CN116515904A - Red-green light switching type traffic signal lamp gene editing positive report enrichment system - Google Patents
Red-green light switching type traffic signal lamp gene editing positive report enrichment system Download PDFInfo
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Abstract
The invention discloses a red-green light switching type traffic signal lamp gene editing positive report enrichment system. Novel red-green light 'switching' report vectors and corresponding expression vectors of CRISPR/Cas systems for verification are constructed. And simultaneously targeting the genome and the report vector through the CRISPR/Cas system, and repairing the double-fluorescence report gene and resistance gene integrated expression cassette in the report vector by using an SSA repairing mechanism. The report enrichment system can simply and rapidly detect nuclease activity in transfected cells, and efficiently enrich gene editing positive cells.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and relates to a red-green light 'switching' traffic signal lamp (Traffic Light Reporter, TLR) gene editing positive report enrichment system which can be used for CRISPR/Cas system mediated gene editing positive cell enrichment.
Background
The CRISPR/Cas9 gene editing technique is a new generation gene editing technique following ZFNs (nc-finger nucleic) and TALENs (Transcription activator-like effector nuclease). It was first reported from 2012 that the use of proteins derived from the streptococcus pyogenes(s) type II CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats, CRISPR/CRISPR-associated protein-9 nucleic, cas9) and guide RNAs (guide RNAs, grnas) can target sheared plasmid DNA in vitro, with elucidation of the mechanism of CRISPR/Cas9 and successful gene editing in mammals, gene editing applications based on the RNA-mediated acquired immune system in this bacteria of CRISPR/Cas are expanding.
The CRISPR/Cas9 gene editing system mainly comprises: a CRISPR-associated gene (Cas) encoding a Cas 9nuclease and a guide RNA (sgRNA) comprised of a simplified tracrRNA/crRNA binary complex. CRISPR/Cas9 systems can utilize guide RNA sequences to guide Cas9 nucleases to create DNA double strand breaks (double strand breaks, DSBs) at specific sites in the genome, triggering DNA repair mechanisms of the cell itself. The DNA fragmentation repair mechanism consists essentially of both Non-homologous end joining (Non-homo-logous end joining, NHEJ) independent of homologous sequences and homologous recombination (Homologous recombination, HR) dependent on homologous sequences. Among them, NHEJ includes intracellular classical non-homologous end joining (C-NHEJ) and selective non-homologous end joining (A-NHEJ); HR includes homologous-directed repair (HDR) and single strand annealing repair (SSA).
The NHEJ repair mechanism is a main mechanism for repairing DSBs in mammalian cells, and the repair mode does not need the participation of homologous DNA, directly depends on DNA ligase to connect the ends of the broken DNA double chains to realize repair, and generally introduces insertion or deletion (Insertions and deletions, indels) of a small number of nucleotide bases, thereby possibly leading to frame shift mutation and causing gene damage and even gene knockout. Compared with the NHEJ pathway, the HDR repair mechanism is more complex, a large amount of biochemical factors are needed, and the HDR repair efficiency is far lower than that of NHEJ repair, which limits the wide application of the HDR repair mechanism in genetic engineering technology. However, if there are repetitive elements near the ends of the DNA double strand breaks, the repair pattern of SSA will exhibit higher repair efficiency. Therefore, SSA can be widely used as an efficient double-strand repair mechanism in report screening systems.
Screening of Gene editing Positive clones was mainly accomplished by using fluorescent marker genes (EGFP, mRFP, dsRed, mCherry, etc.) and drug screening genes (Puro R 、Zeo R 、Neo R 、Hygro R ) And screening and enriching target cells by using the screening markers. The vectors used for reporting screening mainly include: the enrichment vector for serially expressing nuclease and screening genes and for screening transfected positive cells, and the resistance gene report screening integrated vector constructed based on different repair principles of double strand breaks and used for screening nuclease positive cells. Since nuclease activity is relatively low in transfected cells, screening only cells that are positive for transfection is not very efficient for gene editing. The report screening integrated vector can be further divided into: report carrier based on NHEJ repair, SSA repair, and HDR repair. Report vehicles based on NHEJ repair theoretically only have a maximum of two-thirds of repair yieldThe substance can restore the function of the reporter gene; however, although the report vector based on HDR repair can accurately edit genome, the repair efficiency of the report vector is inconsistent with the editing efficiency of the cell genome due to low repair efficiency of the cell; meanwhile, the reported report carrier based on HDR repair has single screening mode and has no specificity consistent with the gene editing site, and the reasons limit the wide application of the report system.
Compared to report vectors based on NHEJ, HDR repair, report vectors based on SSA repair may be more than ten times more efficient than NHEJ in the presence of homologous repeats. Although the report vector constructed by using the SSA repair principle is suitable for screening the gene editing positive cells of all editing types, the existing report vector based on SSA repair is double expression frames, so that the vector is larger, and the cell transfection efficiency is reduced; meanwhile, the expression efficiency of different expression frames in the double expression frames is inconsistent, so that the enrichment efficiency of the gene editing positive cells is reduced.
Disclosure of Invention
The invention aims to provide a red-green light switching traffic light gene editing positive report enrichment system, which can screen nuclease positive cells through fluorescence sorting or medicines and can obtain gene editing positive cells through simpler and efficient enrichment.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a gene editing positive cell report enrichment system comprising a site-specific report carrier; the reporter vector comprises a dual fluorescent reporter gene and resistance gene integrated expression cassette, the integrated expression cassette comprises an upstream fragment which is positioned in a transcription region of the integrated expression cassette and is homologous to a resistance gene coding sequence, a downstream fragment which is homologous to the resistance gene coding sequence, a first fluorescent protein (used for marking transfected positive cells; transfection positive means that plasmids are transfected into cells and can express corresponding fluorescent proteins as screening tags, but plasmids are not edited), a gene coding sequence of a second fluorescent protein (used for marking nuclease positive cells; nuclease positive means that plasmids are transfected into cells, and the plasmids can express the corresponding fluorescent proteins as screening tags after being edited by a Cas protein with nuclease cleavage activity), and a first target sequence and a second target sequence which are recognized by guide RNA, the first target sequence is positioned between the upstream fragment and the gene coding sequence of the first fluorescent protein, the second target sequence is positioned between the downstream fragment and the gene coding sequence of the first fluorescent protein, and the upstream fragment and the downstream fragment comprise a repair sequence used for carrying out repeated annealing from the downstream fragment and the corresponding fluorescent protein expression to the single-stranded fluorescent protein after the integrated expression cassette is repeatedly expressed.
