Direct sgRNA capture probe for screening and sequencing space CRISPR (clustered regularly interspaced short palindromic repeats) and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a direct sgRNA capture probe for screening and sequencing space CRISPR and application of the direct sgRNA capture probe. The direct capture sgRNA probe is designed based on a sgRNA sequence of a specific gene of a target species and comprises a left side probe and a right side probe; the left side probe consists of a nucleic acid sequence 1 and a nucleic acid sequence 2; the right side probe consists of a nucleic acid sequence 3, a nucleic acid sequence 4 and a poly-A tail; the nucleotide sequence 1 is a Read2 sequence required by next-generation sequencing; the nucleic acid sequence 2 is reversely complementary with a 6-30 bp sequence at the 5'end of a scaffold sequence of the sgRNA, the nucleic acid sequence 3 is reversely complementary with an initial 5 bp sequence at the 5 'end of the scaffold sequence of the sgRNA, and the nucleic acid sequence 4 is reversely complementary with an original interval sequence of the sgRNA. The probe has high specificity, breaks through plasmid design limitation when being applied to space CRISPR screening and sequencing, is compatible with various related platforms and is suitable for gene function research.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a direct capture sgRNA probe for spatial CRISPR screening sequencing and its application. Background Art

[0002] Biological systems inherently exhibit significant characteristics of spatial orderliness, and the interactions between cells and various processes at the molecular level all occur in specific spatial environments. Among the numerous advanced research methods in the field of spatial biology, spatial transcriptomics has fully demonstrated its powerful research capabilities. It can not only deeply explore the tissue structure and gene expression patterns in the spatial dimension, but also conduct detailed research on cell populations and the interactions between cells. However, currently, the tools capable of establishing a causal relationship between gene expression and the functional impact of spatial phenotypes are still lacking, and at the same time, the regulatory pathways driving these biological processes have far from been fully explored.

[0003] The CRISPR-Cas9 technology realizes precise genome editing through sgRNA-mediated Cas9 protein, bringing a revolutionary breakthrough to the research of functional genomics. This technology can directly associate gene perturbation with specific phenotypes (such as cell growth, survival rate, marker gene expression, etc.), greatly promoting the research of gene function. With the development of CRISPR screening technology towards the high-throughput level, researchers have been able to systematically explore gene function. In addition, the emergence of single-cell RNA sequencing (single-cell RNA-seq) technology has made it possible to conduct whole-transcriptome gene expression analysis under single-cell perturbation conditions. Among them, Perturb-seq, as a typical platform mediated by CRISPR, although it has achieved the integration of gene perturbation and transcriptome analysis at the single-cell level, its application is often limited by the specific plasmid system design and requires carrying the sequence corresponding to sgRNA in mRNA. The Perturb-seq technology developed by 10×Genomics, which directly captures sgRNA, has broken through this limitation and is compatible with almost all types of sgRNA expression plasmids, promoting the wide application of single-cell Perturb-seq technology, but there are still obvious limitations in its application at the spatial transcription level.

[0004] Currently, there is still a lack of high-throughput spatial CRISPR screening sequencing methods with single-cell whole-transcriptome as the readout output, and there is also a lack of methods for directly capturing sgRNA at the spatial level, which brings challenges to the further research of spatial biology while also providing opportunities for its innovative development. Summary of the Invention

[0005] Technical Problem The present invention aims to develop an ultra-high weight targeted capture probe, which is applied to spatial CRISPR screening sequencing by combining spatial transcriptomics.

[0006] Technical solution In the first aspect of the present invention, a direct capture sgRNA probe for spatial CRISPR screening sequencing is provided. The direct capture sgRNA probe is designed based on the sgRNA sequence of a specific gene of a target species. Among them, the direct capture sgRNA probe includes a left probe and a right probe. The sgRNA contains a scaffold sequence and a protospacer sequence. Among them, the left probe is composed of nucleic acid sequence 1 and nucleic acid sequence 2. The right probe is composed of nucleic acid sequence 3, nucleic acid sequence 4 and a poly-A tail. Among them, nucleic acid sequence 1 is the Read2 sequence required for next-generation sequencing. Nucleic acid sequence 2 is reverse complementary to the 6-30 bp sequence at the 5' end of the scaffold sequence of the sgRNA. Nucleic acid sequence 3 is reverse complementary to the starting 5 bp sequence at the 5' end of the scaffold sequence of the sgRNA. Nucleic acid sequence 4 is reverse complementary to the protospacer sequence of the sgRNA.

