CN110438161A - Utilize the method for Cas12a protein screening diallele mutant clone - Google Patents
Utilize the method for Cas12a protein screening diallele mutant clone Download PDFInfo
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Abstract
The present invention relates to a kind of methods using Cas12a protein screening diallele mutant clone, are mainly used for screening based on the edited diallele mutant clone of CRISPR/Cas9;It solves diallele mutant clone and is difficult to the technical problem filtered out.The present invention is based on the nucleic acid detection techniques that Cas12a albumen (including all bacterial origins) is developed can simple, efficiently, steadily screen the edited diallele mutation positive cell of CRISPR/Cas9, this method and PCR sequencing PCR can be partially replaced, substantially reduce the cost of screening.
Description
Technical field
The present invention relates to medicine, biological field, it is related to a kind of being based on CRISPR-Cas9 system using Cas12a protein screening
The method of diallele mutant clone after gene editing.
Background technique
CRISPR/Cas9 is a kind of adaptive immunity defence that bacterium and archeobacteria are formed during long-term evolution, can
For fighting the viral and exogenous DNA of invasion.CRISPR/Cas9 system is by the way that the segment for invading exogenous DNA to be integrated into
In CRISPR, and the degradation of homologous sequence is instructed using corresponding CRISPR RNAs (crRNAs), to provide immunity.
By the transformation of scientist, CRISPR/Cas9 is widely used in bacterium, animals and plants, the gene editing of mammalian cell.
The gene mutant animals of this method building, which have, is significantly higher than the system genitale transfer ability of conventional method, be it is a kind of it is reliable, efficiently,
The quickly new method of building knock-out animal model shows great potentiality in biology, medical domain.The work of this system is former
Reason is that crRNA (CRISPR-derived RNA) is tied by base pairing and tracrRNA (trans-activating RNA)
Conjunction forms tracrRNA/crRNA compound, this compound guides sequence target position of the nuclease Cas9 albumen again with crRNA pairing
Point shearing double-stranded DNA.And tracrRNA/crRNA is transformed by the SgRNA with guiding function by engineer
(single-guide RNA), guidance Cas9 cut the fixed point of DNA.Host cell mainly passes through nonhomologous end reparation
(NHEJ) mechanism repairs the edited generation DNA notch of Cas9, will be inserted into wrong DNA sequence dna at DNA break, lead to base
The frameshift mutation of cause, to realize the knockout to target gene.
Preliminary screening currently used for the edited positive cell of CRISPR-Cas9 system gene is mainly in T7E1 nucleic acid
Enzyme cutting restriction analysis, this enzyme identify and cut incomplete pairing DNA, cross-shaped configuration DNA, Holliday structure or intersect DNA,
Heteroduplex DNA or with slower speed cutting or nicking double-stranded DNA.It is amplified by PCR reaction with mutational site
Then DNA fragmentation is incubated for T7E1, electrophoresis detection DNA cuts situation to judge gene knockout result.The method takes time and effort,
Unstable result, and monoallelic mutation and diallele knockout strain cannot be distinguished, it obtains through subsequent sequence verification, this
The just greatly cost of increased time and money.
Summary of the invention
For the shortcomings of the prior art, the present invention, which provides, a kind of to be based on using Cas12a protein screening
The method of the edited diallele mutant clone of CRISPR-Cas9 system gene.First with CRISPR-Cas9 system
Gene is edited, generate notch in target sequence and insertion mutation base is generated by nonhomologous end reparation, is caused
The mutation of target gene.Be enriched with target gene by PCR amplification and design gRNA for target sequence, by Cas12a albumen and gRNA and
After target gene and ssDNA (ssDNA-FQ) are incubated for altogether, the power for detecting fluorescence can filter out CRISPR/Cas9 and edit
Diallele mutant clone afterwards.
To achieve the above object, the present invention provides a kind of using Cas12a protein screening diallele mutant clone
Method, which comprises the following steps:
Design gRNA sequence: according to the sequence design gRNA of the editing sites of CRISPR/Cas9；
GRNA synthesis: turn green enzyme with T7 and synthesize in vitro；
Design of primers: design primer amplified target-gene sequence；
Cleavage reaction: gRNA guides Cas12a in conjunction with target gene, the trans- cleavage activity of Cas12a is activated, to cut
Fluorescence report substrate single strand DNA-FQ (ssDNA-FQ) generates fluorescence；
Fluorescence detection: the fluorescence after detecting cutting substrate using instrument, is unmutated strain if detecting strong fluorescence
Perhaps monoallelic mutant strain is diallele mutant clone if fluorescence or hypofluorescence is detected then.
