A CRISPR-CAS system for targeted myeloid cell gene editing and its construction method

Abstract

The present invention relates to a CRISPR-CAS system for targeting myeloid cell gene editing and a construction method thereof. This scheme uses molecular biological technology to replace the promoter of the Cas9 nuclease in the CRISPR / Cas9 system with a chimeric promoter specifically recognized by myeloid cells, so that the Cas9 nuclease with cutting function is only expressed in myeloid cells; and guide RNA is designed to construct a CRISPR / Cas9 plasmid targeting the target gene, which is then packaged and produced by lentiviral transfection. This scheme can be used to study the role of different genes in myeloid cells regulating the tumor microenvironment.

Description

Technical Field

[0001] The present invention relates to the technical field of gene editing, and in particular to a CRISPR-CAS system for targeted myeloid cell gene editing and a construction method thereof. Background Art

[0002] During human hematopoiesis, myeloid cells are derived from common myeloid progenitors (CMPs) in the bone marrow. This lineage, which includes monocytes, granulocytes, erythrocytes, and platelets, is a major component of the innate immune system and serves as the first line of defense against infection. Myeloid cells also have a profound impact on tumor development. Tumor-associated myeloid cells (TAMCs) are heterogeneous and have distinct or even opposing effects on tumor cells and the tumor microenvironment (TME). In addition, myeloid cells play a key role in regulating lymphocyte behavior, resulting in either an immunostimulatory or immunosuppressive TME, thereby inhibiting or promoting tumor development.

[0003] The most studied TAMCs include monocytes, tumor-associated macrophages (TAMs), dendritic cells (DCs), tumor-associated neutrophils (TANs), and myeloid-derived suppressor cells (MDSCs). They participate in pleiotropic processes, including tumor cell growth, survival, differentiation, dissemination and metastasis, angiogenesis, TME remodeling, immune regulation, and response to cancer therapy. Understanding the role and mechanism of TAMCs in tumors will help discover new treatments.

[0004] The 2020 Nobel Prize in Chemistry was awarded to two scientists, Emmanuelle Charpentier and Jennifer Doudna, for their contributions to CRISPR / Cas9 gene editing technology. CRISPR-Cas9 is the third generation of gene editing technology after the introduction of ZFN, TALENs and other gene editing technologies. Due to its high efficiency, simple operation, low cost and easy to use, the application of CRISPR-Cas9 has swept the world in just a few years and has become the most mainstream gene editing system today.

[0005] CRISPR-Cas9 gene editing technology combines Cas9 protein with artificially designed single guide RNA (sgRNA), and then the independently designed sgRNA specifically targets the target sequence, causing double-strand breaks in the target gene DNA. The broken double-stranded DNA is repaired using non-homologous end joining (NHEJ) or homology-directed repair (HDR) DNA repair mechanisms, thereby achieving knockout or knock-in of the target gene fragment.

[0006] However, there is currently a lack of effective means for targeted treatment of myeloid cells. The current CRISPR-Cas9 system does not have cellular selectivity and lacks targeting specificity, making it difficult to achieve gene knockout in specific areas, which has created certain obstacles for scientific research and disease treatment in the field of targeted myeloid cells. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a CRISPR-CAS system for targeted myeloid cell gene editing and a method for constructing the same, so as to solve the problem of insufficient targeting specificity of the CRISPR / Cas9 system in the related art.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The first aspect of the present invention is to provide a method for constructing a CRISPR-CAS system for targeted myeloid cell gene editing, comprising the following steps:

[0010] S1: Synthetic chimeric promoter, which is an improved Cas9 promoter and is only activated in myeloid cells;

[0011] S2: Insert the chimeric promoter into the destination vector;

[0012] S3: synthetic guide RNA (sgRNA);

[0013] S4: inserting the guide RNA into the target vector containing the chimeric promoter obtained in step S2 to obtain a recombinant plasmid;

[0014] S5: Transfection of recombinant plasmid.