Preferably, the upstream fragment, the first target sequence, the gene coding sequence of the first fluorescent protein, the second target sequence, the downstream fragment and the gene coding sequence of the second fluorescent protein are in the same open reading frame, which is separated by a stop codon disposed between the second target sequence and the downstream fragment.
Preferably, the same direction repeated sequence is 200-350 bp. The homodromous repeat sequence is located at the rear of the upstream fragment (specifically the 3 'end) and the front of the downstream fragment (specifically the 5' end), respectively; too long homodromous repeated sequences can influence the cell transfection efficiency of the report carrier, and too short homodromous repeated sequences can influence the single-chain annealing repair efficiency of the report carrier, so that the enrichment efficiency of the gene editing positive cells can be directly influenced.
Preferably, the gene coding sequence of the second fluorescent protein is located at the rear side of the downstream fragment, and the transcription region of the integrated expression cassette further comprises a cleavage peptide sequence for linking the downstream fragment and the gene coding sequence of the second fluorescent protein.
Preferably, the first fluorescent protein is a red fluorescent protein (e.g., mCherry protein) and the second fluorescent protein is a green fluorescent protein (e.g., EGFP protein). The report carrier is red-green light switching type, only red fluorescent protein is expressed before repair, and only green fluorescent protein is expressed after repair.
Preferably, the upstream fragment and the downstream fragment form a sequence for rendering a gene editing target cell expressing puromycin (puromycin) resistance after single-strand annealing repair.
Preferably, the system further comprises a site-specific CRISPR/Cas expression vector comprising a sgRNA expression cassette for expressing the guide RNA and a nuclease expression cassette (Cas protein expression cassette for short) for expressing a Cas protein with cleavage activity, the target sequences recognized by the guide RNA (specifically the first and second target sequences described above) being derived from the genome of the gene editing target cell.
Preferably, the gene editing target cell is a eukaryotic cell (e.g., a mammalian cell, etc.).
The preparation method of the gene editing positive cell report enrichment system comprises the following steps:
1) Cloning of a recombinant fragment containing the above-mentioned upstream fragment homologous to the coding sequence of the resistance gene, downstream fragment homologous to the coding sequence of the resistance gene, the cleavage peptide sequence, and the gene coding sequence of the second fluorescent protein (e.g., purol 1-305 -PuroR 105-597 -T2A-EGFP sequence); integrating the recombinant fragment with the gene coding sequence of the first fluorescent protein obtained by cloning after being connected with an expression vector (for example, pcDNA3.1 vector) framework, and enabling the gene coding sequence of the fluorescent protein to be connected between the upstream fragment and the downstream fragment to obtain a double-fluorescence reporter gene and resistance gene integrated recombinant expression vector (for example, pSSA-PMG vector);
2) And (3) replacing the gene coding sequence of the first fluorescent protein in the double-fluorescent reporter gene and resistance gene integrated recombinant expression vector obtained in the step (1) with the cloned gene coding sequence containing the first fluorescent protein and the recombinant fragments (for example, sg.T-hCR 5-mCherry-sg.T-hCR 5 sequences) of the first target sequence and the second target sequence identified by the guide RNA, so as to obtain the site-specific reporter vector.
Preferably, in the step 2, the recombinant fragment obtained by cloning further comprises a stop codon located at the rear side of the second target sequence.
Preferably, the preparation method further comprises the following steps: designing a fitting primer according to a target locus of a target gene, annealing the fitting primer to obtain a primer annealing double strand, connecting the primer annealing double strand with a carrier framework containing the nuclease expression cassette, and forming the sgRNA expression cassette in the carrier framework by using the primer annealing double strand to obtain a locus-specific CRISPR/Cas expression vector.
A method of screening for gene editing positive cells, comprising the steps of:
1) Co-transfecting the constructed site-specific report vector and CRISPR/Cas expression vector to a gene editing target cell;
2) Performing fluorescence sorting by using a flow cytometer after 48-72 hours of transfection to obtain nuclease positive cells, and then performing enrichment culture; or, after 48-72 hours of transfection, screening cells capable of expressing the corresponding resistance genes by using a drug to obtain nuclease positive cells, and then carrying out enrichment culture.
Preferably, the drug (e.g., puromycin) is added to the culture system of transfected cells at a final concentration of 0.2 to 5 μg/mL (depending on the actual resistance of the different cells to the drug).
Preferably, the enrichment culture specifically comprises the following steps: diluting nuclease positive cells obtained by fluorescence sorting or drug screening, and culturing cell monoclonal (for example, culturing in a 96-well plate culture dish for 7-14 days); then carrying out gene editing positive cell identification on the cultured monoclonal; the monoclonal identified as gene editing positive cells are expanded (e.g., transferred to a 24-well plate).
The gene editing positive cell report enrichment system is applied to eukaryotic cell gene editing.
Preferably, the site-specific CRISPR/Cas expression vector and the reporter vector are co-transfected into a cell and then expressed, wherein the double fluorescent reporter gene and the resistance gene of the site-specific reporter vector are integrated into an expression cassette, and Cas protein expressed by the site-specific CRISPR/Cas expression vector is sheared (the shearing position is positioned on a first target sequence and a second target sequence of the integrated expression cassette, and simultaneously Cas protein targets a target site with the same sequence on the genome of the cell) and completes single-strand annealing repair, and the expression of one fluorescent reporter gene (particularly the gene coding sequence of the first fluorescent protein) is shut down (deleted from the transcribed region of the integrated expression cassette due to the shearing and single-strand annealing repair) and the expression of the other fluorescent reporter gene (particularly the gene coding sequence of the second fluorescent protein) are restored, so that the transfected cell can be directly used for screening nuclease-positive cells, and the enrichment efficiency of the gene-editing positive cells is improved.
