Library building method for enriching trace CRISPR (clustered regularly interspaced short palindromic repeats) gene knockout library from space transcriptome

Abstract

The invention discloses a library building method for enriching a trace CRISPR (clustered regularly interspaced short palindromic repeats) gene knockout library from a space transcriptome, and belongs to the technical field of biology. The method comprises the following steps: obtaining a Total cDNA library mixed by a CRISPR gene knockout library and a space transcriptome library, and carrying out inward nested outer amplification on the Total cDNA library aiming at the CRISPR gene knockout library to obtain an outer enriched product; the outer enrichment product is subjected to inward nested Inner enrichment aiming at the CRISPR gene knockout library, and an Inner enrichment product is obtained; adding a sp-TSO cyclization sequence into the Inner enriched product, and then carrying out cyclization reaction, so as to obtain cyclized cDNA (complementary deoxyribonucleic acid); removing the cyclized sequence to obtain annular cDNA (complementary deoxyribonucleic acid); the CRISPR gene knockout library is obtained by performing amplification on the CRISPR gene and the cyclic cDNA to obtain a target fragment library, and adding a second-generation sequencing joint to the target fragment library to obtain the CRISPR gene knockout library which can be used for second-generation sequencing.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for constructing a library for enriching a trace amount of CRISPR gene knockout library from a spatial transcriptome. Background Art

[0002] Spatial transcriptome sequencing is a technology that uses probes on array chips to capture mRNA in permeabilized tissues, reverse transcribes in situ, obtains cDNA libraries with spatial coordinates, and performs high-throughput library sequencing. The spatial transcriptome contains libraries of some mutable genes, such as T cells and B cells in tissues that mutate to obtain different TCR and BCR sequences during their development and maturation.

[0003] Spatial transcriptomes can also be combined with targeted enrichment of certain genes or sequences in overexpression plasmids that are artificially introduced into certain cells in tissues. Enriching barcodes of specific genes from spatial transcriptomes will make it possible to perform personalized sequencing such as recovery of CRISPR screen information and cell type-specific barcodes from spatial transcriptome libraries.

[0004] Since the spatial transcriptome is captured by probes, the limited probes limit the total amount of RNA captured and the amount of cDNA reverse transcribed from the RNA, resulting in a low total amount of cDNA that the experimenter can obtain. The spatial transcriptome captures genes in sliced ​​tissues in an unbiased manner, and the types of genes highly expressed in the tissue account for a high proportion of the cDNA library. For example, compared to highly expressed housekeeping genes like GAPDH, the expression level of transcripts knocked out by CRISPR genes is usually lower.

[0005] Therefore, in order to enrich the sequences of trace CRISPR gene knockout libraries and their spatial position sequences from the spatial transcriptome library, the two problems of low total library volume and low gene proportion must be overcome. Summary of the invention

[0006] In view of the existing technical problems, the technical problem to be solved by the present invention is to provide a method for constructing a library for enriching a trace amount of CRISPR gene knockout library from a spatial transcriptome.

[0007] To solve the existing technical problems, the technical solution of the present invention is as follows:

[0008] A method for constructing a library for enriching a trace amount of CRISPR gene knockout library from a spatial transcriptome comprises: obtaining a Total cDNA library mixed with a CRISPR gene knockout library and a spatial transcriptome library, performing an outer nested amplification on the Total cDNA library for the CRISPR gene knockout library to obtain an outer enriched product; performing an inner nested amplification on the outer enriched product for the CRISPR gene knockout library to obtain an inner enriched product; performing a cyclization reaction on the inner enriched product to obtain a cyclized cDNA; removing uncyclized sequences in the cyclization reaction product to obtain a single-stranded circular cDNA with higher purity; performing amplification with a specific primer on the single-stranded circular DNA to obtain a target fragment library, and adding a second-generation sequencing adapter to the target fragment library by PCR to obtain a CRISPR gene knockout library.

[0009] In some embodiments of the present invention, the preparation method of the Total cDNA library in which the CRISPR gene knockout library and the spatial transcriptome library are mixed is: infecting the target cells with the CRISPR screening library virus; sorting out the positively infected target cells and culturing them for a period of time after the knockout occurs; injecting the obtained target cells into the body of an experimental animal, wherein 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 to the spatial transcriptome slide, and after processing on the machine, obtaining the Total cDNA library in which the CRISPR gene knockout library and the spatial transcriptome library are mixed.

