A method for targeted gene knock-in in stem cells

Abstract

The present invention belongs to the field of gene editing, and specifically discloses a method for targeted gene knock-in in stem cells, comprising: S1, using a genome database to select a small sequence knock-in site and a large sequence knock-in site on a target gene; S2, designing pegRNA according to the small sequence knock-in site, and designing a specific sgRNA according to the large sequence knock-in site; S3, using electroporation to deliver PrimeEditor and pegRNA to stem cells to achieve precise small sequence editing; the present invention combines Prime Editing with the CRISPR / Cas9 system, first uses Prime Editing to achieve precise small sequence editing, and then uses CRISPR / Cas9 to assist in precise knock-in of large sequences, thereby greatly improving the accuracy of gene editing, being able to meet the demand for precise gene modification in stem cell research, and providing strong technical support for building more accurate disease models and conducting cell therapy.

Description

Technical Field

[0001] The present invention belongs to the field of gene editing, and in particular relates to a method for targeted gene knock-in in stem cells. Background Art

[0002] Gene-directed knock-in technology is of great significance in the field of stem cell research. In stem cell research, precise gene editing is essential to maintaining the characteristics and functions of stem cells. For example, when building a disease model, it is necessary to accurately simulate the gene mutation in the patient's body; in cell therapy, ensuring the accuracy and safety of gene editing is a key prerequisite for clinical application;

[0003] However, traditional gene editing methods, such as early homologous recombination technology, although capable of targeted gene knock-in, are extremely inefficient and have difficulty achieving precise editing of small sequences and high-precision knock-in of large sequences. Summary of the invention

[0004] The purpose of the present invention is to provide a method for targeted gene knock-in in stem cells to solve the problem of difficulty in achieving precise editing of small sequences and high-precision knock-in of large sequences.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for targeted gene knock-in in stem cells, comprising:

[0007] S1. Using the genome database, select small sequence knock-in sites and large sequence knock-in sites on the target gene;

[0008] S2. Design pegRNA according to the small sequence knock-in site, and design specific sgRNA according to the large sequence knock-in site;

[0009] S3. Use electroporation to deliver PrimeEditor and pegRNA to stem cells for precise small sequence editing.

[0010] S4, designing specific sgRNA according to the large sequence knock-in site, preparing high-fidelity Cas9 nuclease, and constructing a donor DNA template containing a large sequence insertion fragment and homology arms at both ends;

[0011] S5. Use electroporation to deliver sgRNA, high-fidelity Cas9 nuclease, and donor DNA template to stem cells that have completed small sequence editing, so as to achieve precise knock-in of large sequences into the target site;

[0012] S6. Use thymidine to synchronize stem cells to the S / G2 phase, screen positive cells using a marker-free screening strategy, and perform genome sequencing to verify the accuracy and specificity of the knock-in.

[0013] Preferably, the pegRNA comprises a guide sequence, a primer sequence and an extension sequence;

[0014] The guide sequence is used to accurately match the small sequence editing target;

[0015] The primer sequence is used to provide a site for reverse transcription initiation;

[0016] The extended sequence is used to contain the desired small sequence editing information.

[0017] Preferably, the homology arms at both ends of the donor DNA template are 700 to 1000 bp in length, and the donor DNA template contains a protection sequence to prevent the template from being degraded by nucleases during delivery.

[0018] Preferably, after synchronizing the stem cells to the S / G2 phase using thymidine and then adding an HDR promoter and an NHEJ inhibitor, the thymidine concentration is 2 to 5 mM and the treatment time is 16 to 24 hours.

[0019] Preferably, the HDR promoter is RS-1 at a concentration of 10 to 20 μM, and the NHEJ inhibitor is SCR7 at a concentration of 1 to 5 μM.

[0020] Preferably, the PrimeEditor is a PE3 system, which is used to generate single-stranded DNA breaks at target sites.

[0021] Preferably, the method uses electroporation to deliver PrimeEditor and pegRNA to stem cells to achieve precise small sequence editing, while introducing the RAD51 expression vector into the stem cells.

[0022] Preferably, the method further comprises verifying the gene expression after knocking in the large sequence by RT-qPCR, detecting the proliferation and differentiation abilities of stem cells, and evaluating the effect of the large sequence knock-in operation on the biological characteristics of stem cells.