Preferably, the nuclease-positive cells have a green fluorescent label (e.g., EGFP after transfection of the cells with the vector of the system + ) And drug (e.g., puromycin) resistance; whereas nuclease-negative cells (i.e., transfection-positive cells) have a red fluorescent label (e.g., mCherry + ) And has no drug resistance, and realizes a screening strategy of 'red light stop green light row' for screening nuclease positive cells (namely Traffic Light Reporter) through the expression of different fluorescent reporter genes. At the same time, with a red fluorescent label (e.g., mCherry + ) The ratio of cell number to total number of cells can characterize the efficiency of cell transfection with green fluorescent markers (e.g., EGFP + ) The ratio of the number of cells to the total number of cells may characterize the efficiency of the nuclease in exerting its cleavage activity (i.e., the efficiency of vector repair).
The beneficial effects of the invention are as follows:
the two ends of the corresponding gene coding sequence of the fluorescent protein for marking transfected positive cells in the report carrier are respectively provided with a target sequence for guiding RNA recognition, and the expression structure is positioned between an upstream fragment and a downstream fragment which are homologous with the coding sequence of the resistance gene; co-transfecting the reporter vector with a targeting vector (specifically, CRISPR/Cas expression vector) of a cell genome to form a reporter enrichment system having the same target sequence as the cell genome editing site; the report enrichment system is site-specific, has higher consistency with genome editing efficiency in target cells, can rapidly and simply detect nuclease activity in the target cells through fluorescence sorting or drug screening, and screen out nuclease positive cells, and improves the enrichment efficiency of the gene editing positive cells; and the report enrichment system adopts an SSA repair mode with high sensitivity, and can be widely applied to various cells of animals.
Drawings
FIG. 1 is a schematic diagram of the SSA-PMG report enrichment system operation; in the figure: purol (1-305) represents Purol 1-305 The method comprises the steps of carrying out a first treatment on the surface of the PuroR (105-597) represents PuroR 105-597 。
FIG. 2 is a map of the identification of pX330-U6-Chimeric_dBsai-CBh-hSpCas9 vector cleavage, wherein lane M: trans2K plus II DNA Marker; lanes 1, 2: the vector was digested with Bsa I.
FIG. 3 is a graph showing the sequencing results of phCCR5-sgRNA/Cas9 vector sanger.
FIG. 4 shows the amplification of Purol 1-305 -PuroR 105-597 -PCR product identification map of T2A-EGFP sequence, wherein lane M: trans2K DNAMaroker; lanes 1, 2: purol 1-305 -PuroR 105-597 Amplification results of the T2A-EGFP sequence.
FIG. 5 is a pcDNA3.1-CMV-Purol 1-305 -PuroR 105-597 -T2A-EGFP-polyA vector (plasmid) cleavage map, wherein lane M: trans2K plus II DNAMarker; lanes 1, 2: the plasmid was digested with Hind III and Xba I.
FIG. 6 is a diagram of PCR product identification of amplified mCherry sequences, wherein lane M: trans2K DNAMaroker; lanes 1, 2: amplification results of mCherry sequence.
FIG. 7 is a pcDNA3.1-CMV-Purol 1-305 -(BamHⅠ)mCherry(NotⅠ)-PuroR 105-597 -T2A-EGFP-polyA vector (plasmid) cleavage map, wherein lane M: trans2K plus II DNAMarker; lanes 1, 2: the plasmid was digested with BamH I and Not I.
FIG. 8a is a map of pSSA-PMG vector.
FIG. 8b is a map of the digestion identification of the phCCR5-SSA-PMG vector, lane M: trans2K plus II DNA Marker; lanes 1, 2: the plasmid was digested with BamH I and Not I.
FIG. 8c is a map of the phCCR5-SSA-PMG vector.
FIG. 9 is a diagram of PCR product identification by amplifying the coding sequence of the puromycin resistance gene, wherein lane M: trans2KDNA Marker; lanes 1, 2: amplification results of the coding sequence of the puromycin resistance gene.
FIG. 10 is a diagram of PCR product identification of amplified T2A-EGFP sequence, wherein lane M: trans2K DNA Marker; lanes 1, 2: amplification results of T2A-EGFP sequence.
FIG. 11 is a Overlap Extension PCR product identification plot, wherein lane M: trans2K DNA Marker; lanes 1, 2: fusion results containing Puro-T2A-EGFP sequence.
FIG. 12 is a diagram showing the identification of pcDNA3.1-CMV-Puro-T2A-EGFP-polyA vector (plasmid), wherein lane M: trans2K plus II DNA Marker; lanes 1, 2: the plasmid was digested with Hind III and Xba I.
FIG. 13 is a flow chart of the procedure for gene editing positive cells using a reporter vector-based co-transfection system.
FIG. 14 is a graph of the reporting effect of the SSA-PMG report enrichment system against the human CCR5 gene locus (hCR 5).
FIG. 15 shows the results of sorting HEK293T transfected report vector (positive control) and nuclease positive cells transfected with report vector (double-sided target sequence cleavage) by FACS.
FIG. 16 is a graph showing the results of deep sequencing of gene editing efficiency of hCR 5 sites in nuclease-positive cells sorted by FACS.
FIG. 17 is a graph showing the results of SSA-PMG report enrichment system expression and cell status (after puromycin selection).
FIG. 18 is a graph showing the results of deep sequencing of gene editing efficiency of hCR 5 sites in nuclease-positive cells obtained by enrichment (after screening with puromycin).
