CN114107292B - Gene editing system and method for site-directed insertion of exogenous gene - Google Patents
Gene editing system and method for site-directed insertion of exogenous gene Download PDFInfo
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Abstract
The embodiment of the invention discloses a gene editing system and a method for site-directed insertion of an exogenous gene, wherein the gene editing system comprises Cas9, sgRNA and ssDNA, wherein the Cas9 and the sgRNA form an RNP complex, the sgRNA is an insertion site of the exogenous gene, and the ssDNA is an insertion template of the exogenous gene. The invention mainly uses the gene editing technology, uses ssDNA as an insertion template of an exogenous gene, and successfully realizes the fixed-point knock-in of 4.5kb (aAPC 1: EF1a-CD19-CD86-CD 64-PolyA) at an AAVS1 site under the cooperation of sgRNA; AAVS1 knockin exogenous gene segment has the advantages of stable expression and no influence on transcription of other genes; so that the genetic background of the transformed cells is simpler and unified, and the influence of the insertion site and the copy number on the functions of other genes is eliminated.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a gene editing system and method, and in particular relates to a gene editing system and method for site-specific insertion of exogenous genes.
Background
The stable expression cell strain is also called stable transgenic cell strain, which is one of the most commonly used means of gene function research, model establishment, crop breeding, drug development, gene therapy, etc., and is characterized in that specific gene plasmid DNA is integrated on cell chromosome by a molecular biological method to make the cell stably express for a long time or interfere with gene expression.
The traditional construction method of the stable transgenic cell strain mainly comprises two major types of non-viruses and viruses, wherein the construction of the non-virus stable transgenic cell strain mainly comprises the steps of transfecting plasmids or transposons into cells through electrotransformation or liposome and the like, continuously pressurizing the cells by using the half lethal concentration of antibiotics (such as puromycin), so that the plasmids containing specific genes are integrated into chromosome DNA, and finally obtaining the stable transgenic cell strain through multiple rounds of cell screening; the virus method is mainly to transfect target cells by using retrovirus, lentivirus and the like according to a certain MOI, and screen the cells after 48 hours or 72 hours of transfection to obtain cell strains with stable expression.
However, the stable expression cell strains obtained by the two methods have random integration of the target genes in the chromosome DNA, so that the loss of functions of other genes is easily caused, and the copy number and the position of the insertion are not fixed. Thus, the difference in expression between different cells in the same cell line may be significant, potentially risky, interfering with the final experimental results, and this random integration may also present a potential risk of pathogenicity and carcinogenesis in immunotherapy.
Disclosure of Invention
In view of the above, the present invention aims to provide a gene editing system and method for site-directed insertion of exogenous genes, so that the genetic background of the transformed cells is simpler and unified, and the influence of insertion sites and copy numbers on the functions of other genes is eliminated.
The invention provides a gene editing system for site-directed insertion of an exogenous gene, which comprises Cas9, sgRNA and ssDNA, wherein the Cas9 and the sgRNA form an RNP complex, the sgRNA is an insertion site of the exogenous gene, and the ssDNA is an insertion template of the exogenous gene.
In a preferred embodiment of the invention, the insertion sites include PD1, TIGIT, AAVS1, CTLA-4, tim3 and LAG3.
In a preferred embodiment of the invention, when the insertion site is AAVS1, the nucleotide sequence of the sgRNA is as shown in SEQ ID NO. 1 or a homologous sequence thereof; the homologous sequence of the sgRNA has a homology of 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more with the original sequence.
In a preferred embodiment of the present invention, the exogenous gene is aAPC1, the ssDNA comprises aAPC1 and homology arms arranged at two sides of the aAPC1, the aAPC1 has a structure of EF1a-CD19-CD86-CD64-PolyA, the homology arms at two sides are LHA and RHA, respectively, the LHA is connected with EF1a, and the RHA is connected with PolyA.
In a specific embodiment of the invention, the nucleotide sequence of the aAPC1 is shown in SEQ ID No. 2 or a homologous sequence thereof; the homology of the aAPC1 homologous sequence to the original sequence is 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more; and/or
The nucleotide sequence of the LHA is shown as SEQ ID NO. 3 or a homologous sequence thereof; the LHA homologous sequence has a homology of 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more to the original sequence; and/or
The nucleotide sequence of the RHA is shown as SEQ ID NO. 4 or a homologous sequence thereof; the homology of the homologous sequence of the RHA to the original sequence is 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.
