Mitochondrial gene editing method for seedless gene off-target

Abstract

The invention provides a mitochondrial gene editing method for seedless gene off-target. The mitochondrial gene editing method comprises the following steps: carrying out mitochondrial gene editing in an initial cell; separating the edited mitochondria from the initial cells; and transplanting the edited mitochondria into a target cell. According to the present disclosure, a mitochondrial gene editing method for seedless gene off-target can be provided.

Description

Technical Field

[0001] The present disclosure relates to the field of genetic engineering, and in particular to a mitochondrial gene editing method without nuclear gene off-targeting. Background Art

[0002] Gene editing technology is a gene therapy method that has emerged in recent years, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs) and CRISPR / Cas9 systems. The recent success of gene therapy in multiple clinical trials has inspired people's hope for the treatment of intractable diseases.

[0003] Among them, the zinc finger nuclease system and the transcription activator-like effector nuclease system can achieve the editing of mitochondrial DNA. However, the above systems will induce the degradation of damaged DNA after cutting mtDNA, and cannot achieve precise editing. In 2020, Liu Ruqian's team reported a new mtDNA editing system: DNA deaminase (DddA)-derived cytosine base editor (DdCBE), which can achieve precise editing of mtDNA. DdCBE integrates DddA and transcription activator-like effector (TALE), which can achieve CG to TA mutation in mtDNA without causing double-strand breaks, thereby achieving precise single-base editing of mtDNA. In 2022, Jin-Soo Kim proposed a programmable TALE-linked deaminase that can induce targeted A-to-G editing in human mitochondria, which lays the foundation for disease modeling and clinical research. Subsequently, Liu Ruqian's team improved DdCBE, which used phage assistance to improve editing efficiency and overcome the strict TC sequence constraints of DddA, greatly improving the effectiveness and applicability of base editing.

[0004] However, studies have shown that mitochondrial base editors can induce extensive off-target editing in the nuclear genome. If mitochondrial gene editing is performed directly in target cells, uncontrollable negative effects may be caused to target cells due to the existence of off-target effects. In addition, mitochondria exist in multiple copies in cells, and mitochondrial gene editing is more difficult in certain types of cells (such as primary cells, non-proliferating cells, embryonic stem cells, etc.), and it is difficult to achieve the expected editing efficiency. Summary of the invention

[0005] The present disclosure is completed in view of the above-mentioned state of the prior art, and its purpose is to provide a mitochondrial gene editing method without nuclear gene off-target.

[0006] To this end, the present disclosure provides a mitochondrial gene editing method, comprising the following steps: performing mitochondrial gene editing in an initial cell; separating the edited mitochondria from the initial cell; and transplanting the edited mitochondria into a target cell.

[0007] In the present disclosure, mitochondrial gene editing is first performed in the initial cells, and then the edited mitochondria are transplanted into the target cells. Direct editing is not performed in the target cells, and there is no risk of nuclear gene off-target, and the expected target cells can be stably obtained; and for target cells that are difficult to edit, the editing method disclosed in the present disclosure not only has no risk of nuclear gene off-target, but also first edits the mitochondria in the easy-to-edit initial cells, and then transplants the edited mitochondria into the target cells, which helps to improve the editing efficiency, so that the mitochondrial genes in the target cells can be efficiently edited. Thus, a mitochondrial gene editing method without nuclear gene off-target can be provided.

[0008] In the mitochondrial gene editing method involved in the first aspect of the present disclosure, optionally, the initial cells are selected from any one or more of A549 human lung cancer cell line, HEK293 human kidney epithelial cell line, 293T human kidney epithelial cell line and 3T3 mouse fibroblasts. Thus, the mitochondria in the initial cells can be easily edited.

[0009] In the mitochondrial gene editing method involved in the first aspect of the present disclosure, optionally, the target cell is selected from any one or more of primary cells, non-proliferating cells, embryonic stem cells and cells for disease treatment. Thus, mitochondrial genes in target cells that are difficult to edit or cells for disease treatment can be edited.

