Method for screening by combining space transcriptome and CRISPR (clustered regularly interspaced short palindromic repeats) in same unbiased mode and application of method in gene function verification

Abstract

The invention discloses a method for screening by combining a space transcriptome and CRISPR (clustered regularly interspaced short palindromic repeats) in a same unbiased mode and application of the method in gene function verification, and belongs to the technical field of biology. The method comprises the following steps: infecting target cells with CRISPR screening library viruses; sorting out positively infected target cells, and culturing for a period of time after knockout; injecting the target cells obtained in the previous step into an experimental animal body, wherein the experimental animal is an animal with autoimmunity deficiency; taking out tissues infiltrated by the target cells, and preparing frozen sections; transferring the frozen section to a spatial transcriptome slide, and loading on a machine to prepare a spatial transcriptome cDNA library; constructing a common transcriptome library and a CRISPR sgRNA library by using the space transcriptome cDNA library; and respectively carrying out sequencing and gene sequence comparison on the common transcriptome library and the CRISPR sgRNA library to obtain common transcriptome sequencing data carrying space coordinates and CRISPR sgRNA library sequencing data carrying space coordinates.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice and its application in gene function verification. Background Art

[0002] CRISPR screening is a technology that uses CRISPR knockout or activation gene editing systems and nucleic acid barcodes to perform specific phenotypes. It was developed by Feng Zhang's team at MIT in 2013 (Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells). Currently, the mainstream barcode used in CRISPR screening is to stably transduce the single guide RNA (sgRNA) used for Cas9 protein knockout genes into the genome of the host cell by viral infection. After the screening pressure ends, cells with sgRNA DNA barcodes can obtain the change relationship of specific genes enriched or missing in specific phenotypes before and after screening through genomic PCR.

[0003] Spatial transcriptome sequencing is an emerging sequencing technology in recent years. Based on single-cell transcriptome sequencing, it has achieved the ability to record cells at different spatial locations while measuring the expression of different RNAs. The feasible technologies are mainly divided into two technical routes: imaging and counting after probe in situ hybridization and sequencing and counting after spatial position encoding probe capture, which ultimately obtains transcriptional gene expression at different locations in space. Among them, in situ sequencing technology designs probes for a set of known target genes of interest, and restores spatial single-cell expression by reading the number of spatial in situ signals through a high-resolution microscope. Single-cell transcriptomes using 10X's Vsium technology and BGI's Stereo-seq technology study the heterogeneity between cells, and spatial transcriptome sequencing adds an exploration of the spatial characteristics of the structure of organisms on the basis of single-cell transcriptome sequencing, that is, assuming that the same cells have different gene expressions at different locations and verifying them.

[0004] In 2022, Brown's laboratory developed a technology for spatial CRISPR screen and 10X's Visium spatial transcriptome sequencing in adjacent tumor slices (Spatial CRISPR genomics identifies regulators of the tumor microenvironment, Cell, 2022). It can simultaneously read out the immune infiltration characteristics in the adjacent areas of tumor cells after knocking out different genes at different spatial locations, as well as the transcriptional status of tumor cells in the corresponding areas in adjacent slices. The principle of this technology is to connect sgRNA and a pre-encoded membrane protein sequence to the same plasmid at the same time, so as to overexpress membrane proteins corresponding to different sgRNAs on the surface of different cells. Finally, the membrane protein information of each cell can be read out and the corresponding sgRNA library can be restored through antibody hybridization and spatial proteomics technology. This technology successfully reads out spatial cell relationships and transcriptome characteristics from CRISPR screen, greatly expanding the phenotypic dimension of CRISPR screen screening. However, the limitations and shortcomings of this technology are also obvious. First of all, this technology uses the method of mixing plasmids after constructing plasmids for all sgRNAs separately, which is time-consuming and labor-intensive, and lacks a method for constructing mixed libraries. Secondly, the technology uses a spatial proteomic approach to mark different sgRNA locations and immune cell types, and a neighboring spatial transcriptome sequencing scheme. This determines that it is impossible to define different cell types and their subtypes in different states from the perspective of the unbiased full-space transcriptome on the same slice. In addition, the combination with the transcriptome cannot be implemented on random and highly motile immune cells such as T cells. Finally, since the library construction is carried out in units of single sgRNAs and the readout method is antibody-labeled, the technology has a low throughput and there is an upper limit on the types of sgRNAs that can be detected. Summary of the invention

[0005] In view of the existing technical problems, the technical problem to be solved by the present invention is to provide a method for combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice. Another technical problem to be solved by the present invention is to provide a method for combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice.

[0006] To solve the existing technical problems, the technical solution of the present invention is as follows:

[0007] A method for combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice, comprising the following steps:

[0008] S1: Infect target cells with CRISPR screening library virus;

[0009] S2: positively infected target cells are sorted and cultured for a period of time after knockout occurs;

[0010] S3: injecting the target cells obtained in step S2 into an experimental animal, wherein the experimental animal is an animal with autoimmune deficiency;

[0011] S4: Remove the tissue infiltrated by target cells and prepare frozen sections;

[0012] S5: Transfer the frozen sections to the spatial transcriptome slide and load them onto the machine to prepare the Total cDNA library;

[0013] S6: Construction of common transcriptome library and CRISPR sgRNA library using Total cDNA library;

[0014] S7: The common transcriptome library and CRISPR sgRNA library are sequenced and gene sequences are aligned respectively to obtain the common transcriptome sequencing data and CRISPR sgRNA library sequencing data with spatial coordinates.

