A method for integrating spatial transcriptomics and CRISPR screening in a same-slice unbiased manner and its application in gene function verification

Abstract

The present invention discloses a method for combining spatial transcriptomics and CRISPR screening in a same-slice unbiased manner and its application in gene function verification, belonging to the field of biotechnology. The steps of the present invention include: infecting target cells with a CRISPR screening library virus; sorting out positively infected target cells and culturing them for a period of time after knockout occurs; injecting the target cells obtained in the foregoing steps into an experimental animal, where the experimental animal is an animal with autoimmune deficiency; taking out the tissue infiltrated by the target cells and preparing frozen sections; transferring the frozen sections onto a spatial transcriptomics slide and loading it onto a machine to obtain a spatial transcriptomics cDNA library; constructing a general transcriptome library and a CRISPR sgRNA library using the spatial transcriptomics cDNA library; respectively sequencing and comparing gene sequences of the general transcriptome library and the CRISPR sgRNA library to obtain general transcriptome sequencing data and CRISPR sgRNA library sequencing data carrying spatial coordinates.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for combining spatial transcriptomics 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 technique that uses a gene editing system for CRISPR knockout or activation and nucleic acid barcodes for specific phenotypes, developed by the Zhang Feng 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 a single guide RNA (sgRNA) used for knocking out genes of Cas9 protein into the genome of host cells by means of virus infection. Cells with sgRNA DNA barcodes, after the screening pressure ends, obtain the change relationship of specific genes with enrichment or deletion of specific phenotypes before and after screening through genomic PCR.

[0003] Spatial transcriptome sequencing is a newly emerging sequencing technology in recent years. Based on single-cell transcriptome sequencing, it realizes the simultaneous measurement of the expression of different RNAs while recording the spatial positions of cells. The feasible techniques are mainly divided into two technical routes: imaging and counting after probe in situ hybridization and sequencing and counting after capturing spatially encoded probes, and finally obtain the gene expression at different spatial positions at the transcriptional level. Among them, in situ sequencing technology designs probes for a set of known target genes of interest and restores the spatial single-cell expression by reading the number of spatial in situ signals through a high-resolution microscope. The 10X Genomics' Visium technology and BGI's Stereo-seq technology study the heterogeneity between cells in single-cell transcriptomics, while spatial transcriptome sequencing adds the exploration of the spatial characteristics of the organism's structure on the basis of single-cell transcriptome sequencing, that is, it is assumed that the gene expression of the same type of cells is different at different positions and is verified.

[0004] In 2022, the Brown Laboratory developed a technique that combines spatial CRISPR screening and 10X Genomics' Visium spatial transcriptome sequencing on adjacent tumor sections (Spatial CRISPR genomics identifies regulators of the tumor microenvironment, Cell, 2022). This technique can simultaneously read out the immune infiltration characteristics in the tumor cell neighborhood and the transcriptional status of tumor cells in the corresponding regions of adjacent sections after knocking out different genes at different spatial positions. The principle of this technique is to ligate sgRNA and a pre-encoded membrane protein sequence onto the same plasmid, thus overexpressing membrane proteins corresponding to different sgRNAs on the surfaces of different cells. Finally, through antibody hybridization and spatial proteomics techniques, the membrane protein information of each cell can be read out and the corresponding sgRNA library can be restored. This technique has successfully combined CRISPR screening to read out spatial cell relationships and transcriptome characteristics, greatly expanding the phenotypic dimensions of CRISPR screening. However, the limitations and deficiencies of this technique are also relatively obvious. First, this technique uses the method of separately constructing plasmids for all sgRNAs and then mixing the plasmids, which is time-consuming and laborious, and lacks a method for constructing a mixed library. Second, this technique uses spatial proteomics methods to label the positions of different sgRNAs and the types of immune cells, and the scheme of adjacent-section spatial transcriptome sequencing. This determines that it is impossible to define different cell types and their subtype states in different states from the perspective of unbiased whole-spatial transcriptome in the same section. Moreover, the combination with the transcriptome cannot be implemented on immune cells with high randomness and motility, such as T cells. Finally, since the library construction is carried out in units of single sgRNAs and the readout method is antibody labeling, this technique has a low throughput and there is an upper limit to the types of detectable sgRNAs. Summary of the Invention

[0005] Aiming at the problems of the prior art, 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 within the same section. Another technical problem to be solved by the present invention is to provide the aforesaid method for combining spatial transcriptome and CRISPR screening in an unbiased manner within the same section.

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

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

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

[0009] S2: Sort out positively infected target cells and culture them for a period of time after knockout occurs;

[0010] S3: Inject the target cells obtained in step S2 into an experimental animal, where the experimental animal is an animal lacking autoimmune function;

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

[0012] S5: Transfer the frozen sections onto a spatial transcriptomics slide and load it onto the machine to obtain a Total cDNA library;

[0013] S6: Use the Total cDNA library to construct a general transcriptome library and a CRISPR sgRNA library;

[0014] S7: Sequencing and gene sequence alignment are performed on the general transcriptome library and the CRISPR sgRNA library respectively to obtain general transcriptome sequencing data and CRISPR sgRNA library sequencing data carrying spatial coordinates.

[0015] In the method of combining spatial transcriptomics and CRISPR screening in the same - slide unbiased manner, the target cells are specifically immune cells.

[0016] In the method of combining spatial transcriptomics and CRISPR screening in the same - slide unbiased manner, the immune cells are specifically effector T cells.

[0017] In the method of combining spatial transcriptomics and CRISPR screening in the same - slide unbiased manner, the tissue is tumor tissue or normal tissue.

[0018] In the method of combining spatial transcriptomics and CRISPR screening in the same - slide unbiased manner, the way of injecting into the experimental animal is intravenous injection.

[0019] In the method of combining spatial transcriptomics and CRISPR screening in the same - slide unbiased manner, the construction of the CRISPR sgRNA library using the Total cDNA library is enriched. The enrichment method is specifically: PCR amplify the CRISPR sgRNA library in the Total cDNA with a primer pair. The reverse primer of the primer pair binds to the common sequence at the 3' end of all cDNAs in the sense strand of 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. Perform several rounds of the PCR amplification, and after each PCR amplification, screen the sequence by magnetic bead - bound DNA to gradually increase the concentration of the CRISPR sgRNA library.

[0020] In the method of combining spatial transcriptomics and CRISPR screening in the same-slice unbiased manner, the construction of the CRISPR sgRNA library from the Total cDNA library is enriched. The specific enrichment method is as follows: The two ends of the target fragment are used as circularization sequences, and an oligonucleotide that is complementary to the front half and the back half is added; The circularization is carried out under the catalysis of T4 ligase by the splint-ligation method, and the uncircularized products are digested by DNA exonuclease to obtain a single-stranded circular DNA with higher purity. A target fragment library with higher purity can be obtained by performing double-ended specific primer amplification from the single-stranded circular DNA.

[0021] The method of combining spatial transcriptomics and CRISPR screening in the same-slice unbiased manner further includes performing gene sequence alignment on the ordinary transcriptome sequencing data carrying spatial coordinates and the CRISPR sgRNA library sequencing data respectively, and then merging them into the data of the same chip according to the same spatial coordinates.

[0022] Application of the ordinary transcriptome sequencing data carrying spatial coordinates and the CRISPR sgRNA library sequencing data obtained by any of the above methods in verifying gene functions.

[0023] Furthermore, the specific steps of the above application include: performing unbiased clustering on the ordinary transcriptome sequencing data, performing differential analysis on each clustered category with all other categories, obtaining the signature genes of each category as the key features of each category; According to the CRISPR sgRNA library sequencing data, performing differential analysis 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.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] By combining CRISPR screening and spatial transcriptome sequencing, this technology adds the spatio-temporal dimension of traditional phenotypes and the whole-transcriptome dimension to the screening, which is beneficial to studying the state and positional relationship of genes and cells in the biological structure in tumor biology, immunology, developmental biology or neuroscience. For example, in the spatial transcriptome screening of T cell infiltration into tumors, the present application found that the knockout of the Piezo1 gene significantly enriched T cells in the inner region of tumors with low fibrosis degree, reflecting that the expression of knocking out the membrane protein Piezo1 gene can enable T cells to better cross the fibrotic region and infiltrate into the tumor parenchyma. This result verifies the high possibility of the Piezo1 target being druggable for tumors with high fibrosis and immune rejection. Description of the Drawings

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

[0027] Figure 2 Comparison diagram of the cDNA sequence structures in the ordinary 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 ordinary transcriptome library;

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

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

[0030] Figure 5A Spatial transcriptome map of mouse MC38 subcutaneous tumors;

[0031] Figure 5B Spatial CRISPR screening library map of mouse T cells;

[0032] Figure 6A Spatial transcriptome clustering of mouse MC38 subcutaneous tumors reflecting different intratumoral microenvironments;

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

[0034] Figure 6C Statistical chart showing that T cells after knocking out different genes tend to be enriched and distributed in different intratumoral microenvironments;

[0035] Figure 7A Map depicting the fibroblast characteristic regions of the tumor microenvironment using whole gene transcriptomics;

[0036] Figure 7B Ratio map of T cells with different genes knocked out in regions with high and low fibroblasts. Detailed implementation methods

[0037] For the convenience of describing the technical solution of the application, some concepts, reagents and materials involved in the present application will be described below. To make the technical problems solved by the present invention, the technical solutions adopted and the achieved technical effects clearer, the technical solutions of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. The materials, reagents and equipment used in the examples can be obtained from the market without detailed description.

[0038] Cantilever primer: A part of the PCR primer binds to the DNA template, while the other part cannot bind, resulting in a decrease in the overall affinity of the primer. It is used to reduce the PCR efficiency in the region where all fragments at the 3' end of the library are located, thereby increasing the proportion of the affinity of the gene-specific primer binding at the 5' end in the entire PCR.

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

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

[0041] CRISPR sgRNA knockout library: An sgRNA library synthesized by Genewiz from the designed sgRNA sequences.

[0042] Stereo-seq chip: BGI.

[0043] Reagents and materials:

[0044]

[0045]

[0046] Example 1:

[0047] I. Construction of sgRNA library and infection of effector T cells

[0048] Several genes that can significantly cause immune cells to accumulate in tumors after knockout in immune cells were used as target genes. For each target gene, 4-6 sgRNAs were designed, and a total of 68 sgRNAs including 2 control sgRNAs were designed. The target genes include the Zc3h12a gene and the Gata3 gene. Knocking out these two genes in T cells can significantly enhance the infiltration ability of T cells in tumors.

[0049] The 68 sgRNAs were respectively synthesized into single-stranded oligonucleotide DNA, and were respectively digested and ligated onto the pMYs vector ( Figure 1 ), and then the pMYs vector was packaged into virus particles, namely the sgRNA library virus.

[0050] After sacrificing OT1 / Cas9 mice, spleens were obtained. 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 (from Miltenyi Biotec). The resulting CD8+ T cells expressed OT1 (a TCR that specifically recognizes the OVA antigen of tumor cells) and Cas9 protein.

[0051] The sorted CD8+ T cells were induced to become effector T cells through in vitro activation and culture, and then mixed with all sgRNA library viruses to infect the effector T cells. According to 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 sorting was used to sort out the positively infected T cells. Since the sgRNA transcribed from the virus in the positively infected T cells binds to the Cas9 protein in the T cells to form an RNP complex that targets the target gene, the resulting population of positively infected T cells thus contains T cells with different genes knocked out.

[0052] II. Using tumor tissue spatial heterogeneity as a screening pressure, perform CRISPR screening on the population of positively infected T cells

[0053] There is a certain heterogeneity in different microenvironments (spaces) within tumor tissues, which are different in terms of tumor spatial characteristics such as hypoxia, fibrosis, immune rejection, and interactions between immune cells, and have different effects on different immune cells.

[0054] Six days after the knockout occurred, the positively infected T cells were injected into Rag1 gene-deficient mice (lacking their own T and B cells) with subcutaneous tumors of mouse MC38 (colorectal cancer cell line) implanted 7 days in advance through microvenous injection, enabling the T cells with different genes knocked out to freely move and infiltrate into different spatial domains of the tumor according to the preferences generated after different genes were knocked out during recruitment.

[0055] III. Prepare frozen sections of tumor tissues infiltrated with positive T cells and their spatial transcriptome cDNA libraries

[0056] 4 - 10 days after T cells edited by sgRNA library infiltrated the tumor, the mice were sacrificed, and the intact subcutaneous tumors were removed, embedded in OCT, cryosectioned, and then the freshly cryosectioned samples were loaded onto the prepared Stereo-seq chip. The surface of the chip contains probes with poly dT, which are used to capture mRNAs with polyA in the tissue (including mRNAs embedded with the sgRNA library). After taking GFP fluorescence photos of the sections, according to the Stereo-seq chip instruction manual, the mRNAs captured by the probes with poly dT were reverse-transcribed to obtain a cDNA library, namely the Total cDNA library. The Total cDNA library includes a common transcriptome library and a CRISPR sgRNA library. For all cDNAs in the Total cDNA library, different spatial ID coordinates (CID) encoded by different DNAs are connected after pA (poly dA), and unique molecular names (MID) of different probes under the same coordinate are contained. A fixed sequence Fixed is contained between CID and MID. There is a Read1 sequence for amplification behind CID. There is another TSO sequence for amplification behind the Read1 sequence, and the corresponding other end is the Read2 and TSO sequences. For the cDNAs in the CRISPR sgRNA library, in addition to being embedded with the sgRNA sequence, they are also embedded with common known sequences derived from the pMYs viral vector such as U6, sgRNA, LTR, pA, Unnamed-1, and Unnamed-2 (Unnamed-1 and Unnamed-2 represent two virus infection element sequences without fixed names, and can also be called Virus Related Sequence). Different sgRNAs have different 20bp gene variable sequences, but different sgRNAs have a common and immutable 76bp sequence scaffold. Unknown cDNA represents unknown sequences in the spatial transcriptome library. The structural differences between the cDNAs of the common transcriptome library and the CRISPR sgRNA library are as Figure 2 shown.

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

[0058] IV. Further construction of the common transcriptome library and the CRISPR sgRNA library using the total cDNA library

[0059] The total cDNA library was divided into two parts. One part was used for constructing and sequencing a general transcriptome library, and the other part was used for constructing and sequencing a CRISPR sgRNA library. The general transcriptome sequencing consumed 1 / 10 of the total cDNA library volume, and the remaining library was used for constructing and sequencing the CRISPR sgRNA library.

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

[0061] According to the commercial method of Stereo-seq, about 1 / 10 of the total cDNA library was fragmented into appropriate lengths and then used for library construction and sequencing on BGI MGI second-generation sequencer, so as to obtain the general transcriptome sequencing files (a very small amount of CRISPR sgRNA library contained therein would not affect the sequencing results).

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

[0063] To construct a CRISPR sgRNA library from the total cDNA library, it was necessary to enrich the CRISPR sgRNA library to meet the requirements of sequencing samples. The main difficulties in enriching CRISPR sgRNA or other specific genes in single-cell transcriptome or spatial transcriptome sequencing were: the target fragments were mixed with all gene fragments and the content of the target fragments was extremely low; on the other hand, in order to simultaneously amplify the amplified cell or spatial ID located at the end of the library, one end of the two primers for PCR had to use the amplification primer common to all cDNA of the genes, and it was difficult to amplify by ordinary PCR methods. This application successfully constructed a CRISPR sgRNA library for sequencing by using an enrichment method starting from self-developed ultra-trace templates.

[0064] For the cDNA library, the main strategy for enriching gene knockout barcode fragments was nested PCR specific amplification and magnetic bead fragment screening. All DNAs in the cDNA had a common amplification sequence and corresponding amplification primer at the 3' end of the sense strand. By setting multiple specific primers at the 5' end of the gene knockout barcode fragments, multi-step PCR was carried out, and after each step of PCR, fragment screening with DNA-binding magnetic beads was performed, which could gradually increase the proportion of positive fragments.

[0065] For samples with a low proportion of positive fragments, a circularization method is adopted for further enrichment. The specific circularization method is to use the two ends of the target fragment as the circularization sequences, and add an oligonucleotide that is complementary to the front half and the back half. Circularization is carried out under the catalysis of T4 ligase (Thermo) by the splint-ligation method, and the uncircularized products are digested by DNA exonuclease to obtain single-stranded circular DNA with high purity. A target fragment library with high purity can be obtained by performing double-end specific primer amplification on the single-stranded circular DNA. The second-generation sequencing adapter is added to the target fragment library by PCR, and then it can be sequenced on the machine.

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

[0067] Step 1: (Optional, performed when the total library is insufficient) Perform total amplification on the total cDNA library;

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

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

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

[0071] Step 5: Perform Splint-Ligation circularization;

[0072] Step 6: Perform dual-specific enrichment-1;

[0073] Step 7: Perform dual-specific enrichment-2;

[0074] Step 8: Add the illumina library adapter P5-P7 (ST-P5P7), recover the product, and use it for sequencing on the machine. 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, performed when the total library is insufficient) Perform total library amplification on the total cDNA library: Prepare 5 tubes in the PCR tube, and the system is as follows:

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

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

[0081] Step Temperature Time 1. Pre - denaturation 95℃ 5 min 2. Denaturation 98℃ 20 sec 3. Annealing 58℃ 30 sec 4. Extension 72℃ 2 min 5. Number of cycles 2 - 4 10 times 6. Final extension 72℃ 5 min 7. Storage 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 stand and let stand for 5 min;

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

[0086] (4) Dry until not shiny (do not wait until cracked), and dissolve with 15 μL of enzyme-free water;

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

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

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

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

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

[0092] Step Temperature Time 1. Pre - denaturation 95℃ 5 min 2. Denaturation 98℃ 20 sec 3. Annealing 65℃ 30 sec 4. Extension 72℃ 2 min 5. Number of cycles 2 - 4 8 - 9 times 6. Final extension 72℃ 5 min 7. Storage 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 stand and let stand for 5 min;

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

[0097] (4) Dry until not shiny (do not wait until cracked), and dissolve with 15 μL of enzyme-free water;

[0098] (5) Detect the concentration and fragment size, calculate the yield, which should be about 10%, that is, obtain a concentration of 10 - 20 ng / μL (Nanodrop).

[0099] 3. Inner enrichment of the Outer enrichment product:

[0100] Following the PCR products of the previous step (if the fragment analysis is correct), perform PCR on 5 tubes for each library, and the reaction system is as follows:

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

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

[0103] Step Temperature Time 1. Pre - denaturation 95℃ 5 min 2. Denaturation 98℃ 20 sec 3. Annealing 64℃ 30 sec 4. Extension 72℃ 1 min 5. Number of cycles 2 - 4 8 - 9 times 6. Final extension 72℃ 5 min 7. Storage 4℃ Forever

[0104] Recovery (0.5× purification):

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

[0106] (2) Place it on the magnetic stand and let it stand for 5 min;

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

[0108] (4) Dry until it is not shiny (do not wait until it cracks), and dissolve it with 15 μL of enzyme-free water;

[0109] (5) Detect the concentration and fragment size, calculate the yield, and the concentration of 1 - 5 ng / μL (Nanodrop) can be obtained.

[0110] 4. Add the splint-TSO circularization sequence to the Inner enrichment product:

[0111] Following the PCR products of the previous step (if the fragment analysis is correct), perform PCR on 5 tubes for each library, and the reaction system is as follows:

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

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

[0114] Step Temperature Time 1. Pre - denaturation 95℃ 5 min 2. Denaturation 98℃ 20 sec 3. Annealing 72℃ 30 sec 4. Extension 72℃ 1 min 5. Number of cycles 2 - 4 8 - 9 times 6. Final extension 72℃ 5 min 7. Storage 4℃ Forever

[0115] Recovery (0.5× purification):

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

[0117] (2) Place it on the magnetic stand and let it stand for 5 min

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

[0119] (4) Dry until it is not shiny (do not wait until it cracks), and dissolve it with 15 μL of enzyme-free water

[0120] (5) Detect the concentration and fragment size. The yield is about 20 - 30%. That is, a concentration of 20 - 30 ng / μL (Nanodrop) is obtained. If the total amount is less than 900 ng, perform step 4 (adding splint - TSO circularization sequence) again.

[0121] 5. Circularization sequence:

[0122] (1) Turn on the PCR instrument pre - heated at 95°C.

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

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

[0125] (3) After placing it at 95°C for 5 min, immediately place it on a - 30°C ice brick to cool down and put it into a - 30°C refrigerator (place it for 1 - 2 min. The purpose is to cool down, not to freeze solidify), and proceed to the next ligation:

[0126] (4) Ligation. The system is as follows

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

[0128] 37°C ligation for 1 h program:

[0129] Step Temperature Time 1. Ligation 37℃ 1 hour 2. Storage 4℃ Forever

[0130] (5) Digestion. The system is as follows:

[0131] Reagents Stock Amount Ligation product of step (4) 60 μL Exonuclease I 1.95 μL Exonuclease III 0.65 μL Buffer 1.4 μL Total 64 μL

[0132] The program is as follows:

[0133] Step Temperature Time 1. Ligation 37℃ 30 min 2. Storage 4℃ Forever

[0134] After the program ends, 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 it stand at room temperature for 10 min;

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

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

[0139] (4) Dry to a non-reflective state (do not wait until it cracks), and dissolve in 15 μL of enzyme-free water;

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

[0141] 6. Bispecific Enrichment - 1

[0142] Following the PCR product from the previous step (if the fragment analysis is correct), prepare 5 tubes for each PCR library, and the reaction system is as follows:

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

[0144] Take out the magnetic beads 30 minutes in advance and set the PCR program (Bispecific Enrichment - 1):

[0145] Step Temperature Time 1. Pre-denaturation 95℃ 5 min 2. Denaturation 98℃ 20 sec 3. Annealing 72℃ 30 sec 4. Extension 72℃ 30 sec 5. Number of cycles of 2 - 4 12 times 6. Final extension 72℃ 5 min 7. Storage 4℃ Forever

[0146] Recovery (1.5× purification):

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

[0148] (2) Place it on the magnetic rack and let it stand for 5 min;

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

[0150] (4) Dry to a non-reflective state (do not wait until it cracks), and dissolve in 15 μL of enzyme-free water;

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

[0152] If there is no obvious band but no background bands, then repeat the same steps of Bispecific Enrichment - 1 once more. The product can be named Direct - 6 - 2, and the concentration should be 30 - 80 ng / μL:

[0153] 7. Bispecific Enrichment - 2

[0154] Following the PCR product from the previous step (if the fragment analysis is correct), prepare 5 tubes for each PCR library, and the reaction system is as follows:

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

[0156] Take out the magnetic beads 30 minutes in advance and set the PCR program (Bispecific 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 stand and let stand for 5 min;

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

[0163] (4) Dry until not shiny (do not wait until cracked), and dissolve in 15 μL of enzyme-free water;

[0164] (5) Measure the concentration and fragment size to obtain a product with a concentration of 60 - 80 ng / μL (Nanodrop).

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

[0166] Following the PCR product from the previous step (if the fragment analysis is correct), perform PCR in 5 tubes for each library. The P5 - P7 (ST - P5P) system is as follows:

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

[0168] Take out the magnetic beads 30 minutes in advance and set the PCR program (bispecific enrichment - 1):

[0169] Step Temperature Time 1. Pre-denaturation 95℃ 5 min 2. Denaturation 98℃ 20 sec 3. Annealing 72℃ 30 sec 4. Extension 72℃ 30 sec 5. Number of cycles of 2 - 4 5 times 6. Final extension 72℃ 10 min 7. Storage 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 stand and let stand for 5 min

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

[0174] (4) Dry until not shiny (do not wait until cracked), and dissolve in 15 μL of enzyme-free water

[0175] (5) Measure 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 on the machine.

[0176] V. Perform next-generation sequencing on the CRISPR sgRNA library to obtain the data off the machine

[0177] By performing second-generation sequencing on the enriched CRISPR sgRNA library, the sequences of CRISPR sgRNAs in the sequencing library that overlap with the ordinary transcriptome and their corresponding spatial IDs for each specific sequence can be obtained.

[0178] Send for second-generation sequencing. The total data volume is 10G. The CRISPR sgRNA library is sequenced on the Illumina NovaSeq 6000 mainstream NGS (second-generation sequencing) platform.

[0179] VI. Perform bioinformatics analysis on the off-machine data of the CRISPR sgRNA library and the ordinary transcriptome library to obtain transcriptome data with spatial coordinates

[0180] (1) Main steps of bioinformatics analysis of the ordinary transcriptome library

[0181] For the sequencing results of the ordinary transcriptome, use the analysis software SAW (Stereo-seq Analysis Workflow) of BGI's spatial transcriptome to align the gene sequences of different reads in the sequencing results to the reference genome to determine gene expression levels, align the corresponding CIDs to the pre-encoded spatial coordinate library to determine their spatial positions, and use the MID to deduplicate reads with the same CID. The unique reads expression level information of different genes at all coordinates can be obtained ( Figure 5A ), and after the alignment, the ordinary transcriptome sequencing data file is obtained.

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

[0183] Align and rename the sequencing data. For the off-machine data of MGI2000, this step is not required; for the second-generation sequencing data produced by Illumina and other platforms, first open the corresponding *.fq1.gz and *.fq2.gz files for off-machine data, read in the file content. For the read-in file, after finding the sequence name line according to the @ symbol, for the lines from *.fq1.gz, add " / 1"; for the lines from *.fq2.gz, add " / 2". Write out the new *.fq1.gz and *.fq2.gz files. This step is called fromillumina2MGI.

[0184] Split sequencing data: For the sequenced *.fq1.gz and *fq2.gz files, first screen the sequences in the *fq1.gz file that have a Levenshtein distance less than or equal to 1 from the fixed sequence. If such sequences are found, extract the first 25 and the last 10 bases of the fixed sequence as the coordinate identifier and molecular identifiers respectively; for the corresponding *fq2.gz file, find the first 21 bases before the sgRNA scaffold fixed sequence (the length is dynamically adjusted according to the sgRNA library, which is the maximum sgRNA sequence length plus 1). If it does not meet this standard, remove this sequence and the corresponding sequence in the *fq2.gz file. This step is named seekseq.

[0185] Perform SAW alignment on the obtained *.fq1.gz and *fq2.gz above, and finally obtain the corresponding *.gem file with the spatial position coordinates of sgRNA. The content structure of the *.gem file is

[0186] #FileFormat=GEMv1.0

[0187] #SortedBy=None

[0188] #STOmicsChip=

[0189] #OffsetX=

[0190] #OffsetY=

[0191] geneID x y MIDCount ExonCount

[0192] sgCd44_1_gene 1123 3482 1 1

[0193] Among them, the first line of the annotation row 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 where the sgRNA is distributed 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 position coordinates of the gRNA, and the fourth and fifth columns are the number of times this type of sgRNA is detected at this position; merge this CRISPR sgRNA library sequencing data file with the ordinary transcriptome sequencing data file to obtain the final file of sgRNA and RNA expression ( Figure 5A , Figure 5B ).

[0194] VII. Perform downstream analysis on ordinary transcriptome sequencing data and CRISPR sgRNA library sequencing data carrying spatial coordinates to achieve spatial visualization of gene expression and verify the functions of genes knocked out by sgRNAs

[0195] Perform downstream analysis on the data in the above-mentioned files of sgRNA and RNA expression containing spatial coordinates using Scanpy V1.10.3:

[0196] a. After reading in the data, cut the whole slice into small regions (bin50) with a side length of 0.025 mm, as Figure 5A 、 Figure 5B shown. Figure 5A shows a viewable graph of the whole-transcriptome expression of the unbiasedly sequenced tumor microenvironment on the same slice, while Figure 5B is a viewable graph of the distribution of the sgRNA library of the unbiasedly sequenced tumor microenvironment T cell CRISPR screening in tissue space. Among them, each color represents a sgRNA knockout information.

[0197] b. Unbiasedly cluster all bin50 regions of the whole gene transcriptome (ordinary transcriptome sequencing data) according to gene expression type and expression level to obtain the result graph of unbiased clustering, as Figure 6A shown. Different categories in the graph are shown in different colors, and the same color represents the same tumor microenvironment.

[0198] c. Perform an overall 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.

[0199] d. According to the same spatial coordinates, migrate the clustering information of genes to the bin50 containing the sgRNA library ( Figure 6B ).

[0200] e. Perform a differential analysis on the expression levels of sgRNAs in different clusters to obtain which type of T cells tend to be enriched in which type of tumor microenvironment after a certain gene is knocked out, as Figure 6C shown. This graph shows that, according to the different spatial gene expression regions delimited by unbiased clustering for spatial transcriptomics, compared with the control sgRNA (sgNTC, Non Targeting Control, sgRNA that does not target any gene sequence on the genome), the enrichment or depletion distribution characteristics presented by the sgRNAs with gene knockouts gradually change to blue, indicating that the knockout of this sgRNA makes T cells more depleted in a certain region, while the gradual change to red indicates that this sgRNA makes T cells more concentrated in a certain region. In this graph, it can be seen that sgCd44 is significantly enriched in the region of cluster3.

[0201] f. A 2022 literature published in Cell (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 parenchyma, and the core role of Piezo1 in triggering T cell exhaustion during this process. In this example, the spatial transcriptome of T cell infiltration into tumors was screened for the infiltration of T cells in the fibrotic region. First, several tumor fibrosis marker genes were selected from the literature reports (GO gene annotation Term: ID fibroblast proliferation; GO: 0048144; including genes such as Egfr). After normalizing the expression levels of these marker genes in each bin50 region with the total expression levels of all genes, the relative levels of fibrosis in different regions were scored, thereby characterizing the fibrotic region characteristics in the MC38 tumor, such as Figure 7A shown. The figure shows a visual diagram depicting the fibroblast characteristic regions of the tumor microenvironment using the whole gene transcriptome. Among them, the score bar on the right gradually changes from dark green to yellow while the score gradually increases. The larger the score value, the greater the expression level of the tumor fibrosis marker gene and the higher the degree of fibrosis. Figure 7B shows the enrichment diagrams of T cells with different genes knocked out in the high fibroblast region and the low fibroblast region. Taking Figure 7A the regions with scores greater than 2.2 in the figure as the high fibroblast regions and the regions with scores less than 0.8 as the low fibroblast regions, the measured number of sgRNAs was used to represent the enrichment amount of T cells with the corresponding genes knocked out. Figure 7B Figure A in it shows the ratio ranking of the enrichment amounts of T cells in the high and low fibroblast regions. The higher the value on the vertical axis, the more the T cells with a certain gene knocked out are enriched in the high fibroblast region; conversely, Figure 7B Figure B in it shows the ratio ranking of the enrichment amounts of T cells in the low and high fibroblast regions. The higher the value on the vertical axis, the more the T cells with a certain gene knocked out are enriched in the low fibroblast region. Figure 7B It can be clearly seen from the figure that the T cells with the Piezo1 gene knocked out are more enriched in the low fibroblast region, while the control group sgNTC (sgnon-targeting_2_gene in the figure) is significantly enriched in the high fibrosis region, which is consistent with the conclusion in the literature published in Cell. And combined with Figure 7AIt is not difficult to see that the regions with high fibrosis are concentrated at the tumor margin, while the regions with low fibrosis are concentrated in the tumor parenchyma. This indicates that knocking out the Piezo1 gene (sgPiezo1) enables T cells to be more enriched in the inner regions of the tumor with low fibrosis, reflecting that knocking out the expression of the membrane protein Piezo1 gene allows T cells to better cross the fibrotic regions and infiltrate into the tumor parenchyma. This result verifies the high possibility of the Piezo1 target being druggable for tumors with high fibrosis and immune rejection.

[0202] The technology of this application combines CRISPR screening and spatial transcriptome sequencing, adding spatio-temporal dimensions and whole-transcriptome dimensions of traditional phenotypes to screening, which is conducive to studying the state and positional relationship of genes and cells in the structure of organisms in tumor biology, immunology, developmental biology or neuroscience.

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: Sequencing and gene sequence alignment of the common transcriptome library and CRISPR sgRNA library are performed respectively to obtain common transcriptome sequencing data and CRISPR sgRNA library sequencing data with spatial coordinates; Among them, the CRISPR sgRNA library constructed using the Total cDNA library is enriched, and the enrichment method is specifically as follows: the CRISPR sgRNA library in the Total cDNA is amplified by PCR using a primer pair, the reverse primer of the primer pair is combined with the common sequence of the 3' end of the sense chain of all cDNAs in the Total cDNA library, and the forward primer in the primer pair is combined with 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; the two ends of the target fragment are used as cyclization sequences, and the splint-ligation method is used to perform cyclization under the catalysis of T4 ligase, and the uncyclized product is digested by DNA exonuclease to obtain a single-stranded circular DNA with higher purity, and the single-stranded circular DNA is used for double-end specific primer amplification to obtain a CRISPR sgRNA library with higher purity.

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 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.

7. Application of common transcriptome sequencing data and CRISPR sgRNA library sequencing data carrying spatial coordinates obtained by any of the methods described in claims 1-6 in verifying gene function.

8. The use according to claim 7, 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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