CRISPR editing screening system and application

Abstract

The application provides a CRISPR editing screening system and application, and relates to the technical field of biology.The nucleic acid construct provided by the application is based on a marker gene-ribozyme regulatory sequence, an intron is built into the marker gene, and an expression cassette is reversely inserted in the intron, which is used for independently expressing another marker gene and a drug resistance protein gene.Through the combination of drug screening and flow cytometry, positive cells successfully knocked into the regulation system can be efficiently screened.Through the design of different intron reverse expression cassettes, cell lines carrying single allele or double allele markers can be sorted from a mixed cell pool.The application can realize the precise marking and expression control of single or multiple alleles, and the efficient spatiotemporal specific regulation of the ribozyme regulation system on the expression of the target gene at the RNA level.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a CRISPR editing and screening system and its applications. Background Technology

[0002] Conditional gene expression is a key approach to understanding the functions of genes and proteins in different tissues, lineages, and developmental stages of complex organisms. The control of gene expression is typically achieved using gene regulatory elements with specific spatiotemporal activities. Most genetic information is stored in DNA in sequence form. Following the central dogma, genetic information is transferred from DNA to RNA through transcription and translation, and then from RNA to protein, thus achieving gene expression. Gene expression can be regulated at multiple key stages throughout the entire expression process.

[0003] Ribozyme-mediated gene silencing systems typically involve inserting a self-cleaving ribozyme sequence into the transcript of a target gene, thereby downregulating the expression of the target gene through ribozyme-mediated RNA self-cleavage. Some studies further combine this with antisense oligonucleotides to restore expression, thus forming a reversible regulatory system.

[0004] In CRISPR / Cas9-mediated endogenous gene silencing using ribozymes, target gene expression is often significantly reduced after the ribozyme functions, making traditional screening strategies that rely on target gene fluorescence signals or drug resistance protein expression difficult to use for enriching successfully edited cells. Furthermore, existing gene silencing methods (such as CRISPRi, shRNA, and Cas13) cannot differentially fluorescently label and regulate gene expression at the allele level.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The primary objective of this invention is to provide a nucleic acid construct to address the aforementioned technical problems.

[0007] A second objective of this invention is to provide a CRISPR editing and screening system utilizing an intron-reverse expression cassette.

[0008] A third objective of this invention is to provide a recombinant carrier.

[0009] A fourth objective of this invention is to provide a cell.

[0010] The fifth objective of this invention is to provide a kit for preparing transgenic engineered strains.

[0011] The sixth objective of this invention is to provide the application of the above-mentioned nucleic acid constructs, recombinant vectors, or kits in gene expression regulation for purposes other than disease diagnosis and treatment.

[0012] The seventh objective of this invention is to provide a gene expression regulation method for purposes other than disease diagnosis and treatment.

[0013] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a nucleic acid construct comprising, from the 5' end to the 3' end, an upstream homologous arm of a target gene knock-in site, a DNA fragment of a first marker gene, a DNA fragment of a ribozyme gene, and a downstream homologous arm of a target gene knock-in site, which are connected sequentially. The DNA fragment of the first marker gene contains an intron sequence inserted; An intron reverse expression cassette is inserted into the intron sequence; the expression direction of the intron reverse expression cassette is opposite to that of the first marker gene; from the 5' end to the 3' end, the intron reverse expression cassette sequentially includes: a promoter, a DNA fragment of the second marker gene, a DNA fragment of a short peptide that mediates self-splicing, a DNA fragment of a drug resistance protein gene, and a transcription terminator; The intron sequence is used for the cleavage of the reverse expression cassette during mRNA processing and maturation.

[0014] As a further technical solution, the first marker gene and the second marker gene are selected from different fluorescent protein genes; The fluorescent proteins include red fluorescent protein, green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, or cyan fluorescent protein.

[0015] As a further technical solution, the ribozyme includes T3H38 or T3H48.

[0016] As a further technical solution, the short peptide that mediates self-cleavage includes T2A or P2A.

[0017] As a further technical solution, the drug resistance protein gene includes a puromycin resistance gene or a hygromycin resistance gene.

[0018] In a second aspect, the present invention provides a CRISPR editing and screening system utilizing an intron reverse expression cassette, comprising the above-mentioned nucleic acid constructs; The nucleic acid construct is used to insert into the 3' end of the target gene to enable the target gene, the first marker gene, and the ribozyme gene to be expressed in a fusion manner. Based on the expression of the first marker gene, the second marker gene, and the drug resistance protein gene, cells that have been successfully edited by CRISPR are selected.

[0019] Thirdly, the present invention provides a recombinant vector containing the above-mentioned nucleic acid construct.

[0020] Fourthly, the present invention provides a cell, characterized in that the above-mentioned nucleic acid constructs are integrated into the genome of the cell.

[0021] Fifthly, the present invention provides a kit for preparing transgenic engineered lines, comprising the above-mentioned nucleic acid constructs, Cas9 protein or its expression plasmid, and sgRNA or its expression plasmid; The sgRNA is used to guide the Cas9 protein to cleave the target gene so that the nucleic acid construct can be inserted into the cleavage site.

[0022] In a sixth aspect, the present invention provides the application of the above-mentioned nucleic acid constructs or CRISPR editing and screening system recombinant vectors or kits in gene expression regulation for purposes other than disease diagnosis and treatment.