FIG. 19 shows the results of sorting nuclease-positive cells after HEK293T transfection of reporter vector (single-sided target sequence cleavage) using FACS.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
Construction of SSA-PMG report enrichment System
1. Construction of CRISPR/Cas expression vectors
1.1 target site selection
Taking the hCR 5 gene as an example, a target site containing PAM (NGG/NGGNG) was first found in the genome. Then, through screening of CRISPR Dsign website (http:// CRISPR. Mit. Edu /), the following sequence is selected as hCR 5 gene target site (nuclease target site of hCCR gene, abbreviated as sg T-hCR 5): (seq. Id. No. 1), the portion of the site that precedes PAM (e.g., AGG within the black wire frame) is cleaved by a Cas protein (e.g., spCas 9).
1.2 primer design and Synthesis
For the target site to design a fit primer, for insertion into the pX 330-U6-chimeric_dbsai-CBh-hscas 9 vector (edge plasmid #42230, i.e., the Bbs I cleavage recognition site in "pX 330-U6-chimeric_dbbsi-CBh-hscas 9 vector" is replaced with a Bsa I cleavage recognition site), the fit primer sequence is as follows (for example sg.t-hCCR 5):
hCCR5-F(Bsa I)：5'-caccCACACTTGTCACCACCCCAA-3'(SEQ.ID.NO.2)
hCCR5-R(Bsa I)：5'-aaacTTGGGGTGGTGACAAGTGTG-3'(SEQ.ID.NO.3)
1.3 primer annealing fitting
The primers hCR 5-F (BsaI) and hCR 5-R (BsaI) were each used at a concentration of 10 pmol/. Mu.L, and were mixed with reference to the components of Table 1, and subjected to annealing fitting by a PCR instrument (see Table 2) to obtain a primer annealed duplex having complementary pairing with the cohesive end after cleavage by BsaI.
TABLE 1 primer annealing System
TABLE 2 primer annealing procedure
1.4 enzyme digestion
Carrying out enzyme digestion on the pX330-U6-Chimeric_dBsai-CBh-hSpCas9 carrier by using Bsa I, wherein a uniformly mixed enzyme digestion system (see table 3) is placed in a constant-temperature water bath kettle at 37 ℃ for reaction for 3-4 h; the digested fragments were detected by agarose gel electrophoresis and the corresponding target bands of the vector backbone were recovered using a DNA recovery kit (see fig. 2).
TABLE 3 enzyme digestion system
1.5 connections
The primer annealed double strand was ligated with the recovered vector backbone using T4 ligase, wherein the ligation system after mixing (see Table 4) was placed in a PCR apparatus and reacted at 25℃for 30min. Then transforming E.coli DH 5-alpha competent cells, and coating LB/AMP + Plate, picking up monoclonal and at LB/AMP + Culturing in liquid culture medium at 37 deg.c for 8-10 hr.
TABLE 4 connection System
1.6 identification of Positive clones
Different monoclonal bacterial liquid plasmids are selected, and the U6 universal sequencing primer is used for sanger sequencing. The correct plasmid for sequencing (see fig. 3), i.e. the CRISPR/Cas expression vector phCCR5-sgRNA/Cas9 constructed for the target site on hCCR5 gene, is used for expressing Cas protein with nuclease (nucleic) cleavage activity (specifically spCas 9) and sgrnas recognizing the target site.
U6 general sequencing primer sequence: 5'-ATGGACTATCATATGCTTACCGTA-3' (SEQ. ID. NO. 4).
2. Construction of reporter vector pSSA-PMG
2.1 amplification of Purol 1-305 -PuroR 105-597 T2A-EGFP sequence
PCR amplification was performed using pSSA-RPG vector (Addgene # 85932) as a template, primers were designed and synthesized (see Table 5). Wherein the primer sequences are as follows:
PMG-F(Hind III)：5'-cccAAGCTTACCATGACCGAGTACAAG-3'(SEQ.ID.NO.5)
PMG-R(Xba I)：5'-cagttaTCTAGATTACTTGTACAGCTC-3'(SEQ.ID.NO.6)
in the above sequences, the lower case letter is a protective base, the upper case bolded italic letter is a restriction enzyme recognition site, and the designed primers were sent to Shanghai Biotechnology Co., ltd for synthesis (the same applies below).
TABLE 5 Purol 1-305 -PuroR 105-597 Cloning and amplifying system for T2A-EGFP sequence
The PCR reaction conditions were: (98 ℃, 10s;55 ℃, 10s;72 ℃, 30 s). Times.33 cycles.
After the PCR reaction is finished, detecting by agarose gel electrophoresis, and recovering the target band by using a DNA recovery kit (see FIG. 4), thereby completing the Purol 1-305 -PuroR 105-597 Cloning of the T2A-EGFP sequence and addition of restriction endonuclease (i.e., hindIII, xba I) recognition sites at both ends of the sequence, the sequence comprising the following elements in order: puromycin resistance gene Puro R Coding region left homology arm (specifically Puro R The upstream sequence segment with 1 to 305 bases in the coding sequence, which is abbreviated as Purol 1-305 ) Puromycin resistance gene Puro R Coding region right homology arm (specifically Puro R The downstream sequence segment of 105 to 597 bases in the coding sequence, puroR for short 105-597 ；PuroR 105 -597 Does not include a stop codon), a cleavage peptide sequence (named for the specific class of cleavage peptide used, e.g., T2A), a green fluorescent protein EGFP gene coding sequence (abbreviated EGFP), wherein Puro R Coding region left homology arm and Puro R The right homology arm of the coding region has a direct repeat of about 200 bp.
2.2 construction of pcDNA3.1-CMV-Purol 1-305 -PuroR 105-597 T2A-EGFP-polyA vector
pcDNA3.1 vector (Invitrogen V79520) and amplified Purol-containing vector by restriction endonuclease 1-305 -PuroR 105-597 The target fragments of the-T2A-EGFP sequence are respectively subjected to double enzyme digestion, wherein the enzyme digestion system (see table 6) is evenly mixed and then placed in a constant temperature water bath kettle at 37 ℃ for reaction for 2-4 hours.