[0015] In the method of combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice, the target cells are specifically immune cells.

[0016] In the method of combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice, the immune cells are specifically effector T cells.

[0017] In the method of combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice, the tissue is tumor tissue or normal tissue.

[0018] In the method of combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice, the method of injecting into the experimental animal is intravenous injection.

[0019] In the method of combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice, the CRISPR sgRNA library constructed using the Total cDNA library is enriched, and the enrichment method is specifically: the CRISPR sgRNA library in the Total cDNA is amplified by PCR using a primer pair, the reverse primer of the primer pair binds to the common sequence of the 3' end of the positive chain of all cDNAs in the Total cDNA library, and the forward primer in the primer pair binds to the common specific sequence at the 5' end of the CRISPR sgRNA library, and the PCR amplification is performed several times, and after each PCR amplification, the DNA screening sequence is combined with magnetic beads to gradually increase the concentration of the CRISPR sgRNA library.

[0020] In the method of combining spatial transcriptome and CRISPR screening in an unbiased manner, the CRISPR sgRNA library constructed using the TotalcDNA library is enriched, and the enrichment method is specifically as follows: using the two ends of the target fragment as the cyclization sequence, adding an oligonucleotide that is half complementary to the front end and half complementary to the back end; using the splint-ligation method to perform cyclization under the catalysis of T4 ligase, and digesting the uncyclized product with DNA exonuclease to obtain a single-stranded circular DNA with higher purity. Double-end specific primer amplification can be performed from the single-stranded circular DNA to obtain a target fragment library with higher purity.

[0021] The method of combining spatial transcriptome and CRISPR screening in an unbiased manner on the same chip also includes performing gene sequence alignment on the common transcriptome sequencing data and CRISPR sgRNA library sequencing data carrying spatial coordinates, respectively, and then merging them into data on the same chip based on the same spatial coordinates.

[0022] The application of common transcriptome sequencing data and CRISPR sgRNA library sequencing data carrying spatial coordinates obtained by any of the above methods in verifying gene function.

[0023] Furthermore, the application specifically comprises the following steps: performing unbiased clustering on the common transcriptome sequencing data, performing differential analysis on each clustered class and all other classes, and obtaining the landmark gene of each class as the key feature of each class; performing differential analysis on the expression levels of sgRNA in different clusters according to the CRISPR sgRNA library sequencing data, and obtaining the clusters in which the target cells of the sgRNA knockout gene tend to be enriched, thereby verifying the function of the sgRNA target gene.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This technology combines CRISPR screening and spatial transcriptome sequencing to add the spatiotemporal dimension of traditional phenotypes and the dimension of the whole transcriptome to the screening, which is beneficial to the study of the state and positional relationship between genes and cells in the structure of organisms in tumor biology, immunology, developmental biology or brain science. For example, in the spatial transcriptome screening of T cell-infiltrating tumors, this application found that the knockout of the Piezo1 gene caused T cells to be significantly enriched in the internal areas of tumors with low fibrosis, reflecting that knocking out the expression of the membrane protein Piezo1 gene can enable T cells to better cross the fibrotic area and infiltrate into the tumor substance. This result verifies the high possibility of the Piezo1 target for highly fibrotic and immune-rejected tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Schematic diagram of viral vector design for mRNA-embedded CRISPR sgRNA knockout library;

[0027] Figure 2 This is a comparison diagram of the cDNA sequence structure composition in the common transcriptome library and the CRISPR sgRNA library. In the figure, the upper part is the cDNA sequence structure diagram in the CRISPR sgRNA library, and the lower part is the sequence structure diagram of the common transcriptome library;

[0028] Figure 3 Schematic diagram of the binding position of primer sequences to cDNA in the total library amplification step, the inward nested Outer step, the inward nested Inner step, the circularized adapter, and the phosphorylation step during the CRISPR sgRNA library enrichment process;

[0029] Figure 4 Schematic diagram of the primer sequence and cDNA binding position in the circularization Splint-Oligo step, bispecific enrichment-1 step, bispecific enrichment-2 step, and Illumina library adapter step during the CRISPR sgRNA library enrichment process;

[0030] Figure 5A This is the spatial transcriptome map of mouse MC38 subcutaneous tumor;

[0031] Figure 5B CRISPR screening library map for mouse T cell space;

[0032] Fig. 6A The spatial transcriptome clustering of mouse MC38 subcutaneous tumors reflects the different microenvironments within the tumor;

[0033] Figure 6B Distribution map of mouse T cell spatial CRISPR screening library in different microenvironments;

[0034] Figure 6C This is a statistical diagram showing that T cells after different gene knockouts tend to be enriched and distributed in different intratumor microenvironments;

[0035] Fig. 7A To characterize the fibroblastic characteristic regions of the tumor microenvironment using the whole genome transcriptome;

[0036] Figure 7B The figure shows the ratio of T cells with different genes knocked out in the areas with high fibroblasts to those with low fibroblasts. DETAILED DESCRIPTION

[0037] To facilitate the technical solution of the application, some concepts, reagents and materials involved in the application are first described below. In order to make the technical problems solved by the present invention, the technical solutions adopted and the technical effects achieved clearer, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. The materials, reagents and instruments used in the examples, if not described in detail, can all be purchased from the market.

