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WO2023241051A1 - Arnsg composite marqué par fluorescence, son procédé de préparation et son utilisation - Google Patents

Arnsg composite marqué par fluorescence, son procédé de préparation et son utilisation Download PDF

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WO2023241051A1
WO2023241051A1 PCT/CN2023/072846 CN2023072846W WO2023241051A1 WO 2023241051 A1 WO2023241051 A1 WO 2023241051A1 CN 2023072846 W CN2023072846 W CN 2023072846W WO 2023241051 A1 WO2023241051 A1 WO 2023241051A1
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sgrna
composite
sequence
hulu
fragment
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程永升
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Pixel Biosciences Suzhou Co Ltd
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Pixel Biosciences Suzhou Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers

Definitions

  • the invention belongs to the field of molecular biology, and specifically relates to a fluorescently labeled composite sgRNA and its preparation method and application.
  • DNA FISH is a powerful technique for detecting chromosome structure and has been widely used in the study of chromosome structure and clinical diagnosis of chromosomal abnormalities.
  • DNA FISH technology requires the genomic DNA to be opened through thermal denaturation, and the target region to be detected needs to be long enough (usually greater than 150Kb) to generate a hybridization signal for detection.
  • BAC (bacterial artificial chromosome)-based DNA FISH probes typically span tens or even hundreds of kb in genomic DNA.
  • the genomic DNA cannot be thermally denatured, or a target region that is too long is selected. For example, in gene therapy or when exogenous genes are introduced into the genome, the target region should be less than 10 kb.
  • the CRISPR-Cas9 (Streptococcus pyogenes) system has been extensively explored in the study of genome structure in vitro and in vivo.
  • the CRISPR-Cas9/sgRNA ribonucleoprotein complex can target a specific site on the genome and mark the site or open the genome for hybridization.
  • chemically synthesized sgRNA labeling efficiency is not high, especially when the sgRNA length exceeds 100 bp.
  • the chemical synthesis conditions of long RNA are demanding and the yield is less than 10%, and additional fluorescent labeling will make the yield even lower.
  • the advantage of the CRISPR-Cas9/sgRNA-based FISH detection method compared to traditional FISH is that the detection target area can be more precise.
  • the fluorescent group labeled on the Cas9 will cause the same sgRNA to have different bands due to the dissociation and recombination of the Cas9 protein and sgRNA. Fluorescence, it is impossible to distinguish under a microscope which fluorescent signal belongs to which sgRNA corresponds to the target.
  • the present invention provides a fluorescently labeled composite sgRNA and its preparation method and application.
  • the present invention provides a fluorescently labeled composite sgRNA, which includes a targeting sgRNA specific to a target nucleic acid sequence, Hulu connected to the targeting sgRNA, and a Hulu for connecting the target nucleic acid sequence.
  • the linker sequence of the targeting sgRNA and the Hulu which is a DNA oligonucleotide labeled with at least one fluorescent group;
  • the targeting sgRNA includes a guide RNA sequence and a backbone sequence that are complementary to the target nucleic acid sequence.
  • the 3' end of the guide RNA sequence is connected to the 5' end of the backbone sequence; the Hulu is connected to the backbone sequence through a linker sequence.
  • the gibbs free energy of the secondary structure is >-10kJ/mol (for example -5kJ/mol, 0kJ/mol, 5kJ/mol); 80 to 100% (for example, 85%, 90%, 95%) of the nucleosides in Hulu
  • the bases of acids are H (H is A, C, and T), B (H is C, G, and T), V (H is A, C, and G), or D (H is A, G, and T);
  • the homologous sequence of Hulu does not exist in the genome or transcriptome sequence of the gene where the guide RNA sequence is complementary to the target nucleotide sequence.
  • the homologous sequence of Hulu refers to the homology with the Hulu within More than 80% of the sequences.
  • the Gibbs free energy (Gibbs free energy deltaG) of the secondary structure formed by the Hulu nucleotide sequence in the present invention can be predicted using UNAfold software.
  • linker sequence used to connect Hulu and the backbone sequence in the present invention can be designed according to the conventional requirements for connecting DNA and RNA.
  • the linker sequence is as shown in the sequence list SEQ ID NO: 12.
  • the nucleotide length of the Hulu is 5 to 450bp (such as 10bp, 15bp, 20bp, 25bp, 30bp, 35bp, 40bp, 45bp, 50bp, 100bp, 150bp , 200bp, 250bp, 300bp, 350bp, 400bp), more preferably 10 to 135bp (for example, 15bp, 30bp, 60bp, 90bp, 130bp).
