Method, probe and kit for screening dominant phenotypes of low-frequency exogenous introduced genes

Abstract

The embodiment of the invention provides a method, a probe and a kit for screening dominant phenotypes of low-frequency exogenous introduction genes, relates to the technical field of biomedicine, and is used for targeted capture of transcription sequences of the exogenous introduction genes and single cell transcriptome sequencing. The probe comprises a first probe sequence and a second probe sequence, the first probe sequence is configured to capture a transcription sequence of an exogenous introduction gene in a targeted manner, and the second probe sequence is configured to perform single cell transcriptome sequencing.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedical technology, and in particular to a method, a probe and a kit for screening a dominant phenotype of a low-frequency exogenously introduced gene. Background Art

[0002] Chimeric Antigen Receptor (CAR) cell therapy refers to the use of gene modification technology to transfer genetic material with specific antigen recognition domains and T cell activation signals into human immune cells (such as T cells and NK cells), so that they can directly bind to specific antigens on the surface of tumor cells and be activated. They can directly kill tumor cells by releasing perforin, granzyme B, etc., and at the same time release cytokines to recruit endogenous immune cells in the human body to kill tumor cells, thereby achieving the purpose of treating tumors. In addition, immune memory cells can also be formed to obtain a specific long-term anti-tumor mechanism. Summary of the invention

[0003] The purpose of the embodiments of the present disclosure is to provide a method, probe and kit for screening the dominant phenotype of low-frequency exogenously introduced genes, wherein the probe is used for targeted capture of transcription sequences of exogenously introduced genes and single-cell transcriptome sequencing.

[0004] To achieve the above objectives, the embodiments of the present disclosure provide the following technical solutions:

[0005] On the one hand, a method for screening the dominant phenotype of low-frequency exogenously introduced genes is provided, comprising: preparing a single cell suspension. Encapsulating a single cell and a probe into a microdroplet; wherein the probe comprises: a first probe sequence and a second probe sequence, wherein the first probe sequence is configured to target and capture the exogenously introduced gene, and the second probe sequence is configured for single-cell transcriptome sequencing; the first probe sequence and the second probe sequence also include a barcode sequence for cell identification and a UMI sequence for single-molecule identification; lysing cells and capturing transcriptome information; obtaining cDNA by reverse transcription and amplifying it, and then performing high-throughput sequencing and analyzing the dominant phenotype of low-frequency exogenously introduced genes.

[0006] The above method for screening the dominant phenotype of low-frequency exogenously introduced genes uses probes including: a first probe sequence that targets and captures the transcription sequence of the exogenously introduced gene and a second probe sequence for single-cell transcriptome sequencing, which can capture single-cell transcripts and the transcription sequence of the exogenously introduced gene, thereby achieving simultaneous capture of single-cell transcripts and the transcription sequence of the exogenously introduced gene. This can effectively avoid the problem of loss of transcription sequence information of the exogenously introduced gene.

[0007] In some embodiments, the first probe sequence is configured to target and capture a universal sequence after transcription of an exogenously introduced gene.

[0008] On the other hand, a probe is provided, comprising: a first probe sequence and a second probe sequence, wherein the first probe sequence is configured to target and capture the transcription sequence of an exogenously introduced gene, and the second probe sequence is configured for single-cell transcriptome sequencing.

[0009] The first probe sequence in the above probes can directly target and capture the transcription sequence of low-copy exogenously introduced genes in cells, which can effectively avoid the problem of loss of transcription sequence information of exogenously introduced genes. The second probe sequence can be used for single-cell transcriptome sequencing.

[0010] In some embodiments, the first probe sequence is configured to target and capture a universal sequence after transcription of an exogenously introduced gene.

[0011] In some embodiments, the first probe sequence targets the transcribed sequence of the WPRE gene fragment of the lentiviral vector.

[0012] In some embodiments, the first probe sequence includes bases ranging from 13 to 200. The melting temperature of the first probe sequence ranges from 68° C. to 70° C. In the first probe sequence, the number of consecutive identical bases does not exceed 3.

[0013] In some embodiments, the first probe sequence is used to target and capture the CAR transcript.

[0014] In some embodiments, the first probe sequence comprises: a sequence having at least 70% identity to the sequence shown in SEQ ID No. 1; and / or a sequence having at least 70% identity to the sequence shown in SEQ ID No. 2; and / or a sequence having at least 70% identity to the sequence shown in SEQ ID No. 3. Wherein, SEQ ID No. 1 is: 5'-GCCACGGCGGAACTCATCGCCGCCTGCC-3'; SEQ ID No. 2 is: 5'-CCCGTATGGCTTTCATTTTCTCCTCCTTG-3'; SEQ ID No. 3 is: 5'-CGCCTTCGCCCTCAGACGAGTCGGATCTCCC-3'.

[0015] In some embodiments, the probe further comprises: a second probe sequence, wherein the second probe sequence is a PolyT sequence for targeted capture of single-cell transcripts.

[0016] In some embodiments, the first probe sequence is bound to the magnetic beads in a covalently modified manner; and the PolyT sequence is bound to the magnetic beads in a covalently modified manner.

[0017] In some embodiments, the upstream of the first probe sequence contains a barcode sequence for cell identification and a UMI sequence for single molecule identification; and the upstream of the second probe sequence contains a barcode sequence for cell identification and a UMI sequence for single molecule identification.

[0018] In another aspect, a kit is provided, comprising: the probe as described in any one of the above embodiments, and the above probe preservation solution for preserving the probe.

[0019] The application of the above-mentioned kit in CAR transcript capture has the same beneficial technical effects as the probes provided in some of the above-mentioned embodiments, which will not be repeated here. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the present disclosure, the following briefly introduces the drawings required to be used in some embodiments of the present disclosure. Obviously, the drawings described below are only drawings of some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can also be obtained based on these drawings. In addition, the drawings described below can be regarded as schematic diagrams, and are not limitations on the actual size of the product involved in the embodiments of the present disclosure, the actual process of the method, the actual timing of the signal, etc.

[0021] Figure 1 A structural diagram of a probe provided according to some embodiments of the present disclosure;

[0022] Figure 2 The diagrams show the results of single cell RNA sequencing (scRNA-seq) experiments and flow cytometry (Fluorescence activated Cell Sorting, FACS) experiments provided according to some embodiments of the present disclosure. DETAILED DESCRIPTION

[0023] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in some embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present disclosure.

[0024] Unless the context requires otherwise, throughout the specification and claims, the term "including" is to be interpreted as an open, inclusive meaning, that is, "including, but not limited to". In the description of the specification, the terms "one embodiment", "some embodiments", "exemplary embodiments", "examples" or "some examples" and the like are intended to indicate that specific features, structures, materials or characteristics associated with the embodiment or example are included in at least one embodiment or example of the present disclosure. The schematic representations of the above terms do not necessarily refer to the same embodiment or example. In addition, the specific features, structures, materials or characteristics described may be included in any one or more embodiments or examples in any appropriate manner.

[0025] In the following, the terms "first" and "second" are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the embodiments of the present disclosure, unless otherwise specified, "plurality" means two or more.

[0026] “At least one of A, B, and C” has the same meaning as “at least one of A, B, or C” and both include the following combinations of A, B, and C: A only, B only, C only, the combination of A and B, the combination of A and C, the combination of B and C, and the combination of A, B, and C.

[0027] “A and / or B” includes the following three combinations: A only, B only, and a combination of A and B.

[0028] Additionally, the use of “based on” is meant to be open and inclusive, as a process, step, calculation, or other action “based on” one or more stated conditions or values ​​may, in practice, be based on additional conditions or values ​​beyond those stated.

[0029] As used herein, "about," "substantially," or "approximately" includes the stated value and an average value that is within an acceptable range of variation from the particular value as determined by one of ordinary skill in the art taking into account the measurements in question and the errors associated with the measurement of the particular quantity (i.e., the limitations of the measurement system).

[0030] In the present application, DNA (deoxyribonucleic acid) is a long-chain polymer, the constituent units of which are four deoxynucleotides, namely, adenine deoxynucleotide (dATP), thymine deoxynucleotide (dTTP), cytosine deoxynucleotide (dCTP), and guanine deoxynucleotide (dGTP). Deoxynucleotides are composed of phosphate, deoxyribose, and bases; among them, there are four main bases, namely A (adenine), G (guanine), C (cytosine), and T (thymine).

[0031] In this application, RNA (Ribonucleic Acid) is a genetic information carrier present in biological cells and some viruses and viroids. Its main function in the body is to guide protein synthesis. RNA is a macromolecular polymer composed of ribonucleotides, which are composed of phosphate, ribose and bases; among them, there are four main bases, namely A (adenine), G (guanine), C (cytosine) and U (uracil).

[0032] In the present application, a transcript is one or more mature mRNAs (Messenger RNA) that can encode proteins formed by transcription of a gene. Among them, mRNA is a type of single-stranded ribonucleic acid that is transcribed from a strand of DNA as a template and carries genetic information and can guide protein synthesis. In the description of the disclosed embodiments, single-cell transcripts include all types of mRNAs of single cells, and CAR transcripts are one type of single-cell transcripts.

[0033] Chimeric Antigen Receptor (CAR) cells are usually composed of an extracellular antigen binding domain (providing targeting and specific recognition of tumor-associated antigens, such as single-chain antibodies that recognize CD19), a hinge region (promoting the binding of antigen receptors to tumor antigens), a transmembrane region (used to fix CAR), a T cell activation domain (CD3ζ, providing the first signal for T cell activation) and one or more intracellular co-stimulatory domains (CD28 / 4-1BB, providing the second signal for T cell activation). "Chimeric" because it combines the activation function of the original T cell with the new antigen recognition function.

[0034] In recent years, CAR-T cell therapy represented by T cells has shown significant efficacy in the treatment of acute leukemia and non-Hodgkin's lymphoma, and is considered one of the most promising tumor treatments. However, complications related to CAR-T cell therapy, such as cytokine release syndrome (Immune Effector Cell-Associated Neurotoxicity Syndrome, ICANS) and hematological toxicity, have become important factors restricting its application. In addition, compared with blood tumors, solid tumors are more complex, and the problems caused by tumor resistance, antigen heterogeneity and immune escape still need to be solved.

[0035] In the past, people generally believed that the characteristics of individual cells and groups of cells were consistent. As the study of tumor diseases continues to deepen, people's understanding of tumors is also deepening. Researchers' research on tumors has gradually moved from the macroscopic level to the single cell level, revealing the heterogeneity between tumor cells.

[0036] Single cell RNA sequencing (scRNA-seq) technology can explain the heterogeneity between different cells at different levels such as genome, epigenomics, transcriptomics or proteomics. By drawing cell maps, cell types can be identified, rare functional subgroups can be discovered, pathogenesis can be studied, and drug design and in-depth exploration of therapeutic targets can be assisted. Therefore, conducting studies on cell gene expression and cell functional activities at the single cell level is of great significance for revealing the heterogeneity between cells and explaining certain physiological behaviors.

[0037] Single-cell RNA sequencing is a high-throughput experimental technology that uses RNA sequencing to quantify the gene expression profile of a specific cell population at the single-cell level. After the tissue to be tested undergoes single-cell isolation, RNA capture, reverse transcription, library construction, and high-throughput sequencing, the gene expression profile of multiple cells can be obtained using bioinformatics analysis.

[0038] In the process of single-cell RNA sequencing, the most core and technically difficult steps are single-cell isolation and RNA capture. Currently, platforms that can achieve single-cell RNA sequencing include Fluidigm C1 Chromium platform (10X Genomics), BD rhapsody Cytoseq (BD Biosciences) and other derivatives based on these three platforms achieve single-cell capture based on microvalve, droplet microfluidics and microplate technology respectively.

[0039] Regardless of the platform, RNA capture is based on Barcoded Beads technology, which isolates individual microbeads and cells into a micro-reaction system (formed by micro-droplets, micro-wells, etc.). Each microbead has a specific DNA fragment on its surface, which is mainly composed of three parts: Barcode (cell index), UMI (molecular label), and PolyT.