[0023] In a seventh aspect, the present invention provides a gene expression regulation method for purposes other than disease diagnosis and treatment, wherein the above-mentioned nucleic acid construct is inserted into the 3' end of the target gene in the cell through gene editing, so as to achieve the fusion expression of the target gene, the first marker gene and the ribozyme gene; If the cells are not treated with morpholine, the ribozyme will undergo self-cleavage after transcription, inhibiting the expression of the target gene.

[0024] If cells are treated with morpholine, the ribozyme is specifically bound to morpholine after transcription, preventing self-cleavage and allowing the target gene to be expressed normally.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The nucleic acid construct provided by this invention, based on a marker gene-ribozyme regulatory sequence, incorporates an intron within the marker gene and inserts an expression cassette in reverse within it, for the independent expression of another marker gene and a drug resistance protein gene. By combining co-culturing with corresponding cytotoxic drugs and flow cytometry sorting of labeled fluorescently positive cells, positive cells successfully knocked into the regulatory system can be efficiently screened. Through different intron inverse expression cassette designs, bialele-labeled cell lines can be sorted from mixed cell pools. The advantages of using this nucleic acid construct for gene editing are: by inserting an expression cassette in reverse within the intron, the expression of the other marker gene and drug resistance protein gene used for screening is independent and does not interfere with ribozyme regulation; it enables precise labeling and expression manipulation of single or multiple alleles; and it preserves the efficient spatiotemporal specific regulation of target gene expression at the RNA level by the ribozyme regulatory system. Attached Figure Description

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 This is a template design for a ribozyme regulatory system with an intron reverse expression cassette. The figure shows two adapted intron reverse expression cassette designs: (a) The first design uses the miniCMV promoter, applies NLS nuclear localization signals to guide the nuclear localization of BFP, and expresses puromycin resistance (PuroR); (b) The second design uses the SFFV promoter, applies NLS nuclear localization signals to guide the nuclear localization of BFP, and expresses hygromycin resistance (HygroR). Figure 2 This is a schematic diagram of the positive cell screening process for the knock-in regulatory system; its core steps should include: I. CRISPR knock-in of the reverse expression cassette design template; II. Screening the knock-in cell pool for corresponding drugs and using flow cytometry to sort and enrich fluorescently labeled positive cells; III. Treating the sorted cell lines with morpholine and examining the gene expression regulation efficiency using live cell confocal imaging. Figure 3 This study tested the efficiency of ribozyme editing systems integrating different intron reverse expression cassettes in regulating the expression of the target gene (H2B) in 293FT cells; (a) Confocal fluorescence images showing the regulation of H2B expression in cells by different donor plasmid editing systems (GFP labeled target protein H2B, BFP used to label CRISPR knock-in positive cells); scale bar: 10 μm; (b) Quantitative fluorescence analysis of H2B expression levels regulated by different labeling systems in figure a; each point represents the relative fluorescence intensity of H2B-GFP expression in a cell, and cells in each group were randomly selected for statistical analysis, n=100; the fold change is indicated in the figure captions; Figure 4 This test examines the efficiency of different intron reverse expression cassette ribozyme editing systems in regulating the expression of the target gene (H2B) in HeLa cells; (a) Confocal fluorescence images show the H2B expression in cells regulated by different donor plasmid editing systems (GFP labeled target protein H2B, BFP used to label CRISPR knock-in positive cells); Scale bar: 10 μm; (b) Fluorescence quantification of H2B expression levels regulated by different intron systems in Figure a; Each point represents the relative fluorescence intensity of H2B-GFP expression in a cell, and cells in each group were randomly selected for statistical analysis, from left to right: n=318, 318, 315, 318, 314, 317, 318; Figure 5 This is a schematic diagram of the design of a dual intron reverse expression cassette ribozyme regulatory system; (a) The first method uses the miniCMV promoter, applies the NES nuclear localization signal to guide BFP to the cytoplasm, and expresses puromycin resistance (PuroR); (b) The second method uses the miniCMV promoter to express mCherry and hygromycin resistance (HygroR). Figure 6 This is a flowchart of the establishment of biallelic marker cell lines and the regulation of allele expression; its core steps should include: I. Designing templates for two reverse expression cassettes and simultaneously performing CRISPR knock-in; II. Screening the knock-in cell pools for corresponding drugs and using flow cytometry to sort CRISPR knock-in positive cells based on labeled fluorescence; III. Treating the sorted cell lines with morpholine and examining the gene expression regulation efficiency using live cell confocal imaging. Figure 7 The regulation of H2B gene expression is achieved through a dual intron reverse expression cassette ribozyme regulatory system; (a) using Figure 6 The design and cell screening strategy successfully isolated single-cell clones with at least two H2B alleles labeled by the intron reverse expression cassette ribozyme regulatory system; among them, BFP and mCherry were two types of positive cells successfully labeled by fluorescent protein-ribozyme; confocal imaging showed the expression of H2B-GFP in single-cell clones with and without morpholine; scale bar: 10 μm; (b) fluorescence quantification of H2B expression level in single-cell clones of the dual intron reverse expression cassette ribozyme regulatory system under morpholine regulation; the above relevant statistics were n = 100 cells per group, and each point represented one cell; ns: not significant; **** p < 0.0001 (using Tukey corrected one-sided ANOVA), all values ​​are expressed as mean ± SEM. Detailed Implementation

