To determine whether scale has developmental defect.
2. a kind of scale loss zebra fish pattern by Crispr/Cas9 inductions that method as described in claim 1 is established is answered
With, it is characterised in that：The scale loss zebra fish pattern is applied to the verification of appendages of skin related gene function and ectoderm hair
Educate the screening of bad drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510071972.4A CN104928321B (en) | 2015-02-12 | 2015-02-12 | A kind of scale loss zebra fish pattern and method for building up by Crispr/Cas9 inductions |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510071972.4A CN104928321B (en) | 2015-02-12 | 2015-02-12 | A kind of scale loss zebra fish pattern and method for building up by Crispr/Cas9 inductions |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104928321A CN104928321A (en) | 2015-09-23 |
| CN104928321B true CN104928321B (en) | 2018-06-01 |
Family
ID=54115748
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510071972.4A Expired - Fee Related CN104928321B (en) | 2015-02-12 | 2015-02-12 | A kind of scale loss zebra fish pattern and method for building up by Crispr/Cas9 inductions |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104928321B (en) |
Families Citing this family (35)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2012333134B2 (en) | 2011-07-22 | 2017-05-25 | John Paul Guilinger | Evaluation and improvement of nuclease cleavage specificity |
| US20150044192A1 (en) | 2013-08-09 | 2015-02-12 | President And Fellows Of Harvard College | Methods for identifying a target site of a cas9 nuclease |
| US9359599B2 (en) | 2013-08-22 | 2016-06-07 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US9322037B2 (en) | 2013-09-06 | 2016-04-26 | President And Fellows Of Harvard College | Cas9-FokI fusion proteins and uses thereof |
| US9340799B2 (en) | 2013-09-06 | 2016-05-17 | President And Fellows Of Harvard College | MRNA-sensing switchable gRNAs |
| US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| JP2016536021A (en) | 2013-11-07 | 2016-11-24 | エディタス・メディシン，インコーポレイテッド | CRISPR-related methods and compositions with governing gRNA |
| US20150166982A1 (en) | 2013-12-12 | 2015-06-18 | President And Fellows Of Harvard College | Methods for correcting pi3k point mutations |
| EP3177718B1 (en) | 2014-07-30 | 2022-03-16 | President and Fellows of Harvard College | Cas9 proteins including ligand-dependent inteins |
| KR20180069898A (en) | 2015-10-23 | 2018-06-25 | 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 | Nucleobase editing agents and uses thereof |
| CN105594664B (en) * | 2016-02-16 | 2018-10-02 | 湖南师范大学 | A kind of method of gene knockout selection and breeding stat1a Gene Deletion zebra fish |
| KR102547316B1 (en) | 2016-08-03 | 2023-06-23 | 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 | Adenosine nucleobase editing agents and uses thereof |
| AU2017308889B2 (en) | 2016-08-09 | 2023-11-09 | President And Fellows Of Harvard College | Programmable Cas9-recombinase fusion proteins and uses thereof |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| CA3039928A1 (en) | 2016-10-14 | 2018-04-19 | President And Fellows Of Harvard College | Aav delivery of nucleobase editors |
| WO2018119359A1 (en) | 2016-12-23 | 2018-06-28 | President And Fellows Of Harvard College | Editing of ccr5 receptor gene to protect against hiv infection |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| WO2018165629A1 (en) | 2017-03-10 | 2018-09-13 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| IL269458B2 (en) | 2017-03-23 | 2024-02-01 | Harvard College | Nucleobase editors comprising nucleic acid programmable dna binding proteins |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| CN106978445A (en) * | 2017-06-12 | 2017-07-25 | 内蒙古大学 | The method of the goat EDAR gene knockouts of CRISPER Cas9 System-mediateds |
| CN107217075B (en) * | 2017-06-28 | 2021-07-02 | 西安交通大学医学院第一附属医院 | A method for constructing EPO gene knockout zebrafish animal model, primers, plasmids and preparation method |
| CN109207523A (en) * | 2017-06-29 | 2019-01-15 | 南京尧顺禹生物科技有限公司 | The foundation and application of human obesity's zebra fish model based on UCP1 gene |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| WO2019139645A2 (en) | 2017-08-30 | 2019-07-18 | President And Fellows Of Harvard College | High efficiency base editors comprising gam |
| CN111757937A (en) | 2017-10-16 | 2020-10-09 | 布罗德研究所股份有限公司 | Uses of adenosine base editors |
| US12157760B2 (en) | 2018-05-23 | 2024-12-03 | The Broad Institute, Inc. | Base editors and uses thereof |
| CN108707628B (en) * | 2018-05-28 | 2021-11-23 | 上海海洋大学 | Preparation method of zebra fish notch2 gene mutant |
| WO2020092453A1 (en) | 2018-10-29 | 2020-05-07 | The Broad Institute, Inc. | Nucleobase editors comprising geocas9 and uses thereof |
| WO2020154500A1 (en) | 2019-01-23 | 2020-07-30 | The Broad Institute, Inc. | Supernegatively charged proteins and uses thereof |
| BR112021018607A2 (en) | 2019-03-19 | 2021-11-23 | Massachusetts Inst Technology | Methods and compositions for editing nucleotide sequences |
