High-throughput method for screening regulatory factors for heterogeneity of Th17 cells by using CRISPR (clustered regularly interspaced short palindromic repeats)

Abstract

The invention discloses a high-throughput method for screening a regulatory factor of Th17 cell heterogeneity by using CRISPR (clustered regularly interspaced short palindromic repeats), and relates to the technical field of CRISPR screening, the method comprises the following steps: constructing a CRISPR library; carrying out Il17aCreRosa26YFP / Cas92D2T cell transduction, so as to obtain an Il17aCreRosa26YFP cell; carrying out in-vivo transplantation and constructing an EAE model; and single cell transcriptome sequencing and single cell CRISPR screening are carried out. The screening efficiency is remarkably improved by utilizing the high-throughput capacity of a CRISPR-Cas9 technology, and the detection possibility and accuracy of a heterogeneity regulation transcription factor of the Th17 cell are improved: the expression of YFP and CAS9 is controlled by utilizing an Il17aCreRosa26YFP / Cas92D2 cell and the expression of IL-17a-Cre, and YFP < + > and Thy1.1 < + > cells are determined to be sgRNA-transduced Th17 cells, so that only genes in the YFP < + >, Thy1.1 < + >, Th17 or ex-Th17 cells can be edited, and the screening efficiency is improved. And sgRNA libraries in different ranges can be selected, the whole gene or transcription factor range can be covered, an in-vivo experimental model is closer to a real immune environment, the result has biological significance, and a brand-new target spot and an intervention strategy can be provided for diagnosis and treatment of autoimmune diseases.

Description

Technical Field

[0001] The present invention relates to the field of CRISPR screening technology, and in particular to a high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR. Background Art

[0002] T helper 17 (Th17) cells play an important role in the immune system. They can protect barrier tissues by secreting IL-17, and can also induce autoimmune diseases such as multiple sclerosis, rheumatoid arthritis and psoriasis through inflammatory programs under certain conditions. Therefore, due to differences in the environment and regulatory molecules, Th17 cells show a great degree of heterogeneity and plasticity in metabolic activity, transcription factor expression and functional programs. However, the intracellular mechanisms and regulatory molecules that control Th17 cells to enter different programs in vivo are not well understood.

[0003] Existing technologies mainly rely on in vitro culture models and single gene knockout analysis methods to study the decisive transcription factors of Th17 cells. These methods are usually inefficient and difficult to reflect the impact of the complex intercellular environment in the body on gene regulation. At the same time, the lack of effective high-throughput methods to systematically screen the key factors that determine the heterogeneity of Th17 cells has hindered the comprehensive understanding of the functional diversity of Th17 cells and the development of targeted treatment strategies for related diseases. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR, and the specific technical solution is as follows:

[0005] A high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR, comprising the following steps:

[0006] Step 1: CRISPR library construction;

[0007] Step 2: Il17a Cre Rosa26 YFP / Cas9 2D2 T cell transduction;

[0008] Step 3: in vivo transplantation and EAE model construction;

[0009] Step 4: Single-cell transcriptome sequencing and single-cell CRISPR screening.

[0010] Preferably, step 1 specifically includes: designing a sgRNA library covering the target gene for the entire transcription factor group of mice, and integrating the designed sgRNA library into a retroviral vector.

[0011] Preferably, the design of the sgRNA library covering the target gene specifically includes:

[0012] Select target gene set;

[0013] Use bioinformatics tools to design sgRNA sequences targeting target genes;

[0014] Synthesize sgRNA libraries by synthesizing short sequences.

[0015] Preferably, step 2 specifically includes the following sub-steps:

[0016] Step 21: Build Il17a Cre Rosa26 YFP / Cas9 2D2 mouse model;

[0017] Step 22: From Il17a Cre Rosa26 YFP / Cas9 Naive 2D2 cells were isolated from the spleen and peripheral lymph nodes of 2D2 mice;

[0018] Step 23: Activation and transduction of naive 2D2 cells;

[0019] Step 24: Screening transduced cells;

[0020] Step 25: Construction of transduced Th17 cell bank.

[0021] Preferably, step 23 specifically includes:

[0022] Naive 2D2 cells were activated with anti-CD3 and anti-CD28 for 30-35 h, and then the CRISPR library was transduced into the cells by viral infection;

[0023] The cells were cultured with IL-6, IL-23 and IL-1β for 2 to 4 days to induce their differentiation into Th17 cells.

