CN109266651A - SgRNA based on CRISPR/Cas9 technical editor's HBB-41/42 deletion mutation site - Google Patents
SgRNA based on CRISPR/Cas9 technical editor's HBB-41/42 deletion mutation site Download PDFInfo
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Abstract
The present invention provides a kind of sgRNA based on 41/42 deletion mutation site of CRISPR/Cas9 technical editor beta globin genes, and nucleotide sequence is as shown in SEQ ID NO.1.The present invention also provides relative carrier, cell, gene editing kit and its applications, the method for being based particularly on 41/42 deletion mutation site of CRISPR/Cas9 technical editor beta globin genes comprising construct CRISPR/Cas9 expression vector using nucleotide sequence sgRNA as shown in SEQ ID NO.1.The present invention is capable of the seamless directional transformation of gene mutation site in induction 41/42 thalassemia of beta globin genes of specificity, can introduce monoallelic with high efficiency and precision selective and diallele sequence changes, be greatly promoted the raising accurately repaired.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to one kind is for beta globin genes 41/42 (HBB-41/42)
Single base mutation is efficiently repaired in the CRISPR plasmid construction method of the pathogenic point mutation of middle sea anaemia, and design quasi-ly
The scheme of donor.
Background technique
Beta Thalassemia disease (β poor) is to constitute hemoglobin caused by mutation or missing due to beta globin genes
The peptide chain of globin synthesis of HbA (2 β 2 of α) is reduced or cannot be synthesized, and α is caused to synthesize unbalance caused one kind often dye with β peptide chain
Colour solid recessive hereditary disease.The deletion mutation (- CTTT) of tetra- bases of CDs41/42 of beta globin code area be report earliest with
β one of the relevant deletion mutation of poor disease, the missing can lead to the mRNA stability decline of generation, or formed non-functional
MRNA, so that normal beta globin chain cannot be synthesized.
It is reported that there are about 400,000,000 thalassemia gene carriers in the whole world.In China, which is apt to occur in southern ground
The domestic incidence in area, especially Guangdong Province is significantly larger than other provinces.2012 to districts and cities of 21, Guangdong Province totally 14332 household registers
Give birth in hospital pregnant woman screening the study found that the poor gene carrying rate in ground be 16.8%, i.e., up to 1/6 reproductive population carries poor base
Cause.Every severe the birth of poor infant be up to 100 ten thousand to 300 ten thousand yuan to society and the financial burden of family's bring.At present may be used
By lifelong treatment of blood transfusion thalassemia, however most infants are dead still before adult in the case where active treatment
It dies.Another treatment method is then but to find the allosome contributor to match very by marrow or hematopoietic stem cell transplantation
Difficulty, and it is costly.The genetic diagnosis technology that can only be moved ahead at present by embryo implantation passes through fetal umbilical blood cytogene
Detection technique screens normal embryo or fetus to block the propagation of poor Disease-causing gene.
By repairing patient's body cell beta globin genes using gene editing tool CRISPR fine specificity
(hemoglobin subunit beta, HBB) dcc gene, then by gene editing with repair after somatic induction at more
Energy stem cell, is finally implanted into vivo, so that corresponding functional red blood cell is divided and be divided into, to treat and alleviate in β-ground
Extra large Anemic patients' symptom.Currently, being directed to the gene therapy of β type thalassemia, set about object from induction type multipotential stem cell
(iPS) and candidate stem cell (HSC) is transitioned into human embryos, directly blocks the vertical transmission of the genetic disease.
It is fertilized for the first time using CRISPR/Cas9 gene editing technology to the mankind 3PN to inseminate extremely in the world within 2015
Ovum carries out HBB gene editor reparation, observes that CRISPR/Cas9 reaches 51.9% to target gene cutting efficiency in Human embryo, but
Discovery is missed the target obvious simultaneously.2017, applicant utilized single base editing system (APOBEC1) and nuclear transfer technology again,
Single base editor is realized in human embryos for the first time, it is successfully prominent to a kind of mononucleotide of β-thalassemia is caused
Become -28 mutation to be corrected.This single base editing system is made of the variant of CRISPR-Cas9 with cytosine deaminase,
It is pointing directly at the correct position DNA by being oriented to RNA, replaces guanine (C) with thymidine (T).Gene repair efficiency is only
Have 23%, and obtaining embryo after repairing is still chimera, the blastomere being repaired also only has 20%, is difficult to realize and controls completely
More, it and operates complicated cumbersome.In addition to this, there is also higher off-target rates for this method.