Preferably, in design gRNA sequence step, according to the target gene position of the SgRNA of CRISPR/Cas9 targeting
Point sequence design gRNA, gRNA targeting gene loci before need containing PAM be TTTN or TTN (N be any A, T, G, C alkali
Base), gRNA length is 17~22nt, and gRNA is synthesized in vitro by T7 transcriptase.
Preferably, if there is no TTN before the target gene site that gRNA is directed to, it can be by introducing TTTN in PCR primer
Perhaps TTN motif is so that identify TTTN TTN motif containing Cas12a albumen before the target gene site that gRNA is directed to.
Preferably, by Cas12a+gRNA+target DNA+ssDNA-FQ in 30-50 DEG C incubation 10-40 minutes, simultaneously
Control group is set and carries out Experimental comparison.
Preferably, totally two groups of the control group, including negative control group and positive controls, the negative control group
Are as follows: Cas12a+gRNA+ssDNA-FQ is incubated for 30 minutes at 37 DEG C, the positive controls are as follows: Cas12a+gRNA+target
DNA (DNA without the CRISPR/Cas cell edited)+ssDNA-FQ is incubated for 30 minutes at 37 DEG C.
Preferably, finally also needing to carry out Substrate fluorescence detection, determine whether successfully to be screened
The beneficial effects of the present invention are: the present invention, which provides a kind of efficient, stable screening, is based on CRISPR/Cas9 system
The method of diallele mutant clone after gene editing, what solution diallele mutant clone was difficult to screen asks
Topic.The nucleic acid detection technique developed the present invention is based on Cas12a albumen (including all bacterial origins) can be simple, efficient, steady
Surely it screens CRISPR/Cas9 edited diallele mutation positive cell, this method and can partially replace PCR sequencing PCR, greatly
The big time and money cost for reducing screening.
Detailed description of the invention
Fig. 1 is step and schematic diagram of the invention
Fig. 2 is the design drawing of present invention design gRNA sequence；
Fig. 3 is PCR primer design drawing of the present invention；
Fig. 4 is fluoroscopic examination result figure of the present invention；
Fig. 5 is PCR electrophoresis result figure of the present invention；
The position Fig. 6 sequencing result figure of the present invention；
Fig. 7 is CXCL9ELISA result figure.
Specific embodiment
In order to more clearly state the present invention, the present invention is further described with reference to the accompanying drawing.
Referring to Fig. 1, Cas12a/Cpf1 albumen used in this application also belongs to CRISPR-Cas family, there is class
Like the endonuclease function of Cas9, while the combination of Cas12a-crRNA compound and complementary single-stranded dna or double-stranded DNA
Its powerful trans- cleavage activity of non-specific ssDNA is activated out, by fluorescent quenching (FQ) reporter group on ssDNA label,
Nucleic acid detection instrument can be developed into, when Cas12a-crRNA is matched with detected nucleic acid, it is glimmering that Cas12a cuts ssDNA-
Fluorescence is presented in optical quenching reporter group, if when Cas12a-crRNA and detected nucleic acid mismatch or Incomplete matching,
Cas12a does not cut perhaps part cutting ssDNA- fluorescent quenching reporter group unstressed configuration or faint fluorescence.
Referring to Fig. 1, notch can be generated in target sequence through the edited gene of CRISPR-Cas9/Cpf1 and passed through
Nonhomologous end reparation generates insertion mutation base, causes the mutation of target gene.Therefore, gRNA is designed for target sequence, with
Cas12a, ssDNA-FQ, target-gene sequence detect fluorescence after being incubated for it may determine that CRISPR-Cas9/Cpf1 is edited double
Allelic mutation cell strain.
Experimental method in the following example is unless otherwise specified conventional method；Used material, reagent etc.,
If commercially being obtained without specified otherwise.
