CN116218911A - CRISPR-CAS system for targeted marrow cell gene editing and construction method thereof - Google Patents

Abstract

The invention relates to a CRISPR-CAS system for targeted marrow cell gene editing and a construction method thereof. The scheme utilizes molecular biology technology to replace a promoter of Cas9 nuclease in a CRISPR/Cas9 system with a chimeric promoter specifically identified by myeloid cells, so that the Cas9 nuclease with a cleavage function is expressed only in the myeloid cells; and designing a guide RNA to construct a CRISPR/Cas9 plasmid targeting a target gene, and then packaging and producing by lentiviral transfection. The scheme can be used for researching the effect of different genes in the marrow cell regulating tumor microenvironment.

Description

CRISPR-CAS system for targeted marrow cell gene editing and construction method thereof

Technical Field

The invention relates to the technical field of gene editing, in particular to a CRISPR-CAS system for targeted marrow cell gene editing and a construction method thereof.

Background

During hematopoiesis in humans, myeloid cells are derived from myeloid progenitor Cells (CMP), which are common in bone marrow. This lineage, including monocytes, granulocytes, erythrocytes and platelets, is a major component of the natural immune system and serves as a first line of defense against infection. Myeloid cells also have a great impact on tumor development. Tumor-associated myeloid lineage cells (TAMCs) are heterogeneous, producing a distinct and even opposite effect on tumor cells and Tumor Microenvironment (TME). In addition, myeloid lineage cells play a key role in regulating lymphocyte behavior, leading to immunostimulatory or immunosuppressive TMEs, thereby inhibiting or promoting tumor progression.

The most studied TAMCs include monocytes, tumor-associated macrophages (TAMs), dendritic Cells (DCs), tumor-associated neutrophils (TANs), and myeloid-derived suppressor cells (MDSCs). They are involved in pleiotropic processes including tumor cell growth, survival, differentiation, dissemination and metastasis, angiogenesis, TME remodeling, immunomodulation and response to cancer treatment. Understanding the role and mechanism of TAMCs in tumors would help to discover new therapeutic approaches.

The nobel chemical prize in 2020 awards two scientists, emmoneylor Sha Erpang swiftly and Jane Friedel, to bring out their contributions in CRISPR/Cas9 gene editing technology. CRISPR-Cas9 is a third generation gene editing technology which is introduced by the gene editing technologies such as ZFN, TALENs and the like, and is the most mainstream gene editing system at present because the CRISPR-Cas9 has the advantages of high efficiency, simple operation, low cost, easy operation and the like, and the CRISPR-Cas9 is applied worldwide in a few years.

CRISPR-Cas9 gene editing techniques achieve knockout or knock-in of a gene fragment of interest by combining a Cas9 protein with an artificially designed guide RNA (sgRNA), followed by an autonomously designed sgRNA specific targeting target sequence, thereby double-strand breaking the DNA of the gene of interest, the broken double-stranded DNA being repaired using a non-homologous end joining (NHEJ) or Homology Directed Repair (HDR) DNA repair mechanism.

But currently there is a lack of effective means for targeted therapy of myeloid cells. The existing CRISPR-Cas9 system has no cell-level selectivity, has insufficient targeting specificity, is difficult to realize gene knockout in a specific area, and causes a certain obstacle for scientific research work and disease treatment in the related field of targeted myeloid cells.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, and provides a CRISPR-CAS system for targeted marrow cell gene editing and a construction method thereof, so as to solve the problem of insufficient targeting specificity of the CRISPR/Cas9 system in the related art.

In order to achieve the above purpose, the present invention provides the following technical solutions:

the first aspect of the invention provides a construction method of a CRISPR-CAS system for targeted myeloid cell gene editing, comprising the following steps:

s1: synthesizing a chimeric promoter, wherein the chimeric promoter is an improved Cas9 promoter, and the chimeric promoter is only started in myeloid cells;

s2: inserting the chimeric promoter into a target vector;

s3: synthesizing guide RNA (sgRNA);

s4: inserting the guide RNA into the target vector containing the chimeric promoter obtained in the step S2 to obtain a recombinant plasmid;

s5: and (3) transfecting the recombinant plasmid.