Summary of the invention
It, can be accurately the purpose of the present invention is aiming at the above technical problems to be solved, providing a kind of technical solution
Edit 41/42 type thalassaemia mutations site of beta globin genes.
For this purpose, nucleotide sequence is as shown in SEQ ID NO.1 the present invention provides a kind of sgRNA.
The present invention also provides a kind of carriers comprising nucleotide sequence sgRNA as shown in SEQ ID NO.1.It is preferred that
Ground, the carrier are CRISPR/Cas9 expression vectors.It is highly preferred that the carrier is pX330.
The present invention also provides a kind of cells comprising above-mentioned carrier.
The present invention also provides nucleotide sequence sgRNA as shown in SEQ ID NO.1 for editing HBB-41/42 mutation
The application in site.
The present invention also provides a kind of method based on the mutational site CRISPR/Cas9 technical editor HBB-41/42, packets
It includes and constructs CRISPR/Cas9 expression vector using nucleotide sequence sgRNA as shown in SEQ ID NO.1.
Method according to the present invention based on the mutational site CRISPR/Cas9 technical editor HBB-41/42, it is also preferable to include
It the use of donor (donor) ssDNA, donor ssDNA is the 100bp base sequence near the mutational site HBB-41/42, wherein
HBB-41/42 deletion mutation and nonsense mutation are introduced, and thio-modification is carried out to the base at the both ends donor ssDNA.It is highly preferred that
HBB-41/42 deletion mutation and nonsense mutation are introduced in donor ssDNA, i.e., by two exon 33- of beta globin genes
36 bit bases missing, and the 40th bit base of two exon of beta globin genes sports C (cytimidine) by T (thymidine).It is excellent
Selection of land, the nucleotide sequence of donor ssDNA carry out thio repair as shown in SEQ ID NO.2, and to the base at the both ends donor ssDNA
Decorations preferably carry out thio-modification at 3 ' ends and 5 ' each two bases in end.
Method according to the present invention based on the mutational site CRISPR/Cas9 technical editor HBB-41/42, it is preferable that packet
Include step in detail below:
(1) sgRNA is designed and synthesized, Cas9 sequence and the sgRNA are cloned on CRISPR/Cas9 carrier,
Construct the CRISPR/Cas9 expression vector containing Cas9 sequence and the sgRNA；
(2) the donor ssDNA, the nucleotide sequence of the donor ssDNA such as SEQ ID NO.2 institute are designed and synthesized
Show；
(3) it by the CRISPR/Cas9 plasmid and the donor ssDNA cotransfection 293T cell, and is cultivated.
The present invention is for this code area deletion mutation of the thalassemia of 41/42 site deletion of beta globin genes mutation
Autosomal recessive hereditary diseases, the CRISPR/Cas for constructing the mutational site in corresponding targeting thalassemia expresses matter
Grain, while using the ssDNA (single-stranded oligonucleotide) of 5 ' and 3 ' the two base thio-modifications in end, and amino acid letter is utilized wherein
And property principle introduces the nonsense mutation for blocking and editing again, donor is synthesized, by CRISPR/Cas expression plasmid and donor bis-
Person is transfected into cell jointly, the base in specific 41/42 site thalassemia of induction beta globin genes in 293T cell
Because of the seamless directional transformation in mutational site, monoallelic and diallele sequence can be introduced with high efficiency and precision selective
Change, is greatly promoted the raising accurately repaired.
Detailed description of the invention
Fig. 1 be normal person (homozygous without mutation), 41/42 site of light-duty beta globin genes poor patient (heterozygous mutant) and
Heavy 41/42 site of beta globin genes the blood DNA sequencing result of poor patient (homozygous mutation) compare.
Fig. 2 shows after CRISPR/Cas9 plasmid and donor-ssDNA cotransfection 293T cell, cell genomic dna
Sequencing result.
