A method of performing gene editing in primary cells

Abstract

The application discloses a method for gene editing in primary cells, and belongs to the technical field of gene editing. The method mixes a Cas9 protein, an sgRNA and a PRD protein, and then transfects the primary cells, so that gene editing of the primary cells is quickly completed. By using the method, long-time pre-assembly is not needed, and the whole assembly process can be completed within 1 minute, so that the delivery efficiency is remarkably improved. By using the method, efficient editing of a target gene can be completed, and the method has important application value in the fields of gene function research and gene therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene editing, in particular to a method for performing gene editing in primary cells. BACKGROUND

[0002] In cancer and autoimmune diseases, killer T cells play a crucial role. With the in-depth research and development of technology, CAR-T therapy, which modifies patient T cells through genetic engineering means to treat diseases, has shown great advantages in the treatment of hematological malignancies, but most of them are still in the clinical research stage. The research on T cell functional genes still has a long way to go. Screening target genes that affect genome expression through functional genes is the basis for treating diseases with genetic engineering methods.

[0003] The CRISPR-Cas9 gene editing system is a revolutionary technology in the fields of scientific research, biotechnology, agriculture and medicine. As a molecular scissors, Cas9 protein induces targeted DNA double-strand breaks at the site specified by the guide RNA (gRNA) spacer sequence, thereby creating an opportunity to modify gene sequences through DNA repair pathways, which can be used to study and screen functional genes.

[0004] Cas9 protein and gRNA can be synthesized and verified in advance and stably stored in a refrigerator at -80℃ for genome editing on demand. Delivering ribonucleoprotein complexes (RNP) pre-assembled by Cas9 protein and single guide RNA (sgRNA) for genome editing is a more and more popular method, which is suitable for cell types that are difficult to be genetically manipulated by traditional plasmid and viral methods.

[0005] However, when delivering RNP into primary cells for gene editing, there are the following main disadvantages: low delivery efficiency, causing great damage to cells, and the need for pre-assembly of RNP to achieve efficient and specific gene editing. SUMMARY

[0006] In order to solve at least one of the above technical problems, the present application aims to provide a method for efficiently delivering RNP to primary cells for gene editing, which can achieve efficient delivery and efficient gene editing without damaging cells as much as possible.

[0007] To achieve this object, the present application provides a method for performing gene editing in primary cells, comprising the following steps:

[0008] S1, configure a transfection mixture:

[0009] S11, adding Cas9 protein with a final concentration of 0.1 μM to 0.15 μM and sgRNA with a final concentration of 0.2 μM to 0.3 μM to a 5% to 20% w / v PEG8000 solution, respectively, wherein the sgRNA targets the target gene;

[0010] S12, adding PRD protein at a final concentration of 5 μM to 20 μM and mixing, wherein the amino acid sequence of the PRD protein is shown in SEQ ID No. 2;

[0011] S13, adding a polyol to a final concentration of 5% to 15% w / v to obtain the transfection mixture;

[0012] S2, the transfection mixture was mixed with 0.3 to 0.5 times the volume of the primary cell suspension to obtain a transfection system, and the mixture was incubated at 35 to 39°C for 10 min to 3 h, wherein the cell density in the primary cell suspension was 5×10 6 ~1×10 7 / mL;

[0013] S3, the transfection system is mixed with 4 to 6 times the volume of the transfection system culture medium, the supernatant is removed by centrifugation, and then 3 to 6 times the volume of the transfection system culture medium is added for culturing, thereby completing the editing of the target gene.

[0014] CRISPR-Cas9 gene editing components can be delivered to target cells via a variety of methods, including viral and non-viral delivery. Those skilled in the art recognize that using RNPs is the most efficient non-viral method for delivering CRISPR components. Because transcription and translation are not required, the Cas9 protein and guide RNA (gRNA) can enter the cell nucleus and immediately initiate gene editing. Furthermore, this method eliminates the risk of exogenous gene integration into the host genome, and the expression of CRISPR components is transient, minimizing off-target effects. Single guide RNA (sgRNA) is a short RNA (typically approximately 100 base pairs) that is a crucial component of the CRISPR-Cas9 gene editing system for both knockout and knock-in applications. The sgRNA serves as the "command center" of the CRISPR-Cas9 gene editing system. The approximately 20-base sequence within the sgRNA guides the Cas9 protein for site-specific editing of the target gene's DNA. Those skilled in the art can design the sgRNA using conventional methods based on the DNA sequence of the target gene to be edited.

[0015] In this application, the final concentration refers to the concentration of a substance in a solution after the substance is added to the solution, and is intended to be used to determine the amount of the substance added. When other substances are added, the concentration of the substance will further change and does not need to be considered.

[0016] In the present application, the inventors have unexpectedly found that when PEG8000 is used, the gene editing efficiency is significantly higher than that of other PEGs with different molecular weights, such as PEG200, PEG600, PEG1000, PEG3350 and PEG20000.

[0017] It is important to note that in the method of the present application, the Cas9 and sgRNA do not need to be pre-assembled for a long time, but can be pre-assembled within 1 min, and the other prerequisite is the presence of PRD protein. The PRD protein is a protein for delivery, which can efficiently deliver RNP.

[0018] In the present application, PEG8000 and PRD protein must be used together and cannot be missing.

[0019] In the present application, the polyol is a transfection-promoting agent. In some embodiments of the present application, the polyol is selected from one or more of glycerol, sucrose and trehalose.

[0020] The cells harvested and inoculated for the first time are called primary cells. In some embodiments of the present application, the primary cells are primary immune cells. In some embodiments of the present application, the primary immune cells are primary T cells.

[0021] In some specific embodiments of the present application, the primary T cells are human primary T cells, in which case the polyol is glycerol, and accordingly, in step S13, glycerol is added at a final concentration of 10% ~ 15% w / v.

[0022] Further, the target gene is human TRAC gene, the sequence of the sgRNA is shown in SEQ ID No. 1. TRAC refers to T cell receptor alpha constant region, and after TRAC knocking out the gene, it means that the T cell receptor (TCR) on the surface of T cells is removed, avoiding the occurrence of graft versus host reaction (GVHD).

[0023] In other specific embodiments of the present application, the primary T cells are mouse primary T cells, in which case the polyol is trehalose, and accordingly, in step S13, trehalose is added at a final concentration of 5% ~ 10% w / v.

[0024] Further, the target gene is mouse Trac gene, the sequence of the sgRNA is shown in SEQ ID No. 5.

[0025] In some embodiments of the present application, step S12 is performed within 1 minute after step S11 of adding the Cas9 protein and the sgRNA and mixing.

[0026] In some embodiments of the present application, in step S2, the transfection system is incubated at 37°C for 10-30 minutes.

[0027] In some embodiments of the present application, in step S3, the culture medium is complete medium.

[0028] In some embodiments of the present application, the method further comprises a step of evaluating the gene editing efficiency.

[0029] In some embodiments of the present application, the detection is performed by flow cytometry, specifically:

[0030] About 1x10 6 cells are collected, 1 mL of PBS is added, centrifugation is performed at 300 g for 5 minutes, the supernatant is removed, the cell precipitate is collected, 50 μL of an antibody of a protein expressed or regulated by the target gene is added, incubation is performed at room temperature for 15 minutes, 1 mL of PBS is added, centrifugation is performed at 300 g for 5 minutes, the supernatant is removed, and flow cytometry is performed after resuspending the cells with 50 μL of PBS.

[0031] In some embodiments of the present application, the detection is performed by sequencing. Specifically:

[0032] About 1x10 5 cells are collected, centrifugation is performed at 300 g for 5 minutes, the supernatant is removed, DNA is extracted from the cells, PCR amplification is performed on the target site, the PCR amplification product is sequenced, and the sequencing results are analyzed.

[0033] In some embodiments of the present application, the primary cells are human primary T cells, the target gene is a human TRAC gene, and the primer pair for PCR amplification is shown in SEQ ID No. 3 and SEQ ID No. 4.

[0034] In some embodiments of the present application, the primary cells are mouse primary T cells, the target gene is a mouse Trac gene, and the primer pair for PCR amplification is shown in SEQ ID No. 6 and SEQ ID No. 7.

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] In the prior art, the RNP transfection method usually requires a pre-assembly time of at least 10 min, while using the method of the application, no long pre-assembly is required, only the Cas9 protein, sgRNA and PRD protein are mixed, and the whole assembly process can be completed within 1 min, which significantly shortens the experimental time for gene editing and improves the delivery efficiency.

[0037] Using the method of the application, the RNP can be efficiently delivered into primary cells while being efficiently released in the cells, ensuring the accuracy and success rate of gene editing.

[0038] Using the method of the application, the gene editing is efficiently completed while the damage to the cells is significantly reduced, which helps to maintain the activity and function of the cells, thereby improving the overall effect of gene editing.

[0039] In summary, using the method of the application, the Cas9 protein and sgRNA can be efficiently, low-toxicity and conveniently delivered into and released from primary cells to accurately complete gene editing and achieve genome modification. Therefore, the method of the application can be used for gene function research, and the gene function and disease mechanism can be explored by editing primary cells. In addition, the method of the application can also play an important role in animal model research, which can efficiently edit mouse primary cells to meet the research needs of CAR-T cell therapy and autoimmune diseases.

[0040] It should be understood that the content described in this section is not intended to identify key or important features of the embodiments of the application, nor is it used to limit the scope of the application. Other features of the application will become apparent from the following description. BRIEF DESCRIPTION OF DRAWINGS

[0041] The above and other objects, features and advantages of the exemplary embodiments of the present application will be more apparent from the following detailed description read in conjunction with the accompanying drawings, in which:

[0042] Figure 1 shows the flow detection results after knocking out the gene in human primary T cells in Example 1 of the application; TRAC

[0043] Figure 2 shows the flow detection results after knocking out the gene in human primary T cells in Example 1 of the application; TRAC

[0044] Figure 3 shows the flow detection results after knocking out the gene in mouse primary T cells in Example 2 of the application; Trac

[0045] ​​​Figure 4 Sequencing analysis results after knockout of genes in mouse primary T cells in Example 2 of the present application are shown. Trac Sequencing analysis results after knockout of genes in mouse primary T cells in Example 2 of the present application are shown.

[0046] Figure 5 Sequencing analysis results after knockout of genes in mouse primary T cells in Example 2 of the present application are shown.

[0047] Figure 6 Effect of non-preassembly and different preassembly times on gene editing efficiency of human primary T cells in Example 3 of the present application is shown.

[0048] Figure 7 Effect of non-preassembly and different preassembly times on gene editing efficiency of mouse primary T cells in Example 3 of the present application is shown.

[0049] Figure 8 Effect of PRD protein input on gene editing efficiency of human primary T cells in Example 4 of the present application is shown.

[0050] Figure 9 Effect of PEG of different molecular weights on gene editing efficiency of human primary T cells in Example 5 of the present application is shown.

[0051] Figure 10 Effect of PEG8000 concentration on gene editing efficiency of human primary T cells in Example 6 of the present application is shown.

[0052] Figure 11 Effect of addition of PRD protein and different proteins on gene editing efficiency of human primary T cells in Example 7 of the present application is shown.

[0053] Figure 12 Effect of Cas9 protein and sgRNA input on gene editing efficiency of human primary T cells in Example 8 of the present application is shown.

[0054] Figure 13 Effect of different polyols on gene editing efficiency of human primary T cells in Example 9 of the present application is shown.

[0055] Figure 14 Effect of different polyols on gene editing efficiency of mouse primary T cells in Example 10 of the present application is shown. DETAILED DESCRIPTION

[0056] In order to make the technical problems, technical solutions and beneficial effects solved by the present application clearer, the present application will be further described in detail below in combination with examples.

[0057] The following examples are put forth so as to demonstrate preferred embodiments of the application. Those of ordinary skill in the art will realize that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the application. However, those of ordinary skill in the art will further appreciate that many of the techniques disclosed in the following examples have suitable alternatives, which have been developed by others and yet are within the spirit and scope of the application. Such alternatives have been developed by others, and can be determined by those of ordinary skill in the art from the detailed description, such as by using logical substitutions, equivalents, and the like. Although the examples provided herein are primarily for use with human cells, the skilled artisan will appreciate that the methods described herein can be used with other cells, such as cells from other mammals, including primates, rodents, and the like.

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the meaning ascribed to in the materials incorporated herein by reference. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The materials, methods, and examples provided herein are illustrative only and not intended to be limiting.

[0059] Those of ordinary skill in the art will realize and appreciate that many embodiments of the application can be adapted to different uses, and of the same or similar nature, without departing from the spirit and scope of the application.

[0060] The experimental methods in the following examples are routine unless otherwise specified. The instruments and equipment used in the following examples are routine laboratory instruments and equipment unless otherwise specified. The test materials used in the following examples are commercially available from routine biochemical reagent stores unless otherwise specified.

[0061] Example 1 Gene editing in human primary T cells

[0062] 1. Isolation and activation of human primary T cells

[0063] Whole blood was drawn from healthy human donors and peripheral blood mononuclear cells (PBMCs) were isolated using the cell isolation and activation kit Dynabeads™ Human T-Expander CD3 / CD28 (Thermo Fisher, 11141D). Cells were placed in culture medium containing 300 IU / mL IL2 and incubated in a 37°C incubator for 2-10 days before transfection.

[0064] 2. Transfection system setup

[0065] To 40 μΐ of 10% (w / v) PEG8000 solution, 5 pmol (about 0.8 μg) of Cas9 protein (Coast Protein, E365-01B) and 10 pmol (about 0.3 μg) of sgRNA were added and mixed well. In this example, the sgRNA targets the human CCR5 gene, the sequence of which is as follows (SEQ ID No. 1): TRAC

[0066] ​UGUGCUAGACAUGAGGUCUAGUUUUAGAGCUAUGCGGUAAACAGCAUAGCAAGUUUAAAUAGGCUAGUCCGUAAUCAACUUGAAAAAGUGGCAUCCGAUCGGUGCUUUUUUU

[0067] To the above mixture, 0.7 μΐ of 286 μΜ PRD protein solution was added. The amino acid sequence of the PRD protein is as follows (SEQ ID No. 2):

[0068] MTAAPASPQQIRDRLLQAIDPQSNIRNMVAVLEVISSLEKYPITKEALEETRLGKLINDVRKKTKNEELAKRAKKLLRSWQKLIEPAHQHEAALRGLAGATGSANGGAHNCRPEVGAAGPPRSIHDLKSRNDLQRLPGQRLDRLGSRKRRGDQRDLGHPGPPPKVSKASHDPLVPNSSPLPTNGISGSPESFASSLDGSGHAGPEGSRLERDENDKHSGKIPVNAVRPHTSSPGLGKPPGPCLQPKASVLQQLDRVDETPGPPHPKGPPRCSFSPRNSRHEGSFARQQSLYAPKGSVPSPSPRPQALDATQVPSPLPLAQPSTPPVRRLELLPSAESPVCWLEQPESHQRLAGPGCKAGLSPAEPLLSRAGFSPDSSKADSDAASSGGSDSKKKKRYRPRDYTVNLDGQVAEAGVKPVRLKERKLTFDPMTRQIKPLTQKEPVRADSPVHMEQQSRTELDKQEAKASLQSPFEQTNWKELSRNEIIQSYLSRQSSLLSSSGAQTPGAHHFMSEYLKQEESTRQGARQLHVLVPQSPPTDLPGLTREVTQDDLDRIQASQWPGVNGCQDTQGNWYDWTQCISLDPHGDDGRLNILPYVCLD

[0069] After the addition of the PRD protein solution, the tip was pipetted up and down 20-30 times or vortexed for 10 sec to mix thoroughly. Then 13.5 μΐ of glycerol solution (60%, w / v) was added and mixed to obtain the transfection mixture.

[0070] 3. Transfection of human primary T cells

[0071] Take the human primary T cells to be transfected, centrifuge at 300 g for 5 min, remove the supernatant, collect the cell precipitate, add Opti-MEM (preheat or restore to room temperature in advance) to resuspend and wash once, and adjust the cell suspension to 5x10 6 6 cells / mL for standby. 7

[0072] Mix the transfection mixture prepared in step 2 of this example with 20 μL of cell suspension and incubate directly in a centrifuge tube. After incubation in a 37°C incubator for 15 min, add about 5 times the volume of the transfection system (250 μL) of complete culture medium, centrifuge at 300 g for 5 min, and discard the supernatant. Add 200 μL of complete culture medium to the precipitate and culture the cells, and after 72 h, analyze the editing efficiency of the target.

[0073] 4. Detection of gene editing efficiency of human primary T cells

[0074] (1) Flow detection

[0075] Collect about 1x10 6 6 cells, add 1 mL of PBS, centrifuge at 300 g for 5 min, remove the supernatant, collect the cell precipitate, add 50 μL of prepared CD3-TCR antibody (Biogend, Cat.300318, 1:100 with PBS), incubate at room temperature for 15 min, add 1 mL of PBS, centrifuge at 300 g for 5 min, remove the supernatant, and resuspend the cells with 50 μL of PBS before flow detection. Use untransfected cells as a control group.

[0076] The flow detection results are shown in Figure 1 , and it can be seen from Figure 1 that about 81.2% of the T cells in the transfection group are CD3 negative, indicating that the genes of about 81% of the cells in the total cell population have been successfully edited. TRAC

[0077] (2) Sequencing detection

[0078] Collect about 1x10 5 6 cells, centrifuge at 300 g for 5 min, remove the supernatant, collect the cell precipitate, and extract DNA for PCR amplification of the target position:

[0079] Forward primer sequence: GCAGTATTATTAAGTAGCCCT (SEQ ID No. 3)

[0080] Reverse primer sequence: AACAAGGCTCACTGTTTCTT (SEQ ID No. 4)

[0081] ​​PCR amplification products were sequenced by Sanger, and the sequencing results were analyzed by the TIDE (Tracking of Indels by DEcomposition) website.

[0082] The TIDE analysis results are shown in Figure 2 , which show that the proportion of target gene edited in the transfection group is about 73.9%, which is equivalent to the knock-out result of protein level detected by flow cytometry (the proportion of CD3-TCR flow cytometry negative).

[0083] The above results show that the above method can efficiently deliver RNP to human primary T cells and efficiently edit the target gene.

[0084] Example 2 Gene editing in mouse primary T cells

[0085] 1. Isolation and activation of mouse primary T cells

[0086] Antibody coating: After washing twice with PBS, add 400-500 μL of antibody (anti-mouse CD3, InvivoMab, BE0002; anti-mouse CD28 Invivo Mab, BE0015-1, diluted 1:1000 in PBS) per well in a 24-well plate, and incubate at 37°C for 5 h.

[0087] The sorted T cells in the mouse spleen (isolation kit: EasySep™ Mouse T Cell Isolation Kit, Stem Cell, 19851) were resuspended with complete culture medium, and the cell density was adjusted to 2×10 6 Take out the coated 24-well plate, discard the coating solution, and add 1 mL of T cell suspension per well, and continue to culture to fully activate the T cells.

[0088] 2. Configuration of transfection system

[0089] Add 5 pmol (about 0.8 μg) Cas9 protein (coastal protein, E365-01B) and 10 pmol (about 0.3 μg) sgRNA in 40 μL of 10% (w / v) PEG8000 solution, and mix well.

[0090] In this example, the sgRNA used targets the mouse Trac gene, the sequence of which is as follows (SEQ ID No. 5):

[0091] UAUGGAUUCCAAGAGCAAUGGUUUUAGAGCUAUGCGGUAAACAGCAUAGCAAGUUUAAAUAGGCUAGUCCGUAAUCAACUUGAAAAAGUGGCAUCCGAUCGGUGCUUUUUUU

[0092] Add 0.7 μL of 286 μM PRD protein solution to the mixture, and mix well by pipetting up and down 20-30 times or vortexing for 10 sec. Add 10 μL of trehalose solution (25%, w / v) and mix well to obtain the transfection mixture.

[0093] 3. Transfection of mouse primary T cells

[0094] Take the mouse primary T cells to be transfected, centrifuge at 300 g for 5 min, remove the supernatant, collect the cell pellet, resuspend and wash once with Opti-MEM (preheated or restored to room temperature), and adjust the cell suspension to 5×10 6 ~1×10 7 / mL for standby.

[0095] Mix the transfection mixture prepared in Step 2 of this example with 20 μL of cell suspension uniformly, and incubate directly in a centrifuge tube. After incubation in a 37°C incubator for 15 min, add about 5 times the volume of the transfection system (250 μL) of complete culture medium, centrifuge at 300 g for 5 min, and discard the supernatant. Add 200 μL of complete culture medium for cell culture, and analyze the editing efficiency of the target after 72 h.

[0096] 4. Detection of gene editing efficiency of mouse primary T cells

[0097] (1) Flow cytometry detection

[0098] Collect about 1×10 6 cells, add 1 mL of PBS, centrifuge at 300 g for 5 min, remove the supernatant, collect the cell pellet, add 50 μL of prepared CD3-TCR antibody (Biogend, Cat. 100236, 1:100 in PBS), incubate at room temperature for 15 min, add 1 mL of PBS, centrifuge at 300 g for 5 min, remove the supernatant, resuspend the cells with 50 μL of PBS, and then perform flow cytometry detection, with untransfected cells as the control group.

[0099] The flow cytometry detection results are shown in Figure 3 From Figure 3 it can be seen that about 93% of the T cells in the transfection group are CD3 negative, indicating that the genes of about 93% of the cells in the total cell population have been successfully edited. Trac ​

[0100] (2) Sequencing detection

[0101] About 1 x 10 5 Cells were collected, centrifuged at 300 g for 5 min, the supernatant was removed, and the cells were collected to extract DNA, and the target site was subjected to PCR amplification:

[0102] Forward primer sequence: CACTGGCATCTGAGTTCTGA (SEQ ID No. 6)

[0103] Reverse primer sequence: AACAAGGCTCACTGTTTCTT (SEQ ID No. 7)

[0104] The PCR amplification product was subjected to Sanger sequencing, and the sequencing results were analyzed by the TIDE (Tracking of Indels by DEcomposition) website.

[0105] The TIDE analysis results are shown in Figure 4 , which shows that the proportion of the target gene edited in the transfection group is about 83.5%, which is equivalent to the knock-out result of the protein level (CD3-TCR flow cytometry negative proportion).

[0106] The above results show that the above method can efficiently deliver RNP to mouse primary T cells and efficiently edit the target gene.

[0107] Further, in order to observe the effect of gene editing on T cell proliferation, the inventors counted the number of cells after 72 h of culture of the transfection group and the control group, and the results are shown in Figure 5 . It is shown that after transfection of RNP into mouse primary T cells by the method, the proliferation of T cells is not affected.

[0108] Example 3 Effect of RNP assembly time on gene editing efficiency of human primary T cells

[0109] The isolation and activation of human and mouse primary T cells were performed according to Example 1 and Example 2, respectively.

[0110] Pre-assembly of the transfection system: 5 pmol (about 0.8 μg) Cas9 protein and 10 pmol (about 0.3 μg) sgRNA were added to 40 μL 10% (w / v) PEG8000 solution and mixed well, and then pre-assembled at 37°C for 10 min, 20 min and 40 min, respectively, followed by the addition of 0.7 μL 286 μM PRD protein solution and 13.5 μL glycerol solution (human primary T cells) or 10 μL trehalose solution (mouse primary T cells), mixed well and then transfected; another group was not pre-assembled, and 0.7 μL 286 μM PRD protein solution and 13.5 μL glycerol solution (human primary T cells) or 10 μL trehalose solution (mouse primary T cells) were directly added, mixed well and then transfected to obtain the transfection mixture. The untransfected cells were used as the control group.

[0111] The transfection operation and detection operation were the same as in Examples 1 and 2.

