CN109797170A - A method of detection targeted gene disruption effect - Google Patents

Abstract

The present invention relates to a kind of methods for detecting targeted gene disruption edit effect.It includes the following steps: processing group: 1) constructing the over-express vector of the expression cassette of the target gene containing Code targets albumen；2) knockout editor's carrier of structure building target gene；3) over-express vector and knockout editor's carrier are transferred to jointly in cell to be edited, obtain cell mass to be detected；4) determine whether the knockout edits carrier effective by the variation of the amount of target proteins described in the detection cell mass to be detected.

Description

A method of detection targeted gene disruption effect

Technical field

The present invention relates to a kind of methods for detecting targeted gene disruption effect.

Background technique

CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats And CRISPR associated) system consists of two parts: it mediates the gRNA (guide RNA) of identification and is responsible for cutting Cas9 albumen.CRISPR/Cas9 gene editing technical application research at present is very extensive.The gene editing skill powerful as one Art, such as gene Knockout, carrying out knockout effect detection to the CRISPR/Cas9 plasmid of building is particularly important.Generally In the case of, after CRISPR/Cas9 plasmid is transferred to cell, if since gene knockout causes cell that a large amount of apoptosis occur, it can not It is to verify by way of template progress PCR amplification fluidic cell screening, unicellular culture or using the genomic DNA of extraction The gene knockouts result such as the no missing for producing base or insertion.The more of a large amount of apoptosis of cell can't be caused for gene knockout In number situation, although stable cell can be being established after repeatedly attempting by the cell of CRISPR/Cas9 gene editing System, but if existed in obtained cell line since CRISPR/Cas9 is unable to specific recognition target fragments, and causing could not Effective CRISPR/Cas9 gene editing is carried out, then the cell line obtained is also no any value.Then, cell is built The process of system is not only time-consuming, laborious, is also unfavorable for the progress of research work.

Summary of the invention

Based on problems of the prior art, inventor also once attempted to solve by other conventional means, so And there is no any progress.For example, the Western for the blank control group not knocked out with conventional gene knockout processing group and gene Blot quantitative comparison is analyzed, however eventually because being difficult to not judge knocking out for target gene due to reaching significant difference Property, even result in the inoperative wrong conclusion of knockout carrier used in obtaining in many cases.

After a variety of trials and exploration, it is determined that technical solution of the present invention: construct target gene to be knocked out It is overexpressed box, and the overexpression box is imported into cell with CRISPR/Cas9 plasmid jointly, then passes through the side such as Western blot Formula detects the variation of target gene expression expressing quantity to be knocked out, detects CRISPR/Cas9 plasmid pair cell with this The editability of middle target gene.Pass through this improvement, on the one hand, a large amount of apoptosis of cell will not be caused for targeted gene disruption In most cases, the sequencing procedure that genome is extracted after establishing stable cell lines, thus time-consuming less, operating method are eliminated It is simpler easy.On the other hand, the case where a large amount of apoptosis of cell being caused for targeted gene disruption, since streaming can not be passed through Cell screening, unicellular culture carry out the prior arts such as PCR amplification using the genomic DNA of extraction as template to verify gene volume It collects effect and seems helpless.Importantly, this ingenious design through the invention makes originally through protein quantification point Undetectable method is analysed to be able to using having dissolved various problems existing in the prior art.Further, since in the method may be used Label is added in being overexpressed box, therefore, target gene expression expressing quantity can be detected indirectly using tag antibody Number so that determine CRISPR/Cas9 system again to the knockout effect of target gene, to achieve the purpose that detection.Therefore, very It is suitble to those not have the target gene to be knocked out of specific antibody.At the same time, it can also be carried out for the later period in living body micro- The operation experiments such as injection provide reliable foundation.

Specifically, the present invention provides a kind of methods for detecting targeted gene disruption effect comprising following steps:

Processing group:

1) over-express vector of the expression cassette of the target gene containing Code targets albumen is constructed；

2) knockout editor's carrier of target gene is constructed；

3) over-express vector and knockout editor's carrier are transferred to jointly in cell to be edited, are obtained to be checked Survey cell mass；

4) determine that the knockout is edited by the variation of the amount of target proteins described in the detection cell mass to be detected Whether carrier is effective.