[0015] Through the above steps, the original promoter on the Cas9 protein in the CRISPR-CAS system is replaced with a chimeric promoter that is only expressed in myeloid cells, allowing gene editing to occur in myeloid cells in a targeted manner.

[0016] Furthermore, the sequence of the chimeric promoter is SEQ ID NO.1 or a sequence having more than 90% homology with SEQ ID NO.1, or a complementary sequence thereof.

[0017] Furthermore, in step S3, primer 1 and primer 2 are used to synthesize the guide RNA, the sequence of primer 1 is SEQ ID NO.2, and the sequence of primer 2 is SEQ ID NO.3.

[0018] Preferably, step S1 further includes:

[0019] After the chimeric promoter was synthesized, it was inserted into the cloning vector pUC57 using EcoR I and Age I as restriction sites at both ends.

[0020] Preferably, the target vector in step S2 is LentiCRISPR V2, and the chimeric promoter is inserted into LentiCRISPR V2.

[0021] Preferably, the step of obtaining the recombinant plasmid in step S4 comprises:

[0022] S4-1: Use EcoR I and AgeI as restriction sites at both ends to digest pUC57 carrying the chimeric promoter, and use EcoR I and AgeI as restriction sites at both ends to digest LentiCRISPR V2; at this time, a chimeric promoter fragment and a LentiCRISPR V2 linear vector will be obtained.

[0023] Preferably, the following steps are further performed after step S4-1:

[0024] S4-2: Ligation of the chimeric promoter into LentiCRISPR V2 using T4 ligase.

[0025] Preferably, in step S5, the recombinant plasmid is transfected with a lentivirus.

[0026] Lentiviral cell transfection has the advantages of strong specificity, high efficiency and stable transfection effect, and can prepare a CRISPR-CAS system with excellent characteristics.

[0027] Preferably, in step S5, psPax2 and pMD2.G are used to package lentivirus.

[0028] The second aspect of the present invention is to provide a CRISPR-CAS system for targeted myeloid cell gene editing, which is prepared by the above-mentioned construction method.

[0029] The beneficial effects of the technical solution provided by this application include:

[0030] The present application provides a CRISPR-CAS system for gene editing targeting myeloid cells and a method for constructing the same. Since the promoter of the Cas9 nuclease in the CRISPR / Cas9 system is replaced with a chimeric promoter specifically recognized by myeloid cells, the type II Cas9 nuclease with cutting function is expressed only in myeloid cells. Therefore, the implementation of this method can limit the expression of Cas9 to myeloid cells, so that the CRISPR / Cas9 system only performs shear repair of the required genes on myeloid cells, thereby achieving the effect of specific gene editing. In terms of application, this method can be used to study the role of different genes in myeloid cells regulating the tumor microenvironment, and by targeting tumor-associated myeloid cells, the purpose of changing the immunosuppressive microenvironment and reshaping the body's anti-tumor immune response can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0032] Figure 1 A flowchart of a method for constructing a CRISPR-CAS system for targeted myeloid cell gene editing provided in an embodiment of the present application.

[0033] Figure 2 This is a graph showing the results of a B16F10-Atg5.KO experiment using Western Blot to detect the knockout effect of a target gene provided in an example of the present application.

[0034] Figure 3 Schematic diagram of a plasmid containing a chimeric promoter and sgRNA provided in the examples of the present application. DETAILED DESCRIPTION

[0035] The technical solution of this patent will be further described in detail below in conjunction with specific implementation methods. It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by ordinary technicians in the technical field to which this application belongs.

[0036] In the field of cancer treatment, myeloid cells are important immune cells, and understanding their immune molecular mechanisms is of great significance for cancer treatment. However, there is currently a lack of effective means for targeted treatment of myeloid cells. The most rapidly developing CRISPR / Cas gene editing technology cannot achieve targeted gene editing, resulting in poor clinical efficacy and prone to side effects; the original promoter can activate the expression of Cas9 in eukaryotic cells, but does not have cell-level selectivity. This has created certain obstacles for scientific research and disease treatment in the field of targeting myeloid cells.