[0028] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0029] In a first aspect, the present invention provides a nucleic acid construct comprising, from the 5' end to the 3' end, an upstream homologous arm of a target gene knock-in site, a DNA fragment of a first marker gene, a DNA fragment of a ribozyme gene, and a downstream homologous arm of a target gene knock-in site, which are connected sequentially. The DNA fragment of the first marker gene contains an intron sequence inserted; An intron reverse expression cassette is inserted into the intron sequence; the expression direction of the intron reverse expression cassette is opposite to that of the first marker gene; from the 5' end to the 3' end, the intron reverse expression cassette sequentially includes: a promoter, a DNA fragment of the second marker gene, a DNA fragment of a short peptide that mediates self-splicing, a DNA fragment of a drug resistance protein gene, and a transcription terminator; The intron sequence is used for the cleavage of the reverse expression cassette during mRNA processing and maturation.

[0030] The main advantages of this invention are: (1) High targeting specificity: RNA interference, CRISPR interference, and Cas13-mediated gene knockout are effective tools for gene regulation at the mRNA level. However, these methods may lead to off-target gene regulation and cannot achieve regulation of specific transcriptional isoforms. The nucleic acid constructs provided by this invention can be based on CRISPR / Cas9-mediated precise gene knock-in, and subsequent PCR and sequencing identification ensures targeting specificity to the target gene and its specific isoforms.

[0031] (2) Independent expression of selection markers and gene regulation: By using expression cassettes inserted in reverse into artificial introns, the regulation of the target gene by ribozymes and the expression of selection markers do not interfere with each other. During the screening process, the "off" state of the target gene and the expression of the selection markers can be synchronized, thereby simplifying the screening process and reducing dependence on morpholine.

[0032] (3) Time window controllable knockdown: It can realize the induced knockdown regulation of target genes under different conditions, thereby accurately exploring the time window for the gene to play its role.

[0033] (4) Differential allele labeling and expression dose regulation: By integrating different designed built-in expression cassette systems for knock-in, differential labeling of multiple alleles can be achieved, directly reflecting the number of labeled alleles in successfully edited cells. At the same time, by manipulating the expression levels of single or multiple alleles, different expression doses can be obtained to determine the number of alleles required to resolve a specific phenotype.

[0034] This invention does not impose specific limitations on the type of marker gene. In an optional embodiment, the first marker gene and the second marker gene are selected from different fluorescent protein genes; The fluorescent proteins include red fluorescent proteins (such as tdTomato, DsRed, mCherry), green fluorescent proteins (such as eGFP, ZsGreen), yellow fluorescent proteins (such as YFP), blue fluorescent proteins (such as BFP), or cyan fluorescent proteins (such as CFP).

[0035] In an optional embodiment, the ribozyme includes ribozymes with high self-cleavage efficiency such as T3H38 or T3H48.

[0036] In an optional embodiment, the ribozyme has tandem CAAA repeat sequences on both sides to optimize its spatial conformation.

[0037] This invention utilizes the existing self-cleaving ribozyme T3H38 and its optimized sequences at both ends, inhibiting transgene expression by approximately 730-fold compared to the catalytically inactivated form of T3H38. Transgene expression can be partially restored by sterically blocking antisense oligonucleotides, which bind to and inactivate the self-cleavage of T3H38. This catalytic self-cleavage structure can be specifically recognized and interfered with by a small oligomeric antisense nucleotide analog (vivo-Morpholino 8, morpholine), thereby blocking RNA cleavage and degradation, achieving a reversal of expression to the target sequence. Preferably, the morpholine-specific recognition sequence is as shown in SEQ ID NO.1: GTACCCGAAGTGGAATCCAGGACGC (SEQ ID NO.1) The optimized nucleotide sequence of the T3H38 ribozyme is shown in SEQ ID NO.2: CAAACAAACAAAGCGCGTCCTGGATTCCACTTCGGGTACATCCAGCTGACGAGTCCCAAATAGGACGAAACGCGCCAAACAAACAAA (SEQ ID NO. 2).

[0038] In an optional implementation, the nucleic acid sequence of the GFP fluorescent protein is shown in SEQ ID NO.3: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA (SEQ ID NO. 3).

[0039] The nucleic acid sequence of BFP fluorescent protein is shown in SEQ ID NO. 4: ATGAGCGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACCGTGGACAACCATCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCAGACCATGAGAATCAAGGTGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCTGGCTACTAGCTTCCTCTACGGCAGCAAGACCTTCATCAACCACACCCAGGGCATCCCCGACTTCTTCAAGCAGTCCTTCCCTGAGGGCTTCACATGGGAGAGAGTCACCACATACGAAGACGGGGGCGTGCTGACCGCTACCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGATCAGAGGGGTGAACTTCACATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGCCTTCACCGAGACGCTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAAACGACATGGCCCTGAAGCTCGTGGGCGGGAGCCATCTGATCGCAAACATCAAGACCACATATAGATCCAAGAAACCCGCTAAGAACCTCAAGATGCCTGGCGTCTACTATGTGGACTACAGACTGGAAAGAATCAAGGAGGCCAACAACGAGACCTACGTCGAGCAGCACGAGGTGGCAGTGGCCAGATACTGCGACCTCCCTAGCAAACTGGGGCACAAGCTTAAT (SEQ ID NO: 4).

[0040] The nucleic acid sequence of mCherry fluorescent protein is shown in SEQ ID NO. 5: ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAA (SEQ ID NO. 5).