In one aspect, the present invention also provides a method for site-directed insertion of an exogenous gene, comprising the steps of:
1) Selecting an insertion site according to an exogenous gene to be inserted, and designing and synthesizing sgRNA and ssDNA according to the insertion site;
2) Then forming an RNP complex between Cas9 and the sgRNA, and then forming a gene editing system (RNP-ssDNA complex) for site-directed insertion of exogenous genes from the RNP complex and the ssDNA;
3) Culturing K562 cells, and inserting the exogenous gene into the K562 cells through the gene editing system for site-specific insertion of the exogenous gene formed in the step 2) to obtain the K562 cells with site-specific insertion of the exogenous gene.
In a preferred embodiment of the invention, in step 1), the insertion site comprises PD1, TIGIT, AAVS1, CTLA-4, tim3 and LAG3.
In a preferred embodiment of the invention, when the insertion site is AAVS1, the nucleotide sequence of the sgRNA is as shown in SEQ ID NO. 1 or a homologous sequence thereof; the homologous sequence of the sgRNA has a homology of 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more with the original sequence.
In a preferred embodiment of the present invention, in step 3), the exogenous gene is inserted into the K562 cells by electrotransfection or lipofection.
In a preferred embodiment of the present invention, the exogenous gene is aAPC1, the ssDNA comprises aAPC1 and homology arms provided on both sides thereof, the aAPC1 has a structure of EF1a-CD19-CD86-CD64-PolyA, the homology arms on both sides are LHA and RHA, respectively, the LHA is connected to EF1a, and the RHA is connected to PolyA.
In a specific embodiment of the invention, the nucleotide sequence of the aAPC1 is shown in SEQ ID No. 2 or a homologous sequence thereof; the homology of the aAPC1 homologous sequence to the original sequence is 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more; and/or
The nucleotide sequence of the LHA is shown as SEQ ID NO. 3 or a homologous sequence thereof; the LHA homologous sequence has a homology of 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more to the original sequence; and/or
The nucleotide sequence of the RHA is shown as SEQ ID NO. 4 or a homologous sequence thereof; the homology of the homologous sequence of the RHA to the original sequence is 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.
The invention has the beneficial effects that the gene editing technology is mainly utilized, ssDNA is used as a repair (insertion) template of the exogenous gene, and under the cooperation of sgRNA, the fixed-point knock-in of 4.5kb (aAPC 1: EF1a-CD19-CD86-CD 64-PolyA) is successfully realized at the AAVS1 site; the AAVS1 locus is a specific sequence positioned on the first intron of the human gene PPP1R12C, and the knock-in exogenous gene fragment in the locus has the advantages of stable expression and no influence on the transcription of other genes; so that the genetic background of the transformed cells is simpler and unified, and the influence of the insertion site and the copy number on the functions of other genes is eliminated. Compared with the traditional stable transgenic cell strain construction method, the stable transgenic cell constructed by the method is more accurate and safer, the genetic background is clearer and more reliable, and the construction difficulty of the large fragment gene fixed-point knock-in cell strain is solved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.
The invention may be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a diagram showing the result of electrophoresis of the target genome amplification in example 1 of the present invention, wherein 1 is Marker and 2 is an amplified fragment of the target gene;
FIG. 2a is an electrophoresis chart of the preparation of the in vitro transcription template of sgRNA in example 1 of the present invention, wherein 1 is the in vitro transcription template of sgRNA and 2 is Marker;
FIG. 2b is an electropherogram of the efficiency verification of sgRNA in example 1 of the present invention, wherein 1 is an amplified fragment of the in vitro transcription template of sgRNA, 2 is an enzyme-sliced fragment of the in vitro transcription template of sgRNA, and M is Marker;
FIG. 3 is a schematic diagram of the foreign gene in example 1 of the present invention;
FIG. 4 shows the results of the detection of the electrotransformation efficiency of aAPC1 site-specific knock-in K562 cells in example 3 of the present invention;
FIG. 5 shows the results of flow-sorting of aAPC1 site-specific knock-in K562 cells in example 4 of the present invention;
FIG. 6 shows the results of detecting the gene expression rate in aAPC1 before and after the site-directed knock-in of aAPC1 into K562 cells in example 5 of the present invention;
FIG. 7 shows the results of genomic level identification of the aAPC1 site-specific knock-in K562 cell subclones 1-3 (Clone 1-Clone 3) aAPC1 fragment in example 6 of the present invention, wherein 1-3 is the amplified AAVS1-P1 fragment, 4-6 is the amplified AAVS1-P2 fragment, 7 is the 1kb Marker, and 8-10 is the amplified AAVS1-P3 fragment;
FIG. 8 shows the results of the identification of the genomic level copy number of the site-directed knock-in aAPC1 of the K562 cells of example 6 of the present invention, wherein 1-3 is subclone 1-3 (Clone 1-Clone 3), and 4 is 2kb Marker;
FIG. 9 shows the genotype results of the site-directed knock-in K562 cells with the aAPC1 gene in example 6 of the present invention, wherein K562-WT-seq is the sequence of the wild-type K562 genome and K562-KI-aAPC1 is the genotype results of the site-directed knock-in K562 cells with the aAPC1 gene (Clone 1-Clone 3);
wherein, K562-control is wild type K562, K562-RNP-ssDNA is RNP-ssDNA transfected K562 cells, and K562-KI-aAPC1 is aAPC1 site-specific knocked-in K562 cells.
Detailed Description
Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings.
The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses.
Techniques, methods, and apparatus known to one of ordinary skill in the relevant art may not be discussed in detail, but should be considered part of the specification where appropriate.
Unless otherwise specified, "ssDNA" herein, i.e., "single strand DNA", specifically refers herein to "insertion (repair) template of foreign gene, including foreign gene and homology arms provided on both sides thereof".
Example 1: design and synthesis of exogenous genes and sgrnas
Design of sgRNA
Using CRISPR/Cas9 technology, sgrnas were designed for AAVS1 region using the Crisprgold website (https:// Crisprgold. Mdc-berlin. De/index. Php), and the selected sgRNA sequence is shown in SEQ ID No. 1 (GGGGCCACTAGGGACAGGAT).
2. Genome amplification of interest
Taking 1×10 6 Cells, genomic DNA was extracted using the Tiangen cell gene extraction kit (DP 304), and genomic PCR amplification was performed using primers AAVS1-F1 (SEQ ID NO:5, GCCTCCCTTTCTTGTAGGCC) and AAVS1-R1 (SEQ ID NO:6, AGCCAAGTTAGACTAGG); the amplified fragment was then subjected to gel cutting using a gel kit (Axygen: AP-GX-250), the target fragment was recovered by gel cutting, the result was as shown in FIG. 1, and the recovered target fragment was stored at-20℃for use.
In vitro preparation and editing efficiency verification of AAVS1 insertion site sgRNA
In vitro transcription of sgrnas: based on the selected sgRNA sequence, primers AAVS1-sgRNA-F (SEQ ID NO:7, taatagactcacttatagagggccactagggacaggattgtttttaggctagaaaaatagcaagtt) AAVS1-sgRNA-R (SEQ ID NO:8, aaaagcaccgactcggtgcactttcaagttgataacgataccttactttattactttacttgctttactttagcttacttgctgctct), in vitro transcription template amplification of sgRNA using a gel kit (Axygen: AP-GX-250), and the target fragment was recovered by cutting gel, and the result is shown in FIG. 2 a.
The NEB in vitro transcription kit (E2050S) is used for in vitro transcription synthesis of sgRNA according to the operation steps of the specification, spCas9 protein is purchased from Nanjing Jinsri (Z03393-100), amplified genome is taken as a template, enzyme digestion is carried out for 30min at 37 ℃, in vitro editing efficiency verification is carried out according to the specification, and the result shows that the selected sgRNA has higher activity in vitro and can be used as a subsequent aAPC1 insertion site as shown in FIG. 2 b.
4. Design and synthesis of exogenous gene
The locus of sgRNA is selected as the insertion locus of aAPC1, about 300-400bp is selected as Homologous Arms (HA) at two ends of the insertion locus, LHA (SEQ ID NO: 3) and RHA (SEQ ID NO: 4) are respectively arranged at the left and right homologous arms, the structure of aAPC1 is EF1a-CD19-CD86-CD64-PolyA-RHR (SEQ ID NO: 2), and the full-length sequence of the exogenous gene (the structure diagram is shown in figure 3) is entrusted with the company Limited of Suzhou gold intelligent biotechnology.