[0010] In some embodiments of the present invention, the target cells are specifically immune cells, such as T cells.

[0011] In some embodiments of the present invention, the tissue is tumor tissue or normal tissue.

[0012] In some embodiments of the present invention, the injection into the experimental animal is performed by intravenous injection.

[0013] In some embodiments of the present invention, before performing the inward nested outer amplification on the Total cDNA library for the CRISPR gene knockout library, the Total cDNA library is fully amplified in advance.

[0014] In some embodiments of the present invention, the method for performing a cyclization reaction on the Inner enriched product is specifically: adding a splint-TSO cyclization sequence to the Inner enriched product and then performing a cyclization reaction.

[0015] In some embodiments of the present invention, the specific method for removing the uncircularized DNA in the cyclization reaction product is: using a DNA exonuclease to digest the uncircularized product.

[0016] In some embodiments of the present invention, the cDNA sequence in the CRISPR gene knockout library is chimeric with an sgRNA sequence, and has Read2 and TSO sequences on one side of the sgRNA sequence, and has LTR, pA, MID, Fixed, CID, TSO and Read1 sequences on the other side of the sgRNA sequence.

[0017] In some embodiments of the present invention, the sgRNA sequence includes a gene variable sequence and a scaffold sequence.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The method of the present invention can effectively enrich a very low proportion of CRISPR gene knockout sequence libraries from the spatial transcriptome library for second-generation sequencing library construction. The present invention is suitable for enriching the library of specific recombinant or mutant genes of interest from its library without affecting the spatial transcriptome sequencing, and performing separate deep saturation sequencing, and finally the spatial position of these specific gene libraries can be restored by means of bioinformatics. Ultra-trace sgRNA sequences at different spatial locations can be enriched from the spatial transcriptome of tumor samples, so that different gene knockout events at different spatial locations, as well as the accompanying transcriptome or cell changes, can be quantified, thereby analyzing the causal relationship between gene knockout and spatial biological events, such as the relationship between cell-cell interactions and cell receptor knockout. The present invention provides an effective technical means for this purpose and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 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;

[0021] Figure 2 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;

[0022] Figure 3 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;

[0023] Figure 4This is the outer enrichment result detection diagram;

[0024] Figure 5 This is the Inner enrichment detection result diagram;

[0025] Figure 6 This is the detection result diagram of the Inner enrichment product after adding the splint-TSO cyclization sequence;

[0026] Figure 7 This is a diagram of the detection results of the bispecific enrichment-1 product;

[0027] Figure 8 This is a detection diagram of two rounds of dual-specific enrichment-1 amplification products;

[0028] Fig. 9 This is a diagram showing the detection results of the bispecific enrichment-2 product;

[0029] Fig.10 Figure 2 shows the detection results after adding the illumina library adapter to the bispecific enrichment-2 product;

[0030] Fig.11 This is a product detection diagram without circularization and only using inward nested PCR enrichment

[0031] Fig.12 This is a product detection diagram for direct circularization and then dual-specific enrichment without performing two cantilever inward nested PCRs;

[0032] Fig.13A Product detection diagram for two cantilever inward nested PCRs for circularization followed by dual-specific enrichment;

[0033] Fig. 13B This is a diagram of product detection without performing two cantilever inward nested PCRs and directly performing circularization and then dual-specific enrichment. DETAILED DESCRIPTION

[0034] 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.

[0035] 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.

[0036] OT1 / Cas9 mice: OT1 / Cas9 mice purchased from Jackson Corporation, USA.

[0037] pMYs vector: mouse retrovirus pMYs purchased from Cell Biolabs, USA.

[0038] CRISPR sgRNA knockout library: The designed sgRNA sequence is synthesized by GENEWIZ.

[0039] Stereo-seq chip: BGI Genomics.

[0040] Reagents and materials:

[0041]

[0042] Embodiment 1:

[0043] 1. sgRNA library construction and infection of effector T cells

[0044] 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. Two sgRNAs were designed for each target gene, and a total of 68 sgRNAs including two 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.

[0045] 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.

[0046] 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.

[0047] 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 a fluorescent protein while uninfected ones cannot, flow cytometry is used to sort out the positively infected T cells, and the obtained T cell population contains T cells with different genes knocked out.