[0007] In some embodiments, the left probe and the right probe are DNA. The length of the left probe is 46 bp, and the length of the right probe is 55 bp. Among them, the length of nucleic acid sequence 1 is 21 bp, the length of nucleic acid sequence 2 is 25 bp, the length of nucleic acid sequence 3 is 5 bp, the length of nucleic acid sequence 4 is 20-30 bp, and the length of the poly-A tail is at least 20 bp.

[0008] In some embodiments, when the target species is a mouse, the length of nucleic acid sequence 4 in the right probe is 20 bp.

[0009] In some embodiments, the left sequence is as shown in SEQ ID NO.1, and the right sequence is as shown in SEQ ID NOs. 2-121.

[0010] In some embodiments, the left probe and the right probe are ligated by a splint under the action of T4 ligase during the spatial CRISPR screening sequencing process.

[0011] The second aspect of the present invention provides a spatial CRISPR screening and sequencing method, which includes the following steps: S1: Inject target cells containing a CRISPR knockout library into an experimental animal; S2: Take out the tissue infiltrated by the target cells, prepare paraffin sections, and perform HE staining and imaging; S3: After imaging, the tissue sections are permeabilized and de-crosslinked, and then hybridized with a whole-transcriptome gene expression probe and the direct capture sgRNA probe described in any one of the above; the left probe and the right probe of the direct capture sgRNA probe after hybridization are ligated by a splint under the action of T4 ligase; S4: Use Visium HD to construct a spatial mRNA library and a spatial sgRNA library; S5: Sequencing and gene sequence alignment are performed on the spatial mRNA library and the spatial sgRNA library respectively to obtain ordinary transcriptome sequencing data and CRISPR sgRNA library sequencing data with spatial coordinates.

[0012] In some embodiments, the target cells containing a CRISPR knockout library are obtained through the following steps: Prepare a CRISPR screening library virus, infect the test cells with the CRISPR screening library virus, sort out the positively infected test cells and culture them for a period of time to obtain the target cells containing a CRISPR knockout library.

[0013] In some embodiments, the method of injecting into the experimental animal is intravenous injection, subcutaneous injection, intramuscular injection or tumor injection.

[0014] In some embodiments, the tissue is tumor tissue or normal tissue.

[0015] In some embodiments, it further includes combining the ordinary transcriptome sequencing data and the CRISPR sgRNA library sequencing data into the data of the same chip according to the same spatial coordinates.

[0016] The third aspect of the present invention provides the application of the direct capture sgRNA probe described in any one of the above in spatial CRISPR screening and sequencing.

[0017] The fourth aspect of the present invention provides the application of the spatial CRISPR screening and sequencing method described in any one of the above in verifying gene functions.

[0018] The fifth aspect of the present invention provides the application of the spatial CRISPR screening and sequencing method described in any one of the above in exploring the mechanism of tumor metastasis.

[0019] In some embodiments, unbiased clustering is performed on the ordinary transcriptome sequencing data, and differential analysis is performed on each clustered category with all other categories to obtain the signature genes of each category as the key features of each category; according to the CRISPR sgRNA library sequencing data, differential analysis is performed on the expression levels of sgRNAs in different clusters to obtain the clusters in which the target cells of the sgRNA knockout genes tend to be enriched, thereby verifying the functions of the sgRNA target genes.

[0020] Technical effects The spatial CRISPR screening sequencing (SPAC-seq) technology developed by the present invention through ultra-high-multiplex targeted capture probes in combination with spatial transcriptomics is a general spatial CRISPR screening technology that is compatible with a variety of high-throughput, single-cell sequencing-based spatial transcriptomics platforms, including 10× Genomics Visium HD.

[0021] The direct capture sgRNA probes provided by the present invention have high specificity. Specifically, among the two probes, one is complementary paired with the 20 bp protospacer of the sgRNA, but there is also a complementary pairing with the mRNA corresponding to the target gene of the sgRNA. To avoid this mismatch, on the one hand, the sgRNA-specific probe still has 5 bp paired with the scaffold of the sgRNA, and more importantly, the other probe that is completely adjacent after complementation (with a 21 bp library construction essential sequence and 25 bp complementary pairing on the scaffold) can be ligated to this sgRNA-specific probe under the catalysis of T4 ligase. The successfully ligated probe, on the one hand, has the specificities of the two probes from both ends complementary paired respectively, and more importantly, the T4 splint ligation ensures that only the probes that are consistent with the designed reference sequence and the two probes are completely adjacent rather than spaced can be ligated. These two aspects of specificity enable the obtained probe product to recognize the protospacer specific sequence of the sgRNA on the one hand, recognize the scaffold sequence of the sgRNA rather than the target gene on the other hand, and on the other hand, the two probes that recognize the specific sequence and the sgRNA scaffold sequence must be completely in line with the expectation in terms of spatial distance. These three specificities together ensure the high specificity of the probe.

[0022] Many single-cell CRISPR screening and spatial CRISPR screening methods are limited by specific plasmid designs. The Direct Capture SPAC-seq provided by the present invention overcomes this limitation, just like the breakthrough of Direct Capture Perturb-seq in the field of single-cell CRISPR screening. This advantage enables SPAC-seq to be applied to future spatial research, such as CRISPRi / a, ORF (open reading frame) screening, or other spatial barcoding screens.

[0023] The Direct Capture SPAC-seq provided by the present invention is a single-cell CRISPR screening and sequencing method based on the spatial level, using the spatial whole transcriptome as the biological readout signal to be studied, and is applicable to studying gene functions, such as tumor metastasis mechanisms. Specifically, the present invention has discovered that Icam1 (intercellular adhesion molecule 1) is a key regulator of immune surveillance after tumor dissemination. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Shown is a schematic diagram of the SPAC-seq workflow; Figure 2 Shown is a schematic diagram of performing pooled spatial CRISPR screening using MC38-Cas9 to study lung metastasis at the spatial level; Figure 3 Shown is a process and sequencing flowchart of spatial transcriptome probe hybridization and direct capture sgRNA probe hybridization; Figure 4A Shown is the spatial visualization of 50 sgRNA clone types therein; Figure 4B Shown are sgRNA clone regions with different entropy complexities; Figure 4C Shown are different numbers of sgRNA clones detected in regions with high and low entropy complexities, respectively; Figure 4D Shown is the spatial visualization of the detected sgNfib and CD8+ T cell marker distributions; Figure 5 Shown is a comparison of the correlation between genomic and mRNA-embedded sgRNA counts in a 70-sgRNA library in vitro, and the correlation between genomic and directly captured sgRNA counts; Figure 6A Shown are representative histological images of tumor lesions and the corresponding spatial CRISPR sgRNA library counts for each clone; Figure 6B Shown are the top-level sgRNA clones and their markers for the tumor microenvironment (TME) niches identified by spatial transcriptomics; Figure 6C Shown is the spatial visualization of immune cell enrichment in sgRNA clones, including non-targeting controls and sgIcam1; Figure 6D Shown is the visualization of the top differentially expressed pathway, the IFN-γ signaling pathway, in immune-excluded tumor niches. Detailed implementation mode

[0025] For the convenience of describing the technical solutions of the invention, the terms and expressions involved in the present invention are generally described and defined below.

[0026] Direct capture sgRNA probe: A probe designed in the present invention that can directly capture sgRNA without embedding the sequence of sgRNA into mRNA. Previous single-cell Perturb-seq or CROP-seq technologies identified which sgRNA each cell expressed, that is, which gene was knocked out, by sequencing the sgRNA sequence embedded in mRNA.

[0027] The left / right probes in the present invention refer to the sequential sorting of direct capture sgRNA probes starting from the 5'-end.

[0028] The splint ligation in the present invention refers to the ligation formed by ligase after hybridization of the two DNA probes, the left / right probes, with sgRNA.

[0029] Spatial CRISPR screening sequencing: A technology that combines spatial transcriptomics and CRISPR screening.

[0030] Spatial mRNA library: A DNA sequencing library obtained from biological tissue sections for characterizing the content and spatial distribution information of the entire mRNA transcriptome; Spatial sgRNA library: A DNA sequencing library obtained from biological tissue sections for characterizing the content and spatial distribution information of sgRNA barcodes for different genes in CRISPR screening.

[0031] The test cells in the present invention refer to the cells to be infected, and the target cells in the present invention refer to the successfully infected cells screened.

[0032] Clone type: Cells and their descendants that have been edited by the same sgRNA gene.

[0033] Entropy complexity: The degree of heterogeneity in the spatial distribution of sgRNA clone types.

[0034] The following further describes a direct capture sgRNA probe for spatial CRISPR screening sequencing provided by the present invention and its applications.

[0035] Example 1: Design and preparation of the probe 1.1 Probe design Previously, through the analysis of the whole transcriptome of pan-cancer expression, 735 immune-related functional genes that may affect the growth of tumor cells were discovered. For the 735 target genes, the present invention designed a total of 1520 sgRNAs according to the reference genome of mice (2 sgRNAs were designed for each gene, a total of 1470 sgRNAs targeting genes, and in addition, 50 control sgRNAs that do not target any genes were combined). Based on each sgRNA sequence including a protospacer sequence (protospacer) and a scaffold sequence (scaffold) (5'-3'). The specific design of the probe for capturing each sgRNA is as follows: Take 20 bp of the protospacer (specific sequence) and the first 5 bp of the scaffold (5'-GUUUU-3') sequence, perform reverse complementation, and add 30 A bases at the 3' end, for a total of 55 bp, as the right probe (as shown in SEQ ID NO.2 - SEQ ID NO.121); then take the 6 - 30 bp (a total of 25 bp, specifically: 5'-AGAGCUAGAAAUAGCAAGUUAAAAU-3') sequence of the scaffold region for reverse complementation, and add the Read2 sequence required for Illumina second-generation sequencing library construction (a total of 21 bp, specifically: 5'-CCTTGGCACCCGAGAATTCCA-3') at the 5' end, for a total of 46 bp, as the left probe (as shown in SEQ ID NO.1).

[0036] 1.2 Probe preparation The right probe was prepared through the 5’ Phos Oligo Pool synthesis service of IDT in the United States. The left probe was synthesized by Beijing Xianghong Biotechnology Co., Ltd.

[0037] Example 2: Using SPAC-seq to explore inhibitory regulatory genes in the process of lung metastasis of tumor cells 2.1 Experimental purpose The workflow of spatial CRISPR screening sequencing (SPAC-seq) in the present invention (as Figure 1As shown in the figure, it starts with the transfection of the pooled sgRNA library into the cell type of interest. After fluorescence screening, the perturbed cells are injected into specific sites or organs of a syngeneic mouse model. Subsequently, a biological selection process occurs in vivo, such as tumor growth, metastasis, and T cell infiltration. The target tissue is then collected, sectioned, and analyzed using a high-resolution spatial transcriptomics platform (such as 10× Genomics Visium HD). After capturing the pooled sgRNA and mRNA libraries in situ, second-generation sequencing technology is used to map the CRISPR screening sgRNA and transcriptome data to their spatial positions to explore immune-related regulatory genes during the process of lung metastasis of tumor cells in mice.

[0038] 2.1 Experimental protocol 2.1.1 Cell preparation 1) Construction of the sgRNA oligo library According to the designed 1,520 sgRNAs, single-stranded oligonucleotide DNA was synthesized according to the method reported in previous CRISPR screen literature (Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening, Feng Zhang, 2017, Nature Protocals) to obtain the sgRNA oligo library. The sgRNA oligos were digested and inserted into plasmid vectors using the GoldenGate method (Golden Gate cloning technology) to construct the sgRNA library for CRISPR KO (CRISPR-mediated Knock-Out).

[0039] 2) Plasmid amplification and virus packaging The constructed sgRNA library vector was amplified in Escherichia coli to obtain sufficient plasmids. The amplified plasmids were purified by phenol-chloroform extraction to ensure no contamination. Subsequently, the sgRNA library plasmids were packaged into virus particles through retroviral packaging, namely the sgRNA library virus.

[0040] 3) Cell infection and screening The generated virus was used to infect MC38-Cas9 (mouse colon adenocarcinoma cell line) cells. Based on the fact that positively infected cells can express the GFP gene carried by the sgRNA library virus as a fluorescent protein while uninfected cells cannot, flow sorting was used to screen and isolate cells that had successfully integrated the sgRNA library. Since the sgRNA transcribed from the virus in positively infected cells forms an RNP complex with the Cas9 protein in the cells and targets the target gene, the obtained population of positively infected cells thus contains cells with different genes knocked out.

[0041] 4) Preparation for animal experiments At least 7 days after the cells were infected with the knockout library, a cell suspension for mouse experiments was prepared.

[0042] 2.1.2 Animal experiments 1) Establishment of mouse tumor models Mice (C57 / BL6 mice, female, 6 - 8 weeks old, immunocompetent) were divided into two groups: subcutaneous injection group and tail vein injection group, with 6 animals in each group. The mouse treatment process is as Figure 2 shown below: On the same day, mice were selected to inject the MC38-Cas9 cell line carrying the knockout library. Among them, for some mice, the cells were injected subcutaneously into the mice, and for another part of the mice, tail vein injection was performed to form lung metastasis foci. Within 14 days after injection, the mice were under specific screening pressure.

[0043] For the subcutaneous injection group, the cells were injected subcutaneously into the mice. Since the subcutaneous tissue is relatively simple with fewer interfering factors, it allows tumor cells to grow in a relatively confined subcutaneous environment, facilitating the observation of the local growth of tumors, and is conducive to studying the biological behavior of tumor cells themselves and the effect of the gene knockout library on local tumor growth. At this time, for the subcutaneous injection group, there is a certain heterogeneity in the different microenvironments (spaces) within the tumor tissue, and there are differences in tumor spatial characteristics such as hypoxia, fibrosis, immune rejection, and interactions between immune cells. Therefore, tumor cells with different gene knockouts in subcutaneous tumors will be subject to immune screening pressure and competition pressure from different dominant clones.

[0044] For the intravenous injection group, through tail vein injection in mice, tumor cells can reach the lungs through the blood circulation, simulating the process of tumor cells detaching from the primary site and metastasizing to the lungs through the blood to form lung metastasis foci. This helps to deeply study the tumor metastasis mechanism, analyze which gene knockouts will affect the metastasis ability of tumor cells, and the colonization and growth of tumor cells in the lung microenvironment. At this time, for the intravenous injection group, tumor cells with different gene knockouts in the lung metastasis foci are subject to screening pressure during migration in blood vessels during the metastasis process and immune screening pressure after colonization in the lung metastasis foci.

[0045] 2) Obtain tissue samples After 14 days, sacrifice the mice, remove the subcutaneous tumors, fix them in 10% neutral formalin fixative for 48 hours, and then perform paraffin embedding.

[0046] 2.1.3 Spatial transcriptomics combined with CRISPR screen sequencing 1) Histochemical staining: For paraffin-embedded tissue samples, use a Leica tissue slicer (Leica Cat#14051756235) to cut sections (5 μm thick), and attach them to SuperFrost Plus slides (fisherscientific Cat#12-550-15). Then, dewax the sections, perform HE (hematoxylin-eosin) staining, and finally image them with a slide scanner.