Specific embodiment is as follows:
Experimental material: finished product FnCas12a (article No.: 32111) thin purchased from port biotechnology company, source of people THP-1 is revealed
Born of the same parents, ssDNA-FQ:5 ' HEX-TTTTTTTTTTTT-3 ' BHQ1 are synthesized by the raw work in Shanghai, edited through CRISPR/Cas9
Thp-1 cell strain (CXCL9 mutated gene) primer tasteless nucleotide chain synthesizes by the raw work in Shanghai, T7High Yield RNA
Transcription Kit and VAHTS RNA Clean Beads are purchased from Nanjing Vazyme Biotechnology Co., Ltd., CXCL9
ELISA kit is purchased from the green skies.
Experimental method:
1, the screening of monoclonal cell:
1) medicine sieve is carried out with puromycin (4ug/ml) to through the edited Thp-1 cell of CRISPR/Cas9, screening is about
14 days, sort monoclonal cell strain；
2) unicellular amplification: the cell mixing in step (1) is diluted in 96 orifice plates according to certain multiple, so that often
Kong Douwei individual cells are cultivated two weeks and carry out unicellular amplification；
3) unicellular to draw 1ul cell when being expanded to 500 cells or so, extract monoclonal cell strain DNA, -80 DEG C of guarantors
It deposits spare.
2, referring to Fig. 2, design gRNA sequence: the target gene site sequence targeted according to the SgRNA of CRISPR/Cas9
Design gRNA, needed before the gene loci of gRNA targeting containing PAM be TTN (N be any A, T, G, C base), gRNA length is
1722nt, if there is no TTN before the target gene site that gRNA is directed to, can by PCR primer introduce TTN motif so that
TTN motif is identified containing Cas12a albumen before the target gene site that gRNA is directed to.
3, synthesis in vitro and purifying gRNA sequence:
GRNA transcription templates sequence:
T7 promoter primer: 5 '-CCCTATAGTGAGTCGTATTAATTTC-3 '
Blocked portion is the targeting boot sequence of gRNA, and the part with background color is the cyclic structure of gRNA, underlined sequence
For T7 promoter sequence.GRNA template, T7 promoter primer are synthesized by the raw work in Shanghai, are synthesized in vitro using T7 polymerase a large amount of
GRNA, briefly steps are as follows:
1) following component is chosen:
2) mix gently each component with pipettor, and it is of short duration be collected by centrifugation, 37 DEG C of incubation 2h；
3) the DNase I, 37 DEG C of incubation 15min that 1 μ l is added in the reaction system digest the DNA profiling of transcription；
4) using Nanjing Nuo Weizan company RNA cleaning magnetic bead kit VAHTS RNA Clean Beads (article No.:
N412-01) the RNA after purifying reaction, (step does not make related elaboration referring to kit specification, the application), Nanodrop
The nucleic acid concentration of 2000 measurements after purification.
4, PCR amplification includes the DNA sequence dna of gene mutation target spot:
1) referring to Fig. 2, PCR primer designs: it must include gene knockout target spot DNA sequence dna when PCR primer designs,
And CXCL9-
GRNA-R:5 '-CCTCCTTCAAAGGTTTGTGG-3 ' is used to expand the DNA sequence dna of gRNA targeting, and TT is artificially to draw in box
The site TTN of the identification of primer Cas12a albumen in the product that the base entered is formed so as to PCR amplification；
2) amplification of target sequence: using the DNA in step 1 (3) as template, primer CXCL9-gRNA-F and CXCL-gRNA-
R, primer carry out the target sequence DNA that PCR amplification includes cleavage site, PCR product electrophoresis detection, it is ensured that the successful expansion of target fragment
Increase.
5, the substrate cleavage reaction that Cas12a is mediated: reaction system according to following component,
Liquid-transfering gun piping and druming mixes, and is incubated for 30 minutes at 37 DEG C.
7, fluorescence detection: detecting the fluorescence intensity of above-mentioned reaction solution using VarioskanFlash instrument, and excitation wavelength is set
It is set to 535nm, launch wavelength is set as 560nm, fluorescent value=initial fluorescence-background fluorescence activity；
8, sequence verification: sequence verification is carried out to the above results, it includes the DNA of target sequence that PCR amplification, which goes out, first, and amplification is drawn
Object are as follows: CXCL9-gRNA-1-F:TCTGTGGCTAACCAATTAAGGA and CXCL-gRNA-1-R:
TGTTTGTATGGGCAAGCACT, the PCR product amplified are sent to the raw work sequencing in Shanghai.
9, ELISA is verified: while we verify from reliability of the protein level to appeal method, sample albumen mentions
It takes and is carried out with the operating method of ELISA experiment according to operating instruction.