[0048] 2. Using tumor tissue spatial heterogeneity as screening pressure to screen for infection-positive T cells using CRISPR

[0049] 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.

[0050] The positively infected T cells were cultured until 6 days after the knockout occurred, and then injected into Rag1 gene-deficient mice (without their own T and B cells) 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.

[0051] 3. Preparation of frozen sections of tumor tissues infiltrated with positive T cells and their spatial transcriptome cDNA library

[0052] 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 polyA mRNA 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 according to the instructions for use of the Stereo-seq chip, which is the Total cDNA library. The Total cDNA library includes a common transcriptome library and a CRISPR gene knockout library (also known as a 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 a unique molecular name (MID) for different probes at the same coordinate. A fixed sequence Fixed is contained between the CID and the MID. There is a Read1 sequence for amplification after CID. There is another TSO sequence for amplification after the Read1 sequence, 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 differ in the 20bp gene variable sequence, 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 1 shown.

[0053] The total amount of the Total cDNA library was quantitatively analyzed using Qubit, and a volume of about 10-20 ng was considered qualified.

[0054] Embodiment 2:

[0055] The Total cDNA library obtained in Example 1 was used as the starting sample, and the CRISPR sgRNA library was obtained through amplification and enrichment steps. The primer sequences involved in the steps are shown in Table 1 below:

[0056] Table 1 Primer sequences

[0057]

[0058]

[0059] illustrate( Figure 2-Figure 3 ):

[0060] The total library amplification primer probe is designed for the common sequence TSO at both ends of all cDNAs in the Total cDNA library. The Total cDNA library is amplified by 20 cycles of PCR using the TSO sequence as a primer to obtain a sufficient amount of cDNA of about 1000 ng in total for subsequent reactions.

[0061] The forward primer and reverse primer amplification of the inner nest Outer are used to specifically amplify the cDNA sequence in the CRISPR sgRNA library. In order to take into account both specificity and amplification efficiency, the forward primer is designed for the special sequence shared by the cDNA in the CRISPR sgRNA library, and the reverse primer is designed for all cDNA common sequences in the Total cDNA library, and it has a cantilever sequence that does not bind to the template. In order to reduce the amplification efficiency of the common sequence, thereby increasing the relative amplification efficiency of the specific product and reducing the amplification efficiency of the non-specific product, a specific cantilever sequence is introduced.

[0062] Inner amplification To further improve the specificity of amplification, the forward primer of the inner nested amplification is designed for the common sequence unique to the CRISPR sgRNA library, and the reverse primer of the inner nested amplification is designed for the overhang sequence of the inner nested Outer amplification product. In order to further reduce nonspecific amplification in the next step, the reverse primer of the inner nested amplification has an overhang sequence that does not bind to the template.

[0063] Using the inward nested Inner amplification product as a template, PCR amplification and phosphorylation treatment are performed to add a circularized adapter. The forward primer sequence is still designed for the common sequence unique to the CRISPR sgRNA library, and has a cantilever for the circularized adapter sequence; the reverse primer sequence is designed for the cantilever sequence introduced into the inward nested Inner amplification product, and also has a cantilever for the circularized adapter sequence.

[0064] The cyclization step is to cyclize the cDNA after adding the cyclization linker and phosphorylation treatment, using Splint-Oligo cyclization oligonucleotide, the left and right halves of the sequence are respectively complementary to the cantilevers at both ends of the target fragment, so as to shorten the spatial distance between the two ends of the target fragment, and then connect the target fragment with T4 ligase to complete the cyclization. After the cyclization is completed, the reaction system is treated with DNA exonuclease I and DNA exonuclease III to digest and remove the uncyclized cDNA, while the cyclized cDNA sequence is not digested, which increases the proportion of CRISPR sgRNA library sequences in the system.

[0065] Bispecific enrichment-1 uses the circularized CRISPR sgRNA library sequence obtained in the circularization step as a template for amplification, amplifying from the inside of the target fragment to the outside, thereby converting the circularized sequence into a linear sequence, and adjusting the position of the sgRNA sequence to one end of the linear cDNA sequence, so that the scaffold region is located at the end of the sequence. The forward primer is designed for the unnamed known consensus sequence between the sgRNA and LTR in the template sequence (Unnnamed-2), and the reverse primer is designed for the LTR.