| CN109880827B (en) * | 2019-03-26 | 2021-05-18 | 杭州师范大学附属医院(杭州市第二人民医院) | Method for establishing hepatolenticular degeneration zebra fish model |
| IL297761A (en) | 2020-05-08 | 2022-12-01 | Broad Inst Inc | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| CN113416787A (en) * | 2021-07-08 | 2021-09-21 | 中国科学院西北高原生物研究所 | Method for detecting mutation of Acsl1a gene of zebra fish |
| CN115521899A (en) * | 2022-05-06 | 2022-12-27 | 西南大学 | An efficient Japanese green culture cell CRISPR/Cas9 genome editing method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103388006A (en) * | 2013-07-26 | 2013-11-13 | 华东师范大学 | Method for constructing gene site-directed mutation |
| CN104195177A (en) * | 2014-08-05 | 2014-12-10 | 南京大学 | Method for remarkably improving fish genome editing efficiency |
-
2015
- 2015-02-12 CN CN201510071972.4A patent/CN104928321B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103388006A (en) * | 2013-07-26 | 2013-11-13 | 华东师范大学 | Method for constructing gene site-directed mutation |
| CN104195177A (en) * | 2014-08-05 | 2014-12-10 | 南京大学 | Method for remarkably improving fish genome editing efficiency |
Non-Patent Citations (2)
| Title |
|---|
| Danio rerio ectodysplasin A (eda), mRNA;NM_001115065.1;《Genbank》;20140928;全文 * |
| Zebrafish eda and edar Mutants Reveal Conserved and Ancestral Roles of Ectodysplasin Signaling in Vertebrates;Matthew P.Harris et al.;《PLOS Genetics》;20081031;第4卷(第10期);第1-15页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104928321A (en) | 2015-09-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104928321B (en) | A kind of scale loss zebra fish pattern and method for building up by Crispr/Cas9 inductions | |
| Gutiérrez-Gil et al. | An interpretive review of selective sweep studies in Bos taurus cattle populations: identification of unique and shared selection signals across breeds | |
| Peifer et al. | The segment polarity gene armadillo encodes a functionally modular protein that is the Drosophila homolog of human plakoglobin | |
| Rossi et al. | Two novel/ancient myosins in mammalian skeletal muscles: MYH14/7b and MYH15 are expressed in extraocular muscles and muscle spindles | |
| Rijnkels et al. | Multispecies comparative analysis of a mammalian-specific genomic domain encoding secretory proteins | |
| Xu et al. | Analysis of the erythropoietin of a Tibetan Plateau schizothoracine fish (Gymnocypris dobula) reveals enhanced cytoprotection function in hypoxic environments | |
| Bian et al. | Morphological characteristics and comparative transcriptome analysis of three different phenotypes of Pristella maxillaris | |
| Lacin et al. | Genome-wide identification of Drosophila Hb9 targets reveals a pivotal role in directing the transcriptome within eight neuronal lineages, including activation of nitric oxide synthase and Fd59a/Fox-D | |
| Farkašová et al. | Identification of a GC-rich leptin gene in chicken | |
| Yergeau et al. | bloodthirsty, an RBCC/TRIM gene required for erythropoiesis in zebrafish | |
| Li et al. | Differential evolution of duplicated medakafish mitf genes | |
| Lang et al. | A BAC library of the East African haplochromine cichlid fish Astatotilapia burtoni | |
| An et al. | The period gene in two species of tephritid fruit fly differentiated by mating behaviour | |
| Zhu et al. | Functional analysis of two MyoDs revealed their role in the activation of myomixer expression in yellowfin seabream (Acanthopagrus latus)(Hottuyn, 1782) | |
| Liu et al. | Genome‐wide identification and transcriptome‐based expression profiling of the Sox gene family in the spinyhead croaker (Collichthys lucidus) | |
| Sano et al. | Evolution of the teleostean zona pellucida gene inferred from the egg envelope protein genes of the Japanese eel, Anguilla japonica | |
| Iwabuchi et al. | Molecular cloning and characterization of avian bombesin‐like peptide receptors: new tools for investigating molecular basis for ligand selectivity | |
| US20060160080A1 (en) | Avian sex determination method | |
| Cheng et al. | Two myeloid differentiation factor 88 (MyD88) isoforms identified in ducks | |
| Huynen et al. | Reconstruction and in vivo analysis of the extinct tbx5 gene from ancient wingless moa (Aves: Dinornithiformes) | |
| Yi et al. | Whole genome sequencing identified genomic diversity and candidated genes associated with economic traits in Northeasern Merino in China | |
| Kajiura-Kobayashi et al. | The cloning of cyclin B3 and its gene expression during hormonally induced spermatogenesis in the teleost, Anguilla japonica | |
| Luo et al. | Utilization of a zebra finch BAC library to determine the structure of an avian androgen receptor genomic region | |
| Beaster-Jones | Cis-regulation and conserved non-coding elements in amphioxus | |
| Gu et al. | Whole-genome bisulfite sequencing reveals the function of DNA methylation in the allotransplantation immunity of pearl oysters |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180601 Termination date: 20190212 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |