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WO2022116139A1 - Procédé de mesure de niveaux de gène de faible abondance endogène cellulaire et de arnlnc - Google Patents

Procédé de mesure de niveaux de gène de faible abondance endogène cellulaire et de arnlnc Download PDF

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WO2022116139A1
WO2022116139A1 PCT/CN2020/133860 CN2020133860W WO2022116139A1 WO 2022116139 A1 WO2022116139 A1 WO 2022116139A1 CN 2020133860 W CN2020133860 W CN 2020133860W WO 2022116139 A1 WO2022116139 A1 WO 2022116139A1
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sgrna
endogenous
nucleic acid
expression
mcherry
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杨辉
周海波
高妮
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Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
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Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a method for detecting the level of endogenous low-abundance genes and lncRNA in cells.
  • sgRNA Single guide RNA
  • Cas nucleases can direct Cas nucleases to target genomic loci with specificity, efficiency and versatility.
  • various inducible sgRNAs have been developed to receive endogenous signals in living cells, these approaches have only been shown to respond to small RNAs.
  • Conventional methods for detecting endogenous gene activity often rely on the precise insertion of fluorescent proteins into protein-coding frames.
  • existing methods are not applicable, and current tools for processing endogenous information are very limited.
  • lncRNAs noncoding RNAs
  • the purpose of the present invention is to provide a broad-spectrum method capable of efficiently detecting the expression levels of endogenous low-abundance genes and lncRNAs in cells.
  • an sgRNA responsive promoter which can respond to the complex formed by the upstream sgRNA whose expression is controlled by the endogenous transcript, dCas9 and a transcriptional activator, and initiate Expression of downstream reporter genes.
  • the sgRNA-responsive promoter is a miniature promoter.
  • the sgRNA-responsive promoter is a miniCMV promoter.
  • the dCas9 is a dSpCas9 protein.
  • the downstream reporter gene is a gene sequence encoding a reporter protein.
  • the reporter protein includes fluorescent protein, luciferase (eg, Luciferase), or any signal element that can be used as a reporting indicator.
  • the fluorescent protein is selected from the group consisting of mCherry protein, GFP protein, ZsGreen, or a combination thereof.
  • the fluorescent protein is mCherry protein.
  • a detection system for endogenous nucleic acid in cells comprising:
  • a target nucleic acid editing module including: sgRNA and tRNA targeting the target nucleic acid of the reporter system;
  • the described target nucleic acid editing module comprises the structure shown in formula I,
  • L 1 is the homology arm at the 5'end
  • R 1 is the homology arm at the 3'end
  • tRNA is an endogenous mechanism that can successfully release functional sgRNA
  • sgRNA is guide RNA
  • n is a positive integer ⁇ 1, preferably 2 ⁇ n ⁇ 10;
  • a target nucleic acid reporter module including: a reporter nucleic acid construct, a dCas protein, and a transcriptional activator;
  • reporter nucleic acid construct has the structure shown in formula II,
  • TS is the binding target site of the sgRNA
  • n is a positive integer ⁇ 0, preferably 2 ⁇ m ⁇ 4;
  • I is the spacer sequence between TS
  • P 1 is the sgRNA responsive promoter according to the first aspect of the present invention.
  • Z 2 is the reporter gene sequence.
  • the cellular endogenous nucleic acid includes cellular endogenous gene or long non-coding RNA (lncRNA).
  • the cell endogenous gene may be a cell endogenous gene with low abundance.
  • the low-abundance gene endogenous in the cell refers to a gene whose intracellular expression level is less than 0.001 when the Gapdh internal reference gene is used for relative quantification.
  • the cell endogenous gene can be selected from the following group: Ascl1, Neurog2, Lmx1b, Nkx2-2, Gata3, Hbb, Tubb3, Actb, Nanog, Esrrb, Sox2, Tet1, Neurog2, Lncenc1, Med7, Tert, Jag1, Slc7a11, Dancr, Firre, Pvt1, Tunar, Miat, or a combination thereof.
  • the expression level of the lncRNA in cells is ⁇ 0.001.
  • the sgRNA in (a) targets the 3'-end untranslated region (3'UTR) of the target nucleic acid.