[0066] Bispecific enrichment-2 is to continue the inward nested amplification of the sgRNA fragments closer to the required sequencing with the products of bispecific enrichment-1, so as to improve the specificity and the total amount of CRISPR sgRNA library. The total amount of CRISPR sgRNA library reaches the total amount of sequencing library. The forward primer of bispecific enrichment-2 is designed for the scaffold region of the sgRNA fragment, and the reverse primer is designed for LTR.

[0067] Using the product of bispecific enrichment-2 as a template, the CRISPR sgRNA library sequence was amplified by adding Illumina library adapters. The forward primer used in this step is still designed for the scaffold region of the sgRNA fragment, and the reverse primer is still designed for LTR.

[0068] Amplification and enrichment steps:

[0069] 1. (Only when the total library is not enough) Amplify the total cDNA library: Prepare 5 PCR tubes, the system is as follows:

[0070] Reagents Stock Amount cDNA 150ng(NanoDrop) XμL Q5 2×Master Mix 15μL Total library amplification primers 10μM 1.2μL <![CDATA[DNase / RNase-free H2O]]> 12.6-XμL Total 30μL

[0071] Take out the magnetic beads (1× magnetic beads purification) 30 minutes in advance and set up the PCR program

[0072] 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

[0073] Recovery (1× purification):

[0074] (1) Add 30 μL of magnetic beads to each tube and let stand at room temperature for 10 min;

[0075] (2) Place on a magnetic rack and let stand for 5 minutes;

[0076] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol;

[0077] (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;

[0078] (5) Detect the concentration and fragment size and calculate the yield (about 2-4 times is a reasonable range).

[0079] 2. Outer enrichment of Total cDNA library:

[0080] Prepare 5 PCR tubes with the following system:

[0081] 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-free H2O]]> 12.6-XμL Total 30μL

[0082] Remove the magnetic beads 30 minutes in advance and set the PCR program (ST-Outer):

[0083]

[0084]

[0085] Recovery (0.5× purification):

[0086] (1) Add 15 μL of magnetic beads to each tube and let stand at room temperature for 10 min;

[0087] (2) Place on a magnetic rack and let stand for 5 minutes;

[0088] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol;

[0089] (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;

[0090] (5) Check the concentration and fragment size and calculate the yield, which should be around 10%, that is, the concentration is 10-20 ng / μL (Nanodrop). Figure 4 shown.

[0091] 3. Inner enrichment of Outer enrichment products:

[0092] 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:

[0093] 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 - free H2O]]> 12.6-XμL Total 30μL

[0094] Remove the magnetic beads 30 minutes in advance and set the PCR program (ST-Outer):

[0095] 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

[0096] Recovery (0.5× purification):

[0097] (1) Add 15 μL of magnetic beads to each 30 μL tube and let stand at room temperature for 10 min;

[0098] (2) Place on a magnetic rack and let stand for 5 minutes;

[0099] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol;

[0100] (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;

[0101] (5) Detect the concentration and fragment size and calculate the yield, that is, the concentration is 1-5 ng / μL (Nanodrop), such as Figure 5 shown.

[0102] 4. Add splint-TSO cyclization sequence to the inner enrichment product:

[0103] 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:

[0104]

[0105]

[0106] Remove the magnetic beads 30 minutes in advance and set the PCR program (ST-Outer):

[0107] 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

[0108] Recovery (0.5× purification):

[0109] (1) Add 15 μL of magnetic beads to each 30 μL tube and let stand at room temperature for 10 min.

[0110] (2) Place on a magnetic rack and let stand for 5 minutes

[0111] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol.

[0112] (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

[0113] (5) Detection of concentration and fragment size. The yield is about 20-30%, that is, the concentration is 20-30 ng / μL (Nanodrop)

[0114] If the total amount is less than 900 ng, perform step 4 again (add splint-TSO cyclization sequence).

[0115] The results of the splint-TSO cyclization sequence are as follows Figure 6 shown.