[0040] The barcode is 16 bases long, and there are 4 million types of barcodes in total. One microbead corresponds to one barcode. Through the 4 million types of barcodes, the gel microbeads can be distinguished, which means that each cell can be distinguished.

[0041] UMI is a random sequence used to mark each initial (c)DNA copy, that is, each initial (c)DNA molecule has a corresponding UMI sequence; each UMI has 10 bases and about 1 million sequence variations. UMI can be used to distinguish between real SNP (Single Nucleotide Polymorphism) sites and mutations caused by PCR (Polymerase Chain Reaction).

[0042] PolyT is located at the 3' end of DNA and is used to capture the PolyA tail of free single-cell transcripts (usually 150-200 adenylate residues on mRNA, also known as a special tail structure). The 3' end of the complete single-cell transcript is PolyA. Based on the principle of base complementary pairing, the PolyA at the 3' end of the single-cell transcript can hybridize with its complementary paired PolyT sequence, thereby capturing the single-cell transcript. Although the PolyT-based single-cell transcript capture technology has been widely used in existing single-cell RNA sequencing platforms, it also has obvious shortcomings.

[0043] First, PolyT can only capture the 3' end of mRNA, so the integrity of mRNA is extremely high, otherwise the sequencing results will be greatly biased. Second, the abundance of mRNA needs to reach a certain value, otherwise information will be lost whether in the capture experiment stage or the quality control stage of bioinformatics analysis data.

[0044] It should be noted that the abundance of mRNA refers to the average number of molecules of a specific mRNA in a cell. In other words, in order to capture CAR transcripts, the PolyT-based mRNA capture technology not only requires extremely high integrity of CAR transcripts, but also requires that the abundance of CAR transcripts in single-cell transcripts reach a certain value.

[0045] Based on this, an embodiment of the present disclosure provides a method for screening a dominant phenotype of a low-frequency exogenously introduced gene, the method comprising steps: M1 to M4.

[0046] M1. Prepare single-cell suspension.

[0047] M2, encapsulating single cells and probes into microdroplets; Figure 1 As shown, the probe 100 includes: a first probe sequence and a second probe sequence, the first probe sequence is configured to target and capture exogenously introduced genes, and the second probe sequence is configured for single-cell transcriptome sequencing; the first probe sequence and the second probe sequence also include a barcode sequence for cell identification and a UMI sequence for single molecule identification.

[0048] M3, lyse cells and capture transcriptome information.

[0049] M4. cDNA is obtained and amplified by reverse transcription, followed by high-throughput sequencing and analysis of the dominant phenotype of low-frequency exogenously introduced genes.

[0050] Among them, cDNA (complementary DNA) refers to complementary DNA, specifically the DNA chain that is complementary to RNA after reverse transcription in vitro.

[0051] like Figure 1 As shown, the first probe sequence is configured to target and capture the universal sequence after transcription of the exogenously introduced gene.

[0052] Among them, the universal sequence refers to a part of DNA or RNA that has a nucleotide sequence that is highly conserved in different organisms. By comparing the sequences of different species, the genetic relationship and evolutionary relationship between them can be determined. The universal sequence can be used to construct a phylogenetic tree to study the origin and evolutionary history of species.

[0053] The method for screening the dominant phenotype of low-frequency exogenously introduced genes provided in the embodiments of the present disclosure uses a probe 100 including: a first probe sequence for targeted capture of exogenously introduced genes and a second probe sequence for single-cell transcriptome sequencing, which can capture single-cell transcripts and transcription sequences of exogenously introduced genes, thereby achieving simultaneous capture of single-cell transcripts and transcription sequences of exogenously introduced genes. This can effectively avoid the problem of loss of transcription sequence information of exogenously introduced genes.

[0054] In some embodiments, Figure 1 As shown, an embodiment of the present disclosure further provides a probe 100, which includes a first probe sequence and a second probe sequence, wherein the first probe sequence is configured to target and capture the transcription sequence of an exogenously introduced gene, and the second probe sequence is configured for single-cell transcriptome sequencing.

[0055] The first probe sequence targets the transcription sequence of the WPRE gene fragment of the lentiviral vector, and the first probe sequence can be used to capture the CAR transcript.

[0056] It should be noted that lentiviral vector refers to a viral vector derived from human immunodeficiency virus-1 (HIV-1). Lentiviral vector contains the genetic information required for packaging, transfection (the technology of introducing foreign molecules such as DNA and RNA into eukaryotic cells), and stable integration, and is the main component of the lentiviral vector system. With the assistance of lentiviral packaging plasmids and cell lines, lentiviral vectors carrying foreign genes are packaged into infectious viral particles through viruses, and through infecting cells or living tissues, the foreign genes are expressed in cells or living tissues.

[0057] The sequence interval in the WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) gene segment refers to a portion of the sequence in the WPRE gene segment, that is, the sequence of the transcription sequence of the WPRE gene segment targeting the lentiviral vector is used as the sequence in the probe 100 for CAR transcript capture. WPRE is a gene segment at the 3′LTR (long terminal repeated) position of the lentiviral vector.

[0058] The first probe sequence targeting the transcription sequence of the WPRE gene fragment of the lentiviral vector can be used as the probe 100 sequence for CAR transcript capture for the following reasons.

[0059] In order to obtain T cells or NK cells that can continuously express CAR proteins, the peripheral blood mononuclear cells (PBMC) from the patient are first purified for T cells or NK cells, and then the plasmid loaded with the CAR gene is delivered into the T cells or NK cells through gene transfection technology. The currently widely used gene transfection technology mainly uses lentivirus as a vector. Lentivirus vectors have a good invasion effect on T cells, so efficient transfection efficiency can be obtained. After the CAR gene enters the cell, it will be integrated into the genome under the action of a specific sequence on the plasmid, and then it will continue to be transcribed and translated to express the CAR protein, and then it will be transported to the cell membrane to exert its effect.