  • the bases of 80 to 100% of the nucleotides in Hulu are H, and the bases in H are adenine (A), cytosine (C), and thymine.
  • the number ratio of (T) is (0.1 ⁇ 10): (0.1 ⁇ 10): (0.1 ⁇ 10) (for example, 1:1:1, 1:1:5, 1:1:10, 1:5:1 , 1:5:5, 1:5:10, 5:1:1, 5:1:5, 10:1:10, 10:1:1, 10:1:10).
  • the Hulu 80 to 100% of the nucleotide bases are B, and the number ratio of cytosine (C), guanine (G), and thymine (T) in B is (0.1 to 10): (0.1 to 10) : (0.1 ⁇ 10) (for example, 1:1:1, 1:1:5, 1:1:10, 1:5:1, 1:5:5, 1:5:10, 5:1:1, 5:1:5, 10:1:10, 10:1:1, 10:1:10).
  • the bases of 80 to 100% of the nucleotides in the Hulu are V, and adenine (A), cytosine (C), and guanine in V
  • the number ratio of (G) is (0.1 ⁇ 10): (0.1 ⁇ 10): (0.1 ⁇ 10) (for example, 1:1:1, 1:1:5, 1:1:10, 1:5:1 , 1:5:5, 1:5:10, 5:1:1, 5:1:5, 10:1:10, 10:1:1, 10:1:10).
  • the bases of 80 to 100% of the nucleotides in Hulu are D, and in D, adenine (A), guanine (G), and thymine
  • the number ratio of (T) is (0.1 ⁇ 10): (0.1 ⁇ 10): (0.1 ⁇ 10) (for example, 1:1:1, 1:1:5, 1:1:10, 1:5:1 , 1:5:5, 1:5:10, 5:1:1, 5:1:5, 10:1:10, 10:1:1, 10:1:10).
  • the backbone sequence is as shown in the sequence list SEQ ID NO: 16.
  • the fluorescent group is selected from the group consisting of Atto series dyes, Alexa series dyes, Cy series dyes, CF series dyes, Dylight series dyes, Abberior series dyes, FAM, ROX , TAMRA, FITC, Rodamine, one or more of JF series dyes, etc.
  • the fluorescent group is connected to the Hulu through the bases in the Hulu nucleotide sequence; the number of nucleotides in the Hulu is determined according to the fluorescence The number of groups is determined. When the number of fluorescent groups increases, the length of Hulu will increase.
  • every 10 bp to 20 bp of Hulu is labeled with one fluorescent group; preferably, when Hulu is labeled with multiple fluorescent groups
  • the fluorescent groups can be the same or different; preferably, the Hulu is composed of 1 to 9 species (for example, 1 species, 2 species, 3 species, 4 species, 5 species, 6 species, 7 species, 8 species, 9 types) Fluorophore-labeled DNA oligonucleotides.
  • the composite sgRNA targets human telomeric DNA
  • the guide RNA sequence complementary to the target nucleic acid sequence in the composite sgRNA is as shown in the sequence list SEQ ID NO: 14 shows that the sequence of Hulu in the composite sgRNA is as shown in the sequence list SEQ ID NO: 13; preferably, the nucleotide sequence of the composite sgRNA is as shown in the sequence list SEQ ID NO: 10.
  • the composite sgRNA Targeting the first exon of the TRAC gene the guide RNA sequence complementary to the target nucleic acid sequence in the composite sgRNA is as shown in the sequence list SEQ ID NO: 15, and the sequence of Hulu in the composite sgRNA is as shown in the sequence list SEQ ID NO: As shown in 13; preferably, the nucleotide sequence of the composite sgRNA is as shown in the sequence list SEQ ID NO: 11.
  • the present invention provides a ribonucleoprotein complex, which includes: Cas enzyme, and the fluorescently labeled composite sgRNA described in the first aspect complexed with the Cas enzyme.
  • the Cas enzyme is dCas9, Cas9 Nickase, or CasX; preferably, the CasX is Cas9, Cas12, or Cas13.
  • the present invention provides a method for preparing the above-mentioned ribonucleoprotein complex, which method includes the following steps in sequence:
  • Synthesis of the fluorescently labeled composite sgRNA Mix the chemically synthesized first fragment of composite sgRNA, the second fragment of composite sgRNA and the Hulu through T4 DNA ligase or T4 RNA ligase 2 to form an enzyme conjugate system, and then proceed Enzyme conjugation reaction, the enzyme conjugation product is purified to obtain a fluorescently labeled complex sgRNA solution;
  • the size of the first fragment of the composite sgRNA and the second fragment of the composite sgRNA are both 40-70bp (for example, 45bp, 50bp, 55bp, 60bp, 65bp), and the first fragment of the composite sgRNA includes the guide RNA sequence and part of the Backbone sequence; wherein, the guide RNA sequence is 18 to 24 (preferably 20) nucleotides from the 5' end of the first fragment of the composite sgRNA; the second fragment of the composite sgRNA includes the remaining backbone sequence as well as the linker sequence.
  • the tetraloop region (located at the 22nd to 51st nucleotides of the backbone sequence) or Stem1 (located at the 52nd to 61st nucleotides of the backbone sequence) in the secondary structure of the sgRNA is selected.
  • the targeting sgRNA is divided into the first fragment of the composite sgRNA and the second fragment of the composite sgRNA to convert the long targeting sgRNA into short fragments to facilitate chemical synthesis, and then the two and Hulu are connected into the composite sgRNA through ligase (HuluLINK technology) to overcome the problems of low yield and high mutation rate of chemically synthesized overlong RNA or fluorescently labeled composite sgRNA.
  • ligase HuluLINK technology
  • the enzyme conjugate system it also includes a first splint oligonucleotide used to assist in connecting the first fragment of the composite sgRNA and the second fragment of the composite sgRNA, and a second splint oligonucleotide used to assist in connecting the second fragment of the composite sgRNA and Hulu;
  • the length of the first splint oligonucleotide and the second splint oligonucleotide is both 20-100 nucleotides, wherein 30-70% of the first splint oligonucleotide from the 3' end (e.g., 40%, 50%, 60%) of the nucleotides are completely complementary to the sequence starting from the 3' end of the first fragment of the composite sgRNA, except for the nuclei that are complementary to the sequence starting from the 3' end of the first fragment of the composite sgRNA. Except for the nucleotides, the remaining nucleotides in the first splint oligonucleotide are completely complementary to the sequence starting from the 5' end of the second fragment of the composite sgRNA;
  • nucleotides from the 3' end of the second splint oligonucleotide are completely complementary to the sequence from the 3' end of the second fragment of the composite sgRNA, except With the exception of the nucleotides complementary to the sequence starting from the 3' end of the second segment of the composite sgRNA, the remaining nucleotides in the second splint oligonucleotide are completely complementary to the sequence starting from the 5' end of Hulu.
  • the sequence of the first splint oligonucleotide is as shown in SEQ ID NO:9, and the sequence of the second splint oligonucleotide is as shown in SEQ ID NO:8 shown.
  • the 3' ends of the first fragment of the composite sgRNA, the second fragment of the composite sgRNA and Hulu are all hydroxyl groups, and the 5' ends are all phosphorylated.
  • the composite sgRNA targets human telomeric DNA.
  • the RNA sequence of the first fragment of the composite sgRNA is as shown in SEQ ID NO: 1
  • the first fragment of the composite sgRNA is as shown in SEQ ID NO: 1.
  • the RNA sequence of the two fragments is shown in SEQ ID NO:3; the complete nucleotide sequence of the composite sgRNA is shown in the sequence list SEQ ID NO:10.
  • the composite sgRNA targets the first exon of the TRAC gene.
  • the RNA sequence of the first fragment of the composite sgRNA is as shown in SEQ ID NO: 2
  • the RNA sequence of the second fragment of the composite sgRNA is shown in SEQ ID NO:3
  • the complete nucleotide sequence of the composite sgRNA is shown in the sequence list SEQ ID NO:11.
  • the binding buffer includes: HEPES with a final concentration of 18-22mM (for example, 19mM, 20mM, 21mM), and a final concentration of 90-110mM (for example, 95mM, 100mM).
  • the binding buffer includes: HEPES with a final concentration of 20mM, KCl with a final concentration of 100mM, MgCl2 with a final concentration of 5mM, and a final concentration of 5% by volume.
  • the final concentration is Tween-20 with a volume percentage of 0.1%
  • the final concentration is bovine serum albumin
  • the pH of the HEPES buffer used to prepare the binding buffer is 7 to 8, preferably 7.5.
  • step S1 the purification is performed by using PAGE gel or HPLC to obtain the fluorescently labeled complex sgRNA solution.