[0024] Preferably, step 24 specifically comprises: purifying the transduced cells using a Thy1.1+ cell separation kit.

[0025] Preferably, step 3 specifically includes the following sub-steps:

[0026] Step 31: Sublethal radiation is given to recipient mice to remove their immune cells and provide an immune-tolerant environment for transplanted cells;

[0027] Step 32: Transplant 2D2 Th17 cells transformed with the CRISPR library into irradiated mice via intravenous injection;

[0028] Step 33: After transplantation, mice were immunized with MOG to induce EAE;

[0029] Step 34: 18 to 21 days after transplantation, collect transduced Thy1.1+, YFP+ cells from the spleen and submandibular lymph nodes of the mice.

[0030] Preferably:

[0031] The number of 2D2 Th17 cells transplanted in step 32 was (5±0.2)×10 6 indivual;

[0032] The number of YFP+ cells recovered in step 34 was (1.2±0.2)×10 6 indivual.

[0033] Preferably, step 4 specifically includes the following sub-steps:

[0034] Step 41: sorting out transduced YFP+ cells;

[0035] Step 42: Use the Chromium Next GEM Single Cell 3′ kit to prepare a single cell library for the sorted YFP+ cells;

[0036] Step 43: Sequencing the transcriptome library and CRISPR screening library respectively to obtain gene expression data and sgRNA editing information;

[0037] Step 44: Use Cell Ranger to align the single-cell transcriptome sequencing data with the mouse genome to generate a gene expression matrix for each cell;

[0038] Step 45: Align the sequencing reads of the CRISPR screening library with the previously transduced sgRNA library to determine the sgRNA corresponding to each cell;

[0039] Step 46: Use Seurat to analyze the filtered expression matrix and sgRNA reads for cell clustering, gene expression analysis, and integration of CRISPR screening data;

[0040] Step 47: Identify phenotypic changes or cell subsets associated with specific sgRNAs and screen candidate genes or regulatory factors associated with Th17 cell heterogeneity.

[0041] Preferably, step 47 further includes the following steps:

[0042] Step 48: Perform phenotypic experiments on the screened candidate genes or regulatory factors to verify the effect of the corresponding sgRNA on Th17 cell heterogeneity.

[0043] The high-throughput method provided by the present invention for screening regulatory factors of Th17 cell heterogeneity using CRISPR has the following beneficial effects:

[0044] 1. The high-throughput capability of CRISPR-Cas9 technology significantly improves screening efficiency and increases the possibility and accuracy of detecting Th17 cell heterogeneity-regulating transcription factors: using Il17a Cre Rosa26 YFP / Cas9 2D2 cells, in which the expression of IL-17a-Cre controls the expression of YFP and CAS9, determining that YFP+, Thy1.1+ cells are Th17 cells transduced with sgRNA, so that only genes in YFP+, Thy1.1+, Th17 or ex-Th17 cells will be edited, thereby improving the specificity and efficiency of the screening;

[0045] 2. Different ranges of sgRNA libraries can be selected to cover the entire gene or transcription factor range;

[0046] 3. The in vivo experimental model is closer to the real immune environment, and the results are more biologically meaningful;

[0047] 4. The fate-determining transcription factors of Th17 cell heterogeneity can be screened;

[0048] 5. It can provide new targets and intervention strategies for the diagnosis and treatment of autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0050] Figure 1 A technical flow chart of steps 1, 2 and 3 of a high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR provided in an embodiment of the present invention;

[0051] Figure 2 A technical flow chart of single-cell transcriptome sequencing in step 4 of a high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR provided in an embodiment of the present invention;

[0052] Figure 3 Schematic diagram for identifying the perturbation phenotype or cell subpopulation corresponding to the sgRNA;

[0053] Figure 4 Il17a Cre Rosa26 YFP / Cas9 Real-time expression ratio of IL-17a in 2D2 cells. DETAILED DESCRIPTION

[0054] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below. The description in this section is only exemplary and explanatory and should not have any limiting effect on the scope of protection of the present invention.

[0055] See also Figures 1 to 4 This embodiment provides a high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR, comprising the following steps:

[0056] Step 1: CRISPR library construction.

[0057] Step 2: Il17a Cre Rosa26 YFP / Cas9 2D2 T cell transduction.

[0058] Step 3: In vivo transplantation and EAE model establishment.

[0059] Step 4: Single-cell transcriptome sequencing and single-cell CRISPR screening.