  • the tRNA can be precisely cleaved by endonucleases RNaseP and RNaseZ.
  • nucleotide sequence of the tRNA is shown in SEQ ID NO: 143 (AACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCA).
  • the length of L 1 is 15-50 bp, preferably 25-45 bp, more preferably 35 bp.
  • the length of the R 1 is 15-50 bp, preferably 25-45 bp, more preferably 35 bp.
  • nucleic acid homologous recombinase in (a) is selected from the following group: SaKKHCas9, SaCas9.
  • the endonuclease in (a) is selected from the group consisting of tRNA, ribosome, or a combination thereof.
  • nucleotide sequence of the sgRNA is shown in any of SEQ ID NOs: 1-49 or 50-61 or 62-88.
  • the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 50 or 51.
  • the cell endogenous gene to be detected is Neurog2
  • the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 52 or 53.
  • the nucleotide sequence of the sgRNA is as shown in SEQ ID NO: 54 or 55.
  • the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 56 or 57.
  • the cell endogenous gene to be detected is Gata3
  • the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 58 or 59.
  • the nucleotide sequence of the sgRNA is shown in SEQ ID NO:60.
  • the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 61.
  • the target nucleic acid editing module includes the nucleotide sequence shown in SEQ ID NO: 165 or 166.
  • the dCas9 is a dSpCas9 protein.
  • the transcriptional activator is selected from the group consisting of HSF1, P65, or a combination thereof.
  • the length of the I is 0-100bp, preferably 15-50bp, more preferably 30-40bp, and most preferably 35bp.
  • the sgRNA responsive promoter P 1 is miniCMV, and its nucleotide sequence is shown in SEQ ID NO: 144 (TAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGC).
  • nucleotide sequence of the (TS-I) m -TS-P1 part of the formula II is shown in any one of SEQ ID NOs: 151-160; preferably SEQ ID NO :159.
  • nucleotide sequence of the reporter nucleic acid construct is shown in SEQ ID NO: 150 or 162.
  • the expression of the dCas protein and the transcription activator in the target nucleic acid reporter module is regulated by the same promoter P2.
  • the promoter P 2 is a CAG promoter.
  • the target nucleic acid reporter module comprises the coding sequence for expressing dCas protein and transcriptional activator and its regulatory sequence, the sequence of which is shown in SEQ ID NO: 146 or 161.
  • kits for detecting endogenous nucleic acid in cells comprising the detection system according to the second aspect of the present invention.
  • the kit further includes a reagent for detecting the reporter gene or its encoded product in the target nucleic acid reporter module.
  • the kit further includes a detection system for detecting the housekeeping gene or the internal reference gene in the cell, and the corresponding target nucleic acid editing module comprises a detection system targeting the housekeeping gene or the internal reference gene.
  • sgRNA precursors in the 3'UTR region are examples of sgRNA precursors in the 3'UTR region.
  • the housekeeping gene or internal reference gene is Gapdh.
  • a method for detecting whether a target endogenous nucleic acid exists in a cell sample comprising the steps of:
  • the method includes qualitative detection and quantitative detection.
  • the method includes dynamically detecting the level of target endogenous nucleic acid in the cell sample.
  • the method is non-diagnostic and/or non-therapeutic.
  • the use of the sgRNA responsive promoter according to the first aspect of the present invention or the detection system according to the second aspect of the present invention characterized in that, for preparing a kit, The kit is used to detect endogenous nucleic acid in cells.
  • the kit is a kit for diagnosing a disease, and the disease is a disease related to abnormal expression of endogenous nucleic acid in cells.
  • Figure 1 shows the process and results of developing endogenous switches that directly drive sgRNAs through endogenous promoters.
  • sgRNA a. Schematic showing expression of sgRNA from an endogenous promoter.
  • the sgRNA precursor was targeted for insertion into the transcribed region of the endogenous gene through SaKKHCas9-mediated homology-mediated end joining (HMEJ, 800 bp homology arm) or homologous recombination (HR, 800 bp homology arm). while the sgRNA (orange) flanks (purple)
  • Post-transcriptional cleavage is recognized by endogenous processing machinery.
