Method for screening pathogenic and non-pathogenic Th17 cell regulatory factors by using CRISPR (clustered regularly interspaced short palindromic repeats)

Abstract

The invention discloses a method for screening pathogenic and non-pathogenic Th17 cell regulatory factors by using CRISPR (clustered regularly interspaced short palindromic repeats), and relates to the technical field of CRISPR screening, which 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; sorting the pathogenic Th17 cells and the non-pathogenic Th17 cells; and performing high-throughput sequencing and enrichment analysis and identification. 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. In addition, an in-vivo experiment model is closer to a real immune environment, and a result has a biological significance; the pathogenic and non-pathogenic fate determinants of the Th17 cells can be obtained through screening.

Description

Technical Field

[0001] The present invention relates to the field of CRISPR screening technology, and in particular to a method for screening pathogenic and non-pathogenic Th17 cell regulatory factors 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. Recent studies have found that Th17 cells can be divided into pathogenic and non-pathogenic subpopulations according to their functions. These two Th17 cell subpopulations selectively express SLAMF6 and CXCR6: SLAMF6- and CXCR6+ are pathogenic, while SLAMF6+ and CXCR6- are non-pathogenic. The two subpopulations show significant differences in metabolic activity, transcription factor expression and functional programs. However, the intracellular mechanisms and regulatory molecules that control Th17 cells to enter these two different programs in vivo are not very clear.

[0003] Existing technologies mainly rely on in vitro culture models and single gene knockout analysis methods to study the fate determinants 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 fate of pathogenic and non-pathogenic 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] The present invention provides a method for screening pathogenic and non-pathogenic Th17 cell regulatory factors using CRISPR, aiming to solve the following technical problems:

[0005] 1. Improve the screening efficiency of Th17 cell pathogenic and non-pathogenic fate determinants;

[0006] 2. Identify key genes that affect the fate of Th17 cells in the complex immune environment in vivo;

[0007] 3. Provide high-throughput tools for systematic analysis of Th17 cell gene functions and promote the development of new treatment strategies for autoimmune diseases.

[0008] The specific technical solution is:

[0009] A method for screening pathogenic and non-pathogenic Th17 cell regulatory factors using CRISPR, comprising the following steps:

[0010] Step 1: CRISPR library construction;

[0011] Step 2: Il17a Cre Rosa26 YFP / Cas9 2D2 T cell transduction;

[0012] Step 3: In vivo transplantation and EAE model construction;

[0013] Step 4: Isolation of pathogenic and non-pathogenic Th17 cells;

[0014] Step 5: High-throughput sequencing and enrichment analysis and identification.

[0015] 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.

[0016] Preferably, the design of the sgRNA library covering the target gene specifically includes:

[0017] Select target gene set;

[0018] Use bioinformatics tools to design sgRNA sequences targeting target genes;

[0019] Synthesize sgRNA libraries by synthesizing short sequences.

[0020] Preferably, step 2 specifically includes the following sub-steps:

[0021] Step 21: Build Il17a Cre Rosa26 YFP / Cas9 2D2 mouse model;

[0022] Step 22: From Il17a Cre Rosa26 YFP / Cas9 Naive 2D2 cells were isolated from the spleen and peripheral lymph nodes of 2D2 mice;

[0023] Step 23: Activation and transduction of naive 2D2 cells;

[0024] Step 24: Screening transduced cells;

[0025] Step 25: Construction of transduced Th17 cell bank.

[0026] Preferably:

[0027] Step 23 specifically includes: activating naive 2D2 cells with anti-CD3 and anti-CD28 for 30 to 35 hours, and then transducing the CRISPR library into the cells through viral infection; culturing the cells with IL-6, IL-23 and IL-1β for 2 to 4 days to induce differentiation into Th17 cells;

[0028] Step 24 specifically includes: purifying the transduced cells using a Thy1.1+ cell separation kit.

[0029] Preferably, step 3 specifically includes the following sub-steps:

[0030] Step 31: Sublethal radiation is given to recipient mice to remove their immune cells and provide an immune-tolerant environment for transplanted cells;

[0031] Step 32: Transplant 2D2 Th17 cells transformed with the CRISPR library into irradiated mice via intravenous injection;

[0032] Step 33: After transplantation, mice were immunized with MOG to induce EAE;

[0033] Step 34: 18 to 21 days after transplantation, collect transduced Thy1.1+, YFP+ cells from the spleen and submandibular lymph nodes of the mice.

[0034] Preferably, it is characterized in that:

[0035] The number of 2D2 Th17 cells transplanted in step 32 was (5±0.2)×10 6 indivual;

[0036] The number of YFP+ cells recovered in step 34 was (1.2±0.2)×10 6 indivual.

[0037] Preferably, step 4 specifically comprises: using flow cytometry to sort the cell population according to the expression pattern of CXCR6 and SLAMF6, wherein CXCR6+ and SLAMF6- are pathogenic Th17 cells, and CXCR6- and SLAMF6+ are non-pathogenic Th17 cells.

[0038] Preferably, step 5 specifically includes the following sub-steps:

[0039] Step 51: extracting genomic DNA from the sorted cells;

[0040] Step 52: Use Nextera primers for high-throughput sequencing. The sequencing results are used to analyze the enrichment of sgRNA. Nextera primers are designed as follows:

[0041] Nextera NGS(-F):

[0042] TCGTCGGCAGCGTCAGAT-GTGTATAAGAGACAGttgtggaaaggacgaaacaccg

[0043] Nextera NGS(-R):

[0044] GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGccactttttcaagttgataacgg;

[0045] Step 53: Compare the sequencing results with the sequences in the sgRNA library, calculate the read counts of sgRNA in each sample, and obtain the enrichment of each sgRNA after normalization;

[0046] Step 54: Perform log2 transformation on each sgRNA, calculate its enrichment fold change, and compare between different experimental groups based on the enrichment of sgRNA;

[0047] Step 55: Rank and identify candidate genes that regulate Th17 cell fate based on the average enrichment of gene-specific sgRNAs in SLAMF6+ cells relative to CXCR6+.

[0048] Preferably, step 55 further includes the following steps:

[0049] Step 56: Perform phenotypic experiments on the screened candidate genes to verify their effects on the fate of Th17 cells.

[0050] The method provided by the present invention for screening pathogenic and non-pathogenic Th17 cell regulatory factors using CRISPR has the following beneficial effects:

[0051] 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;

[0052] 2. Different ranges of sgRNA libraries can be selected to cover the entire gene or transcription factor range;

[0053] 3. The in vivo experimental model is closer to the real immune environment, and the results are more biologically meaningful;

[0054] 4. The fate determinants of pathogenicity and non-pathogenicity of Th17 cells can be screened;

[0055] 5. It can provide new targets and intervention strategies for the diagnosis and treatment of autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] 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.

[0057] Figure 1 A flowchart of the steps of the method for screening pathogenic and non-pathogenic Th17 cell regulatory factors using CRISPR provided in an embodiment of the present invention;

[0058] Figure 2 A technical flow chart of flow cytometry sorting in a method for screening pathogenic and non-pathogenic Th17 cell regulatory factors using CRISPR provided in an embodiment of the present invention;

[0059] Figure 3 A diagram showing the high-throughput sequencing results of a method for screening pathogenic and non-pathogenic Th17 cell regulatory factors using CRISPR provided in an embodiment of the present invention;

[0060] Figure 4 Schematic diagram of gRNA enrichment fold change of CXCR6+ and SLAMF6+ cells in the method of using CRISPR to screen pathogenic and non-pathogenic Th17 cell regulatory factors provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0061] 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.

[0062] This embodiment provides a method for screening pathogenic and non-pathogenic Th17 cell regulatory factors using CRISPR, comprising the following steps:

[0063] Step 1: CRISPR library construction.

[0064] Step 2: Il17a Cre Rosa26 YFP / Cas9 2D2 T cell transduction.

[0065] Step 3: In vivo transplantation and EAE model establishment.

[0066] Step 4: Isolation of pathogenic and non-pathogenic Th17 cells.

[0067] Step 5: High-throughput sequencing and enrichment analysis and identification.

[0068] Among them, in step 2, Il17aCre 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] In step 4, flow cytometry can be used to sort pathogenic Th17 cells and non-pathogenic Th17 cells for further analysis. Specifically, cells are sorted according to their surface markers (CXCR6 and SLAMF6) by flow cytometry. Cells marked with CXCR6+ and SLAMF6- are pathogenic Th17 cells, while cells marked with CXCR6- and SLAMF6+ are non-pathogenic Th17 cells. The sorted cells can be used for phenotypic analysis, genomic analysis, and functional verification, respectively.

[0073] In step 5, the sgRNA enrichment of each cell population can be analyzed by high-throughput sequencing technology to identify key genes that regulate the fate of Th17 cells. Specifically, genomic DNA can be extracted from the sorted cells, and sgRNA can be deeply sequenced using Nextera primers, and 50-bp single-end sequencing can be performed on the HiSeq platform to obtain a large amount of genomic data. The enrichment of each sgRNA is calculated by matching the sequencing results with the sgRNA sequence in the library, and the number of reads of the sgRNA is normalized with the total number of reads, and the fold change (log2 ratio) of each sgRNA between different experimental groups is calculated to determine which genes or genomic regions have different enrichments in pathogenic or non-pathogenic Th17 cells, so that the key genes that may regulate the fate of Th17 cells can be screened out through the enrichment analysis of sgRNA, and these genes can be used as candidates for subsequent functional verification.

[0074] The method for screening pathogenic and non-pathogenic Th17 cell regulatory factors using CRISPR provided in this embodiment has the following beneficial effects:

[0075] 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.

[0076] 2. Different ranges of sgRNA libraries can be selected to cover the entire gene or transcription factor range.

[0077] 3. The in vivo experimental model is closer to the real immune environment, and the results are more biologically meaningful.

[0078] 4. The fate-determining factors of pathogenicity and non-pathogenicity of Th17 cells can be screened.

[0079] 5. It can provide new targets and intervention strategies for the diagnosis and treatment of autoimmune diseases.

[0080] 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.

[0081] Furthermore, the design of the sgRNA library covering the target gene specifically includes:

[0082] Select target gene set.

[0083] Use bioinformatics tools to design sgRNA sequences targeting target genes.

[0084] Synthesize sgRNA libraries by synthesizing short sequences.

[0085] Furthermore, step 2 specifically includes the following sub-steps:

[0086] Step 21: Build Il17a Cre Rosa26 YFP / Cas9 2D2 mouse model.

[0087] Step 22: From Il17a Cre Rosa26 YFP / Cas9 Naive 2D2 cells were isolated from spleen and peripheral lymph nodes of 2D2 mice.

[0088] Step 23: Activation and transduction of naive 2D2 cells.

[0089] Step 24: Screening of transduced cells.

[0090] Step 25: Construction of transduced Th17 cell bank.

[0091] Further:

[0092] Step 23 specifically includes: activating naive 2D2 cells with anti-CD3 and anti-CD28 for 30 to 35 hours, and then transducing the CRISPR library into the cells through viral infection; culturing the cells with IL-6, IL-23 and IL-1β for 2 to 4 days to induce them to differentiate into Th17 cells.

[0093] Step 24 specifically includes: purifying the transduced cells using a Thy1.1+ cell separation kit.

[0094] Furthermore, step 3 specifically includes the following sub-steps:

[0095] Step 31: The recipient mice are irradiated with sublethal doses to remove their immune cells and provide an immune-tolerant environment for the transplanted cells.

[0096] Step 32: 2D2 Th17 cells transformed with the CRISPR library were transplanted into irradiated mice via intravenous injection.

[0097] Step 33: After transplantation, mice were induced to develop EAE by MOG immunization.

[0098] Step 34: 18 to 21 days after transplantation, collect transduced Thy1.1+, YFP+ cells from the spleen and submandibular lymph nodes of the mice.

[0099] Furthermore, it is characterized in that:

[0100] The number of 2D2 Th17 cells transplanted in step 32 was (5±0.2)×10 6 indivual.

[0101] The number of YFP+ cells recovered in step 34 was (1.2±0.2)×10 6 indivual.

[0102] Furthermore, step 4 specifically includes: using flow cytometry to sort the cell population according to the expression pattern of CXCR6 and SLAMF6, wherein CXCR6+ and SLAMF6- are pathogenic Th17 cells, and CXCR6- and SLAMF6+ are non-pathogenic Th17 cells.

[0103] Furthermore, step 5 specifically includes the following sub-steps:

[0104] Step 51: Extract genomic DNA from the sorted cells.

[0105] Step 52: Use Nextera primers for high-throughput sequencing. The sequencing results are used to analyze the enrichment of sgRNA. Nextera primers are designed as follows:

[0106] Nextera NGS(-F):

[0107] TCGTCGGCAGCGTCAGAT-GTGTATAAGAGACAGttgtggaaaggacgaaacaccg

[0108] Nextera NGS(-R):

[0109] GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGccactttttcaagttgataacgg.

[0110] Step 53: Align the sequencing results with the sequences in the sgRNA library, calculate the read counts of sgRNA in each sample, and obtain the enrichment of each sgRNA after normalization.

[0111] Step 54: Perform log2 transformation on each sgRNA, calculate its enrichment fold change, and compare between different experimental groups based on the enrichment of sgRNA.

[0112] Step 55: Rank and identify candidate genes that regulate Th17 cell fate based on the average enrichment of gene-specific sgRNAs in SLAMF6+ cells relative to CXCR6+.