Example 2: site-directed insertion of exogenous gene into K562 cells
K562 cell resuscitating culture
Starting a water bath kettle in advance, setting the temperature to be 37 ℃, taking out an IMDM-Full culture medium for preheating, taking NK-92 cells out of a liquid nitrogen tank, immediately putting the cells into the water bath kettle at the temperature of 37 ℃, taking a 15ml centrifuge tube, and adding 8ml of preheated K562 culture medium; placing the cells in a water bath kettle at 37 ℃ and gently shaking until the cells are completely melted, wiping the outer wall of a freezing storage tube by using non-woven fabrics with alcohol, slowly adding the cell suspension into a 15ml centrifuge tube (the process is controlled within 3 min), centrifuging at 1000rpm for 5min after uniformly mixing, and discarding the supernatant; 1mL of the culture medium is taken to resuspend the cell sediment, transferred to a T75 culture bottle containing 30mL of IMDM-Full culture medium for culture, and the cells are passaged according to a certain density, so that the proper cell growth density is maintained.
K562 cell RNP-ssDNA Complex electrotransformation
Adding 10ml of IMDM-Full culture medium into a T25 culture flask in advance, placing the culture flask in an incubator for preheating, and taking out electrotransfer liquid from a refrigerator at 4 ℃ to room temperature; taking a proper amount of cultured K562 cells into a 50ml centrifuge tube, centrifuging at 1500rpm for 5min, and discarding the supernatant; then adding 20ml PBS to resuspend the cell pellet, centrifuging at 1500rpm for 5min, and discarding the supernatant; then adding a proper amount of PBS to re-suspend the cell sediment, counting the cells, and 1X 10 cells per tube 6 The cells were aliquoted into 1.5ml centrifuge tubes, centrifuged at 2500rpm for 5min and the supernatant discarded.
After 20min before electrotransformation, 9 μg of SpCas9 and 6 μg of sgRNA prepared in example 1 are respectively sucked by a pipette and placed in a PCR tube, incubated at room temperature for 10min, 3 μg of ssDNA (synthesized by Jin Weizhi) is added, and after uniform mixing, the mixture is placed at room temperature for 5min, so that an RNP-ssDNA complex (namely a gene editing system for site-directed insertion of exogenous genes) is obtained.
Then taking equal amount of AB electrotransfer liquid to re-suspend cell sediment, adding the incubated RNP-ssDNA complex, and carrying out electrotransfer by using a Celetix (CTX-1500A-LE) electrotransfer instrument; after the electric transformation is finished, K562 cells with exogenous genes inserted at fixed points are obtained, the K562 cells are transferred into a preheated T25 culture flask and placed at 37 ℃ and 5 percent CO 2 Culturing in an incubator to obtain the aAPC1 fixed-point knocked-in K562 cells.
Example 3: aAPC1 fixed-point knock-in K562 cell electrotransformation efficiency detection
Taking K562 cells which are knocked in at the aAPC1 fixed point after the partial transfection for 24 hours in the example 2, transferring the cells into a 15ml centrifuge tube, centrifuging at 1500rpm for 5min, and discarding the supernatant; after washing the cell pellet with 1ml PBS, transferring to a 1.5ml EP tube, centrifuging at 1500rpm for 5min, discarding the supernatant, repeating the operation once, and selecting a FITC channel for electrotransfection efficiency detection using a flow-through (ACEA: NOVOCyte 3130), the result is shown in FIG. 4, from which it can be seen that the RNP complex electrotransfected K562 cells have a transfection efficiency of 49.71%.
Example 4: sorting of aAPC1 site-directed knock-in K562 cells
Taking the aAPC1 fixed-point knocked-in K562 cells transfected for 5-7 days in the example 2, transferring the cells into a 50ml centrifuge tube, centrifuging at 1500rpm for 5min, and discarding the supernatant to obtain cell sediment; then 20ml PBS is added to clean the cell sediment, the cell sediment is centrifuged at 1500rpm for 5min, and the supernatant is discarded; adding a proper amount of PBS to resuspend the cell pellet, adding a proper amount of Anti-CD19 antibody (BioLegend: cat. No. 392503) and dyeing for 20min in a dark place; after the staining is finished, 20ml PBS is added to clean the cells, the cells are centrifuged at 1500rpm for 5min, and the supernatant is discarded; then 20ml PBS is added to clean the cell sediment, the cell sediment is centrifuged at 1500rpm for 5min, and the supernatant is discarded; then adding a proper amount of PBS plus 0.2% FBS to resuspend cell sediment, filtering the cell suspension by a 40 mu m filter screen, transferring the cell suspension to a T25 culture flask for culture, and after the cell grows up, carrying out flow type second flow type separation, wherein the steps are the same as the steps, the flow type separation results before and after the electric transfer are shown in figure 5, and the knocking-in efficiency of the aAPC1 fixed-point knocked-in K562 cells is about 0.99%, and after two rounds of cell separation, the aAPC1 fixed-point knocked-in K562 cells with the purity of more than 99% are obtained.