[0023] Preferably, the electric field strength of the electroporation is 100-300 V / cm, and the pulse time is 20-50 ms.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention combines Prime Editing with the CRISPR / Cas9 system, first using Prime Editing to achieve precise editing of small sequences, and then using CRISPR / Cas9 to assist in precise knock-in of large sequences, greatly improving the accuracy of gene editing. It can meet the needs of precise gene modification in stem cell research and provide strong technical support for building more accurate disease models and conducting cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0027] Figure 1 The figure is a flowchart of the method steps of the present invention. DETAILED DESCRIPTION

[0028] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings.

[0029] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0030] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0031] As attached Figure 1 As shown:

[0032] Embodiment 1: This embodiment provides a method for targeted gene knock-in in stem cells, comprising:

[0033] S1. Using the genome database, select small sequence knock-in sites and large sequence knock-in sites on the target gene. The small sequence knock-in site is located in an area where the gene function is relatively clear and has no obvious interference with the subsequent large sequence knock-in site. The large sequence knock-in site needs to consider the overall structure and function of the gene to avoid affecting the important regulatory elements or functional domains of the gene. At the same time, the distance between the large sequence knock-in site and the small sequence knock-in site will not have a negative impact on their respective editing processes.

[0034] S2. Design pegRNA according to the small sequence knock-in site, and design specific sgRNA according to the large sequence knock-in site. PrimeEditor is a PE3 system, which is used to generate single-stranded DNA breaks at the target site, which helps to improve editing efficiency;

[0035] S3. Use electroporation to deliver PrimeEditor and pegRNA to stem cells to achieve precise small sequence editing. In cells, PrimeEditor forms a complex with pegRNA, and pegRNA guides the complex to bind to the small sequence editing target. PrimeEditor generates single-stranded DNA breaks at the target site, and cells use the extended sequence of pegRNA as a template for reverse transcription to achieve precise small sequence editing.

[0036] S4. Design specific sgRNA according to the large sequence knock-in site, prepare high-fidelity Cas9 nuclease, and construct a donor DNA template containing a large sequence insertion fragment and homology arms at both ends. The specific sgRNA can guide the high-fidelity Cas9 nuclease to accurately recognize and bind to the site;

[0037] S5. Use electroporation to deliver sgRNA, high-fidelity Cas9 nuclease, and donor DNA template to stem cells that have completed small sequence editing, so as to accurately knock in the large sequence into the target site. sgRNA guides the high-fidelity Cas9 nuclease to cut DNA at the large sequence knock-in target site, forming double-strand breaks, and the cell initiates the repair mechanism. In this process, the donor DNA template undergoes homologous recombination with the homologous regions on both sides of the large sequence knock-in site of the genome through the homologous arms at both ends, thereby accurately inserting the large sequence into the target site.

[0038] S6. Use thymidine to synchronize stem cells to the S / G2 phase. During this phase, the cell DNA replication and repair mechanisms are active, which is beneficial to improving the efficiency of homologous recombination and promoting the precise knock-in of large sequences. Positive cells are screened through a marker-free screening strategy, such as PCR detection, to screen positive cells that may have undergone precise knock-in of large sequences. Specific primers are designed for the large sequence knock-in region. If the expected large sequence knock-in occurs in the cell genome, DNA fragments of a specific length can be obtained after PCR amplification. Genome sequencing is then performed to verify the accuracy and specificity of the knock-in, to verify whether the large sequence is accurately inserted into the predetermined large sequence knock-in site, and to check whether other locations in the genome have undergone unexpected changes due to the editing operation.

[0039] Specifically, the pegRNA includes a guide sequence, a primer sequence, and an extension sequence;

[0040] Contains guide sequence for precise matching of small sequence editing targets;

[0041] The primer sequence is used to provide a site for reverse transcription initiation;

[0042] The extension sequence is used to contain the desired small sequence editing information.

[0043] Specifically, the homology arms at both ends of the donor DNA template are 700 to 1000 bp in length and are homologous to the target region. The donor DNA template contains a protection sequence to prevent the template from being degraded by nucleases during delivery.

[0044] Specifically, thymidine is used to synchronize stem cells to the S / G2 phase, and then an HDR promoter and an NHEJ inhibitor are added. The thymidine concentration is 2 to 5 mM, and the treatment time is 16 to 24 hours.

[0045] Specifically, the HDR promoter is RS-1 at a concentration of 10 to 20 μM, and the NHEJ inhibitor is SCR7 at a concentration of 1 to 5 μM, which inhibits the NHEJ pathway and enhances HDR repair.