By the above steps, the original promoter on CAS9 protein in CRISPR-CAS system is replaced with chimeric promoter expressed only in myeloid cells, enabling gene editing to occur directionally in myeloid cells.

Further, 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.

Further, in step S3, a guide RNA is synthesized using primer 1 and primer 2, the sequence of primer 1 is SEQ ID NO.2, and the sequence of primer 2 is SEQ ID NO.3.

Preferably, step S1 further comprises:

after synthesis of the chimeric promoter, the chimeric promoter was inserted into the cloning vector pUC57 using EcoR I and Age I as both cleavage sites.

Preferably, the targeting vector in step S2 is a pantigrispr V2, and the chimeric promoter is inserted into the pantigrispr V2.

Preferably, the step of obtaining a recombinant plasmid in step S4 comprises:

s4-1: pUC57 loaded with the chimeric promoter was digested with EcoR I and AgeI as both cleavage sites, while LeniCRISPR V2 was digested with EcoR I and AgeI as both cleavage sites; chimeric promoter fragments and LentidISPR V2 linear vectors will be obtained at this point.

Preferably, the following steps are further provided after step S4-1:

s4-2: the chimeric promoter was ligated into LentiCRISPR V2 by T4 ligase.

Preferably, the recombinant plasmid is transfected with lentivirus in step S5.

The lentivirus transfected cells have the advantages of strong specificity, high efficiency, stable transfection effect and the like, and can prepare the CRISPR-CAS system with excellent characteristics.

Preferably, the lentiviruses are packaged in step S5 using psPax2 and pmd2. G.

In a second aspect, the present invention provides a CRISPR-CAS system for targeted editing of myeloid lineage cell genes, the system being prepared by the above-described construction method.

The beneficial effects that technical scheme that this application provided brought include:

the application provides a CRISPR-CAS system for targeted gene editing of myeloid cells and a construction method thereof, wherein, the promoter of Cas9 nuclease in the CRISPR-Cas9 system is replaced by a chimeric promoter specifically identified by the myeloid cells, so that the type II Cas9 nuclease with a cutting function is expressed only in the myeloid cells. Therefore, by implementing the method, the Cas9 can be limited in the myeloid cells to start expression, so that the CRISPR/Cas9 system only aims at the myeloid cells to carry out the shearing repair of the required genes, thereby achieving the effect of editing specific genes. In application, the method can be used for researching the effect of different genes in regulating tumor microenvironment by the myeloid cells, and achieving the purposes of changing the immunosuppressive microenvironment and remodelling the anti-tumor immune response of the organism by targeting the tumor-related myeloid cells.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a flow chart of a method for constructing a CRISPR-CAS system for targeted myeloid cell gene editing provided in embodiments of the present application.

FIG. 2 is a graph of B16F10-Atg5.KO experimental results of detecting target gene knockout effect by using Western Blot provided in the example of the present application.

FIG. 3 is a schematic diagram of a plasmid containing a chimeric promoter and sgRNA as provided in the examples of the present application.

Detailed Description

The technical solutions of the present patent will be described in further detail below with reference to specific embodiments, and it should be noted that the following detailed description is exemplary, and is intended to provide further description of the present application. 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.

In the field of cancer treatment, myeloid cells are important immune cells, and understanding the molecular mechanism of their immunity is important for cancer treatment, but there is currently a lack of effective means for targeted therapy of myeloid cells. The most rapidly developed CRISPR/Cas gene editing technology at present has poor clinical curative effect and is easy to produce side effect because of being incapable of realizing directed gene editing; the original promoter can start the expression of Cas9 in eukaryotic cells and has no cell-level selectivity. This presents a barrier to research work and disease treatment in the area of targeting myeloid lineage cells.