[0060] For different specific antigens, the sequences of CAR genes may be quite different. Therefore, if the probe 100 used for capturing CAR transcripts is designed only for CAR genes, it can only capture CARs of specific sequences and is not compatible with other types of CARs. In addition, if the capture probe 100 is designed for the CAR gene, the CAR gene sequence needs to be known in advance, and the CAR gene sequences of different CAR drugs are often different, and the CAR gene sequence cannot be obtained.

[0061] However, when the CAR gene is inserted into the host genome, it is often accompanied by the simultaneous insertion of the WPRE gene.

[0062] Therefore, the design of the probe 100 for CAR transcript capture provided in the embodiments of the present disclosure is mainly based on the partial conserved sequence design on the lentiviral vector plasmid used (a very small circular DNA molecule that can replicate autonomously outside the cell chromosome), namely the WPRE gene fragment at the 3′LTR position.

[0063] It should be noted that conservative sequences refer to molecular sequences with high similarity or identity. These sequences can be nucleic acid sequences (such as RNA or DNA sequences), protein sequences, protein structures or sequences in carbohydrates. These sequences are highly similar, but come from different species or different molecules produced by the same organism. From the perspective of cross-species conservation, the existence of such sequences means that a special gene sequence has been preserved during the evolutionary process of forming different species. By analyzing the genomes of organisms at different evolutionary stages, it was found that some sequences in the genomes of different organisms are very similar. These similar sequences are the so-called conservative sequences.

[0064] The WPRE gene fragment is a cis-acting RNA element and a post-transcriptional regulatory sequence. Placing it before PolyA can significantly increase the expression level and translation efficiency of mRNA, thereby enhancing gene expression. Inserting WPRE into a viral vector can also greatly increase the titer of viral packaging. The complete sequence of the WPRE gene is:

[0065] , a total of 592 bases.

[0066] Therefore, the sequence of the transcription sequence of the WPRE gene fragment of the targeting lentiviral vector is used as the probe 100 sequence for CAR transcript capture. According to the principle of base complementary pairing, the two complementary gene chains of the reverse sequence will be renatured under certain conditions (annealing step, see the subsequent content for details, and will not be described here). In other words, the first probe sequence of the probe 100 will be renatured with the transcript of the WPRE gene fragment under certain conditions. Since the CAR gene is often inserted into the host genome, it is often accompanied by the simultaneous insertion of the WPRE gene. Therefore, when the first probe sequence of the probe 100 is renatured with the transcript of the WPRE gene fragment, the capture of the CAR transcript by the probe 100 can be achieved.

[0067] Moreover, by using the transcription sequence of the WPRE gene fragment targeting the lentiviral vector as the probe 100 sequence for capturing the CAR transcript, there is no need to distinguish the specific CAR gene sequence, and the probe 100 sequence can capture multiple CAR transcripts.

[0068] Moreover, in the single-cell transcripts, there are mRNAs used to guide the formation of multiple proteins, and CAR transcripts are a type of mRNA with a lower copy number in single-cell transcripts. Since the copy number of CAR transcripts in single-cell transcripts is low, the single-cell transcript capture technology based on PolyT will have the problem of information loss when capturing low-copy CAR transcripts. Among them, the copy number refers to the number of a gene (which can be a plasmid) in the genome of a certain organism.

[0069] By using the probe 100 provided in the embodiment of the present disclosure, low-copy number CAR transcripts can be directly captured, and the problem of CAR transcript information loss can be effectively avoided, thereby distinguishing CAR cells from cell groups, and facilitating further analysis of CAR cells. For example, through subsequent gene library construction, the sequence information and original copy number information of CAR cells can be obtained through single-cell RNA sequencing, and then the relative abundance of CAR cell expression can be known, which solves the problem of easy loss of target transcripts in the current single-cell RNA sequencing technology.

[0070] When the sequence of the transcription sequence of the WPRE gene fragment targeting the lentiviral vector is used as the probe 100 sequence for CAR transcript capture, the selection of the probe sequence needs to meet the following conditions.

[0071] In some examples, the first probe sequence includes a number of bases ranging from 13 to 200.

[0072] For example, the first probe sequence includes 13, 15, 18, 21, 25, 32, 39, 45, 58, 80, 95, 120, 155 or 200 bases, etc., which are not limited here.

[0073] A first probe sequence with 13 to 200 bases can form a stable hybrid with the transcript of the WPRE gene (CAR transcript) to achieve the capture of the CAR transcript.

[0074] In some examples, the melting temperature of the first probe sequence ranges from 68°C to 70°C.

[0075] It should be noted that the melting temperature is the temperature at which 50% of the double-stranded structure of a double-stranded DNA molecule is opened.

[0076] For example, the melting temperature of the first probe sequence is 68° C., 69° C., or 70° C., etc., which is not limited here.

[0077] The melting temperature of the first probe sequence is controlled at 68°C to 70°C because the hybridization reaction temperature of the entire system is within this range. Too high a temperature can easily cause mRNA degradation. In addition, polymerase is used in the steps related to capturing CAR transcripts. The optimal reaction temperature of polymerase is between 68°C and 70°C. Too low a temperature can cause insufficient polymerase activity.

[0078] In some examples, in the first probe sequence, the number of consecutive identical bases does not exceed 3.

[0079] For example, in the first probe sequence, the number of identical bases arranged consecutively is 2 or 3.

[0080] Too many consecutive identical bases will cause local hybridization to form a stable structure, thereby capturing the impurity chain through its identical repeating structure, that is, capturing the non-CAR transcript.

[0081] In particular, the number of consecutive G bases cannot be 4 or more, because three hydrogen bonds are formed between the G base and the C base, which makes it easier to form a local stable structure.

