CN110066852B - Method and system for detecting CRISPR/Cas PAM sequence in mammalian cell - Google Patents
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Abstract
The invention belongs to the technical field of gene editing, and particularly relates to a method and a system for detecting a CRISPR/Cas PAM sequence in a mammalian cell. The method comprises the following specific steps: (1) constructing a plasmid library containing a PAM sequence; (2) constructing a lentivirus library comprising a PAM sequence; (3) constructing a cell line comprising a library of PAM sequences; (4) removing false positive background of the fluorescence reporter gene by flow sorting; (5) transfecting CRISPR/Cas to a cell line comprising a PAM sequence library for editing; (6) flow sorting out fluorescence reporter gene positive cells, and constructing a second-generation sequencing library through PCR; (7) bioinformatics analyzed the next generation sequencing results to find PAM sequences that could produce gene edits. The invention can quickly screen out a CRISPR/Cas system capable of generating gene editing in mammalian cells, and find out the needed PAM sequence through sequencing.
Description
Technical Field
The invention belongs to the technical field of gene editing, and particularly relates to a method and a system for detecting a PAM (polyacrylamide sequence) sequence of a CRISPR/Cas gene editing system in a mammalian cell.
Background
The CRISPR/Cas system is an acquired immunity developed by prokaryotes in long-term evolution, and is used for resisting invasion of exogenous plasmids and phages. Researches show that when a PAM (Protospace Adjacent Motif) sequence is required to be arranged near a target sequence, the CRISPR/Cas system can realize the recognition and the cutting of the target site. Thus, accurate identification of the PAM sequence recognized by the CRISPR/Cas system is a prerequisite for gene editing using it.
At present, two methods are mainly used for identifying PAM sequences recognized by a CRISPR/Cas system, one method is to use Cas protein and gRNA to cut target DNA sequences containing various PAMs in vitro, and then perform second-generation sequencing on enzyme digestion products to analyze the recognized PAM sequences; the other is to combine the PAM sequence with a resistance gene in bacteria and identify the PAM sequence through negative selection and second-generation sequencing analysis. However, studies have shown that CRISPR/Cas systems identified using these two methods are not necessarily active in mammalian cells. When the PAM sequence is identified by using an in vitro cleavage experiment, the identification result is influenced by the reaction condition.
The present invention has been made in view of the above problems.
Disclosure of Invention
The invention provides a method and a system for detecting a CRISPR/Cas PAM sequence based on a reporter gene, which can quickly screen out a CRISPR/Cas system capable of generating gene editing in mammalian cells and find out the needed PAM sequence through sequencing. The technical scheme of the invention is specifically introduced as follows.
A method for detecting CRISPR/Cas PAM sequence in mammalian cells comprises the following steps:
(1) construction of a plasmid library comprising a PAM sequence
1) Designing and synthesizing an oligonucleotide single-stranded PAM sequence library containing a gRNA target site and a plurality of degenerate bases; the gRNA target site consists of a gRNA binding sequence and a PAM sequence, wherein the PAM sequence consists of more than 2 bases N which are connected in series, and the base N is A, T, C or G;
2) converting the oligonucleotide single-stranded PAM sequence library into double-stranded DNA by PCR;
3) inserting double-stranded DNA between a promoter and a fluorescent reporter gene, wherein the promoter is used for transcription of a downstream fluorescent reporter gene;
4) extracting plasmids to complete the construction of a plasmid library containing a PAM sequence;
(2) construction of Lentiviral libraries comprising PAM sequences
1) Plating mammalian cells into culture dishes;
2) co-transfecting a plasmid library comprising a PAM sequence with a lentiviral helper plasmid into a mammalian cell;
3) collecting the lentivirus supernatant, and concentrating;
4) determining the titer of the lentivirus, namely completing the construction of the lentivirus library containing the PAM sequence;
(3) constructing a cell line comprising a library of PAM sequences;
(4) removing false positive background of the fluorescence reporter gene by flow sorting;
(5) transfecting a Cas9 protein and a gRNA or a vector expressing the Cas9 protein and the gRNA to a cell line containing a PAM sequence library for editing;
(6) flow sorting out fluorescence reporter gene positive cells, extracting genome DNA, and constructing a second-generation sequencing library by PCR by taking the genome DNA as a template;
(7) bioinformatics analyzed the next generation sequencing results to find PAM sequences that could produce gene edits.
In the present invention, the gRNA target site and degenerate base are located between the initiation codon ATG and the fluorescent reporter gene.
In the present invention, the promoter is selected from any one of CMV, EF1 α, SV40, PGK1, Ubc, CAG, TRE and human beta actin.