In order to solve the problems, the embodiment of the application provides a CRISPR-CAS system for targeting the gene editing of myeloid cells and a construction method thereof. By improving the existing gene editing technology, the expression of Cas9 in the CRISPR/Cas9 system is controlled to occur in the myeloid cells, so that the target gene editing is only carried out on the myeloid cells for gene shearing repair, and the effect of specific gene knockout in the myeloid cells is achieved.

The system can be used for researching the effect of different genes in the marrow cell regulating tumor microenvironment; the system can realize gene knockout of targeted tumor-related myeloid cells so as to change the immune suppression microenvironment in a tumor patient and achieve the aim of remodelling the anti-tumor immune response of the organism.

The technical scheme of the present patent will be described in further detail below in connection with the specific embodiments. The methods used in the examples are conventional methods unless otherwise specified.

Example 1: synthesis of chimeric promoters

The CRISPR/Cas9 system in this approach includes discontinuous repeats, similarly long spacer sequences, leader sequences, and genes encoding Cas-related proteins; in addition, the original promoter on Cas9 in the CRISPR/Cas9 system is replaced by a chimeric promoter, and the chimeric promoter is only started in myeloid cells; the chimeric promoter has the sequence of SEQ ID NO.1 or a sequence with more than 90% homology (preferably 92%, more preferably 95%, even more preferably 98% homology) with SEQ ID NO.1, or the complementary sequence thereof; the CRISPR/Cas9 system of the present approach further comprises a guide RNA (sgRNA).

The original promoter in the Cas9 can start the expression of the Cas9 in eukaryotic cells, and has no cell-level selectivity. In this embodiment, the promoter of the type ii Cas9 nuclease in the CRISPR/Cas9 system is replaced with a chimeric promoter, and since the chimeric promoter is specifically expressed in myeloid cells, the type ii Cas9 nuclease with cleavage function is specifically expressed in myeloid cells, thereby realizing the cell selectivity of gene editing.

The sequence of the chimeric promoter is mainly created by fusion of a plurality of Cathepsin G and c-Fes transcription initiation 5' -terminal shortest sequences. Cathepsin G is a myeloid cell-specific expression protein, and studies have shown that Cathepsin G gene transcription initiates upstream approximately 360 base pairs for its expression to play a critical role. The use of this Cathepsin G transcription initiation sequence enables cell-specific determination of luciferase expression in myeloid lineage. Further studies have found that inclusion of a proximal promoter within 360 bases upstream of the transcription initiation site of Cathepsin G consists essentially of the binding site for PU.1, C-myb, C/EBP-a and a GC-rich sequence.

In addition, the C/EBP binding site, the C-myb binding site and the GC-rich sequence exhibit a distinct functional synergy, allowing the promoter to enhance expression in myeloid cells.

The protein product of protooncogene c-Fes is closely related to the normal development of myeloid cells (e.g., macrophages and neutrophils). The upstream 151 base sequences of the 5' end of the coding region of the c-Fes gene contain a strong 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 together regulate the development of myeloid cells, this enables the chimeric promoter to define specific initiation of Cas9 gene expression in myeloid cells.

The chimeric promoter was synthesized by biosystems, pUC57 was selected as a vector, ecoR I and Age I were selected as both ends cleavage sites, and pUC57 was inserted.

The sequence of the chimeric promoter is as follows:

referring to fig. 1, a flowchart of a CRISPR-CAS system construction method for targeted myeloid cell gene editing is provided in an embodiment of the present application; firstly, chemically synthesizing a chimeric promoter, and replacing an original promoter in the Cas9 with the chimeric promoter; subsequently synthesizing sgRNA, and inserting the sgRNA into a target vector containing a chimeric promoter by using a molecular cloning experiment; then lentivirus is transfected, and purification and concentration are performed.

Example 2: promoter of original Cas9 in replacement plasmid

The chimeric promoter was inserted into LentiCRISPR V2 using molecular cloning procedures.