[0010] In the mitochondrial gene editing method involved in the first aspect of the present disclosure, optionally, the target cells are selected from any one or more of neural cells, leukocytes, H9 human embryonic stem cells and E4 mouse embryonic stem cells. Thus, mitochondrial genes in target cells that are difficult to edit or cells used for disease treatment can be edited.

[0011] In the mitochondrial gene editing method involved in the first aspect of the present disclosure, optionally, performing mitochondrial gene editing includes mutating a target gene in the mitochondria in the initial cell to obtain a target mitochondria, wherein the target mitochondria are mitochondria having a predetermined mutation.

[0012] In the mitochondrial gene editing method involved in the first aspect of the present disclosure, optionally, mitochondrial single base gene editing is performed by transient transfection method. Thus, it is easy to edit the mitochondria in the initial cells to obtain mitochondria with predetermined mutations.

[0013] In the mitochondrial gene editing method involved in the first aspect of the present disclosure, optionally, the edited mitochondria are separated from the initial cells by a mitochondrial extraction kit, thereby facilitating the separation of mitochondria and initial cells.

[0014] In the mitochondrial gene editing method involved in the first aspect of the present disclosure, optionally, the edited mitochondria are detected, the edited mitochondria are determined to be target mitochondria, and then the target mitochondria are transplanted into the target cells. In this case, identifying the edited mitochondria before transplanting can help improve the accuracy of editing.

[0015] In the mitochondrial gene editing method involved in the first aspect of the present disclosure, optionally, transplanting the edited mitochondria into the target cells includes: co-culturing the edited mitochondria with the target cells so that the edited mitochondria enter the target cells. In this case, when the mitochondria are co-cultured with the target cells, the edited mitochondria can fuse into the target cells and gradually replace the original mitochondrial genome in the target cells, thereby achieving gene editing of the mitochondria in the target cells.

[0016] In the mitochondrial gene editing method involved in the first aspect of the present disclosure, optionally, when the edited mitochondria are transplanted into the target cells, the concentration of the mitochondria is positively correlated with the editing efficiency. In this case, the concentration of the mitochondria can be adjusted according to the actual situation to adjust the editing efficiency, for example, by increasing the concentration of the mitochondria to improve the editing efficiency.

[0017] According to the present disclosure, a mitochondrial gene editing method without nuclear gene off-target can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a schematic diagram showing the mitochondrial gene editing method involved in the examples of the present disclosure.

[0019] Figure 2 Schematic diagram showing the operation of achieving MTND5 932G>A single-base gene editing in A549 cells using a transient transfection method in an embodiment of the present disclosure.

[0020] Figure 3 It is a schematic diagram showing the results of staining mitochondria in cells in an embodiment of the present disclosure.

[0021] Figure 4 Schematic diagram showing the fluorescence results of transplanting mitochondria into cells in an embodiment of the present disclosure.

[0022] Figure 5 Schematic diagram showing Sanger sequencing after mitochondria are transplanted into cells in an embodiment of the present disclosure. DETAILED DESCRIPTION

[0023] Hereinafter, the preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same symbols are assigned to the same components, and repeated descriptions are omitted. In addition, the accompanying drawings are only schematic diagrams, and the ratio of the dimensions of the components or the shapes of the components may be different from the actual ones.

[0024] It should be noted that the terms "including" and "having" and any variations thereof in the present disclosure, such as a process, method, system, product or device that includes or has a series of steps or units, are not necessarily limited to those steps or units clearly listed, but may include or have other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0025] The present disclosure relates to a mitochondrial gene editing method without the risk of nuclear gene off-target. That is, the present disclosure can provide a mitochondrial gene editing method without nuclear gene off-target. In the present disclosure, the mitochondrial gene editing method can be referred to as an editing method, and can also be referred to as a mitochondrial editing method or a target cell mitochondrial editing method.