[0038] Cantilever primer: refers to a part of the PCR primer that binds to the DNA template, while the other part cannot bind, which reduces the overall affinity of the primer. It is used to reduce the PCR efficiency in the area where all fragments at the 3' end of the library are contained, thereby increasing the affinity of the target gene near the 5' end and the proportion of the gene-specific primer in the entire PCR.

[0039] OT1 / Cas9 mice: OT1 / Cas9 mice purchased from Jackson Corporation, USA.

[0040] pMYs vector: mouse retrovirus pMYs purchased from Cell Biolabs, USA.

[0041] CRISPR sgRNA knockout library: The designed sgRNA sequence is synthesized by GENEWIZ.

[0042] Stereo-seq chip: BGI Genomics.

[0043] Reagents and materials:

[0044]

[0045]

[0046] Embodiment 1:

[0047] 1. sgRNA library construction and infection of effector T cells

[0048] Several genes that can significantly lead to the enrichment of immune cells in tumors after being knocked out in immune cells were used as target genes. 4-6 sgRNAs were designed for each target gene, and a total of 68 sgRNAs including 2 control sgRNAs were designed. The target genes include Zc3h12a and Gata3 genes, which can significantly enhance the infiltration ability of T cells in tumors after being knocked out in T cells.

[0049] The 68 sgRNAs were synthesized to obtain oligonucleotide single-stranded DNA, which were digested and connected to the pMYs vector ( Figure 1 ), and then the pMYs vector is packaged to form viral particles, namely the sgRNA library virus.

[0050] The spleen of OT1 / Cas9 mice was obtained after being killed. After grinding and filtering, a single cell suspension of spleen cells was obtained, and CD8+T cells were sorted out using a magnetic bead negative selection kit (source: Miltenyi Biotec). The obtained CD8+T cells expressed OT1 (TCR that specifically recognizes the tumor cell OVA antigen) and Cas9 protein.

[0051] The sorted CD8+T cells are activated and induced in vitro to become effector T cells, and then mixed with all sgRNA library viruses at the same time to infect the effector T cells. Based on the fact that positively infected effector T cells can express the GFP gene carried by the sgRNA library virus as fluorescent protein while uninfected ones cannot, flow cytometry is used to sort out positively infected T cells. Since the virus in positively infected T cells transcribes sgRNA and combines with the Cas9 protein in the T cells to form an RNP complex targeting the target gene, the obtained positively infected T cell group contains T cells with different genes knocked out.

[0052] Second, using tumor tissue spatial heterogeneity as a screening pressure to perform CRISPR screening on positively infected T cell populations

[0053] The different microenvironments (spaces) within tumor tissues have a certain degree of heterogeneity, and differ in tumor spatial characteristics such as hypoxia, fibrosis, immune rejection, and interactions between immune cells, and thus have different effects on different immune cells.

[0054] The positively infected T cells were cultured until 6 days after the knockout occurred, and then injected into the bodies of Rag1 gene-deficient mice (self-T and B cell deficiency) that had been implanted with mouse MC38 (colorectal cancer cell line) subcutaneous tumors 7 days in advance through microvenous injection. This allowed the T cells with different genes knocked out to move freely and infiltrate in different spatial neighborhoods of the tumor according to the preferences generated after the knockout of different genes during recruitment.

[0055] 3. Preparation of T cell-infiltrated tumor tissue cryosections and spatial transcriptome cDNA libraries

[0056] 4-10 days after the T cells edited by the sgRNA library infiltrated the tumor, the mice were sacrificed, the intact subcutaneous tumors were removed, OCT embedded, and frozen sections were performed. The fresh frozen sections were then loaded onto the prepared Stereo-seq chip. The chip surface contained a poly dT probe to capture mRNA with polyA in the tissue (including mRNA chimeric with the sgRNA library). After the slices were photographed for GFP fluorescence, the mRNA captured by the poly dT probe was reverse transcribed to obtain a cDNA library, namely the Total cDNA library, according to the instructions for use of the Stereo-seq chip. The Total cDNA library includes the common transcriptome library and the CRISPR sgRNA library. For all cDNAs in the Total cDNA library, pA (poly dA) is followed by a spatial ID coordinate (CID) encoded by different DNA at different positions, and contains the unique molecular name (MID) of different probes at the same coordinate. There is a fixed sequence Fixed between the CID and the MID. There is a Read1 sequence for amplification after the CID. After the Read1 sequence, there is another TSO sequence for amplification, and the corresponding other end is Read2 and TSO sequence. For the cDNA in the CRISPR sgRNA library, in addition to the chimeric sgRNA sequence, it also has chimeric U6, sgRNA, LTR, pA, Unnamed-1 and Unnamed-2, which are common known sequences derived from the pMYs viral vector (Unnamed-1 and Unnamed-2 represent two viral infection element sequences without fixed names, also known as Virus Related Sequence). Different sgRNAs are due to the difference in 20bp of variable gene sequences, but different sgRNAs have a common, immutable sequence scaffold of 76bp. Unknown cDNA indicates an unknown sequence in the spatial transcriptome library. The differences in cDNA structure between ordinary transcriptome libraries and CRISPR sgRNA libraries are as follows: Figure 2 shown.

[0057] The total amount of the Total cDNA library was quantitatively analyzed using Qubit, and a volume of about 10-20 ng was considered qualified.