In a specific embodiment, further include being overexpressed control group in the method: the over-express vector is turned Enter in the cell (cell i.e. identical with cell to be edited), obtains overexpressing cell group；By comparing described to be checked The amount of target proteins described in cell mass and the target proteins in the overexpressing cell group is surveyed to determine that the knockout is compiled Whether effectively to collect.

In a specific embodiment, the amount of the target proteins described in the cell mass to be detected crosses table less than described Up to the target proteins in cell mass amount when, knockout editor is effective.

In a specific embodiment, the amount and the overexpression of the target proteins described in the cell mass to be detected When the amount of the target proteins in cell mass is suitable, the knockout editor is invalid.

It is found by many experiments, the expression quantity for cultivating 60 to 84 hours albumen after transfection is bigger, effective knocking out In the case where, it is detected in this period, extraordinary can distinguish the target egg being overexpressed between control group and processing group The difference of white amount.In a specific embodiment, after being transferred to the carrier, culture measured the target after 60 to 84 hours Mark the amount of albumen.

In a specific embodiment, after being transferred to the carrier, culture measured the target proteins after 72 hours Amount.

In a specific embodiment, the amount of the target proteins is determined by way of Western Blot.

In a specific embodiment, knockout editor's carrier of the target gene is CRISPR/Cas9 system.

In a specific embodiment, the cell is insect cell.

In a specific embodiment, the preferably described cell is that SL cell line Spli221, Spodopterafrugiperda are thin One of born of the same parents system Sf9, Trichopllusia ni cell line High Five.

In a specific embodiment, the CRISPR/Cas9 system is the CRISPR/Cas9 system suitable for insect； It is preferred that the CRISPR/Cas9 system is A3EGFP and IE1Cas9.

In a specific embodiment, the target gene being overexpressed in box and label Gene Fusion.So The fusion protein of targeting proteins and label can be detected by tag antibody.

In a specific embodiment, the label is in histidine tag, Tag label, Flag label, V5 label At least one.

In a specific embodiment, the over-express vector is pIZT/V5-His plasmid in zero load.

In a specific embodiment, further include blank control group in the method: the cell is without any processing, Obtain untreated cell group；It is only transferred to the knockout and edits carrier to the second cell mass to be detected in cell to be edited； Compare the amount of target proteins described in the described second cell mass to be detected and the target proteins in the untreated cell group.

In a specific embodiment, the amount of the target proteins described in the described second cell mass to be detected and it is described not When the amount of the target proteins in processing cell mass is suitable；And the amount for working as target proteins described in the cell mass to be detected is few When the amount of the target proteins in the overexpressing cell group, the knockout editor is effective.

Detailed description of the invention

Figure 1A is Western Blot of the ATPase β albumen after expressing 72h in the first processing group and blank control group As a result.Wherein, it is the first processing group that A3EGFP-ATPase β gRNA plasmid and IE1Cas9 plasmid transfect Spli221 cell jointly, The Spli221 cell of untransfected A3EGFP-ATPase β gRNA plasmid and IE1Cas9 plasmid is blank control group；ATPase β's Size is about 53kDa；M is albumen Marker；GAPDH is the protein for stablizing expression in cell, as internal reference.It can by figure Know, the ATPase β protein expression level in the first processing group is with the ATPase β protein expression level in blank control group visual Indifference in level.

Figure 1B is difference analysis of the ATPase β albumen after expressing 72h in the first processing group and blank control group As a result.According to t examine as a result, NS is indicated in horizontal upper there was no significant difference the property of p < 0.001.Wherein, ATPase β protein expression Level is the average value of independent repeated trials three times.

Fig. 2A is the Western of second processing group and the ATPase β albumen being overexpressed in control group after expressing 72h Blot result.Wherein, pIZT/V5-His-ATPase β is overexpressed plasmid, A3EGFP-ATPase β gRNA plasmid and IE1Cas9 matter The common transfection Spli221 cell of grain is second processing group, and pIZT/V5-His-ATPase β is overexpressed plasmid and individually transfects Spli221 cell is to be overexpressed control group；ATPase β and V5 tag fusion protein size are about 58KDa；M is albumen Marker； GAPDH is the protein for stablizing expression in cell, as internal reference.As seen from the figure, the ATPase β albumen table in second processing group Up to amount significantly lower than the ATPase β protein expression level being overexpressed in control group in visual level.