[0047] 2) Spatial transcriptome sequencing: The specific reagents required are shown in Table 1 below. Specifically, following the procedure in the 10×Visium HD spatial gene expression user guide (10×Genomics User Guide CG000685), the tissue section samples that have completed imaging are permeabilized and de-crosslinked, and then spatial transcriptome probe hybridization and direct capture sgRNA probe hybridization are performed simultaneously on the same tissue sample section. The spatial transcriptome probes are the whole-transcriptome gene expression probes included in the 10×Visium HD kit, and the direct capture sgRNA probes are the synthesized CRISPR screening sgRNA library probes (including left / right probes). Among them, the whole-transcriptome gene expression probes capture mRNA for hybridization, while the CRISPR screening sgRNA library probes hybridize to the protospacer sequence and scaffold sequence of sgRNA. After probe hybridization, following the procedure in the 10×Visium HD spatial gene expression user guide (10×Genomics User Guide CG000685), ligation of the left / right probes is carried out under the action of T4 ligase. Then, the probes hybridized to mRNA or sgRNA are released from the tissue, and the released probes hybridize to the oligonucleotide probes on the spatial transcriptome chip (10×Genomics Visium HD slide). Each oligonucleotide probe consists of four parts: Poly-dT (capturing mRNA or sgRNA with a Poly-A tail), Unique Molecular Identifier (UMI, eliminating PCR amplification bias), spatial coordinate barcode (uniquely identifying the coordinates of this point), and Illumina Read1. After the mRNA and sgRNA probes at different spatial positions with the copied expression levels are obtained on the chip, the probes are eluted from the chip and amplified through Illumina's Read1 and Read2. The spatial mRNA library and spatial sgRNA library (both DNA libraries) are constructed according to the method described in the 10×Visium HD spatial gene expression user guide (10×Genomics User Guide CG000685). The constructed libraries are then sequenced using an Illumina NovaSeq 6000 X plus sequencer. The probe hybridization process and sequencing procedure are as Figure 3 shown.

[0048] Table 1 Reagents and Materials

[0049] 3) Downstream analysis was performed using Scanpy V1.10.3 (a spatial transcriptome analysis software developed by 10× Genomics). It should be noted that the present invention actually designed 1520 direct capture sgRNA probes. However, due to limited space, only 50 groups of sgRNA probes (the right sequences of which are shown in SEQ ID NO.2 to SEQ ID NO.50) were taken as examples to analyze the spatial visualization of sgRNA clonotypes, and 70 groups of probes corresponding to 70 sgRNAs (64 sgRNAs corresponding to 32 genes and 6 Non-targeting control sgRNAs for control genes that do not target genes) (the right sequences of which are shown in SEQ ID NO.51 to SEQ ID NO.121) were taken as examples to analyze the correlation between the genome and the directly captured sgRNA counts: a. After reading in the data, the whole slice was cut into small regions with a side length of 0.025 mm (bin50), Figure 4A which shows a viewable graph of the whole transcriptome expression of the unbiased sequencing tumor microenvironment on the same slice, Figure 4B which is a viewable graph of the distribution of the sgRNA library for CRISPR screening of T cells in the tumor microenvironment by unbiased sequencing on the same slice. Among them, each color represents a kind of sgRNA knockout information (the corresponding 50 genes are as shown in Figure 4B ); for the high-complexity region and low-complexity region of sgRNA, it was found by characterization that the clones with different sgRNA gene knockouts in the high-complexity region showed mixed growth (as shown in Figure 4C ); while the low-sgRNA complexity region showed almost single sgRNA clone growth ( Figure 4C ), indicating that tumor clones with different gene knockouts at different spatial positions have different growth and amplification advantages. For the spatial characterization of the sgRNA expressed by tumor cells and the characteristic genes expressed by CD8+ T cells ( Figure 4D ), it can be found that the distribution of sgRNA can have a high-resolution differentiation from CD8+ T cells in space ( Figure 4D ), which shows the high resolution and high precision of spatial sgRNA library sequencing; b. According to the gene expression type and expression level, unbiased clustering was performed on all bin50 regions of the ordinary transcriptome sequencing data to obtain the result graph of unbiased clustering ( Figure 6B ). Different categories in the graph are shown in different colors, and the same color indicates the same tumor microenvironment; c. Perform a global differential analysis on the gene expression levels and types of each cluster after clustering with all other clusters to obtain the signature genes of each cluster as the key features of each cluster; for adjacent sections of the same sample, perform sgRNA library quantification of the genome (traditional method, gold standard) and the sgRNA quantification described in the present invention, and calculate the correlation of the counts of different sgRNAs in the two methods (as Figure 5 shown, the 38 genes involved are Ccr1, Ccr2, Ccr4, Ccr5, Ccr6, Ccr7, Ccr10, Cxcr1, Cxcr2, Cxcr3, Cxcr4, Ackr3, Cxcr5, Cxcr6, Cd74, Gpr35, Foxp1, Jun, Bcl2, Fos, Batf, Tgif1, Ybx1, Klf2, Bcl2l11, Jund, Elob, Tox, Tcf7, Hopx, Bhlhe40, Ddit4, Non-targeting_1-Non-targeting_6); d. According to the same spatial coordinates, transfer the clustering information of the genes to the bin of 8 μm containing the sgRNA library ( Figure 6B ); e. Perform a differential analysis on the expression levels of sgRNAs in different clusters to obtain which type of tumor microenvironment T cells tend to enrich in after gene knockout, as Figure 6C shown, this figure shows that at different spatial positions of the tumor metastasis foci, the infiltration or rejection of immune cells is different, and a phenomenon related to tumor clones ( Figure 6A ) with different sgRNA gene knockouts is presented.