[0112] The effects of no pre-assembly and different pre-assembly times on the gene editing efficiency of human primary T cells are shown in Table 1. Figure 6 As shown in Table 1, the editing efficiency of the no pre-assembly group was the highest in human primary T cells, with an average of about 67.6%. This indicates that the method of the present application can achieve high-efficiency gene editing without pre-assembly when transfecting human primary T cells.

[0113] The effects of no pre-assembly and different pre-assembly times on the gene editing efficiency of mouse primary T cells are shown in Table 2. Figure 7 As shown in Table 2, the editing efficiency of the no pre-assembly group was the highest in mouse primary T cells, with an average of about 91.2%. This indicates that the method of the present application can achieve high-efficiency gene editing without pre-assembly when transfecting mouse primary T cells.

[0114] The results of this example show that, using the method of the present application, Cas9 protein and sgRNA can be directly transfected without pre-assembly, and the gene editing efficiency is significantly improved.

[0115] Example 4: Effect of PRD protein input amount on gene editing efficiency of primary T cells

[0116] In this example, human primary T cells were used as an example to explore the effect of the input amount of PRD protein on the gene editing efficiency of primary T cells.

[0117] The isolation and activation of human primary T cells were the same as in Example 1.

[0118] Preparation of the transfection system: 5 pmol (about 0.8 μg) Cas9 protein (coastal protein, E365-01B) and 10 pmol (about 0.3 μg) sgRNA (the sequence was the same as in Example 1) were added to 40 μL 10% (w / v) PEG8000 solution and mixed well.

[0119] In different groups, 0.14 μL, 0.35 μL, 0.7 μL, 1.05 μL, 1.4 μL, 2 μL, 2.8 μL of 286 μM PRD protein solution was added to the above mixture, respectively, corresponding to the protein concentration of 1 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 15 μM, 20 μM, respectively, and the mixture was fully mixed by blowing 20-30 times on the gun head or vortexing for 10 sec. Then 13.5 μL of glycerol solution (60%, w / v) was added and mixed to obtain the transfection mixture.

[0120] Human primary T cell transfection and gene editing efficiency flow cytometry detection were the same as in Example 1. The results are shown in Figure 8 It can be seen from Figure 8 that when the amount of PRD protein is below 5 μM, the gene editing efficiency is low; when the amount of PRD protein is 5 μM and above, the gene editing effect is high. This shows that the input of 5 μM PRD protein can achieve efficient gene editing.

[0121] Example 5 Effect of PEG of different molecular weights on gene editing efficiency of primary T cells

[0122] In this example, human primary T cells were used as an example to explore the effect of PEG of different molecular weights on the gene editing efficiency of primary T cells.

[0123] The isolation and activation of human primary T cells were the same as in Example 1.

[0124] Transfection system configuration: 5 pmol (about 0.8 μg) of Cas9 protein (coastal protein, E365-01B) and 10 pmol (about 0.3 μg) of sgRNA (the sequence is the same as in Example 1) were added to 40 μL of 10% (w / v) PEG200, PEG600, PEG1000, PEG3350, PEG8000, PEG20000 solution, respectively, and mixed thoroughly. 0.7 μL of 286 μM PRD protein solution was added to each of the above mixtures, and the mixture was fully mixed by blowing 20-30 times on the gun head or vortexing for 10 sec. Then 13.5 μL of glycerol solution (60%, w / v) was added and mixed to obtain the transfection mixture.

[0125] Human primary T cell transfection and gene editing efficiency flow cytometry detection were the same as in Example 1. The results are shown in Figure 9 It can be seen from Figure 9 that the editing efficiency of the PEG8000 group was significantly higher than that of the other molecular weight PEG solutions. This shows that the configuration of the transfection system in the PEG8000 solution can achieve efficient gene editing.

[0126] Example 6 Effect of PEG8000 concentration on gene editing efficiency of primary T cells

[0127] This example takes human primary T cells as an example to explore the effect of the added amount of PEG8000 concentration on the gene editing efficiency of primary T cells.

[0128] The isolation and activation of human primary T cells are the same as in Example 1.

[0129] Transfection system configuration: 5 pmol (about 0.8 μg) of Cas9 protein (Coast Protein, E365-01B) and 10 pmol (about 0.3 μg) of sgRNA (the sequence is the same as in Example 1) were added to 40 μL of PEG8000 solution with concentrations of 2.5%, 5%, 10%, 15%, and 20% (w / v), respectively, and mixed thoroughly.

[0130] In different groups, 0.7 μL of 286 μM PRD protein solution was added to the above mixture, and the gun head was blown up and down for 20-30 times or vortexed for 10 sec to mix thoroughly. Then 13.5 μL of glycerol solution (60%, w / v) was added and mixed to obtain the transfection mixture.

[0131] The transfection of human primary T cells and the flow cytometry detection of gene editing efficiency are the same as in Example 1. The results are shown in Figure 10 It can be seen from Figure 10 that the effect of PEG8000 concentration on gene editing efficiency shows a trend of increasing with increasing concentration, and then decreasing after reaching a certain concentration (10%, w / v). This indicates that the transfection system configured in 10% PEG8000 solution can achieve efficient gene editing.

[0132] Example 7 Effect of PRD protein on RNP-mediated gene editing efficiency in primary T cells

[0133] This example takes human primary T cells as an example to explore the effect of adding PRD protein, not adding PRD protein, and adding other proteins on the gene editing efficiency of primary T cells.

[0134] The isolation and activation of human primary T cells are the same as in Example 1.

[0135] Transfection system configuration: 5 pmol (about 0.8 μg) of Cas9 protein (Coast Protein, E365-01B) and 10 pmol (about 0.3 μg) of sgRNA (the sequence is the same as in Example 1) were added to 40 μL of PEG8000 solution with concentrations of 2.5%, 5%, 10%, 15%, and 20% (w / v), respectively, and mixed thoroughly.

[0136] Three groups of experimental groups were set up respectively, no protein was added in the first group, 0.6 μL of GFP protein (370 μM, Meibios, GS11110) was added to the above mixture in the second group, and 0.7 μL of PRD protein solution (286 μM) was added to the above mixture in the third group.

[0137] The gun head was blown up and down for 20-30 times or vortexed for 10 sec to mix thoroughly. Then 13.5 μL of glycerol solution (60%, w / v) was added and mixed to obtain the transfection mixture.

[0138] The human primary T cells were transfected and the gene editing efficiency was detected by flow cytometry, as in Example 1. The results are shown in Figure 11 From Figure 11 it can be seen that the RNP itself cannot enter the cells for gene editing, and the GFP protein cannot deliver the RNP into the cells, only the PRD protein can efficiently deliver the RNP to mediate gene editing. The inventors further analyzed that it may be due to the fact that most fragments of the PRD protein are disordered regions, and the isoelectric point of the protein is high, which is positively charged in neutral and acidic environments, and can form agglomerate particles by liquid phase separation assembly.

[0139] Example 8 Effect of Cas9 protein and sgRNA input amount on gene editing efficiency of primary T cells

[0140] In this example, the effect of Cas9 protein and sgRNA input amount on gene editing efficiency of primary T cells was analyzed using human primary T cells as an example.

[0141] The human primary T cells were separated and activated as in Example 1.

[0142] Transfection system configuration: different amounts of Cas9 protein (Coast Protein, E365-01B) and sgRNA (sequence as in Example 1) were added to 40 μL of 10% (w / v) PEG8000 solution and mixed thoroughly, and the groups were as follows:

[0143] 5-5 group: 5 pmol of Cas9 protein and 5 pmol of sgRNA;

[0144] 10-10 group: 10 pmol of Cas9 protein and 10 pmol of sgRNA;

[0145] 10-20 group: 10 pmol of Cas9 protein and 20 pmol of sgRNA;

[0146] 15-15 group: 15 pmol of Cas9 protein and 15 pmol of sgRNA;

[0147] 15-35 group: 15 pmol of Cas9 protein and 30 pmol of sgRNA.

[0148] To each group, add 0.7 μL of 286 μM PRD protein solution to the above mixture and mix thoroughly by pipetting up and down 20-30 times or vortexing for 10 seconds. Then, add 13.5 μL of 60% w / v glycerol solution and mix thoroughly to obtain the transfection mixture.

[0149] Human primary T cell transfection and gene editing efficiency flow cytometry were the same as in Example 1. Figure 12 As shown. Figure 12 As can be seen, Cas9 protein and sgRNA achieve similar editing efficiencies across a wide range of input doses. This suggests that efficient gene editing is achieved with just 5 pmol of Cas9 protein and 5 pmol of sgRNA, and further increases in input do not significantly improve gene editing efficiency.

[0150] Example 9 Effects of different polyols on gene editing efficiency of primary human T cells

[0151] This example explores the effects of different polyols on the gene editing efficiency of primary human T cells.

[0152] The isolation and activation of primary human T cells were the same as in Example 1.

[0153] Transfection system configuration: Add 5 pmol (approximately 0.8 µg) of Cas9 protein (Nearshore protein, E365-01B) and 10 pmol (approximately 0.3 µg) of sgRNA (sequence same as Example 1) to 40 µL of 10% (w / v) PEG8000 solution, respectively, and mix thoroughly. Add 0.7 µL of 286 µM PRD protein solution to each of these mixtures and mix thoroughly by pipetting up and down 20-30 times or vortexing for 10 seconds. Then, add different types or amounts of polyol solution to different groups and mix thoroughly to obtain the transfection mixtures. The groups are as follows:

[0154] 5% trehalose: add 10 μL of trehalose solution (25%, w / v);

[0155] 10% trehalose: add 26.5 μL trehalose solution (25%, w / v);

[0156] 10% glycerol: add 8 μL of 60% glycerol solution (w / v);

[0157] 15% glycerol: Add 13.5 μL of 60% w / v glycerol solution.