[0037] In order to solve the above problems, the embodiments of the present application provide a CRISPR-CAS system for gene editing targeting myeloid cells and a method for constructing the same. By improving the existing gene editing technology, the expression of Cas9 in the CRISPR / Cas9 system is controlled to occur in myeloid cells, so that the target gene editing is only targeted at myeloid cells for gene shear repair, achieving the effect of specific gene knockout in myeloid cells.

[0038] This system can be used to study the role of different genes in myeloid cells regulating the tumor microenvironment; this system can achieve targeted gene knockout in tumor-related myeloid cells to change the immunosuppressive microenvironment in tumor patients and achieve the purpose of reshaping the body's anti-tumor immune response.

[0039] The technical solution of this patent will be further described in detail below in conjunction with specific implementation methods. Among them, the methods used in the embodiments are all conventional methods unless otherwise specified.

[0040] Example 1: Synthesis of chimeric promoters

[0041] The CRISPR / Cas9 system in this embodiment includes discontinuous repeat sequences, spacer sequences of similar length, leader sequences, and genes encoding CAS-related proteins; and, in the CRISPR / Cas9 system in this embodiment, the original promoter on Cas9 is replaced by a chimeric promoter, which is only activated in myeloid cells; the sequence of the chimeric promoter is SEQ ID NO.1 or a sequence with more than 90% homology to SEQ ID NO.1 (preferably 92%, more preferably 95% homology, and further preferably 98% homology), or its complementary sequence; the CRISPR / Cas9 system in this embodiment also includes a guide RNA (single guide RNA, sgRNA).

[0042] The original promoter in Cas9 can initiate the expression of Cas9 in eukaryotic cells and does not have cell-level selectivity. In this implementation, the promoter of the type II Cas9 nuclease in the CRISPR / Cas9 system is replaced with a chimeric promoter. Since the chimeric promoter is specifically expressed in myeloid cells, the type II Cas9 nuclease with cutting function is specifically expressed in myeloid cells, thereby achieving cell selectivity for gene editing.

[0043] The sequence of this chimeric promoter is mainly created by fusing multiple Cathepsin G and the shortest sequence at the 5' end of c-Fes transcription start. Cathepsin G is a protein specifically expressed by myeloid cells. Studies have shown that approximately 360 bases upstream of the transcription start of the Cathepsin G gene play a key role in its expression. This Cathepsin G transcription start sequence can be used to determine the expression of luciferase specifically in myeloid cells. Further studies have found that within 360 bases upstream of the Cathepsin G transcription start point, there is a proximal promoter, which is mainly composed of PU.1, C-myb, C / EBP-a binding sites and a GC-rich sequence.

[0044] In addition, C / EBP binding sites, C-myb binding sites, and GC-rich sequences showed obvious functional synergy, enabling the promoter to enhance expression in myeloid cells.

[0045] The protein product of the proto-oncogene c-Fes is closely related to the normal development of myeloid cells (such as macrophages and neutrophils). The 151 base sequences upstream of the 5' end of the c-Fes gene coding region contain a powerful myeloid cell-specific promoter with binding sites for transcription factors such as Sp1, PU.1 or Elf-1. Since PU.1 and C / EBP-a jointly regulate the development of myeloid cells, this enables the chimeric promoter to limit the specific initiation of Cas9 gene expression in myeloid cells.

[0046] The chimeric promoter was delivered to a biological company for synthesis, pUC57 was selected as the vector, and EcoR I and Age I were selected as the restriction sites at both ends to be inserted into pUC57.

[0047] The sequence of the chimeric promoter is as follows:

[0048]

[0049]

[0050] See also Figure 1 , which is a flow chart of a method for constructing a CRISPR-CAS system for targeted myeloid cell gene editing provided in an embodiment of the present application; first, a chimeric promoter is chemically synthesized, and the original promoter in Cas9 is replaced with a chimeric promoter; then sgRNA is synthesized and inserted into a target vector containing a chimeric promoter using a molecular cloning experiment; then lentivirus is transfected, and purification and concentration are performed.