TABLE 6 double enzyme digestion System
Detecting the digested fragments by agarose gel electrophoresis, respectively recovering the digested target bands by using a DNA recovery kit, and recovering the target bands containing Purol 1-305 -PuroR 105-597 The target fragment of the-T2A-EGFP sequence is connected with a carrier framework, wherein the evenly mixed connection system (see table 7) is placed in a PCR instrument for reaction for 30min at 25 ℃.
TABLE 7 connection System
After the reaction, escherichia coli DH 5-alpha competent cells were transformed and coated with LB/AMP + Plate, picking up monoclonal and at LB/AMP + Culturing in liquid culture medium at 37 deg.c for 8-10 hr. Different monoclonal bacterial liquid plasmids were identified using double digestion (see figure)5) Sequencing, sequencing the correct plasmid, namely pcDNA3.1-CMV-Purol 1-305 -PuroR 105-597 T2A-EGF P-polyA vector in which CMV promoter (promoter), purol 1-305 -PuroR 105-597 The T2A-EGFP sequence and polyA (abbreviated as pA) form an expression cassette.
2.3 amplification of mCherry sequences
The pAAV-minCMV-mCherry vector (Addgene#27970) is used as a template, and primers are designed and synthesized for PCR amplification. Wherein the primer sequences are as follows:
mCherry-F(BamHⅠ)：5'-cgcGGATCCATGGTGAGCAAGGGCG-3'(SEQ.ID.NO.7)
mCherry-R(NotⅠ)：5'-tatGCGGCCGCTTACTTGTACAGCTCG-3'(SEQ.ID.NO.8)
after the PCR reaction is finished, agarose gel electrophoresis is adopted for detection, and a DNA recovery kit is used for recovering a target band (see FIG. 6), so that cloning of a red fluorescent protein mCherry gene coding sequence (mCherry sequence or mCherry for short) is completed, and restriction endonuclease (BamHI and NotI) recognition sites are added at two ends of the sequence.
2.4 construction of pcDNA3.1-CMV-Purol 1-305 -(BamHⅠ)mCherry(NotⅠ)-PuroR 105-597 T2A-EGFP-polyA vector (abbreviated as pSSA-PMG vector)
2.2 pcDNA3.1-CMV-Purol 1-305 -PuroR 105-597 The backbone of the T2A-EGFP-polyA vector is ligated to the amplified fragment of interest containing the mCherry sequence after BamHI/NotI double cleavage, whereby the mCherry sequence is inserted into Purol in the above-mentioned expression cassette 1-305 And PuroR 105-597 Meanwhile, corresponding restriction enzyme recognition sites at two ends of the mCherry sequence are reserved to obtain pcDNA3.1-CMV-Purol 1-305 -(BamHⅠ)mCherry(NotⅠ)-PuroR 105-597 T2A-EGFP-polyA vector, was identified using double digestion (see FIG. 7), sequencing.
The constructed pSSA-PMG vector is shown in FIG. 8a (7678 bp), and comprises the following elements in sequence: CMV promoter and coding region homologous sequence Purol of resistance gene 1-305 Coding region homologous sequence PuroR of mCherry and resistance gene 105-597 T2A cleavage peptide, EGFP, polyA.
3. Construction of specific SSA-PMG reporter vectors
3.1 amplification of sg.T-hCR 5-mCherry-sg.T-hCR 5 sequence
Taking hCCR5 as an example, the target site selected when constructing a CRISPR/Cas expression vector (i.e., sg.t-hCCR 5) was taken as the target sequence of a specific SSA-PMG reporter vector, the forward primer was obtained mainly by adding a bamhi recognition site at the 5 'end and a partially complementary sequence of mCherry sequence at the 3' end (specifically referring to a portion of 5 'end of mCherry sequence), and the reverse primer was obtained by adding a partially reverse complementary sequence of mCherry sequence at the 5' end of the target sequence (specifically referring to a portion of 3 'end of mCherry sequence) and a stop codon, not i recognition site at the 3' end. After the primer design and synthesis, PCR amplification was performed using pSSA-PMG vector as a template. The forward and reverse primer sequences are as follows:
in the above reverse primer sequences, the underlined portion is the reverse complement of an additional stop codon (denoted stop codon).
After the PCR reaction was completed, the target band (778 bp) was recovered by agarose gel electrophoresis using a DNA recovery kit, thereby completing cloning of the sg.T-hCR 5-mCherry-sg.T-hCR 5 sequence including the following elements in order, and restriction endonuclease (i.e., bamHI, notI) recognition sites were introduced at both ends of the sequence: sg.T-hCR 5, mCherry, sg.T-hCR 5, stop codon.
3.2 construction of phCCR5-SSA-PMG vector
The pSSA-PMG vector is taken as a framework, the framework vector and the amplified target fragment containing the sg.T-hCCR5-mCherry-sg.T-hCCR5 sequence are respectively connected after BamHI/NotI double enzyme digestion, so that the sg.T-hCCR5-mCherry-sg.T-hCCR5 sequence replaces mCherry in the framework vector, and the phCCR5-SSA-PMG vector is obtained and is identified by double enzyme digestion (see FIG. 8 b) and sequencing verification.
The constructed phCCR5-SSA-PMG vector is shown in FIG. 8c (7725 bp), and comprises the following elements in sequence: CMV promoter and coding region homologous sequence Purol of resistance gene 1-305 sg.T-hCR 5-mCherry-sg.T-hCR 5 sequence, and homologous sequence of resistance gene coding region PuroR 105-597 T2A cleavage peptide, EGFP, polyA. stop codon (specifically TAA) in the sg.T-hCR 5-mCherry-sg.T-hCR 5 sequence is located in PuroR 105-597 Previously, so that:
1) Preventing T2A-EGFP in the vector from acting when not targeting;
2) Purol when targeting vectors 1-305 And PuroR 105-597 About 200bp of the sequence will recombine and therefore, in Purol 1-305 And PuroR 105-597 Intermediate elements, such as mcherry, are able to express normally when the vector is not targeted.