[0026] Hereinafter, the mitochondrial gene editing method involved in the present disclosure will be described in detail in conjunction with the accompanying drawings.

[0027] In some examples, the mitochondrial gene editing method may include the following steps: performing mitochondrial gene editing in the initial cell (step S10); separating the edited mitochondria from the initial cell (step S20); transplanting the edited mitochondria into the target cell (step S30). In the present disclosure, mitochondrial gene editing is first performed in the initial cell, and then the edited mitochondria are transplanted into the target cell. It is not directly edited in the target cell, and there is no risk of nuclear gene off-target, and the expected target cell can be stably obtained; and for target cells that are difficult to edit, the editing method disclosed in the present disclosure not only has no risk of nuclear gene off-target, but also first edits the mitochondria in the easy-to-edit initial cell, and then transplants the edited mitochondria into the target cell, which also helps to improve the editing efficiency, so that the mitochondrial genes in the target cell can be efficiently edited. Thus, a mitochondrial gene editing method without nuclear gene off-target can be provided.

[0028] In the present disclosure, the edited mitochondria may be referred to as modified mitochondria, mutant mitochondria, target mitochondria, or encoding mitochondria, etc. In the present disclosure, the initial cells may be referred to as original cells, cultured cells, or edited cells, etc.

[0029] In some examples, in step S10, the initial cell can be selected from an easily editable cell line. In some examples, the initial cell can be selected from any one or more of A549 human lung cancer cell line, HEK293 human kidney epithelial cell line, 293T human kidney epithelial cell line, and 3T3 mouse fibroblasts. In some examples, the type of initial cell can be selected based on the type of mitochondria required.

[0030] In some examples, in step S10, performing mitochondrial gene editing may include mutating a target gene in the mitochondria in the initial cell to obtain a target mitochondria, wherein the target mitochondria are mitochondria having a predetermined mutation.

[0031] In some examples, in step S10, a suitable editing method can be selected based on the desired mitochondria. For example, in some examples, in step S10, mitochondrial single base gene editing can be performed by transient transfection method. Thus, it is easy to edit the mitochondria in the initial cells to obtain mitochondria with predetermined mutations.

[0032] In some examples, in step S20, a reagent method may be used to extract mitochondria. In this case, compared with the grinding method which may cause mitochondrial rupture, the reagent method can obtain mitochondria with high integrity.

[0033] In some examples, in step S20, the edited mitochondria can be separated from the initial cells by a mitochondrial extraction kit. Thus, it is easy to separate the mitochondria and the initial cells. In some examples, in step S20, the type of mitochondrial extraction kit can be selected based on the type of mitochondria.

[0034] In some examples, in step S30, the target cells can be selected from any one or more of primary cells, non-proliferating cells, embryonic stem cells, and cells for disease treatment. Thus, mitochondrial genes in target cells that are difficult to edit or cells for disease treatment can be edited. When the target cells are cells for disease treatment, editing using the editing method disclosed herein can obtain therapeutic cells without off-target risks, and the therapeutic cells have higher safety.

[0035] In some examples, the target cells are selected from any one or more of neural cells, leukocytes, H9 human embryonic stem cells, and E4 mouse embryonic stem cells. Thus, mitochondrial genes in target cells that are difficult to edit or cells used for disease treatment can be edited.

[0036] In some examples, in step S30, the edited mitochondria can be detected to determine that the edited mitochondria are target mitochondria, and then the target mitochondria are transplanted into the target cells. In this case, identifying the edited mitochondria before transplanting can help improve the accuracy of editing.

[0037] In some examples, in step S30, transplanting the edited mitochondria into the target cells may include: co-culturing the edited mitochondria with the target cells so that the edited mitochondria enter the target cells. In this case, when the mitochondria are co-cultured with the target cells, the edited mitochondria can fuse into the target cells and gradually replace the original mitochondrial genome in the target cells, thereby achieving gene editing of the mitochondria in the target cells.