[0058] 4. Use the total cDNA library to further construct a common transcriptome library and CRISPR sgRNA library

[0059] The total cDNA library was divided into two parts, one for common transcriptome library construction and sequencing, and the other for CRISPR sgRNA library construction and sequencing. Common transcriptome sequencing consumes 1 / 10 of the total cDNA library, and the remaining library is used for CRISPR sgRNA library construction and sequencing.

[0060] (1) Construction and sequencing of general transcriptome library

[0061] According to the commercial method of Stereo-seq, about 1 / 10 of the total cDNA library was broken into appropriate lengths and then subjected to MGI second-generation sequencing library construction and loading to obtain a common transcriptome sequencing file (the very small amount of CRISPR sgRNA library contained therein will not affect the sequencing results).

[0062] (2) Enriching the sgRNA library using ultra-trace templates to obtain a CRISPR sgRNA library

[0063] Constructing a CRISPR sgRNA library with a total cDNA library requires enrichment of the CRISPR sgRNA library to meet the sequencing sample requirements. The main difficulty in enriching CRISPR sgRNA or other specific genes in single-cell transcriptome or spatial transcriptome sequencing is that the target fragment is mixed with all gene fragments and the target fragment content is extremely low; on the other hand, in order to simultaneously amplify the amplified cell or spatial ID at the very end of the library, one of the two primers of PCR must use an amplification primer common to the cDNA of all genes, and it is difficult to amplify by ordinary PCR methods. This application uses an enrichment method based on an independently developed ultra-micro template to successfully construct a CRISPR sgRNA library for sequencing.

[0064] For cDNA library, the main strategy for enriching gene knockout barcode fragments is nested PCR specific amplification and magnetic bead fragment screening. All DNA in cDNA has a common amplification sequence and corresponding amplification primer at the 3' end of the positive strand. By setting multiple specific primers at the 5' end of the gene knockout barcode fragment, performing multi-step PCR, and screening the fragments by DNA binding to magnetic beads after each step of PCR, the proportion of positive fragments can be gradually increased.

[0065] For samples with a low proportion of positive fragments, a cyclization method is used for further enrichment. The specific method of cyclization is to use the two ends of the target fragment as the cyclization sequence, and add an oligonucleotide that is half complementary to the front end and half complementary to the back end. The splint-ligation method is used to perform cyclization under the catalysis of T4 ligase (Thermo Company), and the uncyclized product is digested by DNA exonuclease to obtain a single-stranded circular DNA with higher purity. Double-end specific primer amplification can be performed from the single-stranded circular DNA to obtain a target fragment library with higher purity. The target fragment library is added with a second-generation sequencing adapter by PCR, and then it can be sequenced on the machine.

[0066] The specific method of enrichment is as follows (eg Figure 3-Figure 4 shown):

[0067] Step 1: (optional, to be done when the total library is not enough) perform total amplification on the total cDNA library;

[0068] Step 2: Perform outer enrichment of the total cDNA library by nested PCR;

[0069] Step 3: Perform inner enrichment of nested PCR on the outer enrichment product;

[0070] Step 4: Add splint-TSO cyclization sequence to the inner enriched product;

[0071] Step 5: Splint-Ligation cyclization;

[0072] Step 6: Bispecific enrichment-1;

[0073] Step 7: Bispecific enrichment-2;

[0074] Step 8: illumina library adapter P5-P7 (ST-P5P7), recover the product, and use it for sequencing. The primer sequences involved in the above steps are shown in Table 1 below:

[0075] Table 1 Primer sequences

[0076]

[0077] Specific steps for amplification and enrichment:

[0078] 1. (Optional, to be performed when the total library is not enough) Amplify the total cDNA library: Prepare 5 PCR tubes, the system is as follows:

[0079] Reagents Stock Amount cDNA 150ng(NanoDrop) XμL Q5 2×Master Mix 15μL Total library amplification primers 10μM 1.2μL <![CDATA[DNAse / RNAse H2O]]> 12.6-XμL Total 30μL

[0080] Take out the magnetic beads (1× magnetic beads purification) 30 minutes in advance and set up the PCR program

[0081] Step Temperature Time 1. Pre-denaturation 95℃ 5min 2. Transformation 98℃ 20sec 3. Annealing 58℃ 30sec 4. Extension 72℃ 2min 5.2-4 Cycle times 10 times 6. Final extension 72℃ 5min 7.Save 4℃ Forever

[0082] Recovery (1× purification):

[0083] (1) Add 30 μL of magnetic beads to each tube and let stand at room temperature for 10 min;

[0084] (2) Place on a magnetic rack and let stand for 5 minutes;

[0085] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol;

[0086] (4) Dry until it is no longer reflective (do not wait until it is dry and cracked), then add 15 μL of enzyme-free water to dissolve;

[0087] (5) Detect the concentration and fragment size and calculate the yield (about 2-4 times is a reasonable range).

[0088] 2. Outer enrichment of Total cDNA library:

[0089] Prepare 5 PCR tubes with the following system:

[0090] Reagents Stock Amount cDNA 150ng(NanoDrop) XμL Q5 2×Master Mix 15μL Outer-F 10μM 1.2μL Outer-R 10μM 1.2μL <![CDATA[DNAse / RNAse H2O]]> 12.6-XμL Total 30μL

[0091] Remove the magnetic beads 30 minutes in advance and set the PCR program (ST-Outer):

[0092] Step Temperature Time 1. Pre-denaturation 95℃ 5min 2. Transformation 98℃ 20sec 3. Annealing 65℃ 30sec 4. Extension 72℃ 2min 5.2-4 Cycle times 8-9 times 6. Final extension 72℃ 5min 7.Save 4℃ Forever

[0093] Recovery (0.5× purification):

[0094] (1) Add 15 μL of magnetic beads to each tube and let stand at room temperature for 10 min;

[0095] (2) Place on a magnetic rack and let stand for 5 minutes;

[0096] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol;

[0097] (4) Dry until it is no longer reflective (do not wait until it is dry and cracked), then add 15 μL of enzyme-free water to dissolve;

[0098] (5) Check the concentration and fragment size and calculate the yield, which should be around 10%, i.e., the concentration should be 10-20 ng / μL (Nanodrop).

[0099] 3. Inner enrichment of Outer enrichment products:

[0100] Next, the PCR products from the previous step (if the fragment analysis is correct) are run for 5 tubes per library. The system is as follows:

[0101] Reagents Stock Amount cDNA 150ng(NanoDrop) XμL Q5 2×Master Mix 15μL Inner-F 10μM 1.2μL Inner-R 10μM 1.2μL <![CDATA[DNAse / RNAse H2O]]> 12.6-XμL Total 30μL

[0102] Remove the magnetic beads 30 minutes in advance and set the PCR program (ST-Outer):

[0103] Step Temperature Time 1. Pre-denaturation 95℃ 5min 2. Transformation 98℃ 20sec 3. Annealing 64℃ 30sec 4. Extension 72℃ 1min 5.2-4 Cycle times 8-9 times 6. Final extension 72℃ 5min 7.Save 4℃ Forever

[0104] Recovery (0.5× purification):

[0105] (1) Add 15 μL of magnetic beads to each 30 μL tube and let stand at room temperature for 10 min;

[0106] (2) Place on a magnetic rack and let stand for 5 minutes;

[0107] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol;

[0108] (4) Dry until it is no longer reflective (do not wait until it is dry and cracked), then add 15 μL of enzyme-free water to dissolve;

[0109] (5) Detect the concentration and fragment size and calculate the yield, that is, the concentration is 1-5 ng / μL (Nanodrop).

[0110] 4. Add splint-TSO cyclization sequence to the inner enrichment product:

[0111] Next, the PCR products from the previous step (if the fragment analysis is correct) are run for 5 tubes per library. The system is as follows:

[0112] Reagents Stock Amount cDNA 150ng(NanoDrop) XμL Q5 2×Master Mix 15μL Circularized linker and phosphorylated-F 10μM 1.2μL Circularized linker and phosphorylated-R 10μM 1.2μL <![CDATA[DNAse / RNAse H2O]]> 12.6-XμL Total 30μL

[0113] Remove the magnetic beads 30 minutes in advance and set the PCR program (ST-Outer):

[0114] Step Temperature Time 1. Pre-denaturation 95℃ 5min 2. Transformation 98℃ 20sec 3. Annealing 72℃ 30sec 4. Extension 72℃ 1min 5.2-4 Cycle times 8-9 times 6. Final extension 72℃ 5min 7.Save 4℃ Forever

[0115] Recovery (0.5× purification):

[0116] (1) Add 15 μL of magnetic beads to each 30 μL tube and let stand at room temperature for 10 min.

[0117] (2) Place on a magnetic rack and let stand for 5 minutes

[0118] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol.

[0119] (4) Dry until it is no longer reflective (do not wait until it is dry and cracked), then add 15 μL of enzyme-free water to dissolve

[0120] (5) Check the concentration and fragment size. The yield is about 20-30%, that is, the concentration is 20-30 ng / μL (Nanodrop). If the total amount is less than 900 ng, perform step 4 again (add splint-TSO cyclization sequence).

[0121] 5. Cyclization sequence:

[0122] (1) Turn on the PCR instrument to preheat at 95°C;

[0123] (2) Take 900 ng of the product of step 4 measured by nanodrop in the previous step, or 400 ng of Qubit, dilute it to 45 μl, then add 5 μl of splint oligo to prepare the following denaturation system:

[0124] Reagents Stock Amount cDNA 900ng(NanoDrop) / 400ng(Qubit) 45μL Cyclization Splint-Oligo 5μL Total 50μL

[0125] (3) After being placed at 95℃ for 5 minutes, it is immediately placed on a -30℃ ice block to cool down and placed in a -30℃ refrigerator (placed for 1-2 minutes, the purpose is to cool down, not to freeze solidify), and then proceed to the next step of connection:

[0126] (4) Connection, the system is as follows

[0127] Reagents Stock Amount Step (2) Denaturation system 50μL Ligation Buffer 5× 9.8μL T4 Ligase 0.2 Total 60μL

[0128] 37 degrees Celsius connection 1h program:

[0129] Step Temperature Time 1. Connect 37℃ 1hour 2.Save 4℃ Forever

[0130] (5) Enzyme digestion, the system is as follows:

[0131] Reagents Stock Amount Step (4) Connecting the product 60μL Exonuclease I 1.95μL Exonuclease III 0.65μL Buffer 1.4μL Total 64μL

[0132] The procedure is as follows:

[0133] Step Temperature Time 1. Connect 37℃ 30min 2.Save 4℃ Forever

[0134] After the program is finished, quickly add 5 μL of 0.1 mM EDTA to terminate (final volume 70 μL)

[0135] Recovery (2.5× purification):

[0136] (1) Add 180 μL of magnetic beads to each 70 μL tube and let stand at room temperature for 10 min;

[0137] (2) Place on a magnetic rack and let stand for 5 minutes;

[0138] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol;

[0139] (4) Dry until it is no longer reflective (do not wait until it is dry and cracked), then add 15 μL of enzyme-free water to dissolve;

[0140] (5) Detect the concentration and fragment size and obtain a concentration of 20-30 ng / μL (Nanodrop).