4. Construction of transfection Positive report vector
4.1 amplification of the coding sequence of the puromycin resistance Gene
The PCR amplification was performed using pSpCas9 (BB) -2A-Puro vector (Addgene 48139) as template, primers were designed and synthesized. Wherein the primer sequences are as follows:
iPuro-F(Hind III)：5'-cttAAGCTTACCATGACCGAGTACAAGC-3'(SEQ.ID.NO.11)
iPuro-R：5'-CCTCTCCACTGCCGGCACCGGGCTTGC-3'(SEQ.ID.NO.12)
after the PCR reaction is finished, agarose gel electrophoresis is adopted for detection, and a DNA recovery kit is used for recovering a target band (see fig. 9), so that cloning of a coding sequence of a puromycin resistance gene (for short Puro) is completed, and a recognition site of restriction enzyme (HindIII) is added at one end of the sequence.
4.2 construction of Puro-T2A-EGFP sequence
The pSSA-RPG vector (Addgene # 85932) was used as a template, and primers were designed and synthesized for PCR amplification. The primer sequences were as follows:
T2A-EGFP-F：5'-GCAAGCCCGGTGCCGGCAGTGGAGAGG-3'(SEQ.ID.NO.13)
T2A-EGFP-R(Xba I)：5'-ttaTCTAGATTACTTGTACAGCTC-3'(SEQ.ID.NO.14)
after the PCR reaction is finished, agarose gel electrophoresis is adopted for detection, and a DNA recovery kit is used for recovering a target band (see fig. 10), so that cloning of a T2A-EGFP sequence is completed, and a restriction enzyme (namely Xba I) recognition site is added to one end of the sequence, wherein the T2A-EGFP sequence consists of the following elements connected in sequence: T2A cleaves peptide, EGFP. The amplified Puro-containing fragment of interest and T2A-EGFP-containing fragment of interest were then fused by Overlap Extension PCR to form a DNA fragment in which the portion between recognition sites for restriction enzymes (i.e., hindIII, xbaI) was designated Puro-T2A-EGFP sequence (see FIG. 11) consisting of the following elements in sequence: puro, T2A cleavage peptide, EGFP.
4.3 construction of pcDNA3.1-CMV-Puro-T2A-EGFP-polyA vector
The pcDNA3.1 vector (Invitrogen V79520) is used as a framework, a fusion product containing the Puro-T2A-EGFP sequence and the framework vector are subjected to double digestion by HindIII/Xba I and then connected, so that the Puro-T2A-EGFP sequence is integrated in the vector, and a pcDNA3.1-CMV-Puro-T2A-EGFP-polyA vector (called as a pPuro-T2A-EGFP vector for short) is obtained, and double digestion identification (see FIG. 12) and sequencing verification (the same as the vector of phCCR5-SSA-PMG subjected to targeting repair) are used.
Working principle and flow of SSA-PMG report enrichment system
1. Principle of operation
The SSA-PMG report enrichment system constructed above is a red-green light "switch" traffic light (Traffic Light Reporter) gene editing reporting system.
The SSA-PMG report enrichment system mainly comprises two parts, namely: CRISPR/Cas expression vectors (e.g., phCCR5-sgRNA/Cas9 vectors) and specific SSA-PMG reporting vectors (e.g., phCCR5-SSA-PMG vectors). The CRISPR/Cas expression vector includes a sgRNA expression cassette and a Cas protein expression cassette; the specific SSA-PMG reporter vector mainly comprises a dual fluorescence reporter system (e.g., a red/green fluorescence reporter system based on mCherry/EGFP) and a drug resistance (e.g., puro-based puromycin resistance) integrated expression cassette. After the SSA-PMG report enrichment system transfects cells, the CRISPR/Cas expression vector can express a gRNA complex (sgRNA) and a Cas protein. Then, by RNA-DNA base pairing using a sequence around 20bp at the 5' end of the sgRNA, cas protein/sgRNA complexes are recruited to specific DNA sequences upstream of PAM, cleaving the target sequence on specific SSA-PMG reporter vectors while cleaving the cell genome target site.
Referring to FIG. 1, following transfection of the specific SSA-PMG reporter vector into cells, it is shown that Purol is located in the integrated expression cassette 1-305 And PuroR 105-597 The two target sequence regions in between are simultaneously sheared, purol can be used 1-305 And PuroR 105-597 SSA repair is carried out on the homologous repeated sequence of the polypeptide, so that the mCherry sequence is knocked out, and the coding sequence of the puromycin resistance gene and the coding sequence of the green fluorescent protein EGFP gene can be translated normally, thereby realizing the expression of puromycin resistance and green fluorescence under the start of CMV. If the above-mentioned clipping does not occur, puroR can be normally translated only 105-597 The previous nucleotide sequence, at this time, the red fluorescent protein mCherry was able to express normally, thus achieving expression of red fluorescence under the initiation of CMV (representing transfection positive). Therefore, through cotransfection of CRISPR/Cas expression vectors, the specific SSA-PMG report vector can be repaired by single-strand annealing, so that the double-fluorescence report system work (two fluorescence conversions) is realized, and puromycin resistance is generated at the same time, so that the method can be used for sorting by a flow cytometer or screening by puromycin drugs, and positive cells are edited by enrichment genes.