[0041] In an alternative embodiment, the DNA fragment of the intron splicing recognition sequence is a modified HSPA5 intron, whose nucleotide sequence is shown in SEQ ID NO. 6: GTAAGTATGAAATTCAGGGATACGGCCACCTTGTTGTCGCCCCACTGTGGGGTGGAGGGGACGAATTCTTCGAACCACCTTGTTGGCATATTTGCCAAATAGTGGAAATGTGAAGTACTGACAAAACTTTTCCCTTTTTCAATCTAATAG (SEQ ID NO. 6).

[0042] An inverted intron expression cassette is inserted between nucleotides 36 and 37 of the HSPA5 intron to enable the cleavage of the inverted intron expression cassette during mRNA processing and maturation.

[0043] This invention does not impose specific limitations on the short peptides that mediate self-cleavage. In an optional embodiment, the short peptide that mediates self-cleavage is T2A or P2A, wherein the nucleotide sequence of T2A is shown in SEQ ID NO.11.

[0044] GAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCA (SEQ ID NO. 11).

[0045] In optional embodiments, the drug resistance protein gene includes, but is not limited to, the puromycin resistance gene or the hygromycin resistance gene, or other drug resistance protein genes well known to those skilled in the art.

[0046] In an optional implementation, the promoter includes a miniCMV promoter (sequence as shown in SEQ ID NO.7) or an SFFV promoter (sequence as shown in SEQ ID NO.8).

[0047] GGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCT (SEQ ID NO. 7).

[0048] GTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAGATATGG CCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGCCCGGG (SEQ ID NO.8).

[0049] In an optional embodiment, the intron reverse expression cassette further includes a short peptide signal NLS (SEQ ID NO. 9) that guides the target protein into the cell nucleus, and a short peptide signal NES (SEQ ID NO. 10) that guides the target protein to be localized in the cytoplasm.

[0050] CCGAAGAAAAAGAGGAAGGTG (SEQ ID NO. 9).

[0051] CTGCCTCCACTTGAAAGACTGACACTG (SEQ ID NO. 10).

[0052] In an optional embodiment, the nucleotide sequence of the terminator is shown in SEQ ID NO.12.

[0053] CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG (SEQ ID NO. 12).

[0054] In a second aspect, the present invention provides a CRISPR editing and screening system utilizing an intron reverse expression cassette, comprising the above-mentioned nucleic acid constructs; The nucleic acid construct is used to insert into the 3' end (preferably located in the 3'UTR) of the target gene to enable the fusion expression of the target gene, the first marker gene, and the ribozyme gene. Based on the expression of the first marker gene, the second marker gene, and the drug resistance protein gene, cells successfully edited by CRISPR are screened.

[0055] The CRISPR editing and screening system provided by this invention is based on existing ribozyme systems. An artificial intron is inserted into a marker gene (e.g., a fluorescent protein gene), and a reverse expression cassette with a selection marker is placed within it. It achieves functional isolation from the ribozyme and reduces the influence of the selection marker on the expression of the target gene through transcriptional direction differences and intron splicing. Regulation at the RNA level can influence the process at an earlier stage of gene expression and can, to some extent, eliminate interference from the stability of different proteins themselves, thus broadening its application range. Furthermore, this system can be used to study the function of a single transcript when multiple transcripts are present, or to achieve functional knockout of all transcripts by inserting an intron at the same terminator location.

[0056] This CRISPR editing and screening system enables efficient and accurate screening of edited cells.

[0057] Thirdly, the present invention provides a recombinant vector containing the above-mentioned nucleic acid construct.

[0058] In an optional embodiment, the recombinant vector includes a plasmid.

[0059] Fourthly, the present invention provides a cell, characterized in that the above-mentioned nucleic acid constructs are integrated into the genome of the cell.

[0060] The cell line of this invention uses CRISPR / Cas system tools for targeted gene knock-in.

[0061] Specifically, in the CRISPR / Cas9 system, the sgRNA of the linearized vector fragment (donor sequence) recognizes and binds to specific sites upstream and downstream of the donor sequence. The sgRNA recognizes and binds to the target sequence of the target gene, guiding Cas9 to cleave the binding site, resulting in donor sequence linearization and double-strand breaks in the cellular genomic DNA. Through intracellular homologous recombination repair (HDR), the exogenous donor DNA (donor sequence) is precisely introduced into the target site of the genome, thereby achieving gene knock-in.

[0062] In a preferred embodiment of the present invention, the present invention provides a method for preparing the stable cell line, the method comprising the steps of: (i) Provide a pool of candidate cells for inserting the first nucleic acid construct downstream of the gene of interest; (ii) Screening for gene knock-in positive cells based on the marker inserted into the first nucleic acid construct; (iii) The expression regulation effect was verified by treating the selected cells with morpholine.

[0063] Fifthly, the present invention provides a kit for preparing transgenic engineered lines, comprising the above-mentioned nucleic acid constructs, Cas9 protein or its expression plasmid, and sgRNA or its expression plasmid; The sgRNA is used to guide the Cas9 protein to cleave the target gene so that the nucleic acid construct can be inserted into the cleavage site.

[0064] This kit can be used to prepare transgenic engineered lines.

[0065] In a sixth aspect, the present invention provides the application of the above-mentioned nucleic acid constructs or CRISPR editing and screening system recombinant vectors or kits in gene expression regulation for purposes other than disease diagnosis and treatment.

[0066] The nucleic acid constructs or CRISPR editing and screening system recombinant vectors or kits provided by this invention can control the expression of target genes through ribozymes.

[0067] In a seventh aspect, the present invention provides a gene expression regulation method for purposes other than disease diagnosis and treatment, wherein the above-mentioned nucleic acid construct is inserted into the 3' end (preferably located in the 3'UTR) of a target gene in a cell through gene editing, so as to achieve the fusion expression of the target gene, the first marker gene and the ribozyme gene; If the cells are not treated with morpholine, the ribozyme will undergo self-cleavage after transcription, inhibiting the expression of the target gene.

[0068] If cells are treated with morpholine, the ribozyme is specifically bound to morpholine after transcription, preventing self-cleavage and allowing the target gene to be expressed normally.

[0069] This regulatory method is simple and convenient, and can efficiently regulate the target gene.

[0070] In an optional implementation, the gene editing method includes CRISPR / Cas9.

[0071] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0072] Example 1: Construction of donor plasmid (1) Construction of donor plasmids for homologous arm-mediated DNA repair (visualization of gene expression): Control group: The following fragments were sequentially spliced ​​from the 5' end to the 3' end into a single fragment using homologous recombination molecular cloning and inserted into a universal vector: Homologous arm approximately 500 bp long at the 5' end of the human histone subunit H2B gene insertion site (SEQ ID NO.13), and homologous arm approximately 500 bp long at the 3' end of the GFP fluorescent protein and human histone subunit H2B gene insertion site (SEQ ID NO.14). Internal control: The following fragments were sequentially spliced ​​from the 5' end to the 3' end into a single fragment using homologous recombination molecular cloning and inserted into a universal vector: The SEQ ID NO. 13 contains a homologous arm approximately 500 bp long at the 5' end of the insertion site of the human histone subunit H2B gene, and a homologous arm approximately 500 bp long at the 3' end of the insertion site of the GFP fluorescent protein and the human histone subunit H2B gene (SEQ ID NO. 14). The nucleotide sequence of the GFP fluorescent protein is shown in SEQ ID NO. 3, with an intron sequence inserted between nucleotides 303 and 304. The intron sequence is shown in SEQ ID NO. 6, with an inverted expression cassette 1 inserted between nucleotides 36 and 37: from the 5' end to the 3' end, it sequentially includes a miniCMV promoter-nuclear localization signal (NLS)-blue fluorescent protein (BFP)-T2A-puromycin resistance protein (PuroR)-TTS. The expression direction of this inverted expression cassette is opposite to that of the GFP fluorescent protein. The nucleotide sequence of the miniCMV promoter is shown in SEQ ID NO. 7, the nucleotide sequence of the nuclear localization signal (NLS) is shown in SEQ ID NO. 9, and the nucleotide sequence of BFP is shown in SEQ ID NO. 14. As shown in NO.4, the nucleic acid sequence of T2A is shown in SEQ ID NO.11, and the nucleic acid sequence of TTS is shown in SEQ ID NO.12; And the following fragments were sequentially spliced ​​from the 5' end to the 3' end into a single fragment using homologous recombination molecular cloning, and then inserted into a universal vector: The 5' homologous arm of the human histone subunit H2B gene insertion site (SEQ ID NO. 13) and the 3' homologous arm of the human histone subunit H2B gene insertion site (SEQ ID NO. 14) are shown. The nucleotide sequence of the GFP fluorescent protein is shown in SEQ ID NO. 3, with an intron sequence inserted between nucleotides 303 and 304. The intron sequence is shown in SEQ ID NO. 6, with an inverted expression cassette 2 inserted between nucleotides 36 and 37: SFFV promoter-nuclear localization signal (NLS)-blue fluorescent protein (BFP)-T2A-hygromycin resistance protein (HygroR)-TTS. The expression direction of this inverted expression cassette is opposite to that of the GFP fluorescent protein. The nucleotide sequence of the SFFV promoter is shown in SEQ ID NO. 8, the nucleotide sequence of the nuclear localization signal (NLS) is shown in SEQ ID NO. 9, the nucleotide sequence of BFP is shown in SEQ ID NO. 4, and the nucleotide sequence of T2A is shown in SEQ ID NO. 9. As shown in NO.11, the nucleic acid sequence of TTS is shown in SEQ ID NO.12.

[0073] Design team (such as) Figure 1 (As shown): The following fragments were sequentially spliced ​​from the 5' end to the 3' end into a single fragment using homologous recombination molecular cloning, and then inserted into a universal vector: The gene contains a homologous arm approximately 500 bp long at the 5' end of the human histone subunit H2B gene insertion site (SEQ ID NO. 13), GFP fluorescent protein, T3H38 ribozyme (sequence shown in SEQ ID NO. 2), and a homologous arm approximately 500 bp long at the 3' end of the human histone subunit H2B gene insertion site (SEQ ID NO. 14). The GFP fluorescent protein's nucleic acid sequence is shown in SEQ ID NO. 3, with an intron sequence inserted between nucleotides 303 and 304. The intron sequence is shown in SEQ ID NO. 6, with a reverse expression cassette 1 inserted between nucleotides 36 and 37: from the 5' end to the 3' end, it sequentially includes a miniCMV promoter-nuclear localization signal (NLS)-blue fluorescent protein (BFP)-T2A-puromycin resistance protein (PuroR)-TTS. The expression direction of this reverse expression cassette is opposite to that of the GFP fluorescent protein. The nucleic acid sequence of the miniCMV promoter is shown in SEQ ID NO. 7, and the nucleic acid sequence of the nuclear localization signal (NLS) is shown in SEQ ID NO. 14. As shown in NO.9, the nucleic acid sequence of BFP is shown in SEQ ID NO.4, the nucleic acid sequence of T2A is shown in SEQ ID NO.11, and the nucleic acid sequence of TTS is shown in SEQ ID NO.12; And the following fragments were sequentially spliced ​​from the 5' end to the 3' end into a single fragment using homologous recombination molecular cloning, and then inserted into a universal vector: The gene contains a homologous arm approximately 500 bp long at the 5' end of the human histone subunit H2B gene insertion site (SEQ ID NO. 13), GFP fluorescent protein, T3H38 ribozyme (sequence shown in SEQ ID NO. 2), and a homologous arm approximately 500 bp long at the 3' end of the human histone subunit H2B gene insertion site (SEQ ID NO. 14). The GFP fluorescent protein's nucleic acid sequence is shown in SEQ ID NO. 3, with an intron sequence inserted between nucleotides 303 and 304. The intron sequence is shown in SEQ ID NO. 6, with an inverted expression cassette 2 inserted between nucleotides 36 and 37: SFFV promoter-nuclear localization signal (NLS)-blue fluorescent protein (BFP)-T2A-hygromycin resistance protein (HygroR)-TTS. The expression direction of this inverted expression cassette is opposite to that of the GFP fluorescent protein. The SFFV promoter's nucleic acid sequence is shown in SEQ ID NO. 8, the nuclear localization signal (NLS)'s nucleic acid sequence is shown in SEQ ID NO. 9, and the BFP's nucleic acid sequence is shown in SEQ ID NO. 14. As shown in ID NO.4, the nucleic acid sequence of T2A is shown in SEQ ID NO.11, and the nucleic acid sequence of TTS is shown in SEQ ID NO.12.

[0074] CTAAGCCGCGTTTGTACTGTGTCTTACCATGCCTGAACCGGCAAAATCCGCTCCGGCCCCTAAAAAGGGCTCCAAGAAAGCCGTCACCAAAGCCCAGAAGAAAGACGGCAAGAAGCGCAAGCGCAGCCGCAAAGAGAGCTACTCCATCTACGTGTACAAGGTGCTGAAGCAGGTCCACCCCGACACCGGCATCTCGTCCAAGGC CATGGGCATCATGAACTCCTTCGTCAACGACATCTTCGAGCGCATCGCGGGAGAGGCTTCCCGCCTGGCGCACTACAACAAGCGCTCCACCATCACATCCCGCGAGATCCAGACGGCGTGCCTGCTGCTGCCGGCGAGCTGGCCAAGCACGCCGTGTCCGAGGGCACCAAGGCGGTCACCAAGTACACCAGCTCCAAG (SEQ ID NO.13) TGAGTaCCTGCCGGGACCTGGCGCTCGCTCGCTCGAGTCGCCGGCTGCTTGACTCCAAAGGCTCTTTTCAGAGCCACCCACCTAATCACTAGAAAAGAGCTTGTTCACTTATTCCCTTAGTTTCTTTTCATAAAGTAAGTTATTTTAGTGTGAAGGTCATGGGAAATGGCATACGTAGCTTTTTAACTATTTGGAACTCGAGGTC CCCAGTGCGTCATTGGATTTGCTTTTGAATCTAGAGCGTGTCTTTACTCATTGTGCTGCTTAGCCTTCCCAGGAGTCGGTTCTCAATTAGGCTGTTGGGAATCCGCCTCTTTACCCGCCCCCACTCCCGCCCCACACGCGCCCTGGTGGCTCCTTGGGTCTGTTTCATTCTAAAACGAAGTGGCTGAGTTCGGCTGTCATTT (SEQ ID NO.14) The plasmid backbone of the aforementioned universal vector carries only AMP resistance and ori elements, and only the AMP resistance sequence carries the promoter sequence to express the AMP resistance protein.

[0075] Example 2: Constructing a cell pool of knock-in single-regulatory systems and screening labeled cells Adherent target cells (293FT cells, HeLa cells) were passaged and cultured in 48-well plates using 10% FBS and 1% PS DMEM in an incubator until they reached 80% confluence as observed under a microscope. 200 ng of the Cas9 expression vector (plasmid #64323 or #62988, Addgene), 125 ng of the linearized guide RNA (GAGCTTACTGAGACTCTTC (SEQ ID NO. 15), used to recognize and bind to specific sites upstream and downstream of the donor sequence to obtain the donor sequence) and 125 ng of the target gene guide RNA (nucleotide sequence GCGAGCGCCAGGTCCCGGCA (SEQ ID NO. 16)) and 500 ng of the HDR knock-in template vector (i.e., the donor plasmid from Example 1) were introduced into the cells. After the cells stabilized and were cultured on a large scale, the internal control and the designed group were cultured for 7 days in medium containing the selection drug (hygromycin B 300ug / ml or puromycin 2ug / ml, selected according to the drug resistance protein gene of the donor plasmid). The surviving cells were passaged into normal culture medium. After the cell morphology returned to normal, the fluorescence signal of the selection marker was examined. The selection procedure is as follows: Figure 2 As shown.