Fig. 3 is T7E1 digestion experimental result.
Fig. 4 is that T7E1 digestion experimental result carries out digesting efficiency calculated and integration efficiency result after gray analysis quantization
Comparison.
Fig. 5 is that the target fragment sequencing result of three kinds of different samples compares.
Specific embodiment
Combined with specific embodiments below, technical solution of the present invention is described in further detail, but the present invention is not limited to
Following embodiment.
As unspecified, related reagent can be bought by commercial sources below.For simplicity, part operation
The parameter, step and used instrument of operation are not described in detail, it should be understood that these are all well known to those skilled in the art and can
Repetition.
CRISPR/Cas9 gene editing system is derived from the bacterial adaptation immune system of micrococcus scarlatinae, by core
The guide RNA (sgRNA) of sour enzyme Cas9 and 20bp is formed.The specificity cutting of CRISPR/Cas9 is mediated by sgRNA,
SgRNA has NGG protospacer-adjacent by RNA-DNA base pair complementarity, with neighbouring on genomic DNA
The site of motif (PAM, preceding region sequence is adjacent to motif) sequence combines, and Cas9 enzyme is oriented to target site.It is this by may be programmed
The simple target navigation that sgRNA is carried out, makes CRISPR/Cas9 system be different from technology (such as Zinc finger nuclease or transcription of early stage
Activity factor sample effect nuclease (TALENs), the assembling that both technologies require complicated DNA binding protein structural domain come
Carry out locus specificity editor).Cas9 needs PAM and sgRNA and combines and could introduce double-strand break in specific position
(DSB), this double-strand break occurs at the 3bp of the upstream PAM.In most cases, these DSB pass through non-homologous end joining
(NHEJ) approach reparation leads to non-specific insertion, missing or other mutation, commonly referred to as " indels " (insertion and deletion).This
It is easy to generate to be mutated in the open reading frame of target gene that codon is caused to terminate translation or erroneous translation in advance, to reach
To the purpose of gene knockout.Change however, NHEJ reparation does not allow to introduce specific sequence, for maintaining normal life function must
The normal gene needed is mutated for the accurate gene therapy for this type of causing a disease and is not suitable for.
Specific sequence is generated to change, for example, be introduced into pathogenic mutation or correct the mutation in patient-derived cell system, it must
With source orientation reparation (HDR), this is a kind of unique cell DNA reparation approach for palpus use.Accomplish this point, the most common side
Method is the DNA recovery template for introducing a modification simultaneously, such as single-stranded oligonucleotide (ssODN), wherein including and base around DSB
Because of the homologous both ends flanking sequence in group region and expected sequence variation.Single stranded DNA (ssDNA) is often as " donor
Template " is widely used in during gene editing, it can be achieved that fixed point is repaired and DNA fragmentation is knocked in, research shows that length
As homology arm there is the reparation of higher fixed point and segment to knock in efficiency for the ssDNA of 70-100bp, is easier to screening and is pinpointed
The mutant that editor and segment are knocked in.In mammalian cells, HDR is relatively rare, and genome edits generation by CRISPR
Double-strand break is mainly repaired by NHEJ, but cell cycle regulating, NHEJ component inhibit or introduce recovery template that the frequency of HDR can be improved
Rate.However, genome editor's event needed for being generated by HDR, it is also desirable to by preventing sgRNA after cutting from targeting combination again
Improve the accuracy of editor, and there is no directly solutions in the research of early stage this point.The editor of these " inaccuracy " comes
Previous edited site is updated derived from CRISPR/Cas9 complex, this will reappear target site, until it
Sufficiently modified.
The present invention is by the way that using the degeneracy of amino acid, it is prominent to introduce nonsense in the target sequence that CRISPR/Cas9 is targeted
Become (blocking base) into HDR-ssDNA recovery template, and to 5 ' and 3 ' ends, two bases progress thio-modifications, it can be with
Undesirable editor (Fig. 2) again is largely prevented, to achieve the purpose that seamless reparation.
