Method for improving gene editing efficiency by using fusion protein

Abstract

The invention discloses a method for improving gene editing efficiency by using fusion protein, and belongs to the technical field of biological medicine. The invention provides a fusion protein, which is a product obtained by fusing a crispr-cas9 protein and a substrate protein of an APC / C compound. The invention also provides a gene editing method based on the CRISPR-Cas9 system, which comprises the following steps: constructing a Cas9 expression vector containing nucleic acid for coding the fusion protein, and co-transfecting the Cas9 expression vector and a gRNA expression vector to a target cell. According to the invention, the crispr protein and the substrate protein of the APC / C compound are fused, so that the crispr mainly plays a role in the G2 / S period, the efficiency of gene knock-in by a crispr method is further improved, and help is provided for application of the crispr in the field of cell therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for improving gene editing efficiency by utilizing fusion proteins. Background Art

[0002] Cell therapy, a treatment approach that primarily involves culturing cells in vitro, genetically editing them, expanding them, and then infusing them back into the patient, has made significant progress in treating cancer and genetic diseases, such as CAR-T cell therapy and stem cell transfusion for thalassemia. However, traditional methods for in vitro cell editing primarily rely on lentiviral infection, which has drawbacks such as random DNA integration (potentially leading to cell canceration) and strong immunogenicity (potentially leading to immune rejection in the re-transfused patient).

[0003] CRISPR, a newly discovered gene-editing tool, can specifically edit specific DNA sites. It also boasts higher editing efficiency than TALEN and ZFN, and is simpler to operate. Therefore, it holds great promise for application in cell therapy. Although CRISPR is highly efficient at gene knockout, it is less efficient at knocking in large fragments, increasing the difficulty and cost of screening for positive cells and significantly reducing its application in cell therapy. Although a variety of small molecules and fusion proteins exist that inhibit NHEJ or activate HR, in addition to DSBs caused by CRISPR, other DNA double-strand breaks (DSBs) can also be generated during cell culture. The presence of these small molecules and fusion proteins can disrupt the cell's normal repair pathways, resulting in a high probability of unnecessary, large-scale chromatin reorganizations, increasing the difficulty and cost of subsequent cell-related screening. Therefore, there is a need to develop a gene-editing tool that can both improve homologous recombination efficiency and not affect the cell's native DSB repair pathway. Summary of the Invention

[0004] In response to the above-mentioned defects in the prior art, in order to solve the problem that CRISPR gene editing tools improve the efficiency of homologous recombination while causing abnormalities in the normal repair pathway of cells, the purpose of the present invention is to design and provide a method for improving gene editing efficiency using fusion proteins.

[0005] The applicant found that the homologous recombination repair pathway only occurs in the G2 / S phase and is dominant, and the M phase becomes highly dense due to the chromosome structure, so it is impossible to effectively perform gene editing. Therefore, as long as the activity of CRISPR is restricted outside the G0 / G1 phase, the knock-in efficiency can be effectively improved, while not affecting the repair pathway in the cell itself, reducing the probability of abnormal mutations. The present invention enables CRISPR to function mainly in the G2 / S phase by fusing the CRISPR protein with another substrate protein of the APC / C complex, thereby improving the efficiency of CRISPR gene knock-in, thereby providing help for the application of CRISPR in the field of cell therapy.

[0006] In order to achieve the above beneficial effects, the present invention adopts the following technical solutions:

[0007] In one aspect, the present invention provides a fusion protein, which is the product of the fusion of the CRISPR-Cas9 protein and the substrate protein of the APC / C complex.

[0008] In the fusion protein, the substrate protein of the APC / C complex is APCS1 protein.

[0009] The fusion protein, wherein the APCS1 protein is the full-length or truncated APCS1 or an amino acid sequence having a sequence similarity of more than 70% thereto;

[0010] Preferably, the amino acid sequence of the APCS1 protein is shown in SEQ ID NO.1.

[0011] In a second aspect, the present invention provides a nucleic acid encoding any one of the fusion proteins.

[0012] In a third aspect, the present invention provides a Cas9 expression vector comprising the nucleic acid sequence of claim 4.

[0013] In a fourth aspect, the present invention provides a CRISPR-Cas9 system, comprising the fusion protein according to any one of claims 1 to 3 or the nucleic acid according to claim 4.

[0014] In a fifth aspect, the present invention provides a gene editing method based on the CRISPR-Cas9 system, constructing the Cas9 expression vector, and co-transfecting the Cas9 expression vector and the gRNA expression vector into the target cells.

[0015] In a sixth aspect, the present invention provides a delivery system comprising (1) a delivery vehicle; and (2) any one of the fusion proteins or the nucleic acid as described in claim 4.

[0016] In a seventh aspect, the present invention provides a pharmaceutical composition comprising any one of the fusion proteins, the nucleic acids, the CRISPR-Cas9 gene editing vectors, the CRISPR-Cas9 system, or the delivery systems.

[0017] In an eighth aspect, the present invention provides the use of the CRISPR-Cas9 system in gene editing;

[0018] Preferably, the gene editing comprises gene editing of immune cells in vitro, gene editing of pluripotent stem cells, or gene editing of primary re-infused cells.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention fuses a protein containing the substrate peptide of the APC / C complex with Cas9, which causes Cas9 to be degraded in the G0 / G1 phase and stably expressed only in the G2 / S phase, where HR is the main repair pathway, thereby improving the gene knock-in efficiency and reducing the risk of abnormal mutations.

[0021] Because the fusion protein is small, less than 100 residues, the fusion of the substrate protein of the APC / C complex and the CRISPR-Cas9 protein does not significantly increase the protein length, and has almost no negative impact on delivery; at the same time, it does not affect the main DSB repair pathway, reducing the probability of abnormal mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the structure diagram of the APCS1-cas9 fusion protein;

[0023] Figure 2 This is the result of flow cytometry detection of 293T knock-in GFP efficiency;

[0024] Figure 3 This is the result of flow cytometry detection of protein cell cycle-dependent expression;

[0025] Figure 4 Schematic diagram of Cas9 post-translational regulation in G1 to enhance homology-directed repair. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to the accompanying drawings and examples.

[0027] Example 1:

[0028] (1) Construction of APCS1-cas9 fusion plasmid by conventional molecular cloning

[0029] APCS1 protein (amino acid sequence is shown in SEQ ID NO. 1), and its structure is shown in Figure 1 Shown: LKHNPSQRAPLGDVSNREVLEHPWITANSSKPSNCQNKESASKQSGGGGSGGGGSGGGGS.

[0030] (2) 293T knock-in experiment: Co-transfect cas9(-APCS1)-gRNA plasmid + donor plasmid (containing green fluorescent protein tag) into 293T cells. The specific steps are as follows:

[0031] Resuscitate 293T cells in a 6-well plate, culture with DMEM complete medium (DMEM + 10% FBS), and passage for more than one generation. Aspirate the cell culture supernatant, add 1 mL of D-PBS to wash the remaining medium, and aspirate and discard. Add 1 mL of 0.25% trypsin, shake the culture flask to evenly distribute the digestion solution on the bottom of the plate, and place the culture flask in a 37°C incubator and incubate for 3 minutes. After digestion, add 1 mL of DMEM complete medium to each culture flask to terminate the digestion. Centrifuge at 300g for 5 minutes, add 1 mL of DMEM complete medium to resuspend and count. Inoculate 2E5 cells into a 6-well plate, add DMEM complete medium to 2 mL, culture overnight, and transfect when the cells grow to 40-50% of their volume. Add 1ug of Cas9 plasmid and 1ug of donor plasmid to DMEM to make the volume 100uL and incubate for 5 minutes. Add 4ug of PEI to DMEM to make the volume 100uL and incubate for 5 minutes. After incubation, mix the PEI and plasmid and incubate for 15 minutes. Aspirate the cell culture supernatant, resuspend the PEI and plasmid mixture in DMEM complete medium, add to the cells, and adjust the volume to 2 mL. Culture the cells daily. Replace the DMEM complete medium daily for expansion. On Day 7, assess knock-in efficiency: harvest the cells at 500 g, wash the cells with PBS at 500 g for 5 minutes, and assess knock-in efficiency and fluorescent protein expression efficiency.

[0032] The results are as follows Figure 2 As shown, WT-cas9 is the original cas9 protein, blank is the untransfected blank cell, and APCS1-cas9 is the fusion protein designed in the above step (1). Figure 2 The bar graph shows the knock-in efficiency of different fusion proteins calculated using WT-cas9 as unit 1. It can be seen that the fusion protein designed using the above step (1) can significantly improve the knock-in efficiency.

[0033] Example 2:

[0034] Cas9 protein degradation experimental system:

[0035] The transfection step was the same as in Example 1 above. Cas9 and APCS1-cas9 plasmids were transfected into 2 wells of 293T cells, respectively, and divided into two groups (WT group, i.e., transfection cas9 group, APCS1 group, i.e., transfection APCS1-cas9 plasmid group). The culture medium was changed 24 hours after transfection. One group added Nocotazole (200 ng / mL) to complete culture medium, and one group replaced the culture medium normally. After 24 hours, cells were collected (collection step was the same as before) for flow cytometry; after enrichment of the cells, 4% PFA (paraformaldehyde) solution was used for fixation at room temperature in the dark for 15 minutes, centrifuged at 4°C 861 × g for 5 minutes, the supernatant was discarded, 1 × permeabilization agent was added to each tube, and the cells were incubated at 4°C for 10 minutes; centrifuged at 4°C 861 × g for 5 minutes, the cells were washed with PBS, fluorescent antibodies were added, and incubated at room temperature for 30 minutes; centrifuged at 861 × g for 5 minutes, and the cells were washed with PBS; the resuspended cells were used for flow cytometry to detect the efficiency of cas9 protein degradation.

[0036] like Figure 3 As shown in the figure, in samples covering all cell cycles, the expression level of APCS1 was significantly lower than that of WT, while in the M phase, the expression levels of WT and APCS1 were basically the same, which proves that APCS1 is significantly degraded in the G0 / G1 phase. Figure 4 Enhanced homology-directed repair for post-translational regulation of Cas9 in G1.

Claims

1. A fusion protein, characterized in that The fusion protein is the product of the fusion of the CRISPR-Cas9 protein and the substrate protein of the APC / C complex.

2. A fusion protein according to claim 1, characterized in that The substrate protein of the APC / C complex is APCS1 protein.

3. A fusion protein according to claim 2, characterized in that The APCS1 protein is the full-length or truncated APCS1 or an amino acid sequence having a sequence similarity of more than 70% thereto; Preferably, the amino acid sequence of the APCS1 protein is shown in SEQ ID NO.

1.

4. A nucleic acid, characterized in that The nucleic acid encodes the fusion protein according to any one of claims 1 to 3.

5. A Cas9 expression vector, characterized in that: The Cas9 expression vector comprises the nucleic acid sequence according to claim 4.

6. A CRISPR-Cas9 system, characterized in that The CRISPR-Cas9 system comprises the fusion protein of any one of claims 1 to 3 or the nucleic acid of claim 4.

7. A gene editing method based on the CRISPR-Cas9 system, characterized in that: Construct the Cas9 expression vector as described in claim 6, and co-transfect the Cas9 expression vector and the gRNA expression vector into the target cells.

8. A delivery system, characterized in that Comprising (1) a delivery vehicle; (2) the fusion protein according to any one of claims 1 to 3 or the nucleic acid according to claim 4.

9. A pharmaceutical composition, characterized in that Comprising the fusion protein according to any one of claims 1 to 3, the nucleic acid according to claim 4, the CRISPR-Cas9 gene editing vector according to claim 5, The CRISPR-Cas9 system of claim 6 or the delivery system of claim 8.

10. Use of the CRISPR-Cas9 system according to claim 6 in gene editing; Preferably, the gene editing comprises gene editing of immune cells in vitro, gene editing of pluripotent stem cells, or gene editing of primary re-infused cells.

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