  • the sgRNA precursor is transcribed along with gene transcription driven by the endogenous promoter, and then the mature sgRNA is formed by the endogenous cleavage program.
  • j Mean fluorescence intensity of mCherry, indicating that tRNA-sgRNA-tRNA induces different levels of mCherry in different introns, and the numbers on the axis represent the number of cell clones in each group.
  • FIG. 2 shows that SPH-OminiCMV-Ents can track low-abundance genes during differentiation.
  • sgRNA precursor targeting mCherry is inserted into the 3' UTR of the endogenous gene, and the sgRNA is released along with the transcription of the endogenous gene.
  • the SPH-sgRNA complex binds upstream of OminiCMV, resulting in the expression of mCherry.
  • Figure 3 shows the results of lncRNA detection with SPH-OminiCMV-Ents.
  • Figure 4 shows that encoding multiple sgRNAs increases the sensitivity of SPH-OminiCMV-Ents and enables multiple transcriptional regulation.
  • c and d Representative images showing mCherry expression in cell lines following insertion of a sgRNA or an sgRNA array in the 3'UTR of lncRNA Tug1 in SPH-OminiCMV-Ents mESCs. Note that Tug1 was detected with an RNA-FISH probe.
  • g Representative images showing mCherry expression during neuronal differentiation of the SPH-OminiCMV-Ents-Tubb3mESC cell line.
  • the sgRNA array contains 8 sgRNA copies. Arrows indicate induced neurons. Note that Tubb3 is lowly expressed in mESCs and highly expressed in neurons.
  • Figure 5 shows homogeneous expression of mcherry by a single promoter driving dCas9 and an activator.
  • Figure 6 shows the mean mCherry intensity induced by different sgRNAs.
  • Figure 7 shows the optimization and results of the miniCMV promoter.
  • FIG. 8 shows that SPH-OminiCMV-induced gene expression levels are higher than SPH-mediated endogenous activation and CMV-mediated overexpression.
  • Figure 9 shows the specificity of SPH-OminiCMV.
  • Figure 10 shows the generation and characterization of SPH-OminiCMV transgenic mESCs.
  • Figure 11 shows that the targeted insertion of tRNA-sgRNA-tRNA into the 3'UTR does not affect normal protein production of the target gene.
  • Figure 12 shows that SPH-OminiCMV-Ents can visualize low abundance genes during cell differentiation.
  • Figure 13 shows that insertion of sgRNA arrays into unexpressed genes does not induce mCherry expression.
  • Figure 14 shows homogeneous expression of mCherry by driving dCas9 and activator under a single promoter.
  • Figure 15 shows FACS analysis of mCherry expression.
  • Figure 16 shows a side-by-side comparison of different strategies and downregulation of mCherry and Nanog at the protein level during differentiation.
  • Figure 17 shows that the SPH(single CAG)-OminiCMV-Ents-sgRNA array induces the highest expression of mCherry.
  • Figure 18 shows quantitative characterization of SPH(single CAG)-OminiCMV-Ents.
  • Ents a universal endogenous transcription gate switch
  • SPH-OminiCMV single guide RNA
  • the methods of the present invention can also monitor the transcriptional activity of long non-coding RNAs (lncRNAs), which are often underexpressed, in living cells.
  • lncRNAs long non-coding RNAs
  • the methods of the present invention are capable of amplifying endogenous signals to visualize the dynamic process of low-abundance genes and lncRNA expression in cells, providing a powerful platform to detect the activity of endogenous genetic elements and their underlying cellular functions.
  • the present invention has been completed on this basis.
  • the term “about” means that the value may vary by no more than 1% from the recited value.
  • the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the terms "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “consisting essentially of,” or “consisting of.”
  • polynucleotide refers to a chain-like compound formed by the polymerization of nucleotides.
  • sgRNA responsive promoter refers to a type of promoter capable of activating a downstream reporter gene in response to the generated sgRNA in the detection of endogenous nucleic acid in cells of the present invention.
  • the sgRNA responsive promoter provided by the present invention can respond to the complex formed by the upstream sgRNA, dCas9 and transcription activator whose expression is controlled by the endogenous transcript, and initiate the expression of the downstream reporter gene.
  • the sgRNA-responsive promoter is a mini-promoter. More preferably, the sgRNA is the miniCMV promoter.
  • a detection system that can be used to detect endogenous nucleic acids in cells, especially genes with low expression abundance and long non-coding RNAs (lncRNAs) is provided.
  • the detection system includes two modules: target nucleic acid editing and target nucleic acid reporting.
  • the cell endogenous low abundance gene refers to a gene whose intracellular expression level is less than 0.001 when the Gapdh internal reference gene is used for relative quantification.
  • the described target nucleic acid editing module includes the structure shown in formula I,
  • L 1 is the homology arm at the 5'end
  • R 1 is the homology arm at the 3'end
  • tRNA is an endogenous mechanism that can successfully release functional sgRNA
  • the sgRNA is a guide RNA, preferably targeted to the 3' untranslated region (3' UTR) of the target nucleic acid;
  • n is a positive integer ⁇ 1, preferably 2 ⁇ n ⁇ 10, more preferably 6 ⁇ n ⁇ 8.
  • the reporter nucleic acid construct has the structure shown in formula II,
  • TS is the binding target site of the sgRNA
  • I is a spacer sequence between TSs, which preferably has a length of 35-37 bp;
  • P 1 is the sgRNA responsive promoter of the present invention
  • Z 2 is a reporter gene sequence, wherein the reporter gene is preferably an encoding gene of mCherry.
  • the expression of the dCas protein and the transcription activator in the target nucleic acid reporter module is regulated by the same promoter P2 ; more preferably, the promoter P2 is a CAG promoter.
  • the detection system provided by the present invention can be used to prepare a kit for detecting endogenous nucleic acid in cells.
  • the kit can be used for disease diagnosis, and the disease is a disease related to abnormal expression of corresponding endogenous nucleic acid in cells.
  • the present invention also provides a method for detecting whether a target endogenous nucleic acid exists in a cell sample, comprising the steps of: (i) introducing the detection system of the present invention into the cell sample to be detected; and (ii) detecting the presence of target endogenous nucleic acid in the detection system Detect the level of the reporter gene or its encoded product in the target nucleic acid reporter module.
  • the method of the present invention includes qualitative detection and quantitative detection.
  • the method includes dynamically detecting the level of target endogenous nucleic acid in a cell sample.
  • the method is non-diagnostic and/or non-therapeutic.
  • the detection system and detection method of the present invention are transcript dependent, and are not dependent on protein expression.
  • the detection system and detection method of the present invention have a strong amplification effect, low-expressed genes and LncRNAs can be detected, and can be used for gene detection of target gene expression level/Gapdh expression level ⁇ 0.001.
  • the detection system and detection method of the present invention can monitor the dynamic expression of low-abundance genes or LncRNA in cells in real time.
  • the key design of the ent-switch is to target the insertion of the sgRNA precursor into the transcribed region of the endogenous gene.
  • the sgRNA precursors are flanked by sequences that are post-transcriptionally recognized and released by endogenous machinery, as shown in Figure 1a.
  • Targeted genes and sgRNA precursors are transcribed into single transcripts driven by endogenous promoters, and then mature sgRNAs are released through an endogenous cleavage program, as shown in Figure 1a.
  • a highly sensitive CRISPR activator-related reporter system is provided to efficiently detect the presence of released sgRNAs.
  • mCherry expression was induced by targeting a minimal promoter (minipromoter) and using the CRISPR-activator Suntag-P65-HSF1 (SPH) activation system previously developed by the inventors.
  • sgRNAs were first pre-screened, and sgRNA 2, which could induce strong mCherry expression, was used in subsequent experiments (Fig. 6a and Table 1).
  • sgRNA 2 which could induce strong mCherry expression
  • Table 1 each sgRNA used to activate the expression of miniCMV-mCherry (named Actb-Intron1, a sequence with a length of 21 bp) is listed, which are all used when knocking Trna-sgRNA into the intron1 position of the actb gene sgRNA.