[0050] 2.1.4 Result Analysis Combined with the growth of mouse subcutaneous tumor clones, compare the growth of lung metastasis foci clones to obtain the specific dominant clones in the lung metastasis foci. Through unsupervised clustering of spatial transcriptomics, obtain the spatial microenvironment classification in the lung metastasis foci, and obtain two important phenotypes of immune rejection and immune infiltration, corresponding to different spatial microenvironment immune classifications of different gene knockout clone types ( Figure 6B ). Through the classification of cell marker gene expression, obtain the types and distributions of immune cells at the corresponding positions of clone types with different gene knockouts in space ( Figure 6C ), which corroborates the spatial microenvironment classification in Figure 6B . At the same time, through the sequential analysis of clone types with single gene knockouts, it is found that in the tumor clones (sgIcam1) formed by knocking out the TCR co-stimulatory factor gene Icam1 (intercellular adhesion molecule 1, which has been reported to bind to the receptor protein encoded by the Lfa-1 gene on the T cell membrane) on the tumor cell membrane surface, the infiltration of T cells is significantly reduced ( Figure 6C). Through the spatial distribution of pathway enrichment, it was found that IFN-γ was also highly expressed in clones with high immune infiltration, while it was lowly expressed in immune rejection-type clones. IFN-γ was significantly lowly expressed in tumor clones (sgIcam1) with knockout of the TCR co-stimulatory factor gene Icam1. Since IFN-γ is the main cytotoxic effector protein for T cells to exert their functions, the rejection phenomenon of T cell infiltration and the downregulation of IFN-γ together prove that knockout of the TCR co-stimulatory factor gene Icam1 clone (sgIcam1) leads to a significant downregulation of the anti-tumor immune response of T cells.

[0051] Overall, through the CRISPR screening method, it was found that spatially distinct CRISPR KO tumor clone types created an immune rejection spatial microenvironment phenotype. From the gene pathway and cell subtype annotation as an explanation for its phenotype, intercellular adhesion molecule 1 (Icam1) was identified as an important target for tumor immune surveillance function dependence. The specific research results are as follows: SPAC-seq precisely mapped the spatial distribution of perturbed tumor clones and their related gene expression programs, and identified Icam1, the deletion of which leads to immune rejection because Icam1, as a TCR (T cell receptor) co-stimulatory signal, can regulate immune surveillance. The deletion of Icam1 leads to impaired T cell activation by inhibiting TCR signal transduction, and an immunosuppressive phenotype with the absence of the IFN-γ (interferon-γ) signaling pathway, enhancing the metastatic colonization potential of its tumor clones (as Figure 6C , Figure 6D shown). In addition, these research results indicate that SPAC-seq found that ligand-receptor interactions are key determinants of tumor immune escape and metastatic progression. This further demonstrates the great potential of spatially resolved CRISPR screening to reveal functional gene networks in complex tissue environments.