[0038] In the present disclosure, the edited mitochondria are transplanted into the target cells without directly performing mitochondrial gene editing in the target cells, thereby avoiding the risk of off-target effects on the nuclear genome in the target cells.

[0039] In some examples, in step S30, when the edited mitochondria are transplanted into the target cells, the concentration of the mitochondria is positively correlated with the editing efficiency. That is, when the edited mitochondria are co-cultured with the target cells, the concentration of the mitochondria is positively correlated with the editing efficiency. In this case, the concentration of the mitochondria can be adjusted according to the actual situation to adjust the editing efficiency, for example, by increasing the concentration of the mitochondria to improve the editing efficiency.

[0040] In some examples, in step S30, the concentration of mitochondria can be set according to the types of target cells and edited mitochondria. Those skilled in the art can adjust the concentration of mitochondria during co-culture based on the types of target cells and edited mitochondria.

[0041] In some examples, in step S30, the edited mitochondria can be washed after being co-cultured with the target cells for a certain period of time to remove the mitochondria that have not entered the cells.

[0042] In some examples, the time for co-culturing the edited mitochondria with the target cells can be 1 hour to 48 hours. For example, the co-culture time can be 1 hour, 2 hours, 4 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 30 hours, 34 hours, 36 hours, or 48 hours.

[0043] In summary, according to the present disclosure, a mitochondrial editing method without nuclear gene off-target can be provided.

[0044] To further illustrate the present disclosure, the mitochondrial gene editing method provided by the present disclosure is described in detail below in conjunction with the examples.

[0045] In the embodiments of the present disclosure, A549 cells (A549 human lung cancer cells) are used as initial cells and 293T cells (293T human renal epithelial cells) are used as target cells for detailed description. It is understood that this does not mean that the initial cells and target cells of the present disclosure can only be the two cells listed in the specific embodiments. In addition, it should be noted that in the embodiments of the present disclosure, unless otherwise specified, the reagents and instruments used are all commercially available products.

[0046] [Example]

[0047] 1. MTND5 932G>A single-base gene editing was achieved in A549 cells (A549 human lung cancer cells) using transient transfection method. Figure 2 Schematic diagram showing the operation of implementing MTND5 932G>A single base gene editing in A549 cells using transient transfection method in the embodiment of the present disclosure. Figure 2 , the specific operations are as follows:

[0048] (1) Prepare cells

[0049] A549 cells were revived and cultured to the third generation. Then, 1×105 cells were inoculated into 6-well plates (JETBIOFIL, China). DMEM complete medium (DMEM containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA)) was added, and the cells were shaken evenly and placed in a 37°C carbon dioxide incubator for overnight culture.

[0050] (2) Cell transfection

[0051] A549 cells were confluent to 70%-80% and transfected with base editors (4.5 μg) using EZtrans cell transfection reagent (LIFE iLAB BIO, China). 72 hours after transfection, the top 15% of GFP and mCherry double-positive cells were harvested from fluorescence activated cell sorting (FACS) for culture.

[0052] Specific methods:

[0053] ①. Solution a: Dilute 2.25 μg of TALEN-Left-mCherry and TALEN-Right-GFP plasmids with 250 μL of serum-free culture medium respectively.

[0054] ②. Solution b: Dilute 9 μL of EZ transfection reagent with 250 μL of serum-free culture medium.

[0055] ③. Mix and shake the ab solution and place it at room temperature for about 15 minutes.

[0056] ④. Take out the cells, add 2 mL of fresh culture medium to each well, and add the mixed ab solution dropwise to each well, shake gently in a cross direction to mix, and culture in a carbon dioxide incubator at 37°C for 72 hours.

[0057] ⑤. Aspirate the culture medium, wash the cells twice with PBS, centrifuge the cells at 800×RPM, add 200μL complete culture medium to resuspend the cells. Use BD AriaIII flow cytometry to sort the top 15% of FITC and PE double positive cells.

[0058] (3) Monoclonal isolation and identification

[0059] The cells sorted by FACS were cultured for three days and then single cells were sorted by FACS into 96-well plates again. The cells were cultured for 15 days and the genotype of the single clones was identified by Sanger sequencing.

[0060] (4) Cultivation of single clones

[0061] The successfully identified single clones were transferred to 24-well plates and continued to be cultured until they expanded to 10 cm culture dishes.

[0062] 2. Mitochondrial Isolation and Purification

[0063] (1) Mitochondrial staining

[0064] Under light-proof conditions, add Mitotracker Green dye to the cells and incubate them in a cell incubator at 37 degrees for 40 minutes. After the incubation, remove the culture medium, wash twice with PBS buffer, and add new PBS buffer. Figure 3 is a schematic diagram showing the results of staining mitochondria in cells in an embodiment of the present disclosure. Figure 3 , green fluorescence can be observed under a fluorescence microscope, proving that the mitochondria are successfully stained. After the mitochondrial extraction step is completed, 2 μl of mitochondrial resuspension solution is taken and observed under a fluorescence microscope, green fluorescence can be observed, proving that the mitochondria are successfully isolated.

[0065] (2) Mitochondrial extraction

[0066] Mitochondria were extracted using a mitochondrial isolation kit produced by Thermo Fisher.

[0067] The specific steps are as follows:

[0068] ①. Add 800 μL Buffer A (make sure PMSF has been added) to the cell pellet, vortex at medium speed for 5 seconds, and incubate on ice for 2 minutes.

[0069] ②. Add 10 μL Buffer B, vortex at maximum speed for 5 seconds, incubate on ice for 5 minutes, vortex once every minute.

[0070] ③. Add 800 μL Buffer C and immediately and gently invert the tube 4-5 times.

[0071] ④. Centrifuge at 4°C and 700×g for 10 minutes and transfer the supernatant to a new 2mL EP tube.

[0072] ⑤. Centrifuge at 4℃, 12000×g for 15 minutes, remove the supernatant and retain the precipitate.

[0073] ⑥. Add 500 μL Buffer C (PMSF has been added) to the mitochondrial pellet to wash the mitochondrial pellet.

[0074] ⑦. Centrifuge at 4°C, 12,000×g for 5 minutes and remove the supernatant.

[0075] ⑧. Add 25 μL of mitochondrial storage reagent to resuspend the mitochondrial pellet. Use Nanodrop A280 or BCA protein quantification kit to measure protein concentration.

[0076] 3. Mitochondrial Transplantation

[0077] 0 μg mitochondrial suspension (experimental group A), 2 μg mitochondrial suspension (experimental group B), and 20 μg mitochondrial suspension (experimental group C) were added to 5 x 10 cells. 4 The edited mitochondria were co-incubated with 293T cells (293T human kidney epithelial cells).

[0078] 4. Mitochondrial transplantation results detection

[0079] After incubating the mitochondrial suspension with 293T cells for 24 hours, the cells were washed twice with PBS and fresh PBS was added. Figure 4 is a schematic diagram showing the fluorescence results of transplanting mitochondria into cells in the embodiment of the present disclosure. Figure 4 In the figure, part A is the fluorescence result of experimental group A, part B is the fluorescence result of experimental group B, and part C is the fluorescence result of experimental group C. Figure 4 As shown, green fluorescence can be observed in the cells of the 2 μg mitochondrial suspension experimental group and the 20 μg mitochondrial suspension experimental group, proving that the isolated edited mitochondria were successfully transferred into the cells.