  • SPH SPH
  • U6-driven sgRNA SPH
  • U6-driven sgRNA SPH
  • U6-driven sgRNA SPH
  • mCherry driven by a mini-promoter Mini-TK, Luc2CP, TRE3G or miniCMV
  • tRNAs can be inserted into foreign genes in the form of sgRNA-tRNA precursors and precisely cleaved by the endonucleases RNase P and RNase Z, which then release the sgRNA.
  • tRNA-sgRNA-tRNAs were inserted into different sites of the Nanog or Actb loci (Fig. 1i and Fig. 11a). Although none of the insertions had a significant effect on Nanog stability at the protein level, only the 3' untranslated region (3' UTR) insertion stably induced high levels of mCherry expression (Fig. 1j-1 and Fig. 11a-c). Therefore, the 3' UTR insertion was selected in subsequent experiments.
  • the inventors also inserted a non-target tRNA-sgRNA-tRNA (sgLacZ) into the 3'UTR of Actb, but no increase in mCherry expression was observed (Fig. 11d, e).
  • sgLacZ non-target tRNA-sgRNA-tRNA
  • Example 2 SPH-OminiCMV-Ents can amplify the signals of low abundance genes and track the dynamic expression of genes during differentiation
  • Fluorescence visualization techniques provide an easy and direct way to capture spatiotemporal information of cellular events.
  • SPH-OminiCMV-Ents can faithfully reflect the expression levels of target genes.
  • tRNA-sgRNA-tRNAs were inserted into the 3'UTR regions of eight differentially expressed genes, including a highly expressed housekeeping gene Actb and seven pluripotent genes. sex-related genes (Fig. 2a).
  • SPH-OminiCMV-Ents induced higher mCherry expression levels than P2A-mCherry strategy and could detect low abundance genes (expression level of target genes/Gapdh ⁇ 0.01, qPCR analysis), such as Sox2, Tet1, Sall4 and Tbx3, these genes were barely visible under the microscope with the P2A-mCherry strategy (Fig. 2b,c and Table 4).
  • SPH-OminiCMV-Ents could track the expression dynamics of endogenous genes during cell differentiation.
  • tRNA-sgRNA-tRNA-inserted SPH-OminiCMV mESCs were cultured in N2B27 medium.
  • genes such as Nanog, Esrrb, Sox2, and Tet1, whose expression levels correlate with pluripotency, decreased after cell differentiation (Fig. 2d-g and Fig. 12a-f, Table 5), which is consistent with previous reports Consistent.
  • mCherry expression decreased along with these pluripotency genes during differentiation, whereas mCherry expression remained stable in SPH-OminiCMV-Ents-Actb mESCs (Fig. 2d-g and Fig. 12a,c, e).
  • LncRNAs Long noncoding RNAs
  • Example 4 Enhancing SPH-OminiCMV-Ents sensitivity by encoding an sgRNA array
  • sgRNA precursor containing six to eight tandem sgRNA copies, each flanked by tRNAs (Fig. 4a).
  • sgRNA arrays were inserted into the 3'UTR of an unexpressed gene Sema3a (0.000005 relative to Gadph), two low-abundance genes Tet1 (0.003195) and Fzd7 (0.000050), and three lncRNAs, Lncenc1 (0.008091), Malat1 (0.003989) and Tug1 (0.003450) (Fig. 4b-e and Table 4).
  • SPH-OminiCMV-Ents sgRNA array
  • the inventors generated SPH-OminiCMV-Ents-Tubb3 (sgRNA array) mESCs and differentiated them into neurons.
  • SPH-OminiCMV-Ents-Tubb3 sgRNA array
  • the results showed that mCherry expression was up-regulated with increased Tubb3 expression during mESCs differentiation (Fig. 4g,h), which was consistent with previous reports.
  • the inventors inserted sgRNA targeting mCherry and multiple sgRNAs of endogenous genes into the 3'UTR of Actb of the SPH-OminiCMV mESC cell line.
  • the present inventors demonstrated that the expression of multiple sgRNAs in sgRNA precursors further enhanced the sensitivity of SPH-OminiCMV-Ents and could perform multiple transcriptional regulation in a cell-type-specific manner.