[0082] Based on the above conditions that need to be met in the design of the first probe sequence, the first probe sequence includes: a sequence having at least 70% identity with the sequence shown in SEQ ID No. 1; and / or, a sequence having at least 70% identity with the sequence shown in SEQ ID No. 2; and / or, a sequence having at least 70% identity with the sequence shown in SEQ ID No. 3. Wherein, SEQ ID No. 1 is: 5'-GCCACGGCGGAACTCATCGCCGCCTGCC-3'; SEQ ID No. 2 is: 5'-CCCGTATGGCTTTCATTTTCTCCTCCTTG-3'; SEQ ID No. 3 is: 5'-CGCCTTCGCCCTCAGACGAGTCGGATCTCCC-3'.

[0083] The above-mentioned SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 are respectively identical to partial sequences in the WPRE gene fragment of the lentiviral vector. Therefore, the probe 100 comprising a sequence having at least 70% identity with the sequence of SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3 can specifically capture the CAR transcript.

[0084] It should be noted that the 5' end refers to the end of a DNA or RNA single strand with a free 5'-hydroxyl or its phosphate. The 3' end refers to the end of a DNA or RNA single strand with a free 3'-hydroxyl or its phosphate. DNA is composed of two nucleotide chains, and the basic units that make up each single strand are four deoxynucleotides. During the DNA synthesis process, multiple deoxynucleotides need to be connected together to form a new DNA single strand. However, this formation process cannot be carried out in any direction, but must start from the 5' end and end at the 3' end.

[0085] In some examples, probe 100 includes one type of first probe sequence.

[0086] For example, the first probe sequence of probe 100 includes a sequence having at least 70% identity to the sequence shown in SEQ ID No.1.

[0087] For example, the first probe sequence of probe 100 includes a sequence having at least 70% identity to the sequence shown in SEQ ID No.2.

[0088] For example, the first probe sequence of probe 100 includes a sequence having at least 70% identity to the sequence shown in SEQ ID No.3.

[0089] In some examples, the probe 100 includes two types of first probe sequences, that is, the probes 100 of these two types of sequences are used simultaneously, and each first probe sequence can capture CAR transcripts. In this way, the efficiency of CAR transcript capture can be improved while ensuring the specificity of CAR transcript capture.

[0090] For example, a sequence having at least 70% identity to the sequence shown in SEQ ID No. 1 and a sequence having at least 70% identity to the sequence shown in SEQ ID No. 2 are used simultaneously. Among them, SEQ ID No. 1: 5'-GCCACGGCGGAACTCATCGCCGCCTGCC-3', SEQ ID No. 2: 5'-CCCGTATGGCTTTCATTTTCTCCTCCTTG-3'.

[0091] For example, a sequence having at least 70% identity to the sequence shown in SEQ ID No. 2 and a sequence having at least 70% identity to the sequence shown in SEQ ID No. 3 are used simultaneously. Among them, SEQ ID No. 2: 5'-CCCGTATGGCTTTCATTTTCTCCTCCTTG-3', SEQ ID No. 3: 5'-CGCCTTCGCCCTCAGACGAGTCGGATCTCCC-3'.

[0092] For example, a sequence having at least 70% identity to the sequence shown in SEQ ID No. 1 and a sequence having at least 70% identity to the sequence shown in SEQ ID No. 3 are used simultaneously. Among them, SEQ ID No. 1: 5'-GCCACGGCGGAACTCATCGCCGCCTGCC-3', SEQ ID No. 3: 5'-CGCCTTCGCCCTCAGACGAGTCGGATCTCCC-3'.

[0093] In some examples, the probe 100 includes three types of first probe sequences, that is, the probes 100 of the three types of sequences are used simultaneously, and each first probe sequence can capture CAR transcripts. In this way, the efficiency of CAR transcript capture can be improved while ensuring the specificity of CAR transcript capture.

[0094] Among them, the three types of sequences include: a sequence having at least 70% identity with the sequence shown in SEQ ID No. 1, a sequence having at least 70% identity with the sequence shown in SEQ ID No. 2, and a sequence having at least 70% identity with the sequence shown in SEQ ID No. 3. SEQ ID No. 1: 5'-GCCACGGCGGAACTCATCGCCGCCTGCC-3', SEQ ID No. 2: 5'-CCCGTATGGCTTTCATTTTCTCCTCCTTG-3', SEQ ID No. 3: 5'-CGCCTTCGCCCTCAGACGAGTCGGATCTCCC-3'.

[0095] In some embodiments, Figure 1 As shown, the probe 100 further includes a second probe sequence, which is a PolyT sequence for targeted capture of single-cell transcripts.

[0096] The 3' end of the single-cell transcript is PolyA. Based on the principle of base complementary pairing, the PolyA at the 3' end of the single-cell transcript can hybridize with its complementary paired PolyT sequence, thereby capturing the single-cell transcript.

[0097] That is, the PolyT sequence of the probe 100 provided in the embodiment of the present disclosure can capture the cell transcripts of cell lysis, and when capturing the cell transcripts, the cell transcripts include some low-copy number CAR transcripts. The first probe sequence of the probe 100 hybridizes with the CAR transcripts to capture the CAR transcripts. The first probe sequence of the probe 100 specifically captures the CAR transcripts, which can avoid the loss of the CAR transcripts.

[0098] In some embodiments, Figure 1 As shown, the first probe sequence is bound to the magnetic beads in a covalently modified manner. The PolyT sequence is bound to the magnetic beads in a covalently modified manner.

[0099] The first probe sequence is bound to the magnetic beads in a covalently modified manner, and the covalently bound connection method can improve the stability of the binding between the first probe sequence and the magnetic beads.

[0100] The PolyT sequence is bound to the magnetic beads in a covalently modified manner, and the covalently bound connection method can improve the stability of the binding between the PolyT sequence and the magnetic beads.

[0101] That is to say, in the embodiments provided in the present disclosure, the surface of the magnetic bead has both a PolyT sequence for capturing single-cell transcripts and a probe 100 for capturing CAR transcripts, thereby achieving simultaneous capture of single-cell transcripts and CAR transcripts. The problem of CAR transcript information loss can be effectively avoided.

[0102] Magnetic beads are used as carriers of the probe 100. The magnetic beads can carry the CAR transcripts captured by the first probe sequence and transfer them in different liquids, making it convenient to purify and separate the CAR transcripts.