  • step S1 the temperature of the enzyme reaction is 35 to 38°C (for example, 36°C, 37°C), and the enzyme connection time is 0.5 to 6h (for example, 1h, 2h ,3h,4h,5h).
  • the molar ratio of the total amount of the first fragment of the composite sgRNA and the second fragment of the composite sgRNA to T4 RNA ligase 2 is (5 ⁇ 100): 1 (for example, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1 , 95:1), or in the enzyme conjugate system, the molar ratio of the total amount of the first fragment of the composite sgRNA and the second fragment of the composite sgRNA to T4 DNA ligase is (0.5 ⁇ 5):1 (for example, 1:1, 1.5:1, 2:1, 3:1, 4:1, 4.5:1).
  • the volume ratio of the fluorescently labeled composite sgRNA solution and the binding buffer is (0.8 ⁇ 1.2):4 (for example, 0.9:4, 1:4 , 1.1:4);
  • the concentration of the fluorescently labeled composite sgRNA is 50-300ng/ ⁇ L (for example, 60ng/ ⁇ L, 70ng/ ⁇ L, 80ng/ ⁇ L, 90ng/ ⁇ L, 100ng/ ⁇ L, 150ng/ ⁇ L, 200ng/ ⁇ L, 250ng/ ⁇ L).
  • the volume ratio of the Cas enzyme protein solution and the binding buffer is (0.8 ⁇ 1.2):4 (for example, 0.9:4, 1:4, 1.1 :4);
  • the mass concentration of the Cas enzyme protein solution is 2 to 40 nM.
  • step S2 the molar ratio of the fluorescently labeled composite sgRNA and Cas enzyme protein in the reaction system is (1-5):1 (for example, 2:1, 3:1, 4:1).
  • the present invention at least has the following beneficial effects:
  • the present invention has 10 times more cost and scale for the preparation of composite sgRNA.
  • the advantages are specifically reflected in:
  • the guide RNA sequence complementary to the target nucleic acid sequence is the specific region of each composite sgRNA, located in the first 18 to 24 nucleotides of the first segment of the composite sgRNA (referred to as sgRNA P1), and is used to produce each customized composite sgRNA. Variable items.
  • the second fragment of composite sgRNA (sgRNA P2 for short) is a universal sequence that can be synthesized in large quantities and used repeatedly to reduce costs. All composite sgRNA molecules required by Cas9 protein can be used to synthesize any new composite sgRNA based on the same universal sgRNA P2.
  • Hulu s nucleotide sequence is also a universal sequence and can be synthesized in large quantities and used repeatedly to reduce costs.
  • sgRNA P1 After short fragments of sgRNA P1, sgRNA P2 and Hulu enzyme are combined to form composite sgRNA, the mutation rate of each nucleic acid is greatly reduced due to the reduction in length.
  • the mutation rate does not increase after the composite splicing of the three, so the synthesis of composite sgRNA is The overall mutation rate is also lower than direct chemical synthesis of composite sgRNA.
  • the preparation efficiency is high.
  • the yield of fluorescently labeled composite sgRNA prepared by the method of the present invention can reach 50%, and the yield of pure chemically synthesized fluorescently labeled composite sgRNA is less than 1%.
  • Figure 1 is a schematic diagram of the synthesis of fluorescently labeled composite sgRNA (referred to as sgRNA-Hulu) through enzyme conjugation.
  • sgRNA-Hulu fluorescently labeled composite sgRNA
  • sgRNA P1 the first fragment of the composite sgRNA
  • sgRNA P2 the second fragment of the composite sgRNA
  • Hulu is a fluorescently labeled DNA oligonucleotide, which is ligated by T4 DNA ligase or T4 RNA ligase 2 with the help of splint molecules (first and second splint oligonucleotides)
  • sgRNA P1 and sgRNA P2 can form a double-stranded structure with the first splint oligonucleotide
  • sgRNAP2 and Hulu can form a double-stranded structure with the second splint oligonucleotide
  • the splint molecules can promote the connection of the connection site.
  • Figure 2 shows CRISPR FISH connected with dCas9 and fluorescently labeled composite sgRNA, that is, a schematic diagram of the ribonucleoprotein complex dCas9-sgRNA-Hulu binding to the target sequence.
  • Figure 3 shows CRISPR FISH (ribonucleoprotein complex) prepared in Example 1 dCas9-sgRNA Telomere -Hulu) human telomere imaging results, the cell nucleus is counter-stained by DA PI (blue), and the human telomere target sequence is stained by dCas9-sgRNA Telomere -Hulu.