Fig. 3-Fig. 6 is please referred to, experimental result is as follows:
1) fluorescence detection result: almost without fluorescence is detected in 11 samples, show to filter out potential 11 CXCL9
Diallele mutant clone；
2) amplification of target sequence: electrophoresis result shows, all 11 monoclonal cell strains filtered out, PCR amplification
Clearly purpose band, primer size 188bp are consistent with estimated stripe size out；
3) for sequence verification as a result, sequencing result is found, there is overlap peak in CRISPR/Cas9 editing sites in 11 samples,
Show that gene is mutated.
4) ELISA result: we verify the reliability of the method screening again on protein level, can from ELISA result
To find out, the Positive mutants monoclonal CXCL9 filtered out by the method is almost without expression or the expression and wild type of polar region
It compares, shows that CXCL9 is successfully mutated, can be used for simple and quick double equipotentials of the screening based on CRISPR-Cas9 system editor
Genetic Mutant Cell strain.
5) the comprehensive method that 3) can prove that this Cas12a is mediated with result 4) can be identified rapidly and accurately
The diallele mutant clone of CRISPR/Cas9 induction.
Disclosed above is only several specific embodiments of the invention, but the present invention is not limited to this, any ability
What the technical staff in domain can think variation should all fall into protection scope of the present invention.
Claims (6)
1. a kind of method using Cas12a protein screening diallele mutant clone, which is characterized in that including following step
It is rapid: design gRNA sequence: according to the sequence design gRNA of the editing sites of CRISPR/Cas9；
GRNA synthesis: turn green enzyme with T7 and synthesize in vitro；
Design of primers: design primer amplified target-gene sequence；
Cleavage reaction: gRNA guides Cas12a in conjunction with target gene, the trans- cleavage activity of Cas12a is activated, to cut fluorescence
Reporter substrate single strand DNA-FQ (ssDNA-FQ) generates fluorescence；
Fluorescence detection: the fluorescence after detecting cutting substrate using instrument, if detecting strong fluorescence be unmutated strain or
Monoallelic mutant strain is diallele mutant clone if fluorescence or hypofluorescence is detected then.
2. the method according to claim 1 using Cas12a protein screening diallele mutant clone, feature
It is, in design gRNA sequence step, is designed according to the target gene site sequence of the SgRNA of CRISPR/Cas9 targeting
Needed before the gene loci of gRNA, gRNA targeting containing PAM be TTTN or TTN (N be any A, T, G, C base), gRNA long
Degree is 17~22nt, and gRNA is synthesized in vitro by T7 transcriptase.
3. the method according to claim 1 using Cas12a protein screening diallele mutant clone, feature
Be, if there is no TTN before the target gene site that gRNA is directed to, can by PCR primer introduce TTTN TTN motif,
So that identifying TTTN TTN motif containing Cas12a albumen before the target gene site that gRNA is directed to.
4. the method according to claim 1 using Cas12a protein screening diallele mutant clone, feature
Be, by Cas12a+gRNA+target DNA+ssDNA-FQ in 30-50 DEG C incubation 10-40 minutes, while be arranged control group into
Row Experimental comparison.
5. the method according to claim 4 using Cas12a protein screening diallele mutant clone, feature
It is, totally two groups of the control group, including negative control group and positive controls, the negative control group are as follows: Cas12a+gRNA
+ ssDNA-FQ is incubated for 30 minutes at 37 DEG C, the positive controls are as follows: Cas12a+gRNA+target DNA is (without CRISPR/
The DNA for the cell that Cas is edited)+ssDNA-FQ 37 DEG C be incubated for 30 minutes.
6. the method according to claim 1 using Cas12a protein screening diallele mutant clone, finally also
Substrate fluorescence detection need to be carried out, determines whether successfully to be screened.
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Cited By (2)
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| CN111455046A (en) * | 2020-03-13 | 2020-07-28 | 深圳市罗湖区人民医院 | Nucleic acid detection method for mutation of ADGRG6 enhancer and kit thereof |
| CN114958834A (en) * | 2020-03-09 | 2022-08-30 | 中山大学 | ADAR protein intracellular efficient binding substrate and application |
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| CN111455046A (en) * | 2020-03-13 | 2020-07-28 | 深圳市罗湖区人民医院 | Nucleic acid detection method for mutation of ADGRG6 enhancer and kit thereof |
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