[0051] Example 2: Replacement of the original Cas9 promoter in the plasmid

[0052] Using molecular cloning techniques, the chimeric promoter was inserted into LentiCRISPR V2.

[0053] In this example, pUC57 and LentiCRISPR V2 loaded with the chimeric promoter were digested with EcoR I and AgeI to obtain the chimeric promoter target fragment and LentiCRISPR V2 linear vector, respectively; and the chimeric promoter was ligated into the target vector using T4 ligase.

[0054] In this example, the original Cas9 promoter in the plasmid was replaced with a chimeric promoter using the above method.

[0055] Example 3: Construction of targeted gene knockout plasmid

[0056] In this example, sgRNA was designed using an online sgRNA design website (Broad Institute GPP), and then sgRNA was synthesized using PCR technology. Then, conventional molecular cloning experiments were used to insert sgRNA into a LentiCRISPR V2 plasmid containing a chimeric promoter.

[0057] The specific steps include:

[0058] (1) Design of sgRNA

[0059] sgRNA was screened through the website (Broad Institute GPP).

[0060] (2) Synthesize sgRNA

[0061] The selected sgRNA was designed according to the following method and synthesized by the biotechnology company GENEWIZ. sgRNA was synthesized using primer 1, oligo 1: 5′-CACC (SEQ ID NO.2) and primer 2, oligo 2: 5′-AAAC (SEQ ID NO.3). Oligo 1→5'-CACCGNNNNNNNNNNNNNNNNNNNN-3'

[0062] 3'-CNNNNNNNNNNNNNNNNNNNNCAAA-5'←Oligo 2

[0063] (3) PCR annealing reaction system

[0064] Dissolve oligo 1 and oligo 2 into 100 μM using enzyme-free water. The annealing reaction system is as follows:

[0065] Annealing reaction system

[0066]

[0067] Place the annealing reaction system into the PCR instrument and perform annealing according to the following procedure:

[0068] Annealing procedure

[0069]

[0070] (4) LentiCRISPR V2 vector digestion

[0071] Enzyme digestion system

[0072]

[0073] The reaction system was then placed in a water bath at 65 °C for 2 h;

[0074] The 1.28KD band was recovered by 1% agarose gel electrophoresis. (5) LentiCRISPR V2 vector ligation

[0075] Connection system

[0076]

[0077]

[0078] Place at room temperature for two hours and inactivate at 65°C for ten minutes.

[0079] (6) Conversion

[0080] Add 100 μl of competent E. coli to the ligation system and mix well. Heat shock at 42°C for 60 seconds, add 500 μl of LB medium without resistance, shake for 1 hour and then apply.

[0081] (7) Single clone picking and identification

[0082] A single clone colony was picked and placed in LB medium with ampicillin resistance, shaken for 16 hours, and the plasmid was extracted and Sanger sequencing was performed.

[0083] This example uses the above method to construct a recombinant vector for targeted gene knockout. The schematic diagram of the obtained recombinant vector is as follows: Figure 3 As shown, Figure 3 Schematic diagram of the plasmid containing the chimeric promoter and sgRNA.

[0084] Example 4: Lentivirus packaging

[0085] In this embodiment, a lentiviral packaging method is used for transfection, and the lentivirus is packaged using lentiviral packaging auxiliary plasmids psPax2 and pMD2.G, and then purified and concentrated.

[0086] psPax2 is a lentiviral packaging auxiliary plasmid, carrying viral gag, pol, rev and tat genes. psPax2 and pMD2.G constitute a lentiviral packaging system for packaging lentiviral vector plasmids. These three plasmids form a three-plasmid system.

[0087] These three plasmids are co-transfected into 293T and other cells to package recombinant lentivirus, and the target cells are infected with this lentivirus to achieve functions such as gene knockout, knockdown and overexpression.

[0088] Finally, the lentivirus was concentrated and purified, and the virus titer was determined.