[0116] 5. Cyclization sequence:

[0117] (1) Turn on the PCR instrument to preheat at 95°C;

[0118] (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:

[0119] Reagents Stock Amount cDNA 900ng(NanoDrop) / 400ng(Qubit) 45μL Cyclization Splint-Oligo 5μL Total 50μL

[0120] (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:

[0121] (4) Connection, the system is as follows

[0122]

[0123]

[0124] 37 degrees Celsius connection 1h program:

[0125] Step Temperature Time 1. Connect 37℃ 1hour 2.Save 4℃ Forever

[0126] (5) Enzyme digestion, the system is as follows:

[0127] Reagents Stock Amount Step (4) Connecting the product 60μL Exonuclease I 1.95μL Exonuclease III 0.65μL Buffer 1.4μL Total 64μL

[0128] The procedure is as follows:

[0129] Step Temperature Time 1. Connect 37℃ 30min 2.Save 4℃ Forever

[0130] After the program is finished, quickly add 5 μL of 0.1 mM EDTA to terminate (final volume 70 μL)

[0131] Recovery (2.5× purification):

[0132] (1) Add 180 μL of magnetic beads to each 70 μL tube and let stand at room temperature for 10 min;

[0133] (2) Place on a magnetic rack and let stand for 5 minutes;

[0134] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol;

[0135] (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;

[0136] (5) Detect the concentration and fragment size and obtain a concentration of 20-30 ng / μL (Nanodrop).

[0137] 6. Bispecific Enrichment-1

[0138] 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:

[0139] 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-free H2O]]> 12.6-XμL Total 30μL

[0140] Remove the magnetic beads 30 minutes in advance and set up the PCR program (dual specific enrichment-1):

[0141]

[0142] Recovery (1.5× purification):

[0143] (1) Add 45 μL of magnetic beads to each 30 μL tube and let stand at room temperature for 10 min;

[0144] (2) Place on a magnetic rack and let stand for 5 minutes;

[0145] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol;

[0146] (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;

[0147] (5) Detect the concentration and fragment size, and obtain a concentration of 1-20 ng / μL (Nanodrop), such as Figure 7 shown.

[0148] If there is no obvious band, but no contaminants, perform another round of the same steps as bispecific enrichment-1. The product can be named Direct-6-2. The concentration should be 30-80 ng / μL. Figure 8 As shown:

[0149] 7. Bispecific Enrichment-2

[0150] 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:

[0151] Reagents Stock Amount cDNA All cDNAs in the previous step XμL Q5 2×Master Mix 15μL Dual Specific Enrichment-2-F 10μM 1.2μL Dual-specific enrichment-2-R 10μM 1.2μL <![CDATA[DNase / RNase - free H2O]]> 12.6-XμL Total 30μL

[0152] Remove the magnetic beads 30 minutes in advance and set up the PCR program (dual specific enrichment-2):

[0153] 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

[0154] Recovery (1.5× purification):

[0155] (1) Add 45 μL of magnetic beads to each 30 μL tube and let stand at room temperature for 10 min;

[0156] (2) Place on a magnetic rack and let stand for 5 minutes;

[0157] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol;

[0158] (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;

[0159] (5) Detection of concentration and fragment size, the concentration is 60-80 ng / μL (Nanodrop), such as Fig. 9 shown.

[0160] 8. Illumina library connector P5-P7 (ST-P5P7)

[0161] 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:

[0162]

[0163]

[0164] Remove the magnetic beads 30 minutes in advance and set up the PCR program (dual specific enrichment-1):

[0165] 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

[0166] Recovery (1.5× purification):

[0167] (1) Add 45 μL of magnetic beads to each 30 μL tube and let stand at room temperature for 10 min.

[0168] (2) Place on a magnetic rack and let stand for 5 minutes

[0169] (3) Discard the supernatant, wash twice with 80% ethanol, and discard all the remaining ethanol.

[0170] (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

[0171] (5) Detect the concentration and fragment size, and obtain a concentration of 100-200 ng / μL (Nanodrop), such as Fig.10 As shown, alternate sequencing.

[0172] 9. High-throughput second-generation library sequencing was performed at Beijing Nouda Gene Technology Co., Ltd. The sequencing results showed that the enriched CRISPR sgRNA library can effectively detect sequence information.

[0173] Comparative Example 1:

[0174] The remaining pre-processing steps were the same as those in Example 2, except that circularization was not performed and only inward nested PCR enrichment was used. The peak specificity of the positive fragment (1100 bp) was poor, as shown in FIG. Fig.11 shown.

[0175] Comparative Example 2:

[0176] The rest of the pre-sequence experimental steps were the same as in Example 2, except that circularization and bispecific enrichment were performed directly without performing two cantilever inward nested PCRs. Although the results were specific, the yield of the second-generation sequencing was too low because the library length was too large. Fig.12 shown.

[0177] Comparative Example 3:

[0178] A 2-step inward nested enrichment of a CRISPR knockout library was performed from a spatial transcriptome library using overhang PCR primers, and an experiment without overhang primers was performed for comparison. Fig.13A It can be seen that the PCR primer with overhang greatly increases the proportion of the target peak (red frame) of the library obtained by the first and second steps of enrichment, and finally there is a clear difference peak signal. Fig. 13B It can be seen that conventional primers without overhangs cannot enrich the CRISPR library, resulting in the target library being mixed in various impurity peaks.

Claims

1. A method for constructing a library for enriching a trace amount of CRISPR gene knockout library from a spatial transcriptome, characterized in that: The steps include: A Total cDNA library mixed with a CRISPR gene knockout library and a spatial transcriptome library is obtained, and the Total cDNA library is subjected to an outer nested amplification for the CRISPR gene knockout library to obtain an outer enriched product; the outer enriched product is subjected to an inner nested amplification for the CRISPR gene knockout library to obtain an inner enriched product; the inner enriched product is subjected to a cyclization reaction to obtain a circularized cDNA, and the uncircularized sequence is removed; PCR amplification is performed using the circular DNA as a template to obtain a target fragment library; and a second-generation sequencing adapter is added to the target fragment library to obtain a CRISPR gene knockout library.

2. The method for constructing a library for enriching a trace amount of CRISPR gene knockout library from a spatial transcriptome according to claim 1, characterized in that: The preparation method of the Total cDNA library in which the CRISPR gene knockout library and the spatial transcriptome library are mixed is as follows: infecting the target cells with the CRISPR screening library virus; sorting out the positively infected target cells and culturing them for a period of time after the knockout occurs; injecting the obtained target cells into the body of an experimental animal, which is an animal with autoimmune deficiency; taking out the tissue infiltrated by the target cells and preparing frozen sections; transferring the frozen sections to a spatial transcriptome slide, and processing them on a machine to obtain the Total cDNA library in which the CRISPR gene knockout library and the spatial transcriptome library are mixed.

3. The method for constructing a library for enriching a trace amount of CRISPR gene knockout library from a spatial transcriptome according to claim 2, characterized in that: The target cells are specifically immune cells.

4. The method for constructing a library for enriching a trace amount of CRISPR gene knockout library from a spatial transcriptome according to claim 2, characterized in that: The tissue is tumor tissue or normal tissue.

5. The method for constructing a library for enriching a trace amount of CRISPR gene knockout library from a spatial transcriptome according to claim 2, characterized in that: The method of injecting into the experimental animal is intravenous injection.

6. The method for constructing a library for enriching a trace amount of CRISPR gene knockout library from a spatial transcriptome according to claim 1, characterized in that: Before performing the inward nested outer amplification on the Total cDNA library for the CRISPR gene knockout library, the Total cDNA library is fully amplified in advance.

7. The method for constructing a library for enriching a trace amount of CRISPR gene knockout library from a spatial transcriptome according to claim 1, characterized in that: The method for performing a cyclization reaction on the Inner enriched product is specifically as follows: adding a splint-TSO cyclization sequence to the Inner enriched product and then performing a cyclization reaction.

8. The method for constructing a library for enriching a trace amount of CRISPR gene knockout library from a spatial transcriptome according to claim 1, characterized in that: The specific method for removing the uncyclized sequence is: using DNA exonuclease to digest the uncyclized sequence.

9. The method for constructing a library for enriching a trace amount of CRISPR gene knockout library from a spatial transcriptome according to claim 1, characterized in that: The cDNA sequence in the CRISPR gene knockout library is chimeric with an sgRNA sequence, and has Read2 and TSO sequences on one side of the sgRNA sequence, and has LTR, pA, MID, Fixed, CID, TSO and Read1 sequences on the other side of the sgRNA sequence.

10. The method for constructing a library for enriching a trace amount of CRISPR gene knockout library from a spatial transcriptome according to claim 9, characterized in that: The sgRNA sequence includes a gene variable sequence and a scaffold sequence.

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