In the present invention, the fluorescent reporter gene is selected from any one of GFP, RFP, BFP, EGFP, mCherry, mStrawberry, mApple, mRuby or EosFP.
In the present invention, the mammalian cells include, but are not limited to, HEK293T cells, HEK293 cells, HeLa cells, HIH3T3 cells, U2-OS osteosarcoma cells, A549 cells, and K562 cells.
In the present invention, in step (3), the method for constructing a cell line comprising a PAM sequence library is as follows:
1) plating mammalian cells into culture dishes;
2) infecting mammalian cells with a lentivirus library comprising a PAM sequence;
3) and screening by using antibiotics to obtain the cell line containing the PAM sequence library.
In the present invention, in step (5), the edition is performed for 2 to 7 days.
The invention also provides a system for detecting CRISPR/Cas PAM sequence in mammalian cells, which comprises a promoter, a gRNA target site, a merged base and a fluorescent reporter gene; the promoter is used for transcription of a downstream fluorescent reporter gene, a gRNA target site consists of a gRNA binding sequence and a PAM sequence, the PAM sequence consists of more than 2 bases N which are connected in series, and the base N is base A, T, C or G; the gRNA target site and degenerate base is located between the ATG initiation codon and the fluorescent reporter gene.
The principle of the invention is as follows: the gRNA target site and the PAM sequence library to be detected are positioned at the 5' end of the fluorescent reporter gene, so that the fluorescent reporter gene is inactivated; if the targeted sequence is subjected to gene editing to generate mutation, part of the fluorescent reporter gene is restored to be normally expressed. The PAM sequence recognized by the Cas protein can be detected by analyzing the PAM sequence contained in the fluorescent reporter positive cell.
Compared with the prior art, the method has the beneficial effects that whether the CRISPR/Cas system can generate gene editing in mammalian cells can be quickly tested, and the needed PAM sequence can be found out through sequencing, which cannot be realized by the prior art; the invention can overcome the defects of protein purification and in vitro gRNA transcription, large workload and high cost in an in vitro cutting experiment; compared with the PAM sequence detected in bacteria, the environment in the bacteria is different from that in mammalian cells, and the detected PAM sequence is not as accurate as that in the cells.
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FIG. 1 is a schematic diagram of a system for detecting CRISPR/Cas PAM sequence based on GFP reporter gene in example 1.
FIG. 2 is a map of pCMV _ Target-PAM _ eGFP _ Puro plasmid.
Fig. 3 is the fluorescence results of a library of cells comprising a PAM sequence before and after transfection with a CRISPR/SpCas9 system.
Fig. 4 shows PAM sequences recognized by SpCas9 protein obtained after bioinformatics analysis.
Detailed Description
The following examples further illustrate the present invention but should not be construed as limiting the invention.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available. The experimental method not specified for the specific conditions is usually carried out under the conventional conditions or the conditions recommended by the manufacturer.
The oligonucleotide single-stranded DNA synthesis, primer sequence synthesis and Sanger sequencing of the invention are synthesized by Jinwei Zhi corporation, Suzhou; gibsonassemby, PmlI, PmeI, DNA polymerase 2 XQ 5Master mix, cohesive end transient ligase from NEB; MegaX DH10BTMT1R ElectrocompTMCells、
2000 from Invitrogen corporation; DMEM high-glucose medium was purchased from Thermo Fisher; fetal bovine serum was purchased from Gibco; agarose gel recovery kits were purchased from Qiagen; blood/cell/tissue genomic DNA was purchased from tiangen biochemistry technology limited; second generation sequencing was done by norstanding provenance.
FIG. 1 is a schematic diagram of a system for detecting CRISPR/Cas PAM sequence based on GFP reporter in example 1, the system is located on a lentiviral vector, wherein a CMV promoter is used for transcribing GFP fluorescent reporter, and a targeting sequence and a PAM sequence to be detected are located at the 5' end of the GFP fluorescent reporter to inactivate GFP; when the CRISPR/Cas system generates mutation due to gene editing, part of GFP recovers normal expression; GFP positive cells are selected, a genome is extracted to perform PCR second-generation sequencing library building, and a PAM sequence identified by the CRISPR/Cas system can be known through performing bioinformatics analysis on sequencing data.
Example 1: the method for detecting the CRISPR/SpCas PAM sequence comprises the following steps:
1. construction of a plasmid library comprising a PAM sequence
(1) An oligonucleotide single-stranded PAM library containing a target site and a plurality of merged bases is designed and synthesized, in the embodiment, the nucleotide sequence is SEQ ID NO. 1, wherein continuous 7 bases N are PAM sequences, and N is any one of A, T, C, G.