Fig. 2 B is second processing group and the ATPase β albumen being overexpressed in the control group otherness after expressing 72h point Analyse result.According to t inspection as a result, * * * is indicated in the horizontal upper significant difference of p < 0.001.Wherein, ATPase β protein expression water It puts down as the average value of independent repeated trials three times.

Specific embodiment

The present invention is further explained in the light of specific embodiments, but following embodiment and not pairs of enough the application Limitation, all includes the technical solution within the scope of spirit herein, within the scope of protection of this application.

Culture medium: the culture medium used is the Insect culture medium that this laboratory is prepared, specific formula: TNM-FH insect culture Base (being purchased from SIGMA, article No. T1032) 50.6g is dissolved in sterilizing ddH₂In O, with 30% NaHCO₃(3g NaHCO₃+ 10ml sterilizing ddH₂O pH to 6.1) is adjusted, is settled to 1L, then uses 0.22 μm of diameter of membrane filtration, packing (90ml/ bottles) is added thereto 10ml Fetal Bovine Serum serum (being purchased from Gibco, article No. 10270), 4 DEG C of preservations.

Embodiment 1

1. being overexpressed the building of plasmid pIZT/V5-His-ATPase β

According to the nucleotide sequence information design primer of FoF1-ATPase β gene (SEQ ID No.1), and in upstream and downstream EcoR I and Not I restriction enzyme site are introduced on primer, the cDNA with the prodenia litura blood lymphocyte obtained by reverse transcription is Template carries out PCR, and PCR product is then obtained pIZT/V5- by being connected on expression vector pIZT/V5-His by digestion His-ATPaseβ。

The design of 2.gRNA scaffold transcription box and primer

The targeting sequence of FoF1-ATPase β gene (SEQ ID No.1) is found out according to http://crispr.mit.edu/ Column, i.e., near the initiation codon of gene and GN is searched in downstream₂₀GG (wherein, N is any base) sequence is as target spot (PAM Identify block), then identify that block determines the sequence (SEQ ID No.2) of gRNA scaffold segment according to the PAM, so Afterwards in design gRNA scaffold transcription box (SEQ ID No.3), transcription box sequence from 5 ' to 3 ' is successively are as follows: Bgl II identification + 3 '+6 T+Bgl II of sequence+U6 promoter+gRNA+5 ' identifies sequence, wherein U6 promoter sequence such as SEQ ID No.4 institute Show；5 '+3 ' sequence is as shown in SEQ ID No.5.Pass through over-lap PCR using the design of primer-design software Primer Premier 5 To expand the primer of the gRNA scaffold transcription box.

The segment gRNA scaffold BUg upstream primer BUg53TB-BUg-F (SEQ of the first part of over-lap PCR ID No.6) and downstream primer gRNA-R (SEQ ID No.9) expand；The segment of the second part of over-lap PCR GRNAscaffold g53TB upstream primer gRNA-F (SEQ ID No.8) and downstream primer BUg53TB-53TB-R (SEQ ID No.7) amplification.All of above primer is synthesized by Shuo Qing Biotechnology Co., Ltd.

The clone of 3.gRNA scaffold transcription box

1) gRNA scaffold BUg segment is cloned

With the plasmid VK001-Inx3 containing Inx3 gene of building laboratory early period, (VK001 is purchased from Beijing only Shang Li Moral insect Cas9/gRNA building kit in carrier, wherein contain U6 promoter) be template, with BUg53TB-BUg-F with GRNA-R is that specific primer progress PCR amplification goes out to obtain gRNA scaffold BUg segment and recycle.

2) gRNA scaffold g53TB segment is cloned

Using VK001-Inx3 as template, PCR amplification is carried out as specific primer using gRNA-F and BUg53TB-53TB-R and is obtained To gRNA scaffold g53TB segment and recycle.

3) it is obtained by gRNA scaffold BUg segment and gRNA scaffold g53TB segment composition complete GRNA scaffold segment transcription box

It is template after above 1) will being mixed with 2) recovery product, is with BUg53TB-BUg-F and BUg53TB-53TB-R Primer carries out the second wheel PCR and this segment is connected to cloning vector after obtaining final gRNA scaffold transcription box On pMD19-T (being purchased from Takara, article No. 6013), pMD19-ATPase β gRNA plasmid is obtained.