[0060] Among them, in step 2, Il17a Cre Rosa26 YFP The YFP (yellow fluorescent protein) reporter gene in the mouse model is driven by the IL-17a-Cre specific promoter, which allows only IL-17-secreting Th17 cells to express YFP, which can very accurately mark Th17 cells. This marker helps to track and screen these specific cell types. YFP / Cas9 2D2 T cell transduction is further enhanced by the introduction of the Cas9 expression system, which enables efficient gene editing in Th17 cells and the screening of the functions of specific genes using CRISPR technology.

[0061] By editing genes in Th17 cells, we can clearly screen out factors that affect the heterogeneity, function and development of Th17 cells, thereby improving the specificity and efficiency of screening. This step enables the subsequent knockout or modification of specific genes through CRISPR technology to directly affect the characteristics of Th17 cells, providing a more reliable model for subsequent research.

[0062] In step 3, the EAE (experimental autoimmune encephalomyelitis) model is a classic animal model for studying multiple sclerosis and immune response. By transplanting transduced Th17 cells into mice, the functions of these cells in vivo can be simulated and studied, thereby revealing their role in immune diseases.

[0063] In the EAE model, observing the changes in Th17 cells in vivo and the impact of CRISPR-edited genes on the disease progression can help understand how specific genes regulate the function and heterogeneity of Th17 cells, further screen out key regulatory factors, and ensure the biological significance of the screened regulatory factors in the disease context.

[0064] In step 4, single-cell transcriptome technology enables the analysis of gene expression profiles at the level of individual cells. Through single-cell RNA sequencing, different subpopulations of Th17 cells can be identified, their molecular characteristics can be explored, and how these characteristics are affected by CRISPR-mediated gene editing. By combining with CRISPR screening, the knockout or editing of specific genes can be directly associated with functional changes in Th17 cells at the single-cell level. This high-throughput screening method makes it possible to simultaneously evaluate the role of multiple genes in the heterogeneity of Th17 cells.

[0065] Single-cell CRISPR screening can more accurately identify which genes are important for the development, differentiation, function or disease relevance of Th17 cells, providing a more refined and targeted screening method than traditional global transcriptome analysis.

[0066] The high-throughput method provided in this embodiment for screening regulatory factors of Th17 cell heterogeneity using CRISPR has the following advantages:

[0067] Beneficial effects:

[0068] 1. The high-throughput capability of CRISPR-Cas9 technology significantly improves screening efficiency and increases the possibility and accuracy of detecting Th17 cell heterogeneity-regulating transcription factors: using Il17a Cre Rosa26 YFP / Cas9 2D2 cells, in which the expression of IL-17a-Cre controls the expression of YFP and CAS9, and YFP+, Thy1.1+ cells are Th17 cells transduced with sgRNA, so only genes in YFP+, Thy1.1+, Th17 or ex-Th17 cells will be edited, thereby improving the specificity and efficiency of screening.

[0069] 2. Different ranges of sgRNA libraries can be selected to cover the entire gene or transcription factor range.

[0070] 3. The in vivo experimental model is closer to the real immune environment, and the results are more biologically meaningful.

[0071] 4. The fate-determining transcription factors of Th17 cell heterogeneity can be screened.

[0072] 5. It can provide new targets and intervention strategies for the diagnosis and treatment of autoimmune diseases.

[0073] Furthermore, step 1 specifically includes: designing a sgRNA library covering the target gene for the entire transcription factor group of mice, and integrating the designed sgRNA library into a retroviral vector.

[0074] Furthermore, designing a sgRNA library covering the target gene specifically includes:

[0075] Select target gene set.

[0076] Use bioinformatics tools to design sgRNA sequences targeting target genes.

[0077] Synthesize sgRNA libraries by synthesizing short sequences.

[0078] Furthermore, step 2 specifically includes the following sub-steps:

[0079] Step 21: Build Il17a Cre Rosa26 YFP / Cas9 2D2 mouse model.

[0080] Step 22: From Il17a Cre Rosa26 YFP / Cas9 Naive 2D2 cells were isolated from spleen and peripheral lymph nodes of 2D2 mice.

[0081] Step 23: Activation and transduction of naive 2D2 cells.

[0082] Step 24: Screening of transduced cells.

[0083] Step 25: Construction of transduced Th17 cell bank.