[0103] Exemplarily, in order to obtain magnetic beads having the first probe sequence and the PolyT sequence on the surface, an in situ solid phase synthesis method or a nucleotide covalent coupling method may be used.

[0104] In some embodiments, Figure 1 As shown, the upstream of the first probe sequence contains a barcode sequence for cell identification and a UMI sequence for single molecule identification; the upstream of the PolyT sequence contains a barcode sequence for cell identification and a UMI sequence for single molecule identification.

[0105] The upstream of a gene refers to the starting end of the gene sequence, while the downstream refers to the ending end of the gene sequence. During DNA synthesis, multiple deoxynucleotides need to be connected starting from the 5' end and ending at the 3' end. Therefore, the upstream of a gene refers to the 5' end of the coding chain.

[0106] The barcode is 16 bases long, and there are 4 million types of barcodes in total. One microbead corresponds to one barcode. Through the 4 million types of barcodes, the gel microbeads can be distinguished, which means that each cell can be distinguished.

[0107] UMI is a random sequence used to mark each initial (c)DNA copy, that is, each initial (c)DNA molecule has a corresponding UMI sequence.

[0108] That is to say, the Barcode sequence of all nucleotides on the surface of each magnetic bead is the same, but the UMI sequence of all nucleotides is different.

[0109] The probe 100 provided in the embodiments of the present disclosure is used to capture CAR transcripts.

[0110] Exemplarily, the CAR protein is introduced into different cells, and after the CAR transcripts are captured by the probe 100 provided in the embodiment of the present disclosure in multiple cells, the CAR transcripts are analyzed, and the multiple cells are classified according to functional types. When verifying the cell function, the corresponding cells can be selected in a targeted manner.

[0111] A method for storing the probe 100 is provided below, including steps: R1 to R4.

[0112] R1. Wash the probe 100 connected with magnetic beads twice with 30 mL of anhydrous ethanol.

[0113] R2. Wash twice with 30 mL of buffer solution. For example, the buffer solution includes 10 mmol / L tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid and Tween 20, and the pH value of the buffer solution is 8.0.

[0114] Tween 20 is polyoxyethylene sorbitan monolaurate, with the molecular formula C 26 H 50 O 10 , also known as Tween 20, is a surfactant, a type of macromolecule that has both a hydrophilic part and a lipophilic part.

[0115] Tris(hydroxymethyl)aminomethane acts as a pH buffer in the system. Ethylenediaminetetraacetic acid acts as a chelating agent for divalent metal ions in the system, and Tween 20 is a surfactant. The main function of these two reagents is to stabilize the microbead system and prevent it from aggregating and precipitating.

[0116] For example, the concentration of EDTA is 0.5 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L or 5 mmol / L, etc., which is not limited here. The mass percentage of Tween 20 is 0.005%, 0.01% or 0.02%), etc., which is not limited here.

[0117] R3. Resuspend the magnetic beads in 10 mL of buffer.

[0118] For the introduction of the buffer solution, please refer to step R2, which will not be repeated here.

[0119] R4, filter into a centrifuge tube and store at 4°C for a long term.

[0120] For example, the filter is a filter mesh of model 352360 of BD Falcon, and the mesh opening of the filter mesh of model 352360 is 100 μm.

[0121] The filter removes the clumps that may be formed in the system, making the magnetic beads more dispersed.

[0122] A method for preparing a sample containing CAR cells is provided below, comprising steps: S1 to S4. However, some embodiments of the present disclosure are not limited thereto.

[0123] S1. The infusion residues of three patients treated with CAR-T were taken. The infusion residues of patients treated with CAR-T contained CAR cells.

[0124] S2. Wash the CAR-T infusion bag after infusion with PBS (phosphate buffer saline), and store the washing solution in the cell freezing solution. First, place the cryopreservation tube containing the cell freezing solution in a 4℃ refrigerator for 2 hours, then in a -20℃ refrigerator for 2 hours, and finally in a -80℃ refrigerator overnight, then take out the cryopreservation tube and place it in a liquid nitrogen container.

[0125] S3. Thaw and wash the cryopreserved solution in the cryopreservation tube, resuspend it in 0.5 mL of culture medium, and culture it in a 48-well plate for 24 hours.

[0126] S4. Antigen stimulation experiment: The infusion residue was co-cultured with target cells MM1.S (myeloma cell line) at a ratio of 1:1, and downstream single-cell RNA sequencing experiments were performed after 20 hours of incubation.

[0127] The culture medium includes: RPMI-1640+10% FBS+1% glutamine. RPMI is the abbreviation of Roswell Park Memorial Institute. RPMI is a type of cell culture medium developed by the institute, and 1640 is the code name of the culture medium. FBS (Fetal Bovine Serum) is a light yellow, clear, non-hemolytic, foreign-free, slightly viscous liquid. Fetal bovine serum should be obtained from fetal bovines delivered by caesarean section.

[0128] The residues are co-cultured with target cells MM1.S in a 1:1 ratio, which is the process of CAR-T activation.

[0129] Before performing a single-cell RNA sequencing experiment, the probe 100 needs to be dispersed in a preservation solution. The specific operation steps and preservation solution are described as follows.

[0130] Specific operation steps: take out a portion of magnetic beads from the centrifuge tube, wash with 500 μL of lysis solution, and then place the magnetic beads in a preservation solution, wherein the preservation solution includes: a mixture of lysis solution and dithiothreitol.

[0131] Lysis buffer washing can suspend the magnetic beads in the lysis environment, making the subsequent mixing of magnetic beads and cells more gentle.

[0132] Exemplarily, the lysis solution includes: tris(hydroxymethyl)aminomethane, polysucrose 400, sodium lauryl sarcosinate and ethylenediaminetetraacetic acid. The molar concentration of tris(hydroxymethyl)aminomethane is in the range of 150mmol / L to 300mmol / L, the mass proportion of polysucrose 400 is in the range of 4% to 15%, the mass proportion of sodium lauryl sarcosinate is in the range of 0.15% to 3%, the molar concentration of ethylenediaminetetraacetic acid is in the range of 15mmol / L to 50mmol / L, and the pH value of the lysis solution is in the range of 7.0 to 7.7.

[0133] For example, the molar concentration of tris(hydroxymethyl)aminomethane is 150 mmol / L, 180 mmol / L, 200 mmol / L, 230 mmol / L, 260 mmol / L or 300 mmol / L, etc., which is not limited here.

[0134] For example, the pH value of the lysate is 7.0, 7.2, 7.4, 7.5, 7.6 or 7.7, etc., which is not limited here.

[0135] For example, the mass percentage of polysucrose 400 is 4%, 6%, 8%, 10%, 11%, 13%, 14% or 15%, etc., which is not limited here.

[0136] For example, the mass percentage of sodium lauryl sarcosinate is 0.15%, 0.2%, 0.3%, 1%, 1.6%, 2%, 2.5% or 3%, etc., which is not limited here.

[0137] For example, the molar concentration of EDTA is 15 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 45 mmol / L or 50 mmol / L, etc., which is not limited here.

[0138] The preservation solution includes: a mixture of lysis solution and dithiothreitol, wherein the molar concentration of dithiothreitol is in the range of 25mmol / L to 100mmol / L.

[0139] That is to say, the preservation solution includes: tris(hydroxymethyl)aminomethane, polysucrose 400, sodium lauryl sarcosinate, ethylenediaminetetraacetic acid and dithiothreitol. Among them, the molar concentration range of tris(hydroxymethyl)aminomethane is 150mmol / L to 300mmol / L, the mass proportion range of polysucrose 400 is 4% to 15%, the mass proportion range of sodium lauryl sarcosinate is 0.15% to 3%, the molar concentration range of ethylenediaminetetraacetic acid is 15mmol / L to 50mmol / L, the molar concentration range of dithiothreitol is 25mmol / L to 100mmol / L, and the pH value range of the probe preservation solution is 7.0 to 7.7.

[0140] For example, the molar concentration of dithiothreitol is 25 mmol / L, 35 mmol / L, 50 mmol / L, 65 mmol / L, 80 mmol / L or 100 mmol / L, etc., which is not limited here.

[0141] For example, the pH value of the probe storage solution is 7.0, 7.2, 7.4, 7.5, 7.6 or 7.7, etc., which is not limited here.

[0142] Exemplarily, the probe preservation solution includes: tris(hydroxymethylaminomethane) with a molar concentration of 200 mmol / L, polysucrose 400 with a mass percentage of 6%, sodium lauryl sarcosine with a mass percentage of 2%, ethylenediaminetetraacetic acid with a molar concentration of 50 mmol / L, dithiothreitol with a molar concentration of 50 mmol / L, and the pH value of the probe preservation solution is 7.5.

[0143] In the preservation solution, tris(hydroxymethyl)aminomethane) acts as a pH buffer, and polysucrose 400 acts as a lysis medium, which can better separate the components after cell lysis. Sodium lauryl sarcosine is a surfactant, which is used to destroy the cell membrane and release the cell contents. Ethylenediaminetetraacetic acid acts as a chelating system for divalent metal ions, and dithiothreitol is a reducing agent. After the cells are lysed, it can prevent the formation of intramolecular or intermolecular disulfide bonds between cysteine ​​in proteins and avoid their aggregation.

[0144] After the magnetic beads are stored in the storage solution, single-cell RNA sequencing experiments are then performed.

[0145] For example, the magnetic beads in the single-cell transcriptome library kit purchased on the market are replaced with the magnetic beads provided by the embodiments of the present disclosure, and the remaining single-cell separation and lysis, mRNA capture, library construction, and high-throughput sequencing are all performed according to the standard process provided by the purchased kit.

[0146] Some embodiments of the present disclosure further provide a kit, which includes the probe 100 provided in any of the above embodiments and the preservation solution provided in the above embodiments, and is used for single-cell RNA sequencing experiments.

[0147] In order to verify that the probe 100 provided in the embodiment of the present disclosure can be used for single-cell RNA sequencing experiments after capturing CAR transcripts, and that the results of the single-cell RNA sequencing experiments are reliable, a reference example of performing a cell flow cytometry experiment on a sample is provided below.

[0148] Flow cytometry experiments analyze and sort cells by detecting cell surface proteins. Since RQR8 is also expressed on the surface of CAR cells, flow cytometry can be used to detect the infection of lentivirus cells.

[0149] Among them, the RQR8 gene is an artificially synthesized CD34-containing protein (CD34 is an immunohistochemical phenotype, CD34 + It contains both a screening marker that can be recognized by the clinical CD34 sorting system and a CD20 membrane protein suicide molecule that can be targeted and bound by the clinical drug rituximab (RTX), which leads to CD20 through complement dependent cytotoxicity (CDC). + (CD20 + indicates positive) cell death.

[0150] Exemplarily, the cell flow cytometry experimental steps include: all samples are counterstained with 7AAD to exclude dead cells, and then incubated with RQR8 at 4°C for 20 minutes before performing flow cytometry analysis.

[0151] Among them, 7AAD is a fluorescent intercalator that undergoes a spectral shift when bound to DNA. The 7-AAD / DNA complex can be excited by a 488nm laser and has a maximum emission wavelength of 647nm, making this nucleic acid stain useful for multicolor fluorescence microscopy and flow cytometry. 7-AAD is usually excluded by living cells, but can be used in fixed and permeabilized cells.

[0152] After single-cell RNA sequencing and flow cytometry experiments on the samples, bioinformatics analysis is performed to obtain the captured CAR sequence and then distinguish the CAR expression of each cell, that is, to perform quality control analysis.

[0153] The specific steps of bioinformatics analysis include: In order to achieve the simultaneous analysis of single-cell transcriptome data and CAR transcript data, a custom reference genome was first designed, mainly including the 1.3kbp sequence upstream of the polyadenylation site of the 3'LTR of the virus used to express CAR. The obtained CAR transcripts were then aligned to the custom genome.