It is worth noting that, according to the present invention be based on CRISPR/Cas9 technical editor beta globin genes 41/42
The method of point is used for non-disease diagnosing and treating purpose, and gene editing, but direct purpose are carried out using human body cell as target
It is to explore gene editing, and does not lie in and directly human body diseases are treated or obtained with diagnostic result, obtains
The health status of human body changes disease event.
Design of primers used in the present embodiment use the online design of primers tool of Primer3Plus (http: //
Www.primer3plus.com/cgi-bin/dev/primer3plus.cgi it) completes；It is limited by Suzhou gold only intelligence biotechnology
Company's synthesis, the thio-modification type donor-ssDNA of integration are synthesized by Suzhou Jin Weizhi Biotechnology Co., Ltd；Identify knife
Engineering enzyme T7E1 enzyme used in whether cutting, is purchased from NEW ENGLAND BIOLAB (article No. #M0302S), identifies donor
Integrate repair engineering enzyme BanII enzyme and matched 10 × Buffer purchased from Thermo Scientific company (article No.:
FD0994).Sanger sequencing commission Sangon Biotech (Shanghai) Co., Ltd.) limited liability company's completion, according to applicants' design
Sequence carries out plasmid construction by Suzhou Jin Weizhi Biotechnology Co., Ltd completion etc..
Detect 41/42 site mutation base of beta globin genes
Take normal person in clinical sample (homozygous without mutation), the poor patient in 41/42 deletion mutation of light-duty beta globin genes ground
(heterozygous mutant) and heavy 41/42 deletion mutation of beta globin genes the blood DNA of poor patient (homozygous mutation) carry out sanger
Sequencing specifies 41/42 deletion mutation of beta globin genes and causes a disease for-CTTT.
Specific step is as follows:
1. taking normal person (homozygous without mutation), the poor trouble in 41/42 deletion mutation of light-duty beta globin genes ground using blood taking needle
Person's (heterozygous mutant) and heavy 41/42 deletion mutation of beta globin genes poor patient (homozygous mutation) blood sample, and use
Full formula gold DNA extraction agent box (article No.: #M10122) extracts the DNA in blood according to operating instruction.
2. the upstream and downstream sequence of 41/42 catastrophe point of beta globin genes is found, in Primer3Plus by ncbi database
Online design of primers tool web site (http://www.primer3plus.com/cgi-bin/dev/primer3plus.cgi)
Design primer, expands the upstream and downstream segment of targeted mutagenesis point, and PCR product serves Hai Sheng work biotech company and carries out Sanger survey
Sequence, specifying 41/42 Characteristics of Mutation of beta globin genes is that four base deletions of-CTTT are mutated (see Fig. 1).Primer sequence are as follows:
Forward primer: GAGGAGAAGTCTGCCGTTACT (SEQ ID NO.3)
Reverse primer: AAACATCAAGCGTCCCATAGA (SEQ ID NO.4)
Resulting homozygous mutation sequencing sequence is as shown in SEQ ID NO.5, wherein HBB in the homozygous base sequence without mutation
Two exon 33-36 bit bases (- CTTT) missing.
The synthesis of sgRNA
The DNA sequence dna that the present invention is directed to are as follows: poor gene base sequence (the ncbi database HGNC:4827or in the ground HBB
GRCh38.p12,GCF_000001405.38).It is online using CRISPR Design (http://crispr.mit.edu/)
SgRNA design tool designs the sgRNA near pathogenic mutation point and passes through for 41/42 type mutational site of beta globin genes
Cell experiment filters out the optimal sgRNA of effect.
According to the design principle of sgRNA, sgRNA is designed near the mutational site of the sequence obtained by sequencing, designs and selects 1
The optimal sgRNA of item constructs CRISPR plasmid accordingly.
The sgRNA sequence selected is as follows:
GAGGTTCTTTGAGTCCTTTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCA
ACTTGAAAAAGTGGCACCGAGTCGGTGC(SEQ ID NO.1)
Wherein, preceding 20 bases are sgRNA sequence, i.e., the sequence in conjunction with target DNA, remaining base portion is
The secondary structure region that scaffold, i.e. sgRNA play a role.