In the embodiment, pUC57 loaded with the chimeric promoter and LeniCRISPR V2 are respectively subjected to enzyme digestion by utilizing EcoR I and AgeI double enzyme digestion to respectively obtain a fragment of the chimeric promoter and a LeniCRISPR V2 linear vector; and then the chimeric promoter is linked into the target vector by using T4 ligase.

This example replaces the original Cas9 promoter in the plasmid with the chimeric promoter in the manner described above.

Example 3: construction of targeting Gene knockout plasmid

In this example, the sgrnas were designed using the website for online design of the sgrnas (Broad Institute GPP), then were synthesized using PCR techniques, and then were inserted into the Lenticrispr V2 plasmid containing the chimeric promoter using conventional molecular cloning experiments.

The method comprises the following specific steps:

(1) Design of sgRNA

Sgrnas were screened through the website (Broad Institute GPP).

(2) Synthesis of sgRNA

The screened sgrnas were sequenced as follows and submitted to gold-only synthesis by biotechnology company. Primer 1, oligo 1, was used: 5' -CACC (SEQ ID NO. 2) with primer 2, oligo 2:5' -AAAC (SEQ ID NO. 3) synthesizes sgRNA. Oligo1→5'-CACCGNNNNNNNNNNNNNNNNNNNN-3'

3’-CNNNNNNNNNNNNNNNNNNNNCAAA-5’←Oligo 2

(3) PCR annealing reaction system

The oligo 1 and oligo 2 were dissolved to 100. Mu.M using enzyme-free water, and the annealing reaction system was as follows:

annealing reaction system

The annealing reaction system is put into a PCR instrument and annealed according to the following procedures:

annealing procedure

(4) Lenigirspr V2 vector cleavage

Enzyme cutting system

Then the reaction system is water-bath for 2 hours under the condition of 65 ℃;

gel recovery was performed by 1% agarose gel electrophoresis for the 1.28KD bands. (5) Lenigirspr V2 vector ligation

Connection system

Left at room temperature for two hours and inactivated at 65℃for ten minutes.

(6) Transformation

100. Mu.l of competent E.coli was added to the ligation system and mixed well. After heat shock at 42℃for 60 seconds, 500. Mu.l of the non-resistant LB medium was added, and the mixture was shaken for 1 hour and then spread.

(7) Monoclonal picking and identification

Monoclonal colonies were picked, placed in LB medium with ampicillin resistance, shaken for 16 hours, plasmids were extracted, and Sanger sequencing was performed.

The embodiment constructs the recombinant vector targeting gene knockout in the above manner. The obtained recombinant vector is schematically shown in FIG. 3, and FIG. 3 is a schematic diagram of a plasmid containing the chimeric promoter and sgRNA.

Example 4: lentivirus package

In this example, lentivirus packaging was used to package lentiviruses using lentivirus packaging helper plasmids psPax2 and pMD2.G, and purification and concentration were performed.

psPax2 is a lentiviral packaging helper plasmid carrying the viral gag, pol, rev and tat genes. The psPax2 and pMD2.G constitute a lentiviral packaging system for packaging lentiviral vector plasmids, and these 3 plasmids constitute a three plasmid system.

The 3 plasmids are transfected together into 293T cells and the like to package recombinant lentiviruses, and the target cells are infected by the lentiviruses, so that the functions of gene knockout, knockout and over-expression and the like can be realized.

Finally, concentration and purification of the lentivirus are carried out, and the virus titer is measured.

Example 5: in vitro targeting validation

The lentivirus was infected with different immortalized immune cell lines including lymphoid cell line El4 and myeloid cell line raw264.7 in this example, and the expression of Cas9 mRNA therein was then determined.

And constructing a cell line for coexpression target gene knockout through drug screening, and verifying whether the target gene knockout effect is limited in a myeloid cell Raw264.7 instead of a lymphoid cell El4 by adopting Western blot.