[0084] Furthermore, step 23 specifically includes:

[0085] Naive 2D2 cells were activated with anti-CD3 and anti-CD28 for 30-35 h, and then the CRISPR library was transduced into the cells by viral infection.

[0086] The cells were cultured with IL-6, IL-23 and IL-1β for 2 to 4 days to induce their differentiation into Th17 cells.

[0087] Furthermore, step 24 specifically includes: purifying the transduced cells using a Thy1.1+ cell separation kit.

[0088] Furthermore, step 3 specifically includes the following sub-steps:

[0089] Step 31: The recipient mice are irradiated at sublethal doses to remove their immune cells and provide an immune-tolerant environment for the transplanted cells.

[0090] Step 32: 2D2 Th17 cells transformed with the CRISPR library were transplanted into irradiated mice via intravenous injection.

[0091] Step 33: After transplantation, mice were induced to develop EAE by MOG immunization.

[0092] Step 34: 18 to 21 days after transplantation, collect transduced Thy1.1+, YFP+ cells from the spleen and submandibular lymph nodes of the mice.

[0093] Further:

[0094] The number of 2D2 Th17 cells transplanted in step 32 was (5±0.2)×10 6 indivual.

[0095] The number of YFP+ cells recovered in step 34 was (1.2±0.2)×10 6 indivual.

[0096] Furthermore, step 4 specifically includes the following sub-steps:

[0097] Step 41: Sorting out transduced YFP+ cells.

[0098] Step 42: Use the Chromium Next GEM Single Cell 3′ Kit to prepare single cell libraries from the sorted YFP+ cells.

[0099] Step 43: Sequence the transcriptome library and CRISPR screening library separately to obtain gene expression data and sgRNA editing information.

[0100] Step 44: Use Cell Ranger to align the single-cell transcriptome sequencing data with the mouse genome to generate a gene expression matrix for each cell.

[0101] Step 45: Align the sequencing reads of the CRISPR screening library with the previously transduced sgRNA library to determine the sgRNA corresponding to each cell.

[0102] Step 46: Use Seurat to analyze the filtered expression matrix and sgRNA reads for cell clustering, gene expression analysis, and integration of CRISPR screening data.

[0103] Step 47: Identify phenotypic changes or cell subsets associated with specific sgRNAs and screen candidate genes or regulatory factors associated with Th17 cell heterogeneity.

[0104] Furthermore, after step 47, the following steps are also included:

[0105] Step 48: Perform phenotypic experiments on the screened candidate genes or regulatory factors to verify the effect of the corresponding sgRNA on Th17 cell heterogeneity.

[0106] Specific examples are provided below. The provided examples may enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way.

[0107] Example:

[0108] Targeting the mouse full transcription factor panel, a sgRNA library covering key genes was designed and integrated into a retroviral vector. sgRNA was designed using an online tool (https: / / portals.broadinstitute.org / gpp / public / analysis-tools / sgrna-design).

[0109] From Il17a Cre Rosa26 YFP / Cas9 Naive 2D2 cells expressing Cas9 were isolated from spleens and peripheral lymph nodes (PLNs) of 2D2 mice. Naive 2D2 cells were activated in vitro for 30 h with anti-CD3 and 1.5 μg / ml anti-CD28 before viral transduction. Cells were cultured with IL-6, IL-23, and IL-1β for 3 days. Transduced cells were purified using a Thy1.1+ cell isolation kit (Miltenyi Biotec) before transplantation into recipients.

[0110] Transformed 2D2 cells (5×10 per recipient) were 6 The transplanted Thy1.1+, YFP+ cells were recovered from the spleen and dLN 20 days after transplantation, with an average of 1.2×10 cells recovered per sample. 6 YFP+ cells (approximately 20× cell coverage per sgRNA) were used for further sequencing.

[0111] The sorted YFP+ cells were prepared using the Chromium Next GEM Single Cell 3′ kit, which uses the characteristic barcoding technology of CRISPR screening (v.3.1; 10x Genomics). The transcriptome library and CRISPR screening library were sequenced separately, and the single-cell transcriptome sequencing was compared with the mouse genome (mm10) using Cell Ranger; the sequencing reads of the CRISPR screening library were compared with the previously transduced sgRNA library, and the filtered expression matrix and sgRNA reads were analyzed using Seurat to identify the perturbation phenotype or cell subpopulation corresponding to the sgRNA. For a schematic diagram of identifying the perturbation phenotype or cell subpopulation corresponding to the sgRNA, please see Figure 3 , Il17a Cre Rosa26 YFP / Cas9 Please refer to the real-time expression ratio of IL-17a in 2D2 cells Figure 4 .