2. Specific enrichment test procedure (see FIG. 13)
(1) Designing a CRISPR/Cas expression vector and a specific SSA-PMG reporting vector for a target site of a target editing Gene (GOI) in a genome of a cell (e.g., HEK293T cell);
(2) Co-transfecting (Co-transfection) cells (e.g., HEK293T cells) with the constructed CRISPR/Cas expression vector and the specific SSA-PMG report vector;
(3) Detecting the efficiency of nuclease to exert the shearing activity by using a flow cytometer (the ratio of nuclease positive cells to transfection positive cells after transfection of a report vector=the report vector repair efficiency/the cell transfection efficiency of a plasmid vector) or sorting by using a flow cytometer to obtain nuclease positive cells;
(4) After 48-72 hours of transfection, puromycin is added into a cell culture medium, and after 3-5 days of puromycin screening, medicine is removed and the cell culture medium is changed into a normal cell culture medium, so that screened cells (namely nuclease positive cells) can be obtained;
(5) Diluting nuclease positive cells by a limiting dilution method, and spreading the diluted nuclease positive cells on a 96-well plate culture dish;
(6) After 7 days, cell monoclonal was randomly selected, and PCR was used to identify cell monoclonal (i.e., gene editing positive cells) in which gene editing (KO) occurred at the genomic target site;
(7) The identified positive cells are monoclonal transferred to 24-well plate, and the cells are subjected to expansion culture, such as HEK293T cells, using DMEM medium containing 10% fetal bovine serum and 100 μg/mL green/streptomycin as cell culture medium, and at 37deg.C, CO 2 The culture was performed in a 5% concentration cell incubator.
Specific application of SSA-PMG report enrichment System in enriching CRISPR/Cas9 Gene editing positive cells Using the hCCR5 gene example described above, the phCCR5-sgRNA/Cas9 vector and phCCR5-SSA-PMG were co-transfected
HEK293T cells of the vector are used as test groups for enriching gene editing positive cells by using an SSA-PMG report enrichment system; HEK293T cells co-transfected with the phCCR5-sgRNA/Cas9 vector and the pPuro-T2A-EGFP vector were used as positive control groups.
The red/green fluorescence of the cells was observed with a fluorescence inverted microscope 48 hours after transfection of each group of cells (see fig. 14). HEK293T cells co-transfected by the pNC-sgRNA/Cas9 vector and the phCCR5-SSA-PMG vector are set as a negative control group (the pNC-sgRNA/Cas9 vector specifically refers to the pX330-U6-Chimeric_dBsai-CBh-hSpCas9 vector). The cells of the test and positive control groups were then digested into 1.5mL EP tubes and centrifuged at 800 rpm for 2 minutes. The cell pellet was resuspended in PBS and the labeled group of centrifuge tubes were inserted into an ice bin. Filtering the cell suspension into a flow tube through a 200-mesh cell screen before loading the sample by a flow cytometer to prevent cell aggregation and blockage of flow type fine particlesA cytometer pipe. FACS: cells were purified by red fluorescence (mCherry in a flow cytometer + ) Green Fluorescence (EGFP) + ) Channel sorting counts, calculating the ratio of nuclease positive cells to transfected positive cells after cotransfection of the test group report vector as follows: 88.67% = report vector repair efficiency (36.39%)/cell transfection efficiency of plasmid vector (41.04%), after which cells in both groups only fluoresce green (EGFP) + ) Collection (see fig. 15), genomic DNA was extracted from both groups of collected cells and tested for genome editing efficiency.
Cells were screened for drug by adding puromycin (3. Mu.g/mL) to the cell culture media of the test and positive control groups 48 hours after transfection of each group. After 3 days of drug screening, the drug was withdrawn and the enriched positive cells were observed with a fluorescent inverted microscope (see fig. 17). And extracting genome DNA of the two groups of cells, and detecting genome editing efficiency.
The cells obtained by enrichment in two different modes of fluorescence sorting and drug screening are subjected to extraction of genome DNA as a template, a detection primer (see Table 8) with a barcode with different sequences is used for amplifying a region (280 bp) near a target site, and the region is sent to the Siam qing department biotechnology company for deep sequencing detection of the amplicon after gel recovery.
TABLE 8 detection primers
After the sequencing result is analyzed by CRISPResso (http:// www.crispresso.rocks) on-line software, the efficiency of editing positive cells by respective enrichment genes of an SSA-PMG report enrichment system and a report enrichment system of a positive control group is counted; statistics were performed using GraphPadPrism 8.0. If no gene editing occurs, the target gene amplified from the genomic DNA of the cell corresponds to the wild-type gene sequence (WT); if gene editing occurs, the gene of interest amplified from the genomic DNA of the cell should not be a 280bp fragment, and the sequencing results show the presence of a deletion or mutation in the gene sequence (see FIGS. 16 and 18).
The results of the positive cell analysis obtained by FACS showed that the positive cell gene editing efficiency obtained using the positive control group report enrichment system (pPuro-T2A-EGFP vector co-transfected with phCCR5-sgRNA/Cas9 vector) was 23.57% (100% -WT%, WT% is the number of cells in which no gene editing occurred), and the positive cell gene editing efficiency obtained using the SSA-PMG report enrichment system was 41.36% (100% -WT%); that is, the efficiency of editing positive cells by nuclease-positive cell enrichment gene was 1.63 times that of editing positive cells by transfection-positive cell enrichment gene (fig. 16).
Analysis of positive cells obtained by puromycin treatment showed that: the positive cell gene editing efficiency obtained using the positive control group report enrichment system (pPuro-T2A-EGFP vector co-transfected with phCCR5-sgRNA/Cas9 vector) was 45.33% (100% -WT%), and the positive cell gene editing efficiency obtained using the SSA-PMG report enrichment system was 66.93% (100% -WT%); that is, the efficiency of editing positive cells by nuclease-positive cell enrichment gene was 1.48 times that of editing positive cells by transfection-positive cell enrichment gene (fig. 18).
The results show that the SSA-PMG report enrichment system can more efficiently enrich and obtain the gene editing positive cells.