[0080] 293T cells were lysed with cell lysis buffer, and PCR amplification was performed using the lysis buffer as a template to amplify the mitochondrial genome fragment at the mutation site. The purified PCR product was subjected to Sanger sequencing. Figure 5 Schematic diagram showing Sanger sequencing after mitochondria are transplanted into cells in the embodiment of the present disclosure. Figure 5In the figure, part A is a schematic diagram of the Sanger sequencing results of experimental group A, part B is a schematic diagram of the Sanger sequencing results of experimental group B, and part C is a schematic diagram of the Sanger sequencing results of experimental group C.

[0081] See also Figure 5 , Sanger sequencing results showed that the G at the 932bp position of the ND5 gene in the 293T cells of experimental group A did not mutate; the sequencing results in experimental group B showed that the G at the 932bp position of the ND5 gene in some 293T cells mutated to A (as can be seen from the changes in the peak graph), which can indicate that there are some edited mitochondria in group B, but the editing ratio is still low; in the 293T cells of experimental group C, the G at the 932bp position of most ND5 mutated to A. It can be seen from the results that the higher the concentration of edited mitochondria during co-culture, the higher the transplantation efficiency, that is, the higher the editing efficiency of the target cells. It shows that the editing of mitochondria in 293T cells is achieved through the mitochondrial editing method disclosed in the present invention. And by deep sequencing of the whole genome of transplanted 293T cells, no off-target effects of the nuclear genome were found.

[0082] Therefore, the edited mitochondria separated by the present disclosure are successfully transplanted into the target cells and are able to gradually replace the original mitochondrial genome in the target cells, indirectly achieving gene editing of the mitochondria in the target cells.

[0083] In summary, the present disclosure can provide a mitochondrial gene editing method without nuclear gene off-target.

[0084] Although the present disclosure is specifically described above in conjunction with the accompanying drawings and examples, it is to be understood that the above description does not limit the present disclosure in any form. Those skilled in the art may modify and change the present disclosure as needed without departing from the essential spirit and scope of the present disclosure, and these modifications and changes all fall within the scope of the present disclosure.

Claims

1. A method for editing mitochondrial genes without nuclear gene off-target, characterized in that: The following steps are involved: Perform mitochondrial gene editing in naive cells; Isolating edited mitochondria from the original cells; as well as Transplant the edited mitochondria into target cells.

2. The mitochondrial gene editing method according to claim 1, characterized in that: The initial cells are selected from any one or more of A549 human lung cancer cell line, HEK293 human kidney epithelial cell line, 293T human kidney epithelial cell line and 3T3 mouse fibroblasts.

3. The mitochondrial gene editing method according to claim 1, characterized in that: The target cells are selected from any one or more of primary cells, non-proliferating cells, embryonic stem cells and cells used for disease treatment.

4. The mitochondrial gene editing method according to claim 1 or 3, characterized in that: The target cells are selected from any one or more of neural cells, leukocytes, H9 human embryonic stem cells and E4 mouse embryonic stem cells.

5. The mitochondrial gene editing method according to claim 1, characterized in that: Performing mitochondrial gene editing includes mutating a target gene in the mitochondria in the initial cell to obtain a target mitochondria, wherein the target mitochondria are mitochondria with predetermined mutations.

6. The mitochondrial gene editing method according to claim 1 or 5, characterized in that: Mitochondrial single-base gene editing by transient transfection method.

7. The mitochondrial gene editing method according to claim 1, characterized in that: The edited mitochondria were isolated from the original cells using a mitochondrial extraction kit.

8. The mitochondrial gene editing method according to claim 1, characterized in that: The edited mitochondria are detected to determine that the edited mitochondria are target mitochondria, and then the target mitochondria are transplanted into the target cells.

9. The mitochondrial gene editing method according to claim 1, characterized in that: Transplanting the edited mitochondria into the target cells includes: co-culturing the edited mitochondria with the target cells so that the edited mitochondria enter the target cells.

10. The mitochondrial gene editing method according to claim 1, characterized in that: When edited mitochondria are transplanted into target cells, the concentration of mitochondria is positively correlated with the editing efficiency.

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