[0154] Among them, the size unit of DNA fragments is base pairs, and commonly used ones are bp (base pairs), Kbp (kilobase pairs) and Mbp (megabase pairs).

[0155] Cellranger (version 2.0.0, a set of analysis processes for alignment, quantification, clustering and gene expression analysis of single-cell RNA sequencing output results) was used for genome and custom genome alignment. Scanpy27 (version 1.5.0, single-cell analysis) was used for downstream analysis. Cell quality control was mainly based on three parameters: 1) at least 1,000 genes were detected per cell; 2) the UMI count associated with mitochondrial genes was less than 8%; 3) the total number of UMIs per cell exceeded 30,000.

[0156] Through the above three parameters, unnecessary cells are eliminated before bioinformatics analysis, and it is determined whether the cells are antigen-positive or antigen-negative.

[0157] In addition, only genes with UMI counts greater than 1 were retained in further analysis, that is, cells with CAR protein on the cell surface were retained. The cells were then normalized to 10,000 UMIs per cell and log-transformed. The “regression-out” function in Scanpy was used to remove read depth, gene number, mitochondrial number, and cell cycle effects.

[0158] After the samples were subjected to single-cell RNA sequencing and flow cytometry experiments, the results after bioinformatics analysis were as follows: Figure 2 shown.

[0159] in, Figure 2 The vertical axis in the figure indicates the percentage of CAR-positive cells. "Positive" indicates antigen-positive. The sample preparation steps for antigen-positive samples include S1 to S4. The difference is that "negative" indicates antigen-negative. The sample for antigen-negative samples does not undergo antigen stimulation experiment in the preparation step S4, that is, target cells MM1.S (myeloma cell line) are not added. Figure 2 The data represented by each rectangle is the data that has been processed with standard deviation, that is, the data of the three samples are processed with standard deviation. Among them, the three samples correspond to the samples prepared from the infusion residues of the three CAR-T treated patients in step S1. For example, the "CAR positive cell percentage" data measured by single-cell RNA sequencing in "negative" is: the samples are prepared from the infusion residues of the three CAR-T treated patients, and the three samples have not been subjected to antigen stimulation experiments, and then single-cell RNA sequencing experiments are performed and the measured data are processed with standard deviation.

[0160] from Figure 2It can be seen that the probe 100 provided in the embodiment of the present disclosure is used to capture the CAR transcript, and the percentage of CAR-expressing cells obtained after the single-cell RNA sequencing experiment is very close to the result of flow cytometry detection of RQR8, indicating that the probe 100 provided in the embodiment of the present disclosure can be used to capture the CAR transcript in single-cell sequencing, and the probe 100 provided in the embodiment of this scheme has great practical value in capturing the CAR transcript.

[0161] The above is only a specific embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any changes or substitutions that can be thought of by any person skilled in the art within the technical scope disclosed in the present disclosure should be included in the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be based on the protection scope of the claims.

Claims

1. A method for screening the dominant phenotype of low-frequency exogenously introduced genes, characterized in that: include: Prepare single cell suspension; Encapsulating a single cell and a probe into a microdroplet; wherein the probe comprises: a first probe sequence and a second probe sequence, wherein the first probe sequence is configured to target and capture the exogenously introduced gene, and the second probe sequence is configured for single-cell transcriptome sequencing; the first probe sequence and the second probe sequence further comprise a barcode sequence for cell identification and a UMI sequence for single-molecule identification; Lyse cells and capture transcriptome information; cDNA is obtained by reverse transcription and amplified, followed by high-throughput sequencing and analysis of the dominant phenotypes of low-frequency exogenously introduced genes.

2. The method for screening the dominant phenotype of low-frequency exogenously introduced genes according to claim 1, characterized in that: The first probe sequence is configured to target and capture a universal sequence after transcription of an exogenously introduced gene.

3. A probe, characterized in that: include: A first probe sequence and a second probe sequence, wherein the first probe sequence is configured to target and capture the transcription sequence of the exogenously introduced gene, and the second probe sequence is configured for single-cell transcriptome sequencing.

4. The probe according to claim 3, characterized in that: The first probe sequence is configured to target and capture a universal sequence after transcription of an exogenously introduced gene.

5. The probe according to claim 3, characterized in that: The first probe sequence targets the transcription sequence of the WPRE gene fragment of the lentiviral vector.

6. The probe according to claim 3, characterized in that: The number of bases included in the first probe sequence ranges from 13 to 200; the melting temperature of the first probe sequence ranges from 68° C. to 70° C.; in the first probe sequence, the number of consecutive identical bases does not exceed 3.

7. The probe according to claim 3, characterized in that: The first probe sequence is used to target and capture CAR transcripts.

8. The probe according to any one of claims 3 to 7, characterized in that: The first probe sequence comprises: A sequence having at least 70% identity to the sequence shown in SEQ ID No. 1; and / or, A sequence having at least 70% identity to the sequence shown in SEQ ID No. 2; and / or, A sequence having at least 70% identity to the sequence shown in SEQ ID No. 3; Among them, SEQ ID No.1 is: 5'-GCCACGGCGGAACTCATCGCCGCCTGCC-3'; SEQ ID No.2 is: 5'-CCCGTATGGCTTTCATTTTCTCCTCCTTG-3'; SEQ ID No.3 is: 5'-CGCCTTCGCCCTCAGACGAGTCGGATCTCCC-3'.

9. The probe according to claim 8, characterized in that: The second probe sequence is a PolyT sequence used for targeted capture of single-cell transcripts.

10. The probe according to claim 9, characterized in that: The first probe sequence is bound to the magnetic beads in a covalently modified manner; The PolyT sequence is bound to the magnetic beads in a covalently modified manner.

11. The probe according to claim 9 or 10, characterized in that: The upstream of the first probe sequence contains a barcode sequence for cell identification and a UMI sequence for single molecule identification; and the upstream of the second probe sequence contains a barcode sequence for cell identification and a UMI sequence for single molecule identification.

12. A kit, characterized in that: include: The probe according to any one of claims 3 to 11; The probe preservation solution is used to preserve the probe.

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