[0158] Human primary T cell transfection and gene editing efficiency flow cytometry were the same as in Example 1. Figure 13 As shown.Figure 13 It can be seen that the editing efficiency of the 10% glycerol and 15% glycerol groups was significantly higher than that of the trehalose group, indicating that the use of 10%-15% glycerol in human T cell RNP transfection can achieve higher efficiency gene editing.

[0159] Example 10 Effects of different polyols on gene editing efficiency of primary mouse T cells

[0160] This example explores the effects of different polyols on the gene editing efficiency of primary mouse T cells.

[0161] The isolation and activation of mouse primary T cells were the same as in Example 2.

[0162] Transfection system configuration: Add 5 pmol (approximately 0.8 µg) of Cas9 protein (Nearshore protein, E365-01B) and 10 pmol (approximately 0.3 µg) of sgRNA (sequence same as Example 2) to 40 µL of 10% (w / v) PEG8000 solution, respectively, and mix thoroughly. Add 0.7 µL of 286 µM PRD protein solution to each of these mixtures and mix thoroughly by pipetting up and down the pipette tip 20-30 times or vortexing for 10 seconds. Then, add different types or amounts of polyol solutions to different groups and mix thoroughly to obtain transfection mixtures. The groups are as follows:

[0163] 5% trehalose: add 10 μL of trehalose solution (25%, w / v);

[0164] 10% trehalose: add 26.5 μL trehalose solution (25%, w / v);

[0165] 10% glycerol: add 8 μL of 60% glycerol solution (w / v);

[0166] 15% glycerol: Add 13.5 μL of 60% w / v glycerol solution.

[0167] Mouse primary T cell transfection and gene editing efficiency flow cytometry were the same as in Example 2. Figure 14 As shown. Figure 14 It can be seen that the editing efficiency of the 5% trehalose and 10% trehalose groups was significantly higher than that of the glycerol group, indicating that the use of 5%-10% trehalose in mouse T cell RNP transfection can achieve higher efficiency gene editing.

[0168] In addition, it should be understood that after reading the above teachings of this application, those skilled in the art may make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for gene editing in primary cells, characterized in that: The following steps are involved: S1, prepare transfection mixture: S11, adding Cas9 protein with a final concentration of 0.1 μM to 0.15 μM and sgRNA with a final concentration of 0.2 μM to 0.3 μM to a 5% to 20% w / v PEG8000 solution, respectively, wherein the sgRNA targets the target gene; S12, adding PRD protein at a final concentration of 5 μM to 20 μM and mixing, wherein the amino acid sequence of the PRD protein is shown in SEQ ID No. 2; S13, adding a polyol with a final concentration of 5% to 15% w / v to obtain the transfection mixture, wherein the polyol is glycerol or trehalose; S2, the transfection mixture was mixed with 0.3 to 0.5 times the volume of the primary cell suspension to obtain a transfection system, and the mixture was incubated at 35 to 39°C for 10 min to 3 h, wherein the cell density in the primary cell suspension was 5×10 6 ~1×10 7 / mL; S3, the transfection system is mixed with 4 to 6 times the volume of the transfection system culture medium, centrifuged to remove the supernatant, and then 3 to 6 times the volume of the transfection system culture medium is added for culturing, thereby completing the editing of the target gene. Wherein, the primary cells are primary immune cells.

2. A method for gene editing in primary cells according to claim 1, characterized in that: The primary immune cells are primary T cells.

3. The method for gene editing in primary cells according to claim 2, wherein: The primary T cells are human primary T cells, the polyol is glycerol, and in step S13, glycerol is added to a final concentration of 10% to 15% w / v.

4. The method for gene editing in primary cells according to claim 3, wherein: The target gene is human TRAC gene, and the sequence of the sgRNA is shown in SEQ ID No.

1.

5. The method for gene editing in primary cells according to claim 2, wherein: The primary T cells are mouse primary T cells, the polyol is trehalose, and in step S13, trehalose is added at a final concentration of 5% to 10% w / v.

6. The method for gene editing in primary cells according to claim 5, wherein: The target gene is mouse Trac gene, and the sequence of the sgRNA is shown as SEQ ID No.

5.

7. The method for gene editing in primary cells according to any one of claims 1 to 6, characterized in that: After adding the Cas9 protein and the sgRNA and mixing them in step S11, step S12 is performed within 1 min.

8. The method for gene editing in primary cells according to any one of claims 1 to 6, wherein: In step S2, the transfection system is incubated at 37° C. for 10 min to 30 min.

9. A method for gene editing in primary cells according to any one of claims 1 to 6, characterized in that: In step S3, the culture medium is a complete culture medium.

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