[0141] 6. Bispecific Enrichment-1

[0142] Next, the PCR products from the previous step (if the fragment analysis is correct) are run for 5 tubes per library. The system is as follows:

[0143] Reagents Stock Amount cDNA All cDNAs in the previous step XμL Q5 2×Master Mix 15μL Bispecific Enrichment-1-F 10μM 1.2μL Dual-specific enrichment-1-R 10μM 1.2μL <![CDATA[DNAse / RNAse H2O]]> 12.6-XμL Total 30μL

[0144] Remove the magnetic beads 30 minutes in advance and set up the PCR program (dual specific enrichment-1):

[0145] Step Temperature Time 1. Pre-denaturation 95℃ 5min 2. Transformation 98℃ 20sec 3. Annealing 72℃ 30sec 4. Extension 72℃ 30sec 5.2-4 Cycle times 12 times 6. Final extension 72℃ 5min 7.Save 4℃ Forever

[0146] Recovery (1.5× purification):

[0147] (1) Add 45 μL of magnetic beads to each 30 μL tube and let stand at room temperature for 10 min;

[0148] (2) Place on a magnetic rack and let stand for 5 minutes;

[0149] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol;

[0150] (4) Dry until it is no longer reflective (do not wait until it is dry and cracked), then add 15 μL of enzyme-free water to dissolve;

[0151] (5) Detect the concentration and fragment size and obtain a concentration of 1-20 ng / μL (Nanodrop).

[0152] If there are no obvious bands, but no contaminants, perform another round of the same steps as bispecific enrichment-1. The product can be named Direct-6-2 and the concentration should be 30-80 ng / μL:

[0153] 7. Bispecific Enrichment-2

[0154] Next, the PCR products from the previous step (if the fragment analysis is correct) are run for 5 tubes per library. The system is as follows:

[0155] Reagents Stock Amount cDNA All cDNAs in the previous step XμL Q5 2×Master Mix 15μL Bispecific Enrichment-2-F 10μM 1.2μL Dual-specific enrichment-2-R 10μM 1.2μL <![CDATA[DNAse / RNAse H2O]]> 12.6-XμL Total 30μL

[0156] Remove the magnetic beads 30 minutes in advance and set up the PCR program (dual specific enrichment-2):

[0157]

[0158]

[0159] Recovery (1.5× purification):

[0160] (1) Add 45 μL of magnetic beads to each 30 μL tube and let stand at room temperature for 10 min;

[0161] (2) Place on a magnetic rack and let stand for 5 minutes;

[0162] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol;

[0163] (4) Dry until it is no longer reflective (do not wait until it is dry and cracked), then add 15 μL of enzyme-free water to dissolve;

[0164] (5) The concentration and fragment size were detected and the concentration was 60-80 ng / μL (Nanodrop).

[0165] 8. Illumina library connector P5-P7 (ST-P5P7)

[0166] Next, the PCR products from the previous step (if the fragment analysis is correct) are used to perform 5 tubes of PCR for each library. The P5-P7 (ST-P5P) system is as follows:

[0167] Reagents Stock Amount cDNA All cDNAs in the previous step XμL Q5 2×Master Mix 15μL Illumina Library Adapter-F 10μM 1.2μL Illumina Library Adapter-R 10μM 1.2μL <![CDATA[DNAse / RNAse H2O]]> 12.6-XμL Total 30μL

[0168] Remove the magnetic beads 30 minutes in advance and set up the PCR program (dual specific enrichment-1):

[0169] Step Temperature Time 1. Pre-denaturation 95℃ 5min 2. Transformation 98℃ 20sec 3. Annealing 72℃ 30sec 4. Extension 72℃ 30sec 5.2-4 Cycle times 5 times 6. Final extension 72℃ 10min 7.Save 4℃ Forever

[0170] Recovery (1.5× purification):

[0171] (1) Add 45 μL of magnetic beads to each 30 μL tube and let stand at room temperature for 10 min.

[0172] (2) Place on a magnetic rack and let stand for 5 minutes

[0173] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol.

[0174] (4) Dry until it is no longer reflective (do not wait until it is dry and cracked), then add 15 μL of enzyme-free water to dissolve

[0175] (5) Detect the concentration and fragment size to obtain a product with a concentration of 100-200 ng / μL (Nanodrop), which can be used for next-generation sequencing.

[0176] 5. Perform next-generation sequencing on the CRISPR sgRNA library to obtain offline data

[0177] By performing next-generation sequencing on the enriched CRISPR sgRNA library, we can obtain the sequence of the CRISPR sgRNA in the same fragment as the common transcriptome in the sequencing library and the spatial ID corresponding to each specific sequence.

[0178] The second-generation sequencing was sent, with a total data volume of 10G. The CRISPR sgRNA library was sequenced using the Illumina NovaSeq 6000 mainstream NGS (second-generation sequencing) platform.