[0046] Specifically, PrimeEditor and pegRNA were delivered to stem cells using electroporation to achieve precise small sequence editing, while the RAD51 expression vector was introduced into the stem cells.

[0047] Specifically, the method also includes verifying the gene expression after knocking in the large sequence through RT-qPCR, and quantitatively analyzing the gene transcription at the mRNA level by RT-qPCR to determine the impact of large sequence knock-in on gene expression, and detecting the proliferation and differentiation capabilities of stem cells, and evaluating the impact of large sequence knock-in operations on the biological characteristics of stem cells, ensuring that stem cells still maintain normal physiological functions after gene editing, and providing a basis for subsequent applications.

[0048] Specifically, the electric field strength of electroporation is 100-300 V / cm, and the pulse time is 20-50 ms.

[0049] Example 2: Experimental Materials

[0050] Stem cell line: human embryonic stem cell line (H9 cell line), purchased from ATCC (American Type Culture Collection).

[0051] Reagents

[0052] PrimeEditor3 (PE3) system-related reagents, including plasmid encoding PE3.

[0053] Raw materials used to synthesize pegRNA and sgRNA, such as dNTPs, primers, etc.

[0054] High-fidelity Cas9 nuclease and corresponding expression plasmid.

[0055] Thymidine, RS-1, SCR7.

[0056] Reagents for electroporation, such as electroporation buffer.

[0057] Culture medium, fetal bovine serum, antibiotics, etc. for cell culture.

[0058] DNA extraction kits, PCR-related reagents, and genome sequencing services.

[0059] RT-qPCR related reagents, including reverse transcription kits, qPCR reagents, etc.

[0060] Instruments and Equipment

[0061] Cell culture incubator (37°C, 5% CO2).

[0062] Electroporator.

[0063] Real-time fluorescence quantitative PCR instrument.

[0064] Centrifuge.

[0065] Clean bench.

[0066] 2. Experimental steps

[0067] (I) Target selection (S1)

[0068] The Oct4 gene in human embryonic stem cells was studied using the NCBI (National Center for Biotechnology Information) genome database. Small sequence knock-in sites and large sequence knock-in sites were selected on the Oct4 gene. The small sequence knock-in site was selected in a region of the 5' non-coding region of the Oct4 gene with a relatively clear function and no obvious interference with the subsequent large sequence knock-in. The large sequence knock-in site was selected in an intron region of the gene coding region, which takes into account the overall structure and function of the gene to avoid affecting important regulatory elements or functional domains. At the same time, it was evaluated that the distance between the large sequence knock-in site and the small sequence knock-in site would not have a negative impact on their respective editing processes.

[0069] (II) Editing tool design (S2)

[0070] pegRNA design: pegRNA is designed based on the selected small sequence knock-in site. Its guide sequence precisely matches the specific DNA sequence of the small sequence knock-in target; the primer sequence provides a suitable site for the subsequent reverse transcription initiation; and the extension sequence contains the small sequence editing information that is expected to be introduced, such as a specific point mutation.

[0071] sgRNA design: Design specific sgRNA for large sequence knock-in sites to ensure that it can guide the high-fidelity Cas9 nuclease to accurately recognize and bind to the site.

[0072] (III) Precise small sequence editing (S3)

[0073] Cell culture: Human embryonic stem cells (H9 cell line) were cultured in a culture system containing fetal bovine serum, basal culture medium and antibiotics in a cell culture incubator at 37°C and 5% CO2. Subsequent operations were performed when the cell density reached 70%-80% fusion.

[0074] Preparation of editing components: Mix the plasmid encoding PE3 with the designed pegRNA and prepare the RAD51 expression vector at the same time.

[0075] Electroporation delivery: The mixed PrimeEditor and pegRNA and RAD51 expression vector are delivered to stem cells using electroporation. The electroporation parameters are set to 200V / cm and 30ms. In the cell, PrimeEditor forms a complex with pegRNA, which guides the complex to bind to the small sequence editing target. PrimeEditor generates single-stranded DNA breaks at the target site, and the cell performs reverse transcription using the extended sequence of pegRNA as a template to achieve precise small sequence editing.

[0076] (IV) Preparation for large sequence knock-in (S4)

[0077] Donor DNA template construction: Construct a donor DNA template containing a large sequence insertion fragment (such as the green fluorescent protein GFP gene) and homology arms at both ends. The homology arm length is designed to be 800bp, which is highly homologous to the target region on both sides of the large sequence knock-in site. At the same time, a protective sequence is added to the donor DNA template to prevent it from being degraded by nucleases during delivery.