The construction of the specific SSA-PMG reporter vector was similar to that described above except that the sg.T-hCR 5-mCherry-sg.T-hCR 5 sequence was changed to the sg.T-hCR 5-mCherry sequence or the mCherry-sg.T-hCR 5 sequence. The thus obtained reporter vector was co-transfected with phCCR5-sgRNA/Cas9 vector respectively into HEK293T cells and vector repair efficiency was examined using FACS, and the results showed that: the report vector was significantly lower in repair efficiency than the specific SSA-PMG report vector with both side target sequences in the case of only single side target sequence cleavage (the repair efficiency of vector phCCR5-SSA-PMG constructed with sg.T-hCR 5-mCherry-sg.T-hCR 5 sequence was 36.39%, FIG. 15, the repair efficiency of report vector constructed with sg.T-hCR 5-mCherry sequence was 21.20%, FIG. 19, and the repair efficiency of report vector constructed with mCherry-sg.T-hCR 5 sequence was 17.28%, FIG. 19). These results indicate that the reporter vector reflects mainly nuclease activity in transfected cells, wherein the specific SSA-PMG reporter vector is more easily recognized by nucleases and sheared, and the specific SSA-PMG reporter vector is more easily repaired after targeting itself, thereby further improving the efficiency of editing positive cells by enriching genes by virtue of self-repair ability.
In a word, the SSA-PMG report enrichment system constructed by the invention can guide a target sequence identified by guide RNA on a specific SSA-PMG report vector to complete the shearing of the vector in a nuclease targeting genome (gene editing) at the same time, and can restore the complete expression sequence of a blocked drug resistance gene through SSA repair, so that transfected cells generate resistance, and simultaneously splice deletion and repair expression are respectively carried out on two expression sequences in a double-fluorescence report system, so that the efficiency of the nuclease in playing shearing activity in transfected cells can be determined by respectively quantifying the expression of the two reporter genes in a TLR type double-fluorescence report system, and nuclease positive cells can be obtained rapidly and simply through fluorescence sorting or drug screening. More importantly, the SSA-PMG report enrichment system can improve the enrichment efficiency of the gene editing positive cells. The invention provides an effective way for promoting the application of the gene editing technology in the aspects of gene function research and animal genetic breeding.
Claims (10)
1. A gene editing positive cell report enrichment system, characterized in that: the system includes a site-specific reporter vector; the report vector comprises a dual fluorescent report gene and resistance gene integrated expression cassette, the integrated expression cassette comprises an upstream segment which is positioned in a transcription region of the expression cassette and is homologous to a resistance gene coding sequence, a downstream segment which is homologous to the resistance gene coding sequence, a gene coding sequence of a first fluorescent protein, a gene coding sequence of a second fluorescent protein, and a first target sequence and a second target sequence which are identified by guide RNA, wherein the first target sequence is positioned between the upstream segment and the gene coding sequence of the first fluorescent protein, the second target sequence is positioned between the downstream segment and the gene coding sequence of the first fluorescent protein, and the upstream segment and the downstream segment comprise identical repeated sequences which are used for carrying out single-strand annealing repair and enabling the integrated expression cassette to be converted from expressing the first fluorescent protein to carrying out tandem expression of the corresponding resistance gene and the second fluorescent protein after repair.
2. The gene editing positive cell reporting enrichment system of claim 1, wherein: the upstream fragment, the first target sequence, the gene coding sequence for the first fluorescent protein, the second target sequence, the downstream fragment and the gene coding sequence for the second fluorescent protein employ the same open reading frame separated by a stop codon disposed between the second target sequence and the downstream fragment.
3. The gene editing positive cell reporting enrichment system of claim 1, wherein: the same direction repeated sequence is 200-350 bp.
4. The gene editing positive cell reporting enrichment system of claim 1, wherein: the gene coding sequence of the second fluorescent protein is positioned at the rear side of the downstream fragment, and the transcription region of the integrated expression cassette further comprises a shear peptide sequence for connecting the downstream fragment and the gene coding sequence of the second fluorescent protein.
5. The gene editing positive cell reporting enrichment system of claim 1, wherein: the first fluorescent protein is red fluorescent protein, and the second fluorescent protein is green fluorescent protein.
6. The gene editing positive cell reporting enrichment system of claim 1, wherein: the upstream fragment and the downstream fragment are subjected to single-strand annealing repair and then used for expressing puromycin resistance.
7. The gene editing positive cell reporting enrichment system of claim 1, wherein: the system further includes a site-specific CRISPR/Cas expression vector comprising a sgRNA expression cassette for expressing the guide RNA and a nuclease expression cassette for expressing a Cas protein, the target sequence recognized by the guide RNA being derived from the genome of a gene editing target cell.
8. The gene editing positive cell reporting enrichment system of claim 7, wherein: the gene editing target cell is a eukaryotic cell.
9. A method of preparing a gene editing positive cell reporting enrichment system according to claim 1, wherein: the method comprises the following steps:
1) Cloning a recombinant fragment comprising an upstream fragment homologous to the coding sequence of the resistance gene, a downstream fragment homologous to the coding sequence of the resistance gene, a cleavage peptide sequence, and a gene coding sequence of a second fluorescent protein; integrating the recombinant segment with a gene coding sequence of a first fluorescent protein obtained by cloning after being connected with an expression vector skeleton, and enabling the gene coding sequence of the fluorescent protein to be connected between the upstream segment and the downstream segment to obtain a double-fluorescence reporter gene and resistance gene integrated recombinant expression vector;
2) And (3) replacing the gene coding sequence of the first fluorescent protein in the double-fluorescent reporter gene and resistance gene integrated recombinant expression vector obtained in the step (1) with the gene coding sequence of the first fluorescent protein obtained by cloning and the recombinant fragments of the first target sequence and the second target sequence identified by the guide RNA to obtain the site-specific reporter vector.
10. A screening method of gene editing positive cells, which is characterized in that: the method comprises the following steps:
1) Transfecting the gene-editing positive cell reporting enrichment system of claim 7 into a gene-editing target cell;
2) Performing fluorescence sorting by using a flow cytometer after 48-72 hours of transfection to obtain nuclease positive cells, and then performing enrichment culture; or, after 48-72 hours of transfection, screening cells expressing the corresponding resistance genes by using a medicament to obtain nuclease positive cells, and then carrying out enrichment culture.
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