  • Example 5 Homogeneous expression of mCherry by driving dCas9 and activator under one promoter
  • the inventors have noticed that certain cell clones show heterogeneous mCherry expression (Fig. 5a and Fig. 14a,b). To explore the reasons for the heterogeneity, the inventors sorted mCherry-high (5% higher) and mCherry-low (5% lower) mESCs and compared the expression of dCas9, P65-HSF1 and sgRNA in these two groups Level.
  • qPCR results showed that dCas9 was significantly downregulated in the mCherry-low cell population (Fig. 14c), indicating that the heterogeneity may be caused by the differential expression of dCas9 and activator in the cells driven by the two CAG promoters, respectively.
  • the inventors constructed a single CAG-SPH vector to express dCas9 and activator under a single promoter (Fig. 5b and Fig. 14d).
  • the results of the present inventors showed that SPH (single CAG)-OminiCMV-Ents induced mCherry expression more uniformly and with higher expression than SPH-OminiCMV-Ents (double CAG) (Fig. 5b-d and Fig. 14e, 10a, 10b) .
  • SPH single CAG
  • sgRNA arrays further improved sensitivity compared to SPH (single CAG)–OminiCMV-Ents–single sgRNA as demonstrated by mCherry expression levels (Fig. 5c,d and Figs. 15a, 15b, 11a -e, 12a).
  • SPH(single CAG)-OminiCMV-Ents-sgRNA arrays can detect low abundance lncRNAs that cannot be seen by SPH(single CAG)-OminiCMV-Ents-single sgRNA ( The expression level of possible target lncRNA/Gapdh ⁇ 0.001, qPCR analysis) (Fig. 5e,f).
  • Nanog-P2A-mCherry cell line decreased Nanog expression was strictly associated with decreased mCherry, whereas there was a time lag between Nanog and mCherry expression in the Ents system (Fig. 18a-d).
  • Tet-On system was designed in which tRNA-sgRNA-tRNA was inserted into the 3'UTR of GFP, and doxycycline (Dox) was added to cell culture to induce GFP and tRNA- sgRNA-tRNA expression (Figure 18e).
  • the present inventors used this system to investigate the time difference between the expression and turn-off of the target gene and the reporter gene.
  • the data show that the time difference between GFP and mCherry expression is approximately 0.5 hours at the mRNA level when turned on (Fig. 18h,i) and 6 hours at the protein level (Fig. 18j); while when turned off, at the mRNA level, there is a time delay Approximately 4.5 hours (Fig. 18k, 1), protein levels were at 18 hours (Fig. 18m).
  • the present invention can visualize the gene (Esrrb) whose expression level is as low as 0.001845 relative to Gadph, and the lncRNA (Malat1) whose expression level is as low as 0.003989 relative to Gadph by using the SPH (single CAG)-OminiCMV-Ents-single sgRNA system.
  • SPH single CAG
  • SPH-OminiCMV-Ents-sgRNA array system gene expression levels as low as 0.000050 (Fzd7) and 1ncRNA (Pvt1) as low as 0.000197 relative to Gapdh were observed.
  • the present invention demonstrates that Ents design enables the construction of multi-input logic operations by integrating multiple sgRNAs targeting different locations into a single precursor for probing endogenous signals and integrating complex cells in specific cell types Functions provide promising tools.

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Abstract

La présente invention concerne un promoteur sensible au ARNsg, un système de mesure contenant le promoteur sensible au ARNsg et utilisé pour détecter l'acide nucléique endogène cellulaire, et un procédé de mesure correspondant. Au moyen du système ou du procédé de mesure, un gène endogène ayant un très faible niveau d'expression intracellulaire peut être détecté de manière fiable, et l'activité transcriptionnelle d'un long ARN non codant (ARNlnc) souvent sous-exprimé peut également être surveillée dans des cellules vivantes. En outre, grâce au procédé, un signal endogène peut être amplifié pour visualiser les processus dynamiques d'expression d'un gène à faible abondance et d'un ARNnlc dans les cellules, et une plate-forme puissante est fournie pour détecter l'activité d'un élément génétique endogène, et sa fonction cellulaire potentielle.
PCT/CN2020/133860 2020-12-04 2020-12-04 Procédé de mesure de niveaux de gène de faible abondance endogène cellulaire et de arnlnc Ceased WO2022116139A1 (fr)

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