[0089] Example 5: In vitro targeting validation

[0090] In this example, different immortalized immune cell lines including lymphoid cell line E14 and myeloid cell line Raw264.7 were infected with lentivirus, and then the expression of Cas9 mRNA therein was determined.

[0091] A cell line co-expressing the target gene knockout was constructed through drug screening, and Western blot was used to verify whether the target gene knockout effect was limited to the myeloid cell Raw264.7 rather than the lymphoid cell El4.

[0092] In this example, a puro-resistant Atg5 stable knockout B16F10 cell line was constructed for in vitro targeting validation. After 5 generations of puromycin gradient screening, the target gene was successfully knocked out by Western Blot. The experimental results are as follows: Figure 2 As shown, Figure 2 This is a diagram of the B16F10-Atg5.KO experimental results using Western Blot to detect the knockout effect of the target gene.

[0093] Example 6: In vivo targeting validation

[0094] The lentivirus targeting a specific target gene was injected intraperitoneally into mice. Three days later, the macrophages and T lymphocytes in the peritoneal cavity were isolated to detect the degree of knockdown of the target gene and to analyze whether the gene knockout was biased towards myeloid cells.

[0095] On this basis, the function of myeloid cell gene knockout was verified using peritoneal tumor-bearing mice to test whether the gene knockout only targets myeloid cells and produces corresponding anti-tumor effects.

[0096] In this example, lentivirus was injected into the peritoneal cavity of mice to analyze the gene knockout effects on myeloid cells and non-myeloid cells.

[0097] In summary, the present invention is a CRISPR-CAS system for targeted myeloid cell gene editing and its construction method, which can achieve the purpose of knocking out specific genes in myeloid cells, while not knocking out non-myeloid cells, thereby avoiding the adverse effects caused by non-myeloid cell gene knockout. This method is also suitable for basic and applied research on myeloid cells, and will be beneficial to the treatment and research of myeloid cell-related immune diseases, such as improving the tumor immunosuppressive microenvironment.

[0098] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A method for constructing a CRISPR-CAS system for targeted myeloid cell gene editing, characterized in that: The following steps are involved: S1: Synthesize a chimeric promoter, which is an improved Cas9 promoter and is activated only in myeloid cells; S2: inserting the chimeric promoter into a target vector; S3: synthetic guide RNA; S4: inserting the guide RNA into the target vector containing the chimeric promoter obtained in step S2 to obtain a recombinant plasmid; S5: transfecting the recombinant plasmid; The sequence of the chimeric promoter is SEQ ID NO.

1.

2. The construction method according to claim 1, characterized in that: In step S3, primer 1 and primer 2 are used to synthesize the guide RNA, the sequence of primer 1 is SEQ ID NO.2, and the sequence of primer 2 is SEQ ID NO.

3.

3. The construction method according to claim 1, characterized in that: The step S1 further comprises: After synthesizing the chimeric promoter, use EcoR I and Age I The chimeric promoter was inserted into the cloning vector pUC57 as restriction sites at both ends.

4. The construction method according to claim 3, characterized in that: The target vector in step S2 is LentiCRISPR V2, and the chimeric promoter is inserted into LentiCRISPR V2.

5. The construction method according to claim 4, characterized in that: The step of obtaining the recombinant plasmid in step S4 comprises: S4-1: Use EcoR I and AgeI pUC57 carrying the chimeric promoter was digested with enzymes at both ends. EcoR I and AgeI LentiCRISPR V2 is digested with enzymes at both ends; at this time, the chimeric promoter fragment and the LentiCRISPR V2 linear vector are obtained.

6. The construction method according to claim 5, characterized in that: After step S4-1, the following steps are also included: S4-2: Ligate the chimeric promoter into LentiCRISPR V2 using T4 ligase.

7. The construction method according to claim 1, characterized in that: In step S5, the recombinant plasmid is transfected with a lentivirus.

8. The construction method according to claim 7, characterized in that: The step S5 uses psPax2 and pMD2.G to package the lentivirus.

9. A CRISPR-CAS system for targeted myeloid cell gene editing, wherein the system is prepared by any construction method described in claims 1-8.

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