(2) The oligonucleotide single-stranded DNA was converted to double-stranded DNA by PCR using, in this example, the following PCR primers: the upstream primer F-CATGCGAGAAAAGCCTTGTTT and the downstream primer R-AGCTCCTCGCCCTTGCTCAC are reacted under the following conditions:
the PCR run program was as follows:
after the PCR was completed, the desired fragment of 76bp was recovered by gel electrophoresis on a 2% agarose gel.
(3) The linearized plasmid pCMV-Filler-GFP-P2A-Puro has the following reaction system:
the enzyme is cut for 1h at 37 ℃, and the target fragment with the size of 8151bp is recovered through 1% agarose gel electrophoresis.
(4) And (3) recombining and connecting the PCR product recovered in the step (2) with the linearized plasmid in the step (3), wherein the reaction system is as follows:
reacting at 50 ℃ for 1h, and carrying out magnetic bead purification on the recombinant ligation product.
(5) Electric conversion: the purified ligation product was used MegaX DH10BTMT1R ElectrocompTMCells (Invitrogen) competent cells were electroporated using Bio-Rad
II electric transfer instrument for 5 timesThe electrotransfer condition is 2.0kV, 200 omega and 25 muF; adding 1ml of recovery culture medium heavy-suspension bacteria liquid into an electrotransfer cup after electrotransfer, transferring the electrotransfer cup to a 15ml centrifuge tube at 225rpm and 37 ℃, and shaking the bacteria for 1 h; supplemented to 100mLLB medium, and cultured overnight at 32 ℃ (low temperature culture reduces plasmid recombination rate).
(6) Plasmid library construction containing the PAM sequence was completed by large extraction of the plasmid by Qiagen's EndoFree plasmid, as shown in fig. 2.
2. Construction of Lentiviral libraries comprising PAM sequences
(1) Day 0, HEK293T cells were plated into 10cm dishes at a density of about 35%;
(2) on day 1, the plasmid library containing the PAM sequence, the viral packaging helper plasmid psPAX2 and pMD2.G were mixed at a ratio of 4:3:1 to 16. mu.g, and used
2000 co-rotation HEK 293T;
(3) after 8-12h of transfection, the medium containing the transfection mixture was discarded and replaced with 10ml DMEM + 10% FBS + penicillin/streptomycin fresh medium at 37 deg.C with 5% CO2Continuously culturing in the incubator;
(4) on day 3, after the solution is changed for 48 hours, collecting a culture medium containing viruses, and centrifuging at 1250rpm for 5min to remove cell masses;
(5) filtering the virus supernatant with 0.45 μm filter membrane;
lentivirus concentration comprises the following steps:
(1)5 XPEG 8000-NaCl preparation: weighing 8.766g of NaCl and 800050 g of PEG, and dissolving in 200ml of Milli-Q pure water; sterilizing at 121 deg.C for 30 min; stored at 4 ℃.
(2) Adding 5ml of 5 XPEG 8000-NaCl mother liquor into every 20ml of filtered virus initial liquor;
(3) the mixture is inverted and mixed once every 20-30 min for 3-5 times, and is placed at 4 ℃ overnight;
(4) centrifuging at 4 deg.C and 4000g for 20 min; sucking and removing the supernatant, standing the tube for 1-2 minutes, and sucking away residual liquid;
(5) adding 1ml of LPBS into the lentivirus sediment for resuspension, and blowing and uniformly mixing by using a liquid transfer gun;
(6) and subpackaging the resuspension solution into Ep tubes with different volumes as required, quickly freezing the resuspension solution by using crushed dry ice, and storing the solution at-80 ℃.
(7) The titer of the concentrated virus was determined to be 109U/mL。
3. Construction of cell lines comprising a PAM sequence library
(1) HEK293T cells were plated into 15cm dishes at 30% cell density;
(2) after 24h, 30 mu L of the concentrated virus obtained in the step 2 is taken to infect HEK293T cells, and 8 mu g/mL polybrene is added to improve the infection efficiency;
(3) and after infection for 24h, changing the solution, adding puromycin with the concentration of 2 mu g/mL, and continuously screening for 5 days to obtain a cell line containing the PAM sequence library.
4. Editing cells comprising a PAM sequence library
In this example, SpCas9 was selected as the subject, the gRNA sequence is shown in SEQ ID NO:2, and the procedure was as follows:
(1) spreading the cells containing the PAM sequence library to a culture dish of 10cm with a cell density of 30%;
(2) passing plasmids expressing SpCas9 protein and gRNA through
2000 transfecting the paved PAM sequence library cells;
(3) after editing for 3 days, the fluorescence ratio was observed by a fluorescence microscope. GFP was bright if SpCas9 and gRNA were transfected, and was not bright if SpCas9 and gRNA were not transfected (figure 3).
5. Preparation of a second Generation sequencing library
(1) Performing flow sorting on the PAM sequence library cells after editing for 3 days;
(2) selecting GFP positive cells, and extracting genome DNA by using a blood/cell/tissue genome DNA kit;
(3) performing PCR for the first round of library construction, wherein an upstream primer F1 of the PCR is shown as SEQ ID NO. 3, a downstream primer R1 is shown as SEQ ID NO. 4, and the reaction system is as follows:
the PCR run program was as follows:
(4) the PCR library construction second round of PCRPCR upstream primer F2 is shown as SEQ ID NO. 5, the downstream primer R2 is shown as SEQ ID NO. 6, and the reaction system is as follows:
the PCR run program was as follows:
(5) and (4) performing agarose gel electrophoresis on the PCR products of the second round, recovering and purifying a target fragment with the size of 366bp by using a gel recovery kit, and finishing the preparation of the second-generation sequencing library.
6. Analysis of the results of the second generation sequencing
(1) The prepared second-generation sequencing library is handed over to HiseqXTen by Nosogenic company for double-end sequencing;
(2) bioinformatics analyzed the results of the secondary sequencing, which showed SpCas9 recognized mainly NGG, but was also able to recognize NAG, NGA, TGT, GGA, GCG, etc. (fig. 4). Many of these PAM sequences have not been reported by others.
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Claims (8)
1. A method for detecting CRISPR/Cas PAM sequence in mammalian cells is characterized by comprising the following steps:
(1) construction of a plasmid library comprising a PAM sequence
1) Designing and synthesizing an oligonucleotide single-stranded PAM sequence library containing a gRNA target site and a plurality of merged bases; the gRNA target site consists of a gRNA binding sequence and a PAM sequence, wherein the PAM sequence consists of more than 2 bases N which are connected in series, and the base N is A, T, C or G;
2) changing the oligonucleotide single-stranded PAM sequence library into double-stranded DNA by PCR;
3) inserting double-stranded DNA between a promoter and a fluorescent reporter gene, wherein the promoter is used for transcription of a downstream fluorescent reporter gene;
4) extracting plasmids, namely completing the construction of a plasmid library containing a PAM sequence;
(2) construction of Lentiviral libraries comprising PAM sequences
1) Plating mammalian cells into culture dishes;
2) co-transfecting a plasmid library comprising a PAM sequence with a lentiviral helper plasmid into a mammalian cell;
3) collecting lentivirus supernatant, and concentrating;
4) determining the titer of the lentivirus, namely completing the construction of the lentivirus library containing the PAM sequence;
(3) constructing a cell line comprising a library of PAM sequences;
(4) removing false positive background of the fluorescence reporter gene by flow sorting;
(5) transfecting a Cas9 protein and a gRNA or a vector expressing the Cas9 protein and the gRNA to a cell line containing a PAM sequence library for editing;
(6) flow sorting out fluorescence reporter gene positive cells, extracting genome DNA, and constructing a second-generation sequencing library by PCR with the genome DNA as a template;
(7) bioinformatics analyzed the next-generation sequencing results to find PAM sequences that could produce gene edits.
2. The method of claim 1, wherein the gRNA target site and degenerate base are located between the initiation codon ATG and the fluorescent reporter.
3. The method of claim 1, wherein the promoter is selected from any one of CMV, EF1 α, SV40, PGK1, Ubc, CAG, TRE or human beta actin.
4. The method of claim 1, wherein the fluorescent reporter gene is selected from any one of GFP, RFP, BFP, EGFP, mCherry, mStrawberry, mApple, mRuby, or EosFP.
5. The method of claim 1, wherein the mammalian cells include, but are not limited to, HEK293T cells, HEK293 cells, HeLa cells, HIH3T3 cells, U2-OS osteosarcoma cells, a549 cells, K562 cells.
6. The method according to claim 1, wherein in step (3), the cell line comprising the PAM sequence library is constructed by the following method:
1) plating mammalian cells into culture dishes;
2) infecting mammalian cells with a lentiviral library comprising a PAM sequence;
3) and screening by using antibiotics to obtain the cell line containing the PAM sequence library.
7. The method according to claim 1, wherein in the step (5), the editing is performed for 2 to 7 days.
8. A system based on the method of claim 1, characterized in that it comprises a promoter, a gRNA target site, a degenerate base and a fluorescent reporter gene; the promoter is used for transcription of a downstream fluorescent reporter gene, a gRNA target site consists of a gRNA binding sequence and a PAM sequence, the PAM sequence consists of more than 2 bases N which are connected in series, and the base N is base A, T, C or G; the gRNA target site and degenerate base is located between the ATG initiation codon and the fluorescent reporter gene.
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