[0076] Example 3: Editing the specific regulation of target gene expression in cells Cells were treated with or without morpholine (15 μM, 48 h) and used as controls for confocal live-cell imaging. For the H2B gene assay, the excitation light for the green fluorescence channel was set to a wavelength of 488 nm with a laser intensity of 80% and an exposure time of 100 ms; the excitation light for the 561 nm laser was set to a laser intensity of 5% with an exposure time of 100 ms. Imaging results are shown below. Figure 3 (a) in the middle Figure 4 As shown in (a) (in the figure, cassette refers to the inverted expression cassette of introns).

[0077] ImageJ was used to measure endogenous protein expression based on fluorescence imaging. The average fluorescence intensity was calculated from all morphologically normal cells in a random field of view, and then the background signal was subtracted. GraphPad analysis software was used to analyze the signal intensity of the GFP protein signal of the gene of interest. The results are as follows: Figure 3 (b) Figure 4 As shown in (b) of the figure. The figure shows the expression regulation effects on cell lines screened by different built-in reverse expression cassettes. Ribozymes cause a decrease in GFP expression, which is partially restored by morpholine treatment. All values ​​are expressed as mean ± sem. n ≥ 100 cells. All fold changes in mean for each group are marked above the corresponding bar pair. In 293FT cells, the regulatory system of the reverse expression cassette 1 design group showed a 193.3-fold decrease in the mean intensity of H2B-labeled green fluorescence compared to the internal control, which returned to a 164.8-fold decrease after the addition of morpholine; the regulatory system of the reverse expression cassette 2 design group showed a 185.0-fold decrease in the mean intensity of H2B-labeled green fluorescence compared to the internal control, which returned to a 130.3-fold decrease after the addition of morpholine. In HeLa cells, the regulatory system of the reverse expression cassette 1 design group showed a 4.7-fold decrease in the mean intensity of H2B-labeled green fluorescence compared to the internal control, which returned to a 2.9-fold decrease after the addition of morpholine; the regulatory system of the reverse expression cassette 2 design group showed a 12.6-fold decrease in the mean intensity of H2B-labeled green fluorescence compared to the internal control, which returned to a 2.0-fold decrease after the addition of morpholine.

[0078] Example 4: Construction of allele differential markers (1) Construction of donor plasmids for homologous arm-mediated DNA repair (visualization of gene expression): like Figure 5 As shown, this donor plasmid is a design group. The following fragments were sequentially spliced ​​from the 5' end to the 3' end into a single fragment using homologous recombination molecular cloning, and then inserted into a universal vector: The gene contains a homologous arm approximately 500 bp long at the 5' end of the human histone subunit H2B gene insertion site (SEQ ID NO. 13), GFP fluorescent protein, T3H38 ribozyme (sequence shown in SEQ ID NO. 2), and a homologous arm approximately 500 bp long at the 3' end of the human histone subunit H2B gene insertion site (SEQ ID NO. 14). The GFP fluorescent protein's nucleic acid sequence is shown in SEQ ID NO. 3, with an intron sequence inserted between nucleotides 303 and 304. The intron sequence is shown in SEQ ID NO. 6, with a reverse expression cassette 3 inserted between nucleotides 36 and 37: from the 5' end to the 3' end, it sequentially includes a miniCMV promoter-nuclear localization signal (NES)-blue fluorescent protein (BFP)-T2A-puromycin resistance protein (PuroR)-TTS. The expression direction of this reverse expression cassette is opposite to that of the GFP fluorescent protein. The nucleic acid sequence of the miniCMV promoter is shown in SEQ ID NO. 7, and the nucleic acid sequence of the nuclear localization signal (NES) is shown in SEQ ID NO. 14. As shown in NO.10, the nucleic acid sequence of BFP is shown in SEQ ID NO.4, the nucleic acid sequence of T2A is shown in SEQ ID NO.11, and the nucleic acid sequence of TTS is shown in SEQ ID NO.12; And the following fragments were sequentially spliced ​​from the 5' end to the 3' end into a single fragment using homologous recombination molecular cloning, and then inserted into a universal vector: The sequence includes a homologous arm approximately 500 bp long at the 5' end of the human histone subunit H2B gene insertion site (SEQ ID NO. 13), GFP fluorescent protein, T3H38 ribozyme (sequence shown in SEQ ID NO. 2), and a homologous arm approximately 500 bp long at the 3' end of the human histone subunit H2B gene insertion site (SEQ ID NO. 14). The GFP fluorescent protein's nucleic acid sequence is shown in SEQ ID NO. 3, with an intron sequence inserted between nucleotides 303 and 304. The intron sequence is shown in SEQ ID NO. 6, with an inverted expression cassette 4 inserted between nucleotides 36 and 37: miniCMV promoter-nuclear localization signal (NLS)-red fluorescent protein (mCherry)-T2A-hygromycin resistance protein (HygroR)-TTS. The expression direction of this inverted expression cassette is opposite to that of the GFP fluorescent protein. The SFFV promoter's nucleic acid sequence is shown in SEQ ID NO. 8, and the nuclear localization signal (NLS)'s nucleic acid sequence is shown in SEQ ID NO. 14. As shown in NO.9, the nucleic acid sequence of red fluorescent protein (mCherry) is shown in SEQ ID NO.5, the nucleic acid sequence of T2A is shown in SEQ ID NO.11, and the nucleic acid sequence of TTS is shown in SEQ ID NO.12.