[0179] 6. Perform bioinformatics analysis on the CRISPR sgRNA library and the common transcriptome library data to obtain transcriptome data with spatial coordinates

[0180] (1) Main steps of bioinformatics analysis of common transcriptome libraries

[0181] For the sequencing results of the common transcriptome, the BGI spatial transcriptome analysis software SAW (Stereo-seqAnalysis Workflow) was used to align the gene sequences of different reads in the sequencing results to the reference genome to determine the gene expression, and the corresponding CID was aligned to the pre-coded spatial coordinate library to determine its spatial position. The MID was used to deduplicate the reads with the same CID, and the unique read expression information of different genes under all coordinates could be obtained ( Figure 5A ), after alignment, the common transcriptome sequencing data file is obtained.

[0182] (2) Main steps of bioinformatics analysis of CRISPR sgRNA libraries

[0183] Compare and rename the sequencing data. For the data from MGI2000, this step is not necessary. For the second-generation sequencing data produced by Illumina and other platforms, first open the *.fq1.gz and *fq2.gz files that were downloaded from the machine, read the file content, and for the read file, find the sequence name line according to the @ symbol, add " / 1" for the line from *.fq1.gz; add " / 2" for the line from *.fq2.gz. Write out new *.fq1.gz and *fq2.gz files. This step is called fromillumina2MGI.

[0184] Sequencing data splitting: For the *.fq1.gz and *.fq2.gz files obtained by sequencing, first screen the *.fq1.gz files for sequences with a Levenshtein distance of less than or equal to 1 to the fixed sequence. If they are present, extract the first 25 and last 10 bases of the fixed sequence as coordinate identities and molecular identifiers; for the corresponding *.fq2.gz files, search for the first 21 bases of the sgRNA scaffold fixed sequence (dynamically adjust the length according to the sgRNA library, which is the maximum sgRNA sequence length plus 1). If they do not meet this standard, remove the sequence and its corresponding sequence in *.fq2.gz. This step is called seekseq.

[0185] The *.fq1.gz and *fq2.gz obtained above are compared by SAW, and finally the corresponding sgRNA spatial position coordinate *.gem file is obtained. The content structure of the *.gem file is

[0186] #FileFormat=GEMv1.0

[0187] #SortedBy=None

[0188] #STOmicsChip=

[0189] #OffsetX=

[0190] #OffsetY=

[0191] geneID xy MIDCount ExonCount

[0192] sgCd44_1_gene 1123 3482 1 1

[0193] The first line of the comment line is the file version output by SAW, the third line is the chip number used, and the fourth and fifth lines are the first non-zero XY coordinates of the sgRNA distribution on the original chip; starting from the sixth line: the first column of each line is the sequence of the sgRNA, the second and third columns are the gRNA position coordinates, and the fourth and fifth columns are the number of sgRNAs of this type measured at this position; the CRISPR sgRNA library sequencing data file is merged with the common transcriptome sequencing data file to obtain the final sgRNA and RNA expression file ( Figure 5A , Figure 5B ).

[0194] 7. Perform downstream analysis on common transcriptome sequencing data and CRISPR sgRNA library sequencing data with spatial coordinates to achieve spatial visualization of gene expression and verify the function of genes knocked out by sgRNA

[0195] Scanpy V1.10.3 was used to perform downstream analysis on the data in the above sgRNA and RNA expression files containing spatial coordinates:

[0196] a. After reading the data, cut the whole piece into small areas (bin50) with a side length of 0.025 mm, such as Figure 5A , Figure 5B As shown, Figure 5A The visualization of the whole transcriptome expression of the tumor microenvironment in unbiased sequencing on the same slide is shown. Figure 5B This is a visualization of the distribution of the sgRNA library in the tissue space by unbiased sequencing of tumor microenvironment T cells using CRISPR, where each color represents a type of sgRNA knockout information.

[0197] b. Perform unbiased clustering of all bin50 regions of the whole gene transcriptome (common transcriptome sequencing data) according to gene expression type and expression level to obtain an unbiased clustering result graph, such as Fig. 6A As shown, different categories are displayed in different colors, and the same color represents the same tumor microenvironment.

[0198] c. Perform an overall difference analysis on the gene expression levels and types of each clustered class and all other classes to obtain the signature genes of each class as the key features of each class.

[0199] d. Based on the same spatial coordinates, the clustering information of the gene is transferred to the library containing sgRNA ( Figure 6B ) in bin50.

[0200] e. Perform differential analysis on the expression levels of sgRNA in different clusters to find out which type of T cells after gene knockout tend to be enriched in which type of tumor microenvironment, such as Figure 6C As shown in the figure, the region of different spatial gene expression delineated by the spatial transcriptome based on unbiased clustering shows the enrichment or loss distribution characteristics of the gene knockout sgRNA relative to the control group sgRNA (sgNTC, Non Targeting Control, sgRNA that does not target any gene sequence on the genome). The gradient to blue represents that the knockout of the sgRNA makes T cells more lost in a certain area, while the gradient to red represents that the sgRNA makes T cells more concentrated in a certain area. It can be seen in the figure that sgCd44 is significantly enriched in the cluster3 region.