[0112] Phenotypic experiments were performed on the screened candidate genes or subpopulations to verify the effect of the corresponding sgRNA on the heterogeneity of Th17 cells.

[0113] The principles and implementation methods of the present invention are described herein using specific examples, and the description of the above embodiments is only used to help understand the method and core ideas of the present invention. The above are only preferred implementations of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, can make equivalent replacements or changes according to the technical solutions and inventive concepts of the present invention, which should be within the protection scope of the present invention.

Claims

1. A high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR, characterized in that: The steps include: S1: CRISPR library construction; S2: Il17a Cre Rosa26 YFP / Cas9 2D2 T cell transduction; S3: In vivo transplantation and EAE model construction; S4: Single-cell transcriptome sequencing and single-cell CRISPR screening.

2. The high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR according to claim 1, characterized in that: Step S1 specifically includes: designing a sgRNA library covering the target gene for the mouse full transcription factor group, and integrating the designed sgRNA library into a retroviral vector.

3. The high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR according to claim 2, characterized in that: The sgRNA library designed to cover the target gene specifically includes: Select target gene set; Use bioinformatics tools to design sgRNA sequences targeting target genes; Synthesize sgRNA libraries by synthesizing short sequences.

4. The high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR according to claim 1, characterized in that: Step S2 specifically includes the following sub-steps: S21: Build Il17a Cre Rosa26 YFP / Cas9 2D2 mouse model; S22: From Il17a Cre Rosa26 YFP / Cas9 Naive 2D2 cells were isolated from the spleen and peripheral lymph nodes of 2D2 mice; S23: Activation and transduction of naive 2D2 cells; S24: screening of transduced cells; S25: Construction of transduced Th17 cell library.

5. The high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR according to claim 4, characterized in that: Step S23 specifically includes: Naive 2D2 cells were activated with anti-CD3 and anti-CD28 for 30-35 h, and then the CRISPR library was transduced into the cells by viral infection; The cells were cultured with IL-6, IL-23 and IL-1β for 2 to 4 days to induce their differentiation into Th17 cells.

6. The high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR according to claim 4, characterized in that: Step S24 specifically includes: purifying the transduced cells using a Thy1.1+ cell separation kit.

7. The high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR according to claim 1, characterized in that: Step S3 specifically includes the following sub-steps: S31: Sublethal radiation is given to recipient mice to remove their immune cells and provide an immune-tolerant environment for transplanted cells; S32: 2D2 Th17 cells transformed with the CRISPR library were transplanted into irradiated mice by intravenous injection; S33: After transplantation, EAE was induced in mice by MOG immunization; S34: 18 to 21 days after transplantation, transduced Thy1.1+, YFP+ cells were collected from the spleen and submandibular lymph nodes of mice.

8. The high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR according to claim 7, characterized in that: The number of 2D2 Th17 cells transplanted in step S32 was (5±0.2)×10 6 indivual; The number of YFP+ cells recovered in step S34 is (1.2±0.2)×10 6 indivual.

9. The high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR according to claim 7, characterized in that: Step S4 specifically includes the following sub-steps: S41: sort out transduced YFP+ cells; S42: Single cell library preparation was performed on sorted YFP+ cells using the Chromium Next GEM Single Cell 3′ kit; S43: Sequence the transcriptome library and CRISPR screening library respectively to obtain gene expression data and sgRNA editing information; S44: Use Cell Ranger to align single-cell transcriptome sequencing data with the mouse genome to generate a gene expression matrix for each cell; S45: Align the sequencing reads of the CRISPR screening library with the previously transduced sgRNA library to determine the corresponding sgRNA for each cell; S46: Analyze the filtered expression matrix and sgRNA reads using Seurat for cell clustering, gene expression analysis, and integration of CRISPR screening data; S47: Identify phenotypic changes or cell subsets associated with specific sgRNAs and screen candidate genes or regulatory factors associated with Th17 cell heterogeneity.

10. The high-throughput method for screening regulatory factors of Th17 cell heterogeneity using CRISPR according to claim 9, characterized in that: After step S47, the following steps are also included: S48: Perform phenotypic experiments on the screened candidate genes or regulatory factors to verify the effect of the corresponding sgRNA on Th17 cell heterogeneity.

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