Construct the expression vector of CRISPR/Cas9
The expression vector establishment method of CRISPR/Cas9 routinely operates completion building with reference to the construction method in quotation,
Cas9 sequence and sgRNA sequence are cloned on expression vector pX330 (Plasmid#42230, addgene), construct CRISPR/
The eukaryotic expression plasmid of Cas9.After the completion of building, by it is conventional be sequenced compare determine carrier construction sequence correctly without mutation,
Right-on clone is picked out to expand and extract plasmid.
The synthesis of donor (donor) sequence
Design and synthesize the donor-ssDNA sequence for pinpointing seamless mutation.
The 100bp base sequence near 41/42 deletion mutation of beta globin genes is intercepted, introduces beta globin genes wherein
41/42 deletion mutation and nonsense mutation (T → C), and thio-modification (shown in *) is carried out at both ends, donor sequence is as follows:
T*A*TTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTT----
GAGCCCTTTGGGGATCTGTCCACTCCTGATG*C*(SEQ ID NO.2)
Sequence submits the synthesis of Suzhou Jin Weizhi Biotechnology Co., Ltd.
Whether the dissection and donor for identifying CRISPR/Cas9 plasmid knife are integrated
After the CRISPR/Cas9 plasmid constructed above and donor-ssDNA cotransfection 293T cell, extract total cellular base
Because whether group dissection of DNA progress Sanger sequence verification CRISPR/Cas9 plasmid knife and donor integrate.
1. cell culture
293T cell line is with the DMEM complete medium containing 10% serum in 37 DEG C, 5%CO2It is cultivated in incubator.To
With after 0.25% pancreatin digestion when cell fusion degree reaches 90%, is terminated and digested with DMEM complete medium, be inoculated into 6 orifice plates
In, continue culture 24 hours.
2. plasmid transfection
After 24 hours, confirmation cell is adherent good, and cell fusion degree reaches 80%, can be transfected.Every hole transfects 2ug
The donor-ssDNA of CRISPR/Cas9 plasmid and 100pmol uses the X-tremeGENE HP DNA of Roche company
Transfection Reagent transfection reagent requires to be transfected to specifications, and the cell after transfection continues in 37 DEG C, 5%
CO2It is cultivated in incubator.
3. genome DNA extraction
After transfection 48 hours, routine uses 0.25% trypsin digestion cell, collects cell into centrifuge tube, normal temperature condition
Lower 300g is centrifuged 5 minutes, and PBS washed once, and 300g is centrifuged 5 minutes under normal temperature condition.Use full formula gold DNA extraction agent box
The reagent component of (Quan Shijin Bioisystech Co., Ltd, article No.: EE101-01), extracts cellular genome in accordance with the following methods
DNA:
(1) 100 μ l cell pyrolysis liquid LB2 are added, mix well, suspension cell, 20 μ l RnaseA and 20 μ l are added
Proteinase K is vortexed and mixes in sample, is incubated at room temperature 2min.
(2) the BB2 reagent of 500 μ l combination DNA is added, is vortexed 5 seconds immediately, is incubated at room temperature 10min.
(3) whole solution is added in centrifugal column, 12000 × g is centrifuged 30 seconds, discards efflux.
(4) 500 μ l solution clean buffer2 (CB2) are added, 12000 × g is centrifuged 30 seconds, discards efflux.Then it weighs
It is multiple primary.
(5) 500 μ l solution wash buffer2 (WB2) are added, 12000 × g is centrifuged 30 seconds, discards efflux.Then it weighs
It is multiple primary.
(6) 12000 × g are centrifuged 2 minutes, completely remove remaining WB2.
(7) centrifugal column is placed in a clean centrifuge tube, column center be added 50 μ l preheating (60-70 DEG C) go from
Sub- water (PH > 7) is stored at room temperature 1 minute, and 12000 × g is centrifuged 1 minute, eluted dna.Measure DNA concentration.