[0078] Preparation of editing components: Prepare high-fidelity Cas9 nuclease and corresponding expression plasmid, as well as specific sgRNA designed for large sequence knock-in sites.

[0079] (V) Precise knock-in of large sequences (S5)

[0080] Cell preparation: The stem cells that have completed small sequence editing are collected and digested with trypsin to form a single cell suspension.

[0081] Electroporation delivery: sgRNA, high-fidelity Cas9 nuclease expression plasmid and donor DNA template are mixed and delivered to the stem cells that have completed small sequence editing by electroporation. The electroporation parameters are the same as the precise small sequence editing step (electric field strength 200V / cm, pulse time 30ms). sgRNA guides the high-fidelity Cas9 nuclease to cut DNA at the large sequence knock-in target site, forming a double-strand break, and the cell initiates the repair mechanism. In this process, the donor DNA template undergoes homologous recombination with the homologous regions on both sides of the large sequence knock-in site of the genome through the homologous arms at both ends, thereby accurately inserting the GFP gene into the target site.

[0082] (VI) Cell cycle synchronization and screening verification (S6)

[0083] Cell cycle synchronization: After completing the knock-in of the large sequence, add thymidine to the cell culture system to a final concentration of 3mM, treat the cells for 20 hours, and synchronize the stem cells to the S / G2 phase. During this stage, the cell DNA replication and repair mechanisms are active, which is conducive to improving the efficiency of homologous recombination and promoting the precise knock-in of large sequences.

[0084] Add repair regulatory reagents: After the cells are synchronized to the S / G2 phase, add 15 μM concentration of RS-1 (HDR promoter) and 3 μM concentration of SCR7 (NHEJ inhibitor) to the culture medium to inhibit the NHEJ pathway and enhance HDR repair.

[0085] Screening positive cells: Through the marker-free screening strategy, PCR detection is used to screen positive cells that may have large sequence knock-in. Design specific primers for the large sequence knock-in region (GFP gene insertion site). If the expected large sequence knock-in occurs in the cell genome, a DNA fragment of a specific length can be obtained after PCR amplification.

[0086] Sequencing verification: Perform genome sequencing on the screened suspected positive cells to verify whether the GFP gene is accurately inserted into the predetermined large sequence knock-in site, and check whether other locations in the genome have produced unexpected changes due to the editing operation.

[0087] (VII) Functional testing

[0088] Gene expression verification: RT-qPCR was used to verify the expression of the Oct4 gene after knocking in the GFP gene. The positive cells after screening and verification were collected, and the total RNA was extracted. The RNA was reverse transcribed into cDNA using a reverse transcription kit, and then amplified using a real-time fluorescence quantitative PCR instrument and qPCR reagents with Oct4 gene-specific primers. The gene transcription was quantitatively analyzed at the mRNA level to determine the effect of large sequence knock-in on Oct4 gene expression.

[0089] Stem cell characteristics detection: Detect the proliferation and differentiation abilities of stem cells. Detect the proliferation ability of stem cells through cell counting and MTT experiments; Induce stem cells to differentiate into different directions such as nerve cells and cardiomyocytes, observe cell morphological changes and detect the expression of related differentiation markers, evaluate the impact of large sequence knock-in operations on the biological characteristics of stem cells, ensure that stem cells still maintain normal physiological functions after gene editing, and provide a basis for subsequent applications.

[0090] 3. Expected Experimental Results

[0091] Through PCR detection and genome sequencing, it was confirmed that the large sequence (GFP gene) was accurately inserted into the predetermined large sequence knock-in site and no unexpected changes occurred in other locations of the genome.

[0092] RT-qPCR results showed that the expression level of Oct4 gene was within an acceptable range after knocking in the large sequence, with no abnormal upregulation or downregulation, indicating that knocking in the large sequence had little effect on Oct4 gene expression.

[0093] The detection of stem cell proliferation ability showed that there was no significant difference in the proliferation rate of gene-edited stem cells compared with that of unedited stem cells.

[0094] The stem cell differentiation ability test showed that the edited stem cells were able to successfully differentiate into different types of cells, and the expression of relevant differentiation markers was normal, indicating that the large sequence knock-in operation did not cause obvious damage to the differentiation ability of stem cells.