[0079] The plasmid backbone of the aforementioned universal vector carries only AMP resistance and ori elements, and only the AMP resistance sequence carries the promoter sequence to express the AMP resistance protein.

[0080] Example 5: Construction of cell lines and expression-specific regulation of differentially expressed allele markers like Figure 6 As shown, target cells (HeLa cells, etc.) were passaged and cultured in 48-well plates using 10% FBS and 1% PSDMEM in an incubator until they reached 80% confluence under a microscope. 200 ng of Cas9 expression vector (plasmid #64323 or #62988, Addgene), 125 ng of linearized guide RNA vector, 125 ng of target gene guide RNA vector, and 250 ng of two HDR knock-in template vectors were coated into the cells using transient conversion reagents (FuGENE, Promega). After cell stabilization and expansion, the cells were cultured for 7 days in medium containing two selection drugs (hygromycin B 300 μg / ml and puromycin 2 μg / ml). Single-cell surviving cells were sorted in normal medium, and the fluorescence signal of the selection markers was examined after cell morphology returned to normal. Cell lines exhibiting fluorescence from both markers were selected.

[0081] Confocal live-cell imaging was performed on cells obtained with or without morpholine (15 μM, 48 h) treatment and as a control. For the H2B gene assay, the excitation light for the green fluorescence channel was set to a wavelength of 488 nm with a laser intensity of 50% and an exposure time of 100 ms; the excitation light for the 561 nm laser was also set to a wavelength of 50% with an exposure time of 100 ms. Imaging results are shown below. Figure 7 As shown in (a) of the diagram.

[0082] ImageJ was used to measure endogenous protein expression based on fluorescence imaging. The average fluorescence intensity was calculated from all morphologically normal cells in a random field of view, and then the background signal was subtracted. GraphPad analysis software was used to analyze the signal intensity of the GFP protein signal of the gene of interest. The results are as follows: Figure 7 As shown in (b) of the figure. The figure shows the expression regulation effects on cell lines screened by different built-in reverse expression cassettes. Ribozymes cause a decrease in GFP expression, which is partially restored by morpholine treatment. All values ​​are expressed as mean ± sem. n = 100 cells. All fold changes in mean for each group are marked above the corresponding bar pair.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nucleic acid construct, characterized in that, From the 5' end to the 3' end, it includes the upstream homologous arm of the target gene knock-in site, the DNA fragment of the first marker gene, the DNA fragment of the ribozyme gene, and the downstream homologous arm of the target gene knock-in site, which are connected in sequence. The DNA fragment of the first marker gene contains an intron sequence inserted; The intron sequence is inserted with an inverse intron expression cassette; The intron reverse expression cassette is expressed in the opposite direction to the first marker gene; from the 5' end to the 3' end, the intron reverse expression cassette sequentially includes: a promoter, a DNA fragment of the second marker gene, a DNA fragment of a short peptide that mediates self-splicing, a DNA fragment of a drug resistance protein gene, and a transcription terminator. The intron sequence is used for the cleavage of the reverse expression cassette during mRNA processing and maturation.

2. The nucleic acid construct according to claim 1, characterized in that, The first marker gene and the second marker gene are selected from different fluorescent protein genes; The fluorescent proteins include red fluorescent protein, green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, or cyan fluorescent protein.

3. The nucleic acid construct according to claim 1, characterized in that, The ribozyme includes T3H38 or T3H48.

4. The nucleic acid construct according to claim 1, characterized in that, The short peptides that mediate self-cleavage include T2A or P2A.

5. A CRISPR editing and screening system utilizing intron-reverse expression cassettes, characterized in that, Includes the nucleic acid constructs according to any one of claims 1-4; The nucleic acid construct is used to insert into the 3' end of the target gene to enable the target gene, the first marker gene, and the ribozyme gene to be expressed in a fusion manner. Based on the expression of the first marker gene, the second marker gene, and the drug resistance protein gene, cells that have been successfully edited by CRISPR are selected.

6. A recombinant vector, characterized in that, Contains the nucleic acid construct according to any one of claims 1-4.

7. A cell, characterized in that, The genome of the cell is integrated with the nucleic acid construct according to any one of claims 1-4.

8. A kit for preparing transgenic engineered strains, characterized in that, Includes the nucleic acid constructs, Cas9 protein or expression plasmid thereof, and sgRNA or expression plasmid thereof as described in any one of claims 1-4; The sgRNA is used to guide the Cas9 protein to cleave the target gene so that the nucleic acid construct can be inserted into the cleavage site.

9. The use of the nucleic acid construct according to any one of claims 1-4, the CRISPR editing and screening system according to claim 5, the recombinant vector according to claim 6, or the kit according to claim 8 in gene expression regulation for purposes other than disease diagnosis and treatment.

10. A method for regulating gene expression for purposes other than disease diagnosis and treatment, characterized in that, By gene editing, the nucleic acid construct according to any one of claims 1-4 is inserted into the 3' end of the target gene in the cell, so that the target gene, the first marker gene and the ribozyme gene are fused and expressed; If the cells are not treated with morpholine, the ribozyme will undergo self-cleavage after transcription, inhibiting the expression of the target gene; If cells are treated with morpholine, the ribozyme is specifically bound to morpholine after transcription, preventing self-cleavage and allowing the target gene to be expressed normally.

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