Claims

1. A direct capture sgRNA probe for spatial CRISPR screening sequencing, characterized in that: The direct capture sgRNA probe is designed based on the sgRNA sequence of a specific gene of the target species; Wherein, the direct capture sgRNA probe includes a left probe and a right probe; the sgRNA includes a scaffold sequence and a protospacer sequence; Wherein, the left probe consists of nucleic acid sequence 1 and nucleic acid sequence 2; the right probe consists of nucleic acid sequence 3, nucleic acid sequence 4 and a poly-A tail; Among them, the nucleic acid sequence 1 is the Read2 sequence required for the second-generation sequencing; the nucleic acid sequence 2 is reverse complementary to the 6-30 bp sequence at the 5' end of the scaffold sequence of the sgRNA; the nucleic acid sequence 3 is reverse complementary to the starting 5 bp sequence at the 5' end of the scaffold sequence of the sgRNA; the nucleic acid sequence 4 is reverse complementary to the original spacer sequence of the sgRNA.

2. The direct capture sgRNA probe for spatial CRISPR screening sequencing according to claim 1, characterized in that The left probe and the right probe are DNA; wherein the nucleic acid sequence 1 is 21 bp in length, the nucleic acid sequence 2 is 25 bp in length, the nucleic acid sequence 3 is 5 bp in length, the nucleic acid sequence 4 is 20-30 bp in length, and the poly-A tail is at least 20 bp in length.

3. A spatial CRISPR screening sequencing method, characterized in that: The steps include: S1: Inject target cells containing the CRISPR knockout library into experimental animals; S2: Remove the tissue infiltrated by target cells, prepare paraffin sections, and perform HE staining and imaging; S3: After the tissue slices have been imaged, they are permeabilized and cross-linked, and then probe hybridization is performed with the whole transcriptome gene expression probe and the direct capture sgRNA probe according to any one of claims 1 to 2; the left probe and the right probe of the direct capture sgRNA probe after hybridization are splinted under the action of T4 ligase; S4: Spatial mRNA library construction and spatial sgRNA library construction using Visium HD; S5: Sequence and align the spatial mRNA library and spatial sgRNA library respectively to obtain common transcriptome sequencing data and CRISPR sgRNA library sequencing data carrying spatial coordinates.

4. The spatial CRISPR screening and sequencing method according to claim 3, characterized in that: The target cells containing the CRISPR knockout library are obtained by the following steps: preparing a CRISPR screening library virus, infecting the test cells with the CRISPR screening library virus, sorting out the positively infected test cells and culturing them for a period of time, and obtaining the target cells containing the CRISPR knockout library.

5. The spatial CRISPR screening sequencing method according to claim 3, characterized in that: The injection into the experimental animal is carried out by intravenous injection, subcutaneous injection, intramuscular injection or tumor injection.

6. The spatial CRISPR screening sequencing method according to claim 3, characterized in that: The tissue is tumor tissue or normal tissue.

7. The spatial CRISPR screening and sequencing method according to claim 3, characterized in that: It also includes merging the common transcriptome sequencing data and the CRISPR sgRNA library sequencing data into data of the same chip according to the same spatial coordinates.

8. Use of the direct capture sgRNA probe according to any one of claims 1-2 in spatial CRISPR screening sequencing.

9. Application of the spatial CRISPR screening sequencing method according to any one of claims 3 to 7 in verifying gene function.

10. The use according to claim 9, characterized in that: The common transcriptome sequencing data are clustered unbiasedly, and each clustered class is differentially analyzed from all other classes to obtain the signature gene of each class as the key feature of each class; based on the CRISPR sgRNA library sequencing data, the expression levels of sgRNA in different clusters are differentially analyzed to obtain clusters in which the target cells of the sgRNA knockout gene tend to be enriched, thereby verifying the function of the sgRNA target gene.

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