Example 5: monoclonal cell selection
The second round of sorted cells from example 4 were selected for subcloning 96-well plates and after the cells were grown up, 3 subcloned cells (Clone 1-Clone 3) were selected for subsequent gene overexpression and genome-level knock-in identification.
The subcloned cells and untransfected K562 cells (control) were used for positive expression rate detection of CD19, CD64 and CD86 cells, and antibody was used as Anti-CD19 (BioLegend, cat# 392503), anti-CD64 (BioLegend, cat# 305005), anti-CD86 (BioLegend, cat# 374203) and the cell treatment was carried out according to the corresponding instructions, and the detection results are shown in FIG. 6, which show that the K562 cells CD19, CD64 and CD86 of the fixed-point knock-in aAPC1 have high expression rate, and the K562 cells stably expressing aAPC1 were successfully obtained.
Example 6: genome horizontal knock-in identification
1X 10 from Clone1-Clone3 6 Extracting genome from the number of cells according to the instruction of a cell genome extraction kit, designing three nested primers of AAVS1-F2 (SEQ ID NO:9, CCTGTGGCCATCTCTCTCGTTTTCT) and AAVS1-R2 (SEQ ID NO:10, GCCTGGGAATCACATGAGG); AAVS1-F3 (SEQ ID NO:11, AGGAACCTCTCTAGTGGTGAAGGT), AAVS1-R3 (SEQ ID NO:12, ACGAGAAGAAGGTCAGCA); AAVS1-F4 (SEQ ID NO:13, ACACACCGTCCTGGTTTCACG), AAVS1-R4 (SEQ ID NO:14, CAAGAGGAGAAGCAGTTTGGA), PCR amplification, detection of site-specific knockin fragments and sites, the corresponding cloned fragments being shown in FIG. 3, respectively, the first fragment (AAVS 1-P1) being located between AAVS1-F2 and AAVS1-R2, the second fragment (AAVS 1-P2) being located between AAVS1-F3 and AAVS1-R3, and the third fragment (AAVS 1-P3) being located between AAVS1-F4 and AAVS 1-R4; the PCR amplification results are shown in FIG. 7, from which it can be seen that subclones Clone1-Clone3 successfully amplified the aAPC1 fragment. The PCR product obtained in the above step was subjected to Sanger sequencing analysis after the target fragment was recovered according to the gel recovery kit procedure, and the result showed that the 4.5kb fragment was successfully knocked into the AAVS1 site.
AAVS1-F5 (SEQ ID NO:15, GGTCCGAGGAGCTCAGGT) primers AAVS1-R5 (SEQ ID NO:16, ACAGGGAGGTGGGGGTTAGA) were used to identify genome-level copy number of Clone1-Clone3 cell lines, and the result is shown in FIG. 8, wherein the amplified band size is 275bp, indicating that LHR-EF1a-CD19-CD64-CD86-PolyA-RHA is single copy knock-in; in addition, the Clone1-Clone3 was sequenced, the genotype of which is shown in FIG. 9, indicating that the genotype of Clone1-Clone3 cell lines was single copy site-directed knock-in.
In summary, the invention mainly utilizes the gene editing technology, takes ssDNA as a repair (insertion) template of the exogenous gene, successfully realizes the fixed-point knock-in of 4.5kb (aAPC 1: EF1a-CD19-CD86-CD 64-PolyA) at the AAVS1 site under the cooperation of sgRNA, and proves that the exogenous gene knock-in is single copy knock-in through experiments; so that the genetic background of the transformed cells is simpler and unified, and the influence of the insertion site and the copy number on the functions of other genes is eliminated. Compared with the traditional stable transgenic cell strain construction method, the stable transgenic cell constructed by the method is more accurate and safer, the genetic background is clearer and more reliable, and meanwhile, the construction difficulty of large fragment gene fixed-point knock-in cell strains is solved.
The description of the present invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.