4. the design of primer
Using the primer for the upstream and downstream segment for expanding targeted mutagenesis point before, across target spot, (product length is excellent at primer both ends
Choosing~500bp, and target spot apart from two sections of primer distance should difference 50bp or more so as to the energy in agarose gel electrophoresis
Effective two small fragments distinguishing digestion and opening).PCR product serves Hai Sheng work biotech company and carries out Sanger sequencing, expands
It is consistent with sequencing primer to increase primer, sequence are as follows:
Forward primer: GAGGAGAAGTCTGCCGTTACT (SEQ ID NO.3)
Reverse primer: AAACATCAAGCGTCCCATAGA (SEQ ID NO.4)
5.PCR reaction
Using the genomic DNA of said extracted as template, PCR reaction is carried out with above-mentioned primer.The high-fidelity that this experiment uses
Archaeal dna polymerase is Beijing Quan Shijin Biotechnology Co., LtdPCR SuperMix (article No.: AS111-
02)。
PCR reaction system is as shown below:
| Component | Volume | Final concentration |
| Template | It is variable | As required |
| Forward primer | 1 | 0.2uM |
| Reverse primer (10uM) | 1 | 0.2uM |
| 2xEasyTaq PCR SuperMix | 25 | 1x |
| Nuclease-free water | It is variable | - |
| Total volume | 50μl | - |
PCR reaction condition is as shown below:
PCR after the reaction was completed, takes a small amount of PCR product to carry out agarose gel electrophoresis, according to electrophoresis result preliminary judgement PCR
Whether the concentration and stripe size of product are correct etc..
6.T7Endonuclease I digestion experiment and polyacrylate hydrogel electrophoresis
The T7Endonuclease I and matched 10 × NEB Buffer 2 that this experiment uses are purchased from New England
Biolabs company (article No.: M0302S).Operating procedure is as follows:
200ng purified pcr product is taken to be reacted as follows:
Reaction system is as shown below:
| 10×NEB Buffer 2 | 2μL |
| Purified pcr product | 200ng |
| H2O | It mends to 19 μ L |
| Total amount | 19μL |
Annealing conditions are as shown below:
| 95℃ | 5min |
| 95-85℃ | -2℃/sec |
| 85-25℃ | -0.1℃/sec |
| 4℃ | It keeps |
After the completion of annealing reaction, 1 μ L of T7Endonuclease I is added at this time, and after concussion mixes, 37 DEG C are incubated for 20 points
Clock completes cutting, and 2 μ L 0.25M EDTA solution are added and terminate endonuclease reaction.After the reaction was completed, using 2% Ago-Gel into
Row electrophoresis.
Agarose gel electrophoresis results show that above-mentioned PCR product can be cut off by T7Endonuclease I, and it is big by one to occur one
Small two band illustrates that CRISPR/Cas9 successfully cuts the gene sequence of 41/42 location proximate of beta globin genes in the cell
Column, are calculated by gray value of the ImageJ software to band, can estimate its cutting efficiency.
Fig. 2 shows after CRISPR/Cas9 plasmid and donor-ssDNA cotransfection 293T cell, extract total cellular gene
Group DNA carries out the dissection of Sanger sequence verification CRISPR/Cas9 plasmid knife and whether donor integrates.
In Fig. 2, WT is represented without transfection CRISPR/Cas9 plasmid；" 41-42-g1 " is represented in beta globin genes 41/42
Mutational site nearby design sgRNA and experimental identification its have cutting effect.Dash area indicates the genome in 293T cell
Upper 41/42 deletion mutation of introducing beta globin genes :-CTTT；The nonsense mutation base that arrow instruction in the rightmost side introduces: T → C, i.e.,
Two exon 33-36 bit base of beta globin genes is lacked, and the 40th bit base of two exon of beta globin genes is by T
(thymidine) sports C (cytimidine)；The gag sequence of frameshit after bottom arrow instruction four bases of missing.
After plasmid and ssDNA are transfected into cell, what plasmid expression came out targets 41/42 mutational site of beta globin genes
CRISPR/Cas9 system can rapidly identify 41/42 mutational site sequence of beta globin genes, play dissection.Cutting
Cell increases the homologous efficiency for repairing HDR when having external source ssDNA as recovery template afterwards, thus reach seamless reparation,
The mutating alkali yl sequence that primer needs simultaneously shows in sequencing peak figure, is shown as 41/42 deletion mutation position of beta globin genes
The frameshift mutation in point and nonsense mutation site covers peak (gap sequence, as shown in bottom arrow).Then go out without phenomenon in WT group
It is existing.