[0095] Importantly, it should be noted that the construction and arrangement of the present application shown in a plurality of different exemplary embodiments are only exemplary. Although only a few embodiments are described in detail in this disclosure, it should be readily understood by those who refer to this disclosure that many modifications are possible (e.g., the size, scale, structure, shape and proportion of various elements, and parameter values ​​(e.g., temperature, pressure, etc.), installation arrangement, use of materials, color, directional changes, etc.) without substantially departing from the novel teachings and advantages of the subject matter described in the application. For example, the element shown as integrally formed can be composed of multiple parts or elements, the position of the element can be inverted or otherwise changed, and the nature or number or position of the discrete element can be changed or changed. Therefore, all such modifications are intended to be included in the scope of the present invention. The order or sequence of any process or method steps can be changed or reordered according to alternative embodiments. In the claims, any "device plus function" clause is intended to cover the structure of performing the function described herein, and is not only structurally equivalent but also equivalent structure. Without departing from the scope of the present invention, other replacements, modifications, changes and omissions can be made in the design, operating conditions and arrangement of the exemplary embodiments. Therefore, the invention is not limited to a specific embodiment, but extends to several modifications still falling within the scope of the appended claims.

[0096] Additionally, in order to provide a concise description of exemplary embodiments, all features of an actual embodiment (ie, those features that are not relevant to the best mode presently contemplated for carrying out the invention or those that are not relevant to implementing the invention) may not be described.

[0097] It will be appreciated that in the development of any actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but will be a routine task of design, fabrication, and production for those of ordinary skill having the benefit of this disclosure without undue experimentation.

[0098] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for targeted gene knock-in in stem cells, characterized in that: include: S1. Using the genome database, select small sequence knock-in sites and large sequence knock-in sites on the target gene; S2. Design pegRNA according to the small sequence knock-in site, and design specific sgRNA according to the large sequence knock-in site; S3. Use electroporation to deliver PrimeEditor and pegRNA to stem cells for precise small sequence editing. S4, designing specific sgRNA according to the large sequence knock-in site, preparing high-fidelity Cas9 nuclease, and constructing a donor DNA template containing a large sequence insertion fragment and homology arms at both ends; S5. Use electroporation to deliver sgRNA, high-fidelity Cas9 nuclease, and donor DNA template to stem cells that have completed small sequence editing, so as to achieve precise knock-in of large sequences into the target site; S6. Use thymidine to synchronize stem cells to the S / G2 phase, screen positive cells using a marker-free screening strategy, and perform genome sequencing to verify the accuracy and specificity of the knock-in.

2. A method for targeted gene knock-in in stem cells according to claim 1, characterized in that: The pegRNA includes a guide sequence, a primer sequence and an extension sequence; The guide sequence is used to accurately match the small sequence editing target; The primer sequence is used to provide a site for reverse transcription initiation; The extended sequence is used to contain the desired small sequence editing information.

3. A method for targeted gene knock-in in stem cells according to claim 1, characterized in that: The length of the homology arms at both ends of the donor DNA template is 700 to 1000 bp, and the donor DNA template contains a protection sequence to prevent the template from being degraded by nuclease during the delivery process.

4. A method for targeted gene knock-in in stem cells according to claim 1, characterized in that: The method uses thymidine to synchronize stem cells to the S / G2 phase, and then adds an HDR promoter and an NHEJ inhibitor. The thymidine concentration is 2 to 5 mM, and the treatment time is 16 to 24 hours.

5. A method for targeted gene knock-in in stem cells according to claim 4, characterized in that: The HDR promoter is RS-1 at a concentration of 10 to 20 μM, and the NHEJ inhibitor is SCR7 at a concentration of 1 to 5 μM.

6. A method for targeted gene knock-in in stem cells according to claim 1, characterized in that: The PrimeEditor is a PE3 system that is used to generate single-stranded DNA breaks at target sites.

7. A method for targeted gene knock-in in stem cells according to claim 1, characterized in that: The method uses electroporation to deliver PrimeEditor and pegRNA to stem cells to achieve precise small sequence editing, while introducing the RAD51 expression vector into the stem cells.

8. A method for targeted gene knock-in in stem cells according to claim 1, characterized in that: The method also includes verifying the gene expression after knocking in the large sequence through RT-qPCR, detecting the proliferation and differentiation abilities of stem cells, and evaluating the effects of the large sequence knock-in operation on the biological characteristics of stem cells.

9. A method for targeted gene knock-in in stem cells according to claim 1, characterized in that: The electric field intensity of the electroporation is 100 to 300 V / cm, and the pulse time is 20 to 50 ms.

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