Sequence listing
<110> Achillea Biotechnology Co., ltd
<120> Gene editing System and method for site-directed insertion of exogenous Gene
<130> 1069
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> SEQ ID NO:1
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ggggccacta gggacaggat 20
<210> 2
<211> 4579
<212> DNA
<213> artificial sequence
<220>
<223> SEQ ID NO:2
<400> 2
ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60
ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120
gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180
gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc 240
gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 300
acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg 360
gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg 420
cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct 480
ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg 540
caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc 600
gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga 660
gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct 720
ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca 780
gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg 840
acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg 900
tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat 960
tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg 1020
gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa 1080
ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca 1140
gtggttcaaa gtttttttct tccatttcag gtgtcgtgag ctagcactag tgccaccatg 1200
ccacctcctc gcctcctctt cttcctcctc ttcctcaccc ccatggaagt caggcccgag 1260
gaacctctag tggtgaaggt ggaagaggga gataacgctg tgctgcagtg cctcaagggg 1320
acctcagatg gccccactca gcagctgacc tggtctcggg agtccccgct taaacccttc 1380
ttaaaactca gcctggggct gccaggcctg ggaatccaca tgaggcccct ggccatctgg 1440
cttttcatct tcaacgtctc tcaacagatg gggggcttct acctgtgcca gccggggccc 1500
ccctctgaga aggcctggca gcctggctgg acagtcaatg tggagggcag cggggagctg 1560
ttccggtgga atgtttcgga cctaggtggc ctgggctgtg gcctgaagaa caggtcctca 1620
gagggcccca gctccccttc cgggaagctc atgagcccca agctgtatgt gtgggccaaa 1680
gaccgccctg agatctggga gggagagcct ccgtgtctcc caccgaggga cagcctgaac 1740
cagagcctca gccaggacct caccatggcc cctggctcca cactctggct gtcctgtggg 1800
gtaccccctg actctgtgtc caggggcccc ctctcctgga cccatgtgca ccccaagggg 1860
cctaagtcat tgctgagcct agagctgaag gacgatcgcc cggccagaga tatgtgggta 1920
atggagacgg gtctgttgtt gccccgggcc acagctcaag acgctggaaa gtattattgt 1980
caccgtggca acctgaccat gtcattccac ctggagatca ctgctcggcc agtactatgg 2040
cactggctgc tgaggactgg tggctggaag gtctcagctg tgactttggc ttatctgatc 2100
ttctgcctgt gttcccttgt gggaattctt catcttgtga aacagacttt gaattttgac 2160
cttctcaagt tggcgggaga cgtggagtcc aacccagggc ccatgtggtt cctgaccaca 2220
ctcctcctct gggtgccagt ggacggacag gtggatacaa ccaaggccgt gatcacactg 2280
cagcctcctt gggtgtccgt gttccaggaa gagaccgtga ccctgcattg cgaggtgctg 2340
catctgccag gatctagcag cacccagtgg ttcctgaacg gcacagccac acagaccagc 2400
acccctagct acaggatcac cagcgccagc gtgaacgata gcggcgagta cagatgccag 2460
aggggtctct caggcagaag cgatcccatc cagctggaga tccatagggg ttggctgctg 2520
ctgcaggtgt caagcagagt gttcaccgag ggcgaacctc tggctctccg ctgtcacgct 2580
tggaaggaca agctggtcta caacgtgctg tactaccgga acggcaaggc cttcaagttc 2640
ttccattgga acagcaacct gaccatcctg aagaccaaca tcagccacaa cggaacctac 2700
cattgcagcg gcatgggcaa gcacagatac accagcgcag gcatcagcgt gaccgtgaag 2760
gagctgtttc ccgctccagt gctgaacgct agcgtgacaa gccctctgct ggagggaaac 2820
ctggtgacac tgagctgcga gaccaagctg ctcctgcaga gaccaggcct gcagctgtac 2880
ttcagcttct acatgggcag caagaccctg aggggcagaa acaccagcag cgagtaccag 2940
atcctgacag ccaggaggga ggatagcggc ctctattggt gcgaagccgc tacagaggac 3000
ggaaacgtgc tgaagaggag cccagagctg gaactgcagg tgctgggact gcagctgcct 3060
acacccgtct ggtttcacgt gctgttctac ctggccgtgg gcatcatgtt cctggtgaac 3120
accgtcctct gggtgaccat ccgcaaggag ctgaagcgga agaagaagtg ggacctggag 3180
atcagcctgg acagcggcca cgagaagaag gtcatcagca gcctgcagga ggacagacac 3240
ctggaagagg agctcaagtg ccaggagcag aaggaggagc agctgcagga aggagtgcac 3300
agaaaggagc cccagggagc taccgccacg aacttctctc tgttaaagca agcaggagac 3360
gtggaagaaa accccggtcc catggacccc cagtgcacca tgggtctgtc caacatcctg 3420
ttcgtgatgg ccttcctgct ctcaggagcc gcccctctga aaattcaggc ctacttcaac 3480
gagaccgccg atctgccttg ccagttcgcc aacagccaga accagagcct gagcgagctg 3540
gtggtgtttt ggcaggacca ggagaacctg gtgctgaacg aggtgtacct gggcaaggag 3600
aagttcgaca gcgtgcacag caagtacatg ggccggacca gcttcgacag cgattcttgg 3660
accctgcggc tgcacaacct gcagatcaag gacaagggcc tctaccagtg catcatccac 3720
cacaagaagc ccaccggcat gatcagaatc caccagatga acagcgagct gagcgtgctg 3780
gccaatttca gccagccaga gatcgtgccc atcagcaaca tcaccgagaa cgtgtacatc 3840
aacctgactt gcagcagcat ccacggctac cccgagccta agaagatgag cgtgctgctg 3900
cggaccaaga acagcaccat cgagtacgac ggcgtgatgc agaagagcca ggacaacgtg 3960
accgagctgt acgacgtgtc catcagcctg agcgtgtcct tcccagacgt gaccagcaac 4020
atgaccatct tctgcatcct ggagaccgac aagaccaggc tgctgtctag ccccttcagc 4080
atcgagctgg aagaccctca gcctcctcca gaccacatcc cttggatcac cgcagtgctg 4140
cccaccgtga tcatttgcgt gatggtgttt tgcctcatcc tctggaagtg gaagaagaag 4200
aagcggcccc ggaacagcta caagtgcggc accaacacca tggagcggga agagagcgag 4260
cagaccaaga agcgggagaa gatccacatc cccgagagaa gcgacgaggc tcagcgggtg 4320
ttcaagagca gcaagaccag cagctgcgac aagagcgaca cttgcttctg actgtgcctt 4380
ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 4440
ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 4500
gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga 4560
atagcaggca tgctgggga 4579
<210> 3
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<213> artificial sequence
<220>
<223> SEQ ID NO:3
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ctggacaacc ccaaagtacc ccgtctccct ggctttagcc acctctccat cctcttgctt 60
tctttgcctg gacaccccgt tctcctgtgg attcgggtca cctctcactc ctttcatttg 120
ggcagctccc ctacccccct tacctctcta gtctgtgcta gctcttccag ccccctgtca 180
tggcatcttc caggggtccg agagctcagc tagtcttctt cctccaaccc gggcccctat 240
gtccacttca ggacagcatg tttgctgcct ccagggatcc tgtgtccccg agctgggacc 300
accttatatt cccagggccg gttaatgtgg ctctggttct gggtactttt atctgtcccc 360
tccaccccac agtggggcca ctagggacag 390
<210> 4
<211> 294
<212> DNA
<213> artificial sequence
<220>
<223> SEQ ID NO:4
<400> 4
gattggtgac agaaaagccc catccttagg cctcctcctt cctagtctcc tgatattggg 60
tctaaccccc acctcctgtt aggcagattc cttatctggt gacacacccc catttcctgg 120
agccatctct ctccttgcca gaacctctaa ggtttgctta cgatggagcc agagaggatc 180
ctgggaggga gagcttggca gggggtggga gggaaggggg ggatgcgtga cctgcccggt 240
tctcagtggc caccctgcgc taccctctcc cagaacctga gctgctctga cgcg 294
<210> 5
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> SEQ ID NO:5
<400> 5
gcctcccctt cttgtaggcc 20
<210> 6
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> SEQ ID NO:6
<400> 6
agccaaagtt agaactcagg 20
<210> 7
<211> 65
<212> DNA
<213> artificial sequence
<220>
<223> SEQ ID NO:7
<400> 7
taatacgact cactataggg gggccactag ggacaggatt gttttagagc tagaaatagc 60
aagtt 65
<210> 8
<211> 75
<212> DNA
<213> artificial sequence
<220>
<223> SEQ ID NO:8
<400> 8
aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt 60
gctatttcta gctct 75
<210> 9
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> SEQ ID NO:9
<400> 9
cctgtgccat ctctcgtttc t 21
<210> 10
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> SEQ ID NO:10
<400> 10
gcctgggaat ccacatgagg 20
<210> 11
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> SEQ ID NO:11
<400> 11
aggaacctct agtggtgaag gt 22
<210> 12
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> SEQ ID NO:12
<400> 12
acgagaagaa ggtcatcagc a 21
<210> 13
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> SEQ ID NO:13
<400> 13
acacccgtct ggtttcacg 19
<210> 14
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> SEQ ID NO:14
<400> 14
caagaggaga agcagtttgg a 21
<210> 15