7. integration identification BanII digestion experiment and polyacrylate hydrogel electrophoresis
Carry out digestion experiment after the cell genomic dna of extracting is carried out the target fragment that PCR amplification includes mutation simultaneously,
The wherein cutting efficiency of T7E1 digestion experimental identification knife, the efficiency of BanII digestion experimental identification donor-ssDNA integration, two
Group digestion experiment control is to test with a batch, and template quantity is consistent.As a result as shown in Figure 3 and Figure 4.It can observe experimental group
There is the digestion band of T7E1 enzyme and BanII enzyme in (41-42-g1).
The BanII enzyme and matched 10 × Buffer used purchased from Thermo Scientific company (article No.:
ER0281)。
Operating procedure is as follows:
200ng purified pcr product is taken to be reacted as follows:
Reaction system is as shown below:
| 10×Buffer 2 | 2μL |
| Purified pcr product | 200ng |
| BanII | 1μl |
| H2O | It mends to 19 μ l |
| Total volume | 20μL |
After mixing, 37 DEG C of incubation completion in 1 hour cuttings are added 2 μ L 0.25M EDTA solution and terminate endonuclease reaction.Reaction
After the completion, electrophoresis is carried out using 2% Ago-Gel.
Agarose gel electrophoresis results show that above-mentioned PCR product can be cut off by BanII, small one and large one two band occur,
After illustrating that CRISPR/Cas9 successfully cuts HBB gene into the cell, the donor-ssDNA of modification has been integrated into target site.Pass through
Image J software calculates the gray value of band, can estimate BanII cutting efficiency, by its with T7E1 digesting efficiency into
Row compares, and can calculate the integration efficiency for obtaining donor.
Fig. 4 is to carry out gray analysis, efficiency value calculated after quantization by the glue figure to Fig. 3.Wherein, editorial efficiency
Calculation formula are as follows: %gene modification=100x (1-(1-fraction cleaved) 1/2).T7E1 digesting efficiency
Reflect the efficiency of knife targeting cutting；BanII digesting efficiency shows when donor is integrated into target position, can be known by this enzyme
Not and cut；The calculation formula of the integration efficiency of donor are as follows: integration efficiency=(BanII digesting efficiency/T7E1 digesting efficiency) ×
100%, this efficiency represents integration and repairs the efficiency for introducing mutation.Fig. 4 shows that donor has effectively been integrated into target position.
Fig. 3 is extract total cellular genomic DNA after CRISPR/Cas9 plasmid and donor-ssDNA cotransfection 293T cell
After carrying out the target fragment that PCR amplification includes mutation, the digestion of progress is tested, the wherein cutting of T7E1 digestion experimental identification knife
Efficiency, the efficiency of BanII digestion experimental identification donor-ssDNA integration, two groups of digestion experiment controls are same a batch experiment, and
Template quantity is consistent.
Nonsense mutation is introduced on donor can produce the restriction enzyme site BanII not having originally simultaneously, improvement
Whether can be integrated by this restriction enzyme site Rapid identification after donor integration, and whether be introduced in genome sequence and block alkali
Base (nonsense mutation) has carried out seamless reparation；Block base can be integrated to avoid donor repair after by same sgRNA target again
To identification.
The identification and screening of the 293T cell line of the mutation of beta globin genes 41/42 and nonsense mutation
Go out the mutation of beta globin genes 41/42 and nonsense mutation by selecting monoclonal cell further evaluation and screening
293T cell line.
Group of cells after taking transfection 48h with 0.25% trypsin digestion and is blown and beaten into individual cells respectively, and carefully
Born of the same parents are suspended in spare in the DMEM culture solution of 10% fetal calf serum.
Cell suspension is made into the dilution of gradient multiple, every group of cell is respectively with the Graded Density of every 50,100,200 cells of ware
It is inoculated in the ware of 37 DEG C of pre-temperature culture solutions containing 10mL, and gently rotates respectively, so that cell is uniformly dispersed, finally make in culture hole
Contain up to 1 cell.Set 37 DEG C, 5%CO2And it is cultivated 2~3 weeks in the cell incubator of saturated humidity.