The building of 4.A3EGFP-ATPase β gRNA carrier

Bgl II is carried out to pMD19-ATPase β gRNA plasmid and carries out digestion, Ago-Gel recycles 580bp segment.Together When, carrier A3EGFP is had by oneself to this laboratory and carries out Bgl II digestion, Ago-Gel recycles 7200bp carrier framework.By gained The ATPase β gRNA segment with restriction enzyme site cohesive end with T4DNA Ligase (being purchased from Takara, article No. 2011A) even It is connected on the linearized vector of A3EGFP.After bacterium solution PCR identification and sequencing identification are correct, plasmid A3EGFP-ATPase β is obtained gRNA。

The knockout of 5.ATPase β gene

5.1. pIZT/V5-His-ATPase β, A3EGFP-ATPase β gRNA and IE1Cas9 plasmid are transfected

(1) prepare the good Spli221 cell of two groups of growth conditions to be layered in 6 orifice plates, every hole about 2 × 10⁵A cell.Its In, first group of transfection A3EGFP-ATPase β gRNA and IE1Cas9 (are studied by Shanghai life science institute plant physiological ecology Institute Huang Yongping professor seminar gifts) two plasmids as the first processing group, do not transfect the Spli221 cell conduct of any plasmid Blank control group, second group of transfection be overexpressed plasmid pIZT/V5-His-ATPase β, A3EGFP-ATPase β gRNA and Tri- plasmids of IE1Cas9 are as second processing group, and only transfection is overexpressed plasmid pIZT/V5-His-ATPase β as overexpression pair According to group.

(2) when the cell density in 6 orifice plates reaches 80%-90%, old culture medium is removed, new culture is added Base gently blows and beats suspension cell with pipette tips, and trypan blue counts；

(3) by the cell after the counting in 6 orifice plates after adhere-wall culture 2h in 27 DEG C of water isolation type constant incubators；With double nothings Culture medium (serum-free, antibiotic-free) is incubated for 30min, and then suction is double without culture medium, and transfection reagent mixed liquor is added dropwise Into overexpression control group and processing group with cell (the bis- no culture mediums of 1ml are only added in blank control group), then it is put into and shakes (about 21rpm/min) is shaked gently on bed, is uniformly mixed transfection reagent mixed liquor, then in 27 DEG C of water isolation type constant incubators Middle standing continues to cultivate.It carries out changing liquid after 4h, i.e., transfection reagent mixed liquor is sucked out, and will be residual in hole with the bis- no culture mediums of 2ml After the transfection reagent mixed liquor stayed cleans up, the culture medium containing 10% serum is added.It is shaked gently on shaking table to training It is uniform to support base, is then placed in 27 DEG C of water isolation type constant incubators, to start timing at this time, cultivates 72h to be used for subsequent step The extraction of total protein of cell.

Wherein, it is formulated as follows for the transfection reagent mixed liquor of 1 hole dosage in 6 orifice plates: drawing the double without training of 100 μ l Feeding base is placed in 1.5ml EP pipe, and the plasmid of 2 μ g is then added, and is mixed；In addition the double of 100 μ l are transfected without culture medium and 6 μ l Reagent C ell fection II (purchased from grace of speeding) is added in the EP pipe of another 1.5ml, and concussion mixes；Two EP are managed In solution merge, 45min is stored at room temperature after mixing, and within the standing phase of the 45min, under every 15min concussion is several, total resonance It swings three times, the double without culture medium of 800 μ l is added thereto later, form transfection reagent mixed liquor.

5.2. the extraction of total protein of cell

(1) after transfecting 72h, culture medium is discarded, is cleaned 1 time with 1 × PBS, 1ml1 × PBS is then added, it is light with liquid-transfering gun Lightly adherent cell is blown afloat, is moved in 1.5ml EP pipe, 3000g, 4 DEG C of centrifugation 5min are discarded supernatant, precipitated；

(2) PMSF is added in RIPA tissue/cell lysate (being purchased from Suo Laibao), obtains efficient RIPA tissue/cell (PMSF is protease inhibitors to lysate, the final concentration of 10 μ l/ml in efficient RIPA tissue/cell lysate, current existing Add PMSF)；Then according to the amount of precipitating, the efficient RIPA tissue/cell cracking of 50-70 μ l is added into the precipitating of step (1) Liquid controls the concentration of total protein within 10mg/ml.

(3) gently pressure-vaccum precipitates, and shakes 30s, makes cell even suspension, and placing 5-10min on ice cracks cell sufficiently； 13000g, 4 DEG C of centrifugation 10min, taking supernatant is total protein.

Determination of protein concentration and denaturation

Egg concentration mensuration is carried out using BCA determination of protein concentration kit:

(I) total protein obtained above is added in 96 orifice plates, 1 μ l sample is loaded in each hole, and it is double to add 9 μ l sterilizing Steam water；It carries out in group three times in parallel to reduce error；

(II) standard curve is drawn with matched standard protein solution B SA in BCA kit (being purchased from ancient cooking vessel state), by albumen mark Quasi- liquid is added separately in 96 orifice plates according to 0,1,2,4,6,8,10 μ l, supplies volume: 10 μ l with sterilizing distilled water；

(III) by after A/B solution in kit proportionally (50 volume A, 1 volume B) mixing, A/B is added in every hole Mixed liquor 200 μ l, 37 DEG C of incubation 30min；

(IV) its light absorption value is measured at microplate reader λ=562nm, and the concentration of total protein is calculated by standard curve；

(V) total protein that above-mentioned steps (3) obtain is mixed in proportion with 5 × SDS-PAGE sample-loading buffer (being purchased from ancient cooking vessel state) After conjunction, 92 DEG C of heat treatment 10min；

(VI) set cooled on ice immediately, then carry out PAGE gel electrophoretic analysis or deposit in -80 DEG C it is spare.

5.3.SDS-PAGE proteins gel electrophoresis

Prepare SDS-PAGE glue and electrophoresis:

The polyacrylamide gel condensed is removed from gel maker, is put into electrophoresis tank, and is added into electrophoresis tank Comb, albumen loading electrophoresis are extracted after entering 1 × PAGE gel electrophoretic buffer.First carry out race glue with 80V, to band excessively on After layer glue, electrophoresis 90min is carried out with 100V voltage.

5.4. transferring film and subsequent operation

(1) pvdf membrane is first impregnated into 1min in methyl alcohol.And pvdf membrane is covered on glue in order.In ice-water bath with 100V voltage carries out 100min；(2) pvdf membrane is taken out after the completion of transferring film, with 5% skimmed milk power confining liquid room temperature close membrane 1h； (3) with 1 × PBST clean closed pvdf membrane twice, each 5min；(4) with the antibody diluent (wherein, one containing primary antibody The anti-volume ratio with antibody diluent is 1:1000, and antibody diluent is purchased from Suo Laibao) it is incubated overnight (4 DEG C)；(5) recycling contains Next the dilution of one antiantibody cleans film three times with 1 × PBST, each 5min；(6) with confining liquid (its containing secondary antibody In, the volume ratio of secondary antibody and confining liquid is 1:5000, and confining liquid is 1 × PBST containing 3% skimmed milk power) incubation at room temperature film 1.5h；(7) film is cleaned three times with 1 × PBST, each 10min；(8) by Pierce ECL Western Blotting After Substrate kit A, B liquid mixes in equal volume, it is added dropwise on pvdf membrane；In FluorChem EFE0511 exposure at Picture；Gray analysis is carried out using Image J software, to determine that expressing quantity changes with the presence or absence of significant otherness.

According to above-mentioned Western Blot operating procedure, result as shown in Figure 1 and Figure 2 is obtained.Wherein, M is albumen Marker, GAPDH are internal reference albumen.Anti-ATPase β (being made by oneself by GL Binchem (shanghai) Ltd) is an antiantibody, Comparative analysis for the first processing group and blank control group；With Anti-V5 (being purchased from invitrogen) for tag antibody, it is used for Second processing group and the comparative analysis for being overexpressed control group；Gray analysis is carried out using Image J software, sees Figure 1B, Fig. 2 B.Root According to the result of Fig. 1 it is found that the ATPase β protein expression level of the first processing group and blank control group is suitable, no significant change.This May be as caused by two reasons: 1) CRISPR/Cas9 system can not knock out target gene；2) endogenous of a large amount of cells ATPase β gene fails to be knocked and stablize expression status in a large amount of, and the cell being knocked is one in cell mass Fraction, so that the first processing group and blank control group ATPase β expressing quantity are not shown when with detection of specific antibody Difference is write, therefore, it is lower that CRISPR/Cas9 system knocks out efficiency.And result further according to fig. 2 is it is found that second processing group ATPase β protein expression level significantly lower than be overexpressed control group., illustrate the external source ATPase β for largely carrying V5 label The cell of gene external source ATPase β gene after using CRISPR/Cas9 system is knocked, and then illustrates knockout carrier energy It is enough to play the function of knocking out target gene.Although can't accurately find the target egg of the first processing group and blank control group at present The reason of white expression indifference, but undoubtedly, no matter theoretically the result of Fig. 2, still experimentally has more convincingness, passes through it As a result it can be determined that knockout carrier has the function of to knock out.Therefore, Fig. 1 is combined with the result of Fig. 2, it is preliminary to illustrate, it causes The suitable reason of the ATPase β protein expression level of first processing group and blank control group may be because of CRISPR/Cas9 system It is lower that system knocks out efficiency, but is easy to make knockout carrier according only to the result of Fig. 1 and can not knock out the wrong conclusion of target gene. Even if it is lower that CRISPR/Cas9 system knocks out efficiency, but under the premise of knocking out more efficient knockout mode without other, makes Use CRISPR/Cas9 system as gene knockout tool be still a kind of selection.

Although the application is described referring to specific embodiment, it should be appreciated by those skilled in the art In the case where no real spirit and scope for being detached from the application, the various changes that can carry out.Furthermore, it is possible to this Shen Main body, spirit and scope please are variously changed to adapt to specific situation, material, material compositions and method.All These changes are included in the range of claims hereof.

Sequence table

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Claims (10)

1. a kind of method for detecting targeted gene disruption effect comprising following steps:

Processing group:

1) over-express vector of the expression cassette of the target gene containing Code targets albumen is constructed；

2) knockout editor's carrier of target gene is constructed；

3) over-express vector and knockout editor's carrier are transferred to jointly in cell to be edited, are obtained to be detected thin Born of the same parents group；

4) determine that the knockout edits carrier by the variation of the amount of target proteins described in the detection cell mass to be detected Whether effectively.

2. the method according to weighing and require 1, which is characterized in that further include being overexpressed control group in the method: by the mistake Expression vector is transferred in the cell, obtains overexpressing cell group；By comparing target egg described in the cell population of interest Whether the white amount with the target proteins in the overexpressing cell is effective to determine the knockout editor.

3. according to power require 2 described in method, which is characterized in that the amount of the target proteins described in the cell mass to be detected is few When the amount of the target proteins in the overexpressing cell group, the knockout editor is effective；And/or

The amounts of the target proteins described in the cell mass to be detected and the target proteins in the overexpressing cell group When measuring suitable, the knockout editor is invalid.

4. the method according to weighing and require any one of 1 to 3, which is characterized in that after being transferred to carrier, culture 60 to 84 is small When after measure the amounts of the target proteins.

5. the method according to weighing and require 4, which is characterized in that after being transferred to carrier, culture measured the target after 72 hours The amount of albumen.

6. method as claimed in any of claims 1 to 5, which is characterized in that the amount of the target proteins passes through The mode of Western Blot determines.

7. method as claimed in any of claims 1 to 6, which is characterized in that the knockout editor of the target gene Carrier is CRISPR/Cas9 system.

8. method as claimed in any of claims 1 to 7, which is characterized in that the cell is insect cell, preferably The cell is SL cell line Spli221, in Spodoptera frugiperda cells Sf9, Trichopllusia ni cell line High Five One kind.

9. according to the method described in claim 8, it is characterized in that, the CRISPR/Cas9 system is suitable for insect CRISPR/Cas9 system；It is preferred that the CRISPR/Cas9 system is A3EGFP and IE1Cas9.

10. method as claimed in any of claims 1 to 9, which is characterized in that the target being overexpressed in box To gene and label Gene Fusion, the preferably described label in histidine tag, Tag label, Flag label, V5 label extremely Few one kind；The more preferable over-express vector is pIZT/V5-His plasmid in zero load.