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> SEQ ID NO:15
<400> 15
ggtccgagag ctcagctagt 20
<210> 16
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> SEQ ID NO:16
<400> 16
acaggaggtg ggggttaga 19
Claims (5)
1. A gene editing system for site-directed insertion of a foreign gene, comprising Cas9, sgrnas and ssDNA, wherein the Cas9 and the sgrnas form an RNP complex, the sgrnas are insertion sites of the foreign gene, and the ssDNA is an insertion template of the foreign gene;
the exogenous gene is aAPC1, the ssDNA comprises aAPC1 and homologous arms arranged at two sides of the aAPC1, the structure of the aAPC1 is EF1a-CD19-CD86-CD64-PolyA, the homologous arms at two sides are LHA and RHA respectively, the LHA is connected with EF1a, and the RHA is connected with the PolyA;
the nucleotide sequence of the aAPC1 is shown as SEQ ID NO. 2; the nucleotide sequence of the LHA is shown as SEQ ID NO. 3; the nucleotide sequence of the RHA is shown as SEQ ID NO. 4;
the insertion site includes AAVS1.
2. The gene editing system for site-directed insertion of exogenous genes according to claim 1, wherein the nucleotide sequence of the sgRNA is as shown in SEQ ID No. 1 or a homologous sequence thereof.
3.A method for site-directed insertion of a foreign gene, comprising the steps of:
1) Selecting an insertion site according to an exogenous gene to be inserted, and designing and synthesizing sgRNA and ssDNA according to the insertion site;
2) Forming an RNP complex between the Cas9 and the sgRNA, and then forming a gene editing system for site-directed insertion of exogenous genes by using the RNP complex and the ssDNA;
3) Culturing K562 cells, and inserting the exogenous gene into the K562 cells through a gene editing system for site-specific insertion of the exogenous gene formed in the step 2) to obtain K562 cells with site-specific insertion of the exogenous gene;
the exogenous gene is aAPC1, the ssDNA comprises aAPC1 and homologous arms arranged at two sides of the aAPC1, the structure of the aAPC1 is EF1a-CD19-CD86-CD64-PolyA, the homologous arms at two sides are LHA and RHA respectively, the LHA is connected with EF1a, and the RHA is connected with the PolyA;
the nucleotide sequence of the aAPC1 is shown as SEQ ID NO. 2; the nucleotide sequence of the LHA is shown as SEQ ID NO. 3; the nucleotide sequence of the RHA is shown as SEQ ID NO. 4;
in step 1), the insertion site comprises AAVS1.
4. The method for site-directed insertion of exogenous genes according to claim 3, wherein the nucleotide sequence of the sgRNA is as shown in SEQ ID NO. 1 or a homologous sequence thereof.
5. The method for site-directed insertion of exogenous genes according to claim 3 or 4, wherein in step 3), the exogenous genes are inserted into the K562 cells by electrotransfection or lipofection.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107326046A (en) * | 2016-04-28 | 2017-11-07 | 上海邦耀生物科技有限公司 | A kind of method for improving foreign gene homologous recombination efficiency |
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| US9234213B2 (en) * | 2013-03-15 | 2016-01-12 | System Biosciences, Llc | Compositions and methods directed to CRISPR/Cas genomic engineering systems |
| CN108368502B (en) * | 2015-06-03 | 2022-03-18 | 内布拉斯加大学董事委员会 | DNA editing using single-stranded DNA |
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| CN107326046A (en) * | 2016-04-28 | 2017-11-07 | 上海邦耀生物科技有限公司 | A kind of method for improving foreign gene homologous recombination efficiency |
| CN111344020A (en) * | 2017-06-15 | 2020-06-26 | 加利福尼亚大学董事会 | Targeted non-viral DNA insertion |
| CN111263808A (en) * | 2017-09-20 | 2020-06-09 | 北京宇繁生物科技有限公司 | gRNA of target HPK1 and method for editing HPK1 gene |
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| CN110885799A (en) * | 2019-10-31 | 2020-03-17 | 南京北恒生物科技有限公司 | Gene editing method based on adenovirus |
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