Often observation, when occurring macroscopic clone in culture dish, amplification be passaged to cell quantity reach 500,000/
Hole, collect a part of cell by preceding method extract cell DNA, by sent after preceding method PCR amplification raw work bioengineering (on
Sea) limited liability company's sequencing.
Monoclonal cell sequencing result by the method success in normal 293T cell as shown in figure 5, the results show that drawn
41/42 deletion mutation (- CTTT) of beta globin genes and nonsense mutation (T → C) are entered, to obtain beta globin genes 41/42
Saltant type 293T cell line shows the successful use of efficient directed mutagenesis method.
Sequence table
<110>Guangzhou Gu Run medical science and technology Co., Ltd
<120>sgRNA based on CRISPR/Cas9 technical editor's HBB-41/42 deletion mutation site
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 96
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gaggttcttt gagtcctttg gttttagagc tagaaatagc aagttaaaat aaggctagtc 60
cgttatcaac ttgaaaaagt ggcaccgagt cggtgc 96
<210> 2
<211> 90
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tattggtcta ttttcccacc cttaggctgc tggtggtcta cccttggacc cagaggttga 60
gccctttggg gatctgtcca ctcctgatgc 90
<210> 3
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gaggagaagt ctgccgttac t 21
<210> 4
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aaacatcaag cgtcccatag a 21
<210> 5
<211> 449
<212> DNA
<213> Homo sapiens
<400> 5
gaggagaagt ctgccgttac tgccctgtgg ggcaaggtga acgtggatga agttggtggt 60
gaggccctgg gcaggttggt atcaaggtta caagacaggt ttaaggagac caatagaaac 120
tgggcatgtg gagacagaga agactcttgg gtttctgata ggcactgact ctctctgcct 180
attggtctat tttcccaccc ttaggctgct ggtggtctac ccttggaccc agaggttgag 240
acctttgggg atctgtccac tcctgatgct gttatgggca accctaaggt gaaggctcat 300
ggcaagaaag tgctcggtgc ctttagtgat ggcctggctc acctggacaa cctcaagggc 360
acctttgcca cactgagtga gctgcactgt gacaagctgc acgtggatcc tgagaacttc 420
agggtgagtc tatgggacgc ttgatgttt 449
Claims (10)
1. a kind of sgRNA based on CRISPR/Cas9 technical editor's HBB-41/42 deletion mutation site, nucleotide sequence is such as
Shown in SEQ ID NO.1.
2. a kind of carrier comprising sgRNA described in claim 1.
3. carrier according to claim 1, which is characterized in that the carrier is CRISPR/Cas9 expression vector.
4. a kind of cell comprising carrier described in claim 2 or 3.
5. a kind of gene editing kit comprising sgRNA described in claim 1.
6. kit according to claim 5, which is characterized in that it further include donor ssDNA, the core of the donor ssDNA
Nucleotide sequence is as shown in SEQ ID NO.2.
7. sgRNA described in claim 1 is in the application for editing HBB-41/42 deletion mutation site.
8. a kind of method based on CRISPR/Cas9 technical editor's HBB-41/42 deletion mutation site comprising use nucleotide
Sequence sgRNA as shown in SEQ ID NO.1 constructs CRISPR/Cas9 expression vector.
9. according to the method described in claim 8, it is characterized in that, further including being using donor ssDNA, the donor ssDNA
HBB-41/42 deletion mutation and nonsense mutation are introduced in the base sequence of HBB-41/42 deletion mutation location proximate, and to institute
It states donor ssDNA both ends base and carries out thio-modification.
10. method according to claim 9, which is characterized in that comprising the following specific steps
(1) sgRNA is designed and synthesized, Cas9 sequence and the sgRNA are cloned on CRISPR/Cas9 carrier, is constructed
CRISPR/Cas9 expression vector containing Cas9 sequence and the sgRNA；
(2) the donor ssDNA is designed and synthesized, the nucleotide sequence of the donor ssDNA is as shown in SEQ ID NO.2；
(3) it by the CRISPR/Cas9 plasmid and the donor ssDNA cotransfection 293T cell, and is cultivated.
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