[0113] Furthermore, after step 55, the following steps are also included:

[0114] Step 56: Perform phenotypic experiments on the screened candidate genes to verify their effects on the fate of Th17 cells.

[0115] 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.

[0116] Example:

[0117] 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).

[0118] 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.

[0119] 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 (~20× cell coverage per sgRNA) for deep sequencing of sgRNAs.

[0120] Flow cytometry was used to sort pathogenic (CXCR6+SLAMF6-) and non-pathogenic (CXCR6-SLAMF6+) Th17 cell subsets. The sorting process is shown in the figure. Figure 2, extract genomic DNA, and use the following Nextera next-generation sequencing (NGS) primers (Nextera NGS(-F):

[0121] TCGTCGGCAGCGTCAGAT-GTGTATAAGAGACAGttgtggaaaggacgaaacaccg; Nextera NGS (-R): GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGccactttttcaagttgataacgg). After purification, a second PCR was performed, and adapters and indexes were added to each sample, followed by Hi-seq 50-bp single-end sequencing (Illumina). The sequencing results are shown in the figure. Figure 3 The sequencing results were matched to the sgRNA sequences in the gRNA library, and the read counts of the sgRNAs were normalized to the total read counts of all samples; the fold change (log2 transformed ratio) of enrichment of each sgRNA was calculated between each biological replicate and experiment; the candidate genes were ranked according to their average enrichment of gene-specific sgRNAs in SLAMF6+ cells relative to CXCR6+. The gRNA enrichment fold change of sorted CXCR6+ and SLAMF6+ cells is shown in Figure 2. Figure 4 .

[0122] Phenotypic experiments were performed on the screened candidate genes to verify their effects on the fate of Th17 cells.

[0123] 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 method for screening pathogenic and non-pathogenic Th17 cell regulatory factors 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: Sorting of pathogenic and non-pathogenic Th17 cells; S5: High-throughput sequencing and enrichment analysis and identification.

2. The method for screening pathogenic and non-pathogenic Th17 cell regulatory factors 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 method for screening pathogenic and non-pathogenic Th17 cell regulatory factors 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 method for screening pathogenic and non-pathogenic Th17 cell regulatory factors 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 method for screening pathogenic and non-pathogenic Th17 cell regulatory factors using CRISPR according to claim 4, characterized in that: Step S23 specifically includes: activating naive 2D2 cells with anti-CD3 and anti-CD28 for 30 to 35 hours, and then transducing the CRISPR library into the cells through viral infection; culturing the cells with IL-6, IL-23 and IL-1β for 2 to 4 days to induce differentiation into Th17 cells; Step S24 specifically includes: purifying the transduced cells using a Thy1.1+ cell separation kit.

6. The method for screening pathogenic and non-pathogenic Th17 cell regulatory factors 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.

7. The method for screening pathogenic and non-pathogenic Th17 cell regulatory factors using CRISPR according to claim 6, 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.

8. The method for screening pathogenic and non-pathogenic Th17 cell regulatory factors using CRISPR according to claim 1, characterized in that: Step S4 specifically includes: using flow cytometry to sort the cell population according to the expression patterns of CXCR6 and SLAMF6, wherein CXCR6+ and SLAMF6- are pathogenic Th17 cells, and CXCR6- and SLAMF6+ are non-pathogenic Th17 cells.

9. The method for screening pathogenic and non-pathogenic Th17 cell regulatory factors using CRISPR according to claim 1, characterized in that: Step S5 specifically includes the following sub-steps: S51: Extract genomic DNA from sorted cells; S52: High-throughput sequencing was performed using Nextera primers. The sequencing results were used to analyze the enrichment of sgRNA. Nextera primers were designed as follows: Nextera NGS(-F): TCGTCGGCAGCGTCAGAT-GTGTATAAGAGACAGttgtggaaaggacgaaacaccg Nextera NGS(-R): GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGccactttttcaagttgataacgg; S53: The sequencing results were compared with the sequences in the sgRNA library, and the enrichment of each sgRNA was obtained by calculating the read counts of sgRNA in each sample and normalizing them; S54: Perform log2 transformation on each sgRNA, calculate its enrichment fold change, and compare between different experimental groups based on the enrichment of sgRNA; S55: Rank and identify candidate genes regulating Th17 cell fate based on the average enrichment of gene-specific sgRNAs in SLAMF6+ cells relative to CXCR6+.

10. The method for screening pathogenic and non-pathogenic Th17 cell regulatory factors using CRISPR according to claim 9, characterized in that: After step S55, the following steps are also included: S56: Perform phenotypic experiments on the screened candidate genes to verify their effects on the fate of Th17 cells.

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