[0201] f. A paper published in Cell in 2022 (Osr2 functions as a biomechanical checkpoint to aggravate CD8+T cell exhaustion in tumor, Cell, 2024) reported the adverse effects of intratumoral fibrosis on T cell crossing and infiltration in the tumor substance, and the core role of Piezo1 in inducing T cell exhaustion in this process. In this example, the spatial transcriptome of T cell-infiltrated tumors was screened for T cell infiltration in fibrotic areas. First, several tumor fibrosis marker genes (GO gene annotation Term: IDfibroblast proliferation; GO:0048144; including genes such as Egfr) were selected from literature reports. The expression level of this type of marker gene in each bin50 region was normalized with the total expression level of all genes to score the relative degree of fibrosis in different regions, thereby depicting the characteristics of fibrotic regions in MC38 tumors, such as Fig. 7A As shown in the figure, a visual graph of the fibroblast characteristic area of ​​the tumor microenvironment is depicted using the whole gene transcriptome, in which the score bar on the right gradually changes from dark green to yellow and the score gradually increases. The larger the score value, the greater the expression of the tumor fibrosis marker gene and the higher the degree of fibrosis. Figure 7B The enrichment of T cells knocked out of different genes in the fibroblast-high area and the fibroblast-low area is shown in the figure. Fig. 7A The area with a score greater than 2.2 is a high fibroblast area, and the area with a score less than 0.8 is a low fibroblast area. The number of sgRNAs measured represents the enrichment of T cells with the corresponding gene knocked out. Figure 7B Figure A in the middle shows the ranking of the ratio of T cell enrichment in areas with high fibroblasts to areas with low fibroblasts. The higher the vertical axis value, the more T cells with a gene knockout are enriched in areas with high fibroblasts; on the contrary, Figure 7B Figure B in the middle shows the ranking of the ratio of T cell enrichment in areas with low fibroblasts to areas with high fibroblasts. The higher the vertical axis value, the more enriched the T cells in areas with low fibroblasts after knocking out a certain gene are. Figure 7B It can be clearly seen that T cells with Piezo1 knockout are more enriched in the low fibroblast area, while the control group sgNTC (sgnon-targeting_2_gene in the figure) is significantly enriched in the high fibrosis area, which is consistent with the conclusions published in the Cell literature. Fig. 7AIt is not difficult to see that the areas with high fibrosis are concentrated at the edge of the tumor, and the areas with low fibrosis are concentrated in the tumor substance. This shows that knocking out the Piezo1 gene (sgPiezo1) makes T cells more enriched in the tumor internal area with low fibrosis, reflecting that knocking out the expression of the membrane protein Piezo1 gene can make T cells better cross the fibrotic area and infiltrate into the tumor substance. This result verifies the high possibility of Piezo1 target for drug development in tumors with high fibrosis and immune rejection.

[0202] The technology of this application combines CRISPR screening and spatial transcriptome sequencing to add the spatiotemporal dimension of traditional phenotypes and the whole transcriptome dimension to screening, which is beneficial for studying the state and positional relationship of genes and cells in the structure of organisms in tumor biology, immunology, developmental biology or brain science.

Claims

1. A method for combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice, characterized in that The steps include: S1: Infect target cells with CRISPR screening library virus; S2: sort out the positively infected target cells and culture them for a period of time; S3: injecting the target cells obtained in step S2 into an experimental animal, wherein the experimental animal is an animal with autoimmune deficiency; S4: Remove the tissue infiltrated by target cells and prepare frozen sections; S5: Transfer the frozen sections to the spatial transcriptome slide and load them onto the machine to prepare the Total cDNA library; S6: Construction of common transcriptome library and CRISPR sgRNA library using Total cDNA library; S7: The common transcriptome library and CRISPR sgRNA library are sequenced and gene sequences are aligned respectively to obtain the common transcriptome sequencing data and CRISPR sgRNA library sequencing data with spatial coordinates.

2. The method for combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice according to claim 1, characterized in that The target cells are specifically immune cells.

3. The method of combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice according to claim 2, characterized in that The immune cells are specifically effector T cells.

4. The method of combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice according to claim 1, characterized in that The tissue is tumor tissue or normal tissue.

5. The method of combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice according to claim 1, characterized in that The method of injecting into the experimental animal is intravenous injection.

6. The method of combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice according to claim 1, characterized in that The CRISPR sgRNA library constructed using the Total cDNA library is enriched, and the enrichment method is specifically: the CRISPR sgRNA library in the Total cDNA is amplified by PCR using a primer pair, the reverse primer of the primer pair binds to the common sequence of the 3' end of the positive chain of all cDNAs in the Total cDNA library, and the forward primer in the primer pair binds to the common specific sequence at the 5' end of the CRISPR sgRNA library, and the PCR amplification is performed several times, and after each PCR amplification, the sequence is screened by magnetic beads to gradually increase the concentration of the CRISPR sgRNA library.

7. The method of combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice according to claim 1, characterized in that The CRISPR sgRNA library constructed using the Total cDNA library is enriched, and the specific method of the enrichment is: using the two ends of the target fragment as circularization sequences, using the splint-ligation method to perform circularization under the catalysis of T4 ligase, and digesting the uncircularized product with DNA exonuclease to obtain a single-stranded circular DNA with higher purity, and performing double-end specific primer amplification with the single-stranded circular DNA to obtain a CRISPR sgRNA library with higher purity.

8. The method of combining spatial transcriptome and CRISPR screening in an unbiased manner on the same slice according to claim 1, 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.

9. Application of common transcriptome sequencing data and CRISPR sgRNA library sequencing data carrying spatial coordinates obtained by any of the methods described in claims 1-8 in verifying gene function.

10. The use according to claim 9, characterized in that: include: 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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