  • Figure 4 is the detection result of the biological activity (gene editing activity) of the ribonucleoprotein complex prepared by the method of the present invention in Example 2.
  • the test uses T7EI to analyze mutations.
  • the leftmost lane is a 1kb Marker (GeneRuler of ThermoScientific) 1Kbplus)
  • lane 1 is the positive control (proves that the kit is working properly)
  • lane 2 is the negative control (no gene editing is performed)
  • lane 3 is Cas9-sgRNA TRAC group 1
  • lane 4 is Cas9-sgRNA TRAC group 2
  • lane 5 is Cas9-sgRNA TRAC -Hulu group 1
  • lane 6 is Cas9-sgRNA TRAC -Hulu group 2.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are all conventional methods unless otherwise specified.
  • the test materials used in the following examples were purchased from conventional biochemical reagent companies unless otherwise specified.
  • the quantitative experiments in the following examples were repeated three times, and the results were averaged.
  • the sgRNA used alone in the following examples or examples refers to the targeting sgRNA that is not connected to Hulu, including the linker sequence or not including the linker sequence.
  • composite sgRNA fluorescently labeled composite sgRNA
  • the full name is used in the following content, or Expressed in the form of sgRNA-Hulu.
  • “Hulu” is a DNA oligonucleotide labeled with at least one fluorophore.
  • the nucleotide sequence of Hulu is generated using UNAfold (http://www.unafold.org/) and RNAfold (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite /RNAfold.cgi) web server is carefully designed and modeled.
  • the design principle is to use the bases H (H for A, C, and T), B (H for C, G, and T), V (H for A, C, and G) in 80-100% of Hulu's nucleotides , or D (H is A, G and T) to avoid the Hulu sequence itself or its formation of strong secondary structure with the sgRNAP1 or sgRNA P2 sequence.
  • Hulu sequences of the required length are randomly generated by computer. These sequences generally lack one of the bases A, T, G, or C.
  • Blast software was used to confirm that there were no obvious homologous sequences between the Hulu sequence and the genome and transcriptome sequences of the target species, and the homology was less than 80%.
  • Hulu labeled with a fluorescent group can be obtained by the following method: first chemically synthesize the Hulu oligonucleotide sequence, and then connect the fluorescent group to the base of Hulu through conventional methods in this field.
  • Targeting sgRNA is an sgRNA that does not include the linker sequence and Hulu, and includes a guide RNA sequence complementary to the target nucleic acid sequence and a backbone sequence, the 3' end of the guide RNA sequence is connected to the 5' end of the backbone sequence.
  • it involves targeting sgRNA targeting different genes.
  • the name of the gene it targets is marked as a subscript on the sgRNA.
  • sgRNA TRAC represents the targeting sgRNA targeting the TRAC gene.
  • Composite sgRNA is a fluorescently labeled sgRNA, also known as DNA FISH, which includes targeting sgRNA, Hulu and a linker sequence.
  • the linker sequence is used to connect the targeting sgRNA and Hulu.
  • it involves composite sgRNA targeting different genes.
  • the name of the gene it targets is marked as a subscript on the sgRNA.
  • sgRNA TRAC -Hulu represents a composite sgRNA targeting the TRAC gene.
  • Linker sequence is used to connect the backbone sequence and the Hulu, the 3' end of the backbone sequence is connected to the 5' end of the linker sequence, and the 3' end of the linker sequence is connected to the 5' end of the Hulu end connected.
  • “Ribonucleoprotein complex” also known as CRISPR FISH, is obtained by complexing Cas enzyme with fluorescently labeled composite sgRNA.
  • the ribonucleoprotein complex obtained by complexing dCas9 with composite sgRNA is represented by dCas9-sgRNA-Hulu.
  • the specific embodiment of the present invention proposes a new method for connecting sgRNA to a variety of fluorescent dyes. By dividing the sgRNA into two sections, chemical synthesis is performed respectively, and at the same time, a 3' end of the sgRNA is added for connection to Hulu.
  • sgRNA P1 the first fragment of composite sgRNA
  • sgRNA P2 the second fragment of composite sgRNA
  • Hulu oligonucleotide labeled with a fluorescent group through T4 DNA ligase or T4 RNA ligase 2 Ligation and synthesis of fluorescently labeled sgRNA, of which sgRNA P1 and sgRNA P2 were chemically synthesