CN110066852A - A kind of method and system detecting CRISPR/Cas PAM sequence in mammalian cells - Google Patents
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Abstract
The invention belongs to gene editing technical field, specially a kind of method and system for detecting CRISPR/Cas PAM sequence in mammalian cells.Specific step is as follows for the method for the present invention: (1) building includes the plasmid library of PAM sequence；(2) building includes the slow virus library of PAM sequence；(3) building includes the cell line of PAM Sequence Library；(4) airflow classification removes fluorescent reporter gene false positive background；(5) the transfection CRISPR/Cas extremely cell line comprising PAM Sequence Library is edited；(6) airflow classification goes out fluorescent reporter gene positive cell, constructs two generation sequencing libraries by PCR；(7) bioinformatic analysis two generations sequencing result finds out the PAM sequence that can generate gene editing.The present invention can generate the CRISPR/Cas system of gene editing by quickly filtering out in mammalian cells, and the PAM sequence of its needs is found out by sequencing.
Description
Technical field
The invention belongs to gene editing technical fields, and in particular to one kind detects CRISPR/Cas in mammalian cells
The method and system of gene editing system PAM sequence.
Background technique
CRISPR/Cas system is a kind of acquired immunity that prokaryotes are formed in long-term evolution, for resisting external source
The invasion of plasmid and bacteriophage.Studies have shown that targeting sequence nearby must have PAM (Protospacer Adjacent Motif)
When sequence, CRISPR/Cas system is just able to achieve identification and cutting to target site.Therefore CRISPR/Cas is accurately identified
The PAM sequence of system identification is that the necessary condition of gene editing is carried out using it.
Currently used for identifying that there are mainly two types of the methods of the identified PAM sequence of CRISPR/Cas system, one is with Cas egg
White and gRNA In vitro digestion contains the targeting DNA sequence dna there are many PAM, and digestion products are then carried out the identification of two generation sequencing analysis
PAM sequence；Another kind is to combine PAM sequence and resistant gene in bacterium, passes through negative sense screening and two generation sequencing analysis
Identify PAM sequence.Studies have shown that using both methods identify come CRISPR/Cas system in mammalian cell
In it is different surely active.When using In vitro digestion experimental identification PAM sequence, the result of identification is influenced by reaction condition.
In view of problem above, the present invention is specifically proposed.
Summary of the invention
The present invention provides a kind of method and system of detection CRISPR/Cas PAM sequence based on reporter gene, the present invention
The CRISPR/Cas system that can generate gene editing in mammalian cells is allowed rapid screening out, and find out it by sequencing to need
The PAM sequence wanted.Technical solution of the present invention is specifically described as follows.
A method of detecting CRISPR/Cas PAM sequence in mammalian cells, the specific steps are as follows:
(1) building includes the plasmid library of PAM sequence
1) the single-stranded PAM Sequence Library of oligonucleotides comprising gRNA target site and several merger bases is designed and synthesized；
GRNA target site is made of gRNA binding sequence and PAM sequence, and PAM sequence is composed in series by 2 or more base N, the base N
For base A, T, C or G；
2) the single-stranded PAM Sequence Library of oligonucleotides is become by double-stranded DNA by PCR；
3) double-stranded DNA is inserted between promoter and fluorescent reporter gene, promoter is used for downstream fluorescent reporter gene
Transcription；
4) plasmid is extracted, that is, completes the building of the plasmid library comprising PAM sequence；
(2) building includes the slow virus library of PAM sequence
1) by mammalian cell bed board into culture dish；
2) by the plasmid library comprising PAM sequence and slow virus helper plasmid cotransfection mammalian cell；
3) slow virus supernatant is collected, and is concentrated；
4) slow virus titre is measured, that is, completes the building in the slow virus library comprising PAM sequence；
(3) building includes the cell line of PAM Sequence Library；
(4) airflow classification removes fluorescent reporter gene false positive background；
(5) carrier for transfecting Cas9 albumen and gRNA or expression Cas9 albumen and gRNA extremely includes the thin of PAM Sequence Library
Born of the same parents system edits；
(6) airflow classification goes out fluorescent reporter gene positive cell, extracts genomic DNA, using genomic DNA as template, leads to
It crosses PCR and constructs two generation sequencing libraries；
(7) bioinformatic analysis two generations sequencing result finds out the PAM sequence that can generate gene editing.
In the present invention, gRNA target site and merger base are between initiation codon ATG and fluorescent reporter gene.
In the present invention, promoter is in CMV, EF1 α, SV40, PGK1, Ubc, CAG, TRE or human beta actin
It is any.
In the present invention, fluorescent reporter gene be selected from GFP, RFP, BFP, EGFP, mCherry, mStrawberry, mApple,
It is any in mRuby or EosFP.
In the present invention, mammalian cell include but is not limited to HEK293T cell, HEK293 cell, HeLa cell,
HIH3T3 cell, U2-OS osteosarcoma cell, A549 cell, K562 cell.
In the present invention, in step (3), the method for cell line of the building comprising PAM Sequence Library is as follows:
1) by mammalian cell bed board into culture dish；
2) by the slow virus library mammalian cell-infecting comprising PAM sequence；
3) after being screened with antibiotic to get arrive the cell line comprising PAM Sequence Library.
In the present invention, in step (5), edit 2 days~7 days.
The present invention also provides a kind of systems for detecting CRISPR/Cas PAM sequence in mammalian cells comprising opens
Mover, annexs base and fluorescent reporter gene at gRNA target site；Promoter is used for the transcription of downstream fluorescent reporter gene, gRNA
Target site is made of gRNA binding sequence and PAM sequence, and PAM sequence is composed in series by 2 or more base N, and the base N is alkali
Base A, T, C or G；GRNA target site and merger base are between ATG initiation codon and fluorescent reporter gene.
The principle of the invention lies in: gRNA target site and PAM Sequence Library to be detected are located at fluorescent reporter gene 5 '
End, inactivates fluorescent reporter gene；Mutation is generated if targeting sequence and gene editing occurring, part fluorescent reporter gene restores just
Often expression.By PAM sequence included in analysis of fluorescence reporter gene positive cell, the identification of Cas albumen can be detected out
PAM sequence.
Compared to the prior art, the beneficial effects of the present invention are the present invention can quickly test a CRISPR/Cas system
Can system generate gene editing in mammalian cells, and the PAM sequence of its needs is found out by sequencing, this is the prior art
It cannot achieve；The present invention can overcome In vitro digestion experiment to need purifying protein and gRNA is transcribed in vitro, and heavy workload is spent high
The shortcomings that；The present invention is different in the environment and mammalian cell in bacterium compared to detecting PAM sequence in bacterium, measures
PAM sequence is not so good as accurate in cell.
Detailed description of the invention
Fig. 1 is the system schematic of the detection CRISPR/Cas PAM sequence in embodiment 1 based on GFP reporter gene.
Fig. 2 is pCMV_Target-PAM_eGFP_Puro plasmid map.
Fig. 3 is fluorescence results of the cell library comprising PAM sequence before and after CRISPR/SpCas9 system transfections.
The PAM sequence that Fig. 4 is identified by the SpCas9 albumen obtained after bioinformatic analysis.
Specific embodiment
Embodiment is further illustrated into the content of present invention below, but should not be construed as limitation of the invention.
Unless stated otherwise, the present invention uses reagent, method and apparatus for the art conventional reagent, method and are set
It is standby.Unless stated otherwise, following embodiment agents useful for same and material are commercially available.Test method without specific conditions is led to
Often according to normal conditions or condition proposed by manufacturer implement.
The synthesis of oligonucleotides single stranded DNA, primer sequence synthesis, Sanger sequencing are closed by Suzhou Jin Weizhi company in the present invention
At；GibsonAssembly, PmlI, PmeI, archaeal dna polymerase 2 × Q5Master mix, the instantaneous ligase of cohesive end are purchased from
NEB company；MegaX DH10BTMT1R ElectrocompTMCells、2000 is public purchased from Invitrogen
Department；DMEM high glucose medium is purchased from Thermo Fisher company；Fetal calf serum is purchased from Gibco company；Agarose gel reclaim reagent
Box is purchased from Qiagen company；Blood/cell/tissue genomic DNA is purchased from Tiangeng biochemical technology Co., Ltd；In two generations, were sequenced by promise
The source He Zhi company completes.
Fig. 1 is the system schematic of the detection CRISPR/Cas PAM sequence in embodiment 1 based on GFP reporter gene, should
System is located on slow virus carrier, and wherein CMV promoter is for transcribing GFP fluorescent reporter gene, targets sequence and to be detected
PAM sequence be located at GFP fluorescent reporter gene 5 ' end, inactivate GFP；It dashes forward when gene editing generation occurs for CRISPR/Cas system
Become, part GFP restores normal expression；GFP positive cell is sorted out, genome is mentioned and carries out bis- generation of PCR and be sequenced and build library, is passed through
The PAM sequence that CRISPR/Cas system is identified is known that sequencing data progress bioinformatic analysis.
Embodiment 1: using the method for present invention detection CRISPR/SpCas PAM sequence, following experiment step is specifically included
It is rapid:
1, building includes the plasmid library of PAM sequence
(1) the single-stranded library PAM of oligonucleotides comprising target site and several merger bases is designed and synthesized, in this implementation
In example, nucleotides sequence is classified as SEQ ID NO:1, wherein continuous 7 base N are PAM sequence, any in N A, T, C, G
Kind.
(2) oligonucleotides single stranded DNA will be become by PCR by double-stranded DNA, in the present embodiment, PCR primer used are as follows:
Upstream primer F-CATGCGAGAAAAGCCTTGTTT, downstream primer R-AGCTCCTCGCCCTTGCTCAC, reaction condition are as follows:
It is as follows that PCR runs program:
After PCR, by 2% agarose gel electrophoresis, glue recycles the target fragment of 76bp size.
(3) linearization plasmid pCMV-Filler-GFP-P2A-Puro, reaction system are as follows:
37 DEG C of digestion 1h recycle the target fragment of 8151bp size by 1% agarose gel electrophoresis.
(4) plasmid linearized in the PCR product recycled in step (2) and step (3) is subjected to recombination connection, reactant
It is as follows:
Recombination connection product is carried out magnetic beads for purifying by 50 DEG C of reaction 1h.
(5) electricity turns: connection product after purification is utilized MegaX DH10BTMT1R ElectrocompTMCells
(Invitrogen) competent cell carries out electricity turn, uses Bio-RadII electroporation carries out 5 electricity and turns, and electricity turns item
Part is the μ F of 2.0kV, 200 Ω, 25；1ml recovery media resuspended bacterium solution is added into electric revolving cup after turning for electricity, is then transferred to 15ml
Centrifuge tube, 225rpm, shake bacterium 1h by 37 DEG C；It adds to 100mLLB culture medium, (Low- temperature culture reduces plasmid weight to 32 DEG C of overnight incubations
Group rate).
(6) plasmid is taken by the big extracting of the EndoFree plasmid of Qiagen, that is, completes the plasmid library structure comprising PAM sequence
It builds, as shown in Figure 2.
2, building includes the slow virus library of PAM sequence
(1) the 0th day, by HEK293T plating cells into 10cm culture dish, density about 35%；
(2) the 1st days, by the plasmid library comprising PAM sequence, virus packaging helper plasmid psPAX2 and pMD2.G with 4:
The ratio of 3:1 is mixed into 16 μ g, uses2000 corotation HEK293T；
(3) after transfecting 8-12h, reject contains the culture medium of transfection mixture, and it is green to be changed to 10ml DMEM+10%FBS+
Mycin/streptomysin fresh culture, 37 DEG C, 5%CO2Continue to cultivate in incubator；
It (4) the 3rd days, after changing liquid 48h, collects and contains virulent culture medium, 1250rpm, centrifugation 5min removes cell mass；
(5) by 0.45 μm of membrane filtration of vial supernatant；
Slow virus concentration the following steps are included:
(1) 5 × PEG8000-NaCl is prepared: weighing NaCl 8.766g, PEG8000 50g is dissolved in 200ml Milli-Q
In pure water；121 DEG C, 30min moist heat sterilization 30min；It is stored in 4 DEG C.
(2) 5 × PEG8000-NaCl mother liquor 5ml is added in the filtered initial liquid of virus of every 20ml；
(3) every 20~30min is mixed by inversion once, is carried out 3~5 times altogether, 4 DEG C stand overnight；
(4) 4 DEG C, 4000g, it is centrifuged 20min；It inhales and abandons supernatant, stand pipe 1~2 minute, siphon away residual liquid；
(5) 1mLPBS is added to be resuspended into slow virus precipitating, liquid-transfering gun piping and druming mixes；
(6) re-suspension liquid is dispensed to different volumes into Ep pipe on demand, is saved with storing -80 DEG C after broken dry ice quick-frozen.
(7) titre for measuring concentrating virus is 109U/mL。
3, building includes the cell line of PAM Sequence Library
(1) by HEK293T plating cells into 15cm culture dish, cell density 30%；
(2) it after for 24 hours, takes the concentrating virus in 30 μ L steps 2 to infect HEK293T cell, adds 8 μ g/mL polybrenes to improve
Efficiency of infection；
(3) after infection for 24 hours, the puromycin for changing liquid and 2 μ g/mL of addition carries out continuing screening 5 days to get to comprising PAM
The cell line of Sequence Library.
4, editor includes the cell of PAM Sequence Library
In the present embodiment, select SpCas9 as research object, gRNA sequence is operation step shown in SEQ ID NO:2
It is rapid as follows:
(1) cell comprising PAM Sequence Library is spread into the culture dish of 10cm, cell density 30%；
(2) plasmid for expressing SpCas9 albumen and gRNA is passed throughThe PAM sequence that 2000 transfections are completed
Column library cell；
(3) after editing 3 days, pass through fluorescence microscope Fluorescence Ratio.If having transfected SpCas9 and gRNA, GFP is just
Bright, if not transfecting SpCas9 and gRNA, GFP would not be bright (Fig. 3).
5, two generation sequencing libraries are prepared
(1) the PAM Sequence Library cell after editing 3 days is subjected to airflow classification；
(2) GFP positive cell is sub-elected, extracts genomic DNA with blood/cell/tissue genomic DNA kit；
(3) it carries out PCR and builds the upstream primer F1 of the library first round PCR, PCR respectively as shown in SEQ ID NO:3, downstream primer
For R1 as shown in SEQ ID NO:4, reaction system is as follows:
It is as follows that PCR runs program:
(4) it carries out PCR and builds the wheel PCRPCR upstream primer F2 of library second as shown in SEQ ID NO:5, downstream primer R2 such as SEQ
Shown in ID NO:6, reaction system is as follows:
It is as follows that PCR runs program:
(5) PCR product of the second wheel is used into plastic recovery kit recovery purifying 366bp by agarose gel electrophoresis
The target fragment of size, the preparation of two generation sequencing libraries finish.
6, two generation sequencing results are analyzed
(1) source Nuo Hezhi company is transferred to carry out both-end sequencing on HiseqXTen the two generation sequencing libraries prepared；
(2) bioinformatic analysis two generations sequencing result, SpCas9 mainly identifies NGG as the result is shown for analysis, but also can
Enough identify NAG, NGA, TGT, GGA, GCG etc. (Fig. 4).Wherein many PAM sequences are that others did not report.
Sequence table
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Claims (8)
1. a kind of method for detecting CRISPR/Cas PAM sequence in mammalian cells, which is characterized in that specific steps are such as
Under:
(1) building includes the plasmid library of PAM sequence
1) the single-stranded PAM Sequence Library of oligonucleotides comprising gRNA target site and several merger bases is designed and synthesized；gRNA
Target site is made of gRNA binding sequence and PAM sequence, and PAM sequence is composed in series by 2 or more base N, and the base N is alkali
Base A, T, C or G；
2) the single-stranded PAM Sequence Library of oligonucleotides is become by double-stranded DNA by PCR；
3) double-stranded DNA is inserted between promoter and fluorescent reporter gene, promoter turns for downstream fluorescent reporter gene
Record；
4) plasmid is extracted, that is, completes the building of the plasmid library comprising PAM sequence；
(2) building includes the slow virus library of PAM sequence
1) by mammalian cell bed board into culture dish；
2) by the plasmid library comprising PAM sequence and slow virus helper plasmid cotransfection mammalian cell；
3) slow virus supernatant is collected, and is concentrated；
4) slow virus titre is measured, that is, completes the building in the slow virus library comprising PAM sequence；
(3) building includes the cell line of PAM Sequence Library；
(4) airflow classification removes fluorescent reporter gene false positive background；
(5) Cas9 albumen and gRNA or expression Cas9 albumen and the carrier extremely cell line comprising PAM Sequence Library of gRNA are transfected
It is edited；
(6) airflow classification goes out fluorescent reporter gene positive cell, extracts genomic DNA using genomic DNA as template and passes through PCR
Construct two generation sequencing libraries；
(7) bioinformatic analysis two generations sequencing result finds out the PAM sequence that can generate gene editing.
2. the method according to claim 1, wherein gRNA target site and merger base are located at initiation codon
Between ATG and fluorescent reporter gene.
3. the method according to claim 1, wherein promoter be selected from CMV, EF1 α, SV40, PGK1, Ubc,
Any one of CAG, TRE or human beta actin.
4. the method according to claim 1, wherein fluorescent reporter gene be selected from GFP, RFP, BFP, EGFP,
It is any in mCherry, mStrawberry, mApple, mRuby or EosFP.
5. the method according to claim 1, wherein mammalian cell includes but is not limited to HEK293T thin
Born of the same parents, HEK293 cell, HeLa cell, HIH3T3 cell, U2-OS osteosarcoma cell, A549 cell, K562 cell.
6. the method according to claim 1, wherein building includes the cell of PAM Sequence Library in step (3)
The method of system is as follows:
1) by mammalian cell bed board into culture dish；
2) by the slow virus library mammalian cell-infecting comprising PAM sequence；
3) after being screened with antibiotic to get arrive the cell line comprising PAM Sequence Library.
7. the method according to claim 1, wherein being edited 2 days~7 days in step (5).
8. a kind of system based on method described in claim 1, which is characterized in that it includes promoter, gRNA target site, simultaneous
And base and fluorescent reporter gene；Promoter is used for the transcription of downstream fluorescent reporter gene, and gRNA target site is by gRNA combination sequence
Column are formed with PAM sequence, and PAM sequence is composed in series by 2 or more base N, and the base N is base A, T, C or G；GRNA target
Site and merger base are between ATG initiation codon and fluorescent reporter gene.
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