In this example, the constructed Atg5 stable knockout B16F10 cell line with puro resistance is used for in vitro targeting verification, puromycin (puromycin) gradient screening is carried out for 5 generations, the target gene is detected to be knocked out successfully by Western Blot, the experimental effect is shown in FIG. 2, and FIG. 2 is a graph of B16F10-Atg5.KO experimental results of detecting the target gene knocked out effect by Western Blot.

Example 6: in vivo targeting validation

Lentiviruses targeting specific genes of interest were intraperitoneally injected into mice, macrophages and T lymphocytes in their peritoneal cavity were isolated three days later, the extent of gene knockdown of interest was detected, and whether gene knockdown was biased into myeloid cells was analyzed.

On the basis, the function verification after the gene knockout of the marrow cells is carried out by using the abdominal cavity tumor-bearing mice, and whether the gene knockout is only aimed at the marrow cells is checked, and the corresponding anti-tumor effect is generated.

In this example, lentiviruses were injected into the abdominal cavity of mice and the gene knockout effect of myeloid cells and non-myeloid cells was analyzed.

In summary, the CRISPR-CAS system for targeted marrow cell gene editing and the construction method thereof can achieve the purpose of knocking out specific genes in marrow cells, and simultaneously does not generate knocking-out effect on non-marrow cells, thereby avoiding adverse effects caused by gene knocking-out of the non-marrow cells. The method is also suitable for the research of the foundation and application of the marrow cell, and is beneficial to the treatment and research of marrow cell related immune diseases, such as improving the tumor immunosuppression microenvironment.

The foregoing is merely a specific embodiment of the application to enable one skilled in the art to understand or practice the application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. The construction method of the CRISPR-CAS system for targeted myeloid cell gene editing is characterized by comprising the following steps:

s1: synthesizing a chimeric promoter that is an improved Cas9 promoter that is only promoted in myeloid lineage cells;

s2: inserting the chimeric promoter into a target vector;

s3: synthesizing a guide RNA;

s4: inserting the guide RNA into the target vector containing the chimeric promoter obtained in the step S2 to obtain a recombinant plasmid;

s5: transfecting the recombinant plasmid.

2. The method of construction of claim 1, wherein: the chimeric promoter has a sequence of SEQ ID NO.1 or a sequence with more than 90 percent of homology with SEQ ID NO.1 or a complementary sequence thereof.

3. The method of construction according to claim 1, wherein the guide RNA is synthesized in step S3 using primer 1 and primer 2, the primer 1 having the sequence of SEQ ID NO.2 and the primer 2 having the sequence of SEQ ID NO.3.

4. The construction method according to claim 1, wherein the step S1 further comprises:

after synthesis of the chimeric promoter, the chimeric promoter was inserted into the cloning vector pUC57 using EcoR I and Age I as both ends cleavage sites.

5. The construction method according to claim 4, wherein the targeting vector in the step S2 is LentiCRISPR V2, and the chimeric promoter is inserted into the LentiCRISPR V2.

6. The construction method according to claim 5, wherein the step of obtaining a recombinant plasmid in the step S4 comprises:

s4-1: pUC57 loaded with the chimeric promoter was digested with EcoR I and AgeI as both cleavage sites, while LeniCRISPR V2 was digested with EcoR I and AgeI as both cleavage sites; the chimeric promoter fragment and the LentiCRISPR V2 linear vector will be obtained at this time.

7. The construction method according to claim 6, further comprising the following steps after said step S4-1:

s4-2: the chimeric promoter was ligated into LenkiCRISPR V2 by T4 ligase.

8. The construction method according to claim 1, wherein the recombinant plasmid is transfected with lentivirus in step S5.

9. The method of construction according to claim 9, wherein step S5 packages lentiviruses using psPax2 and pmd2. G.

10. A CRISPR-CAS system for targeted myeloid cell gene editing, said system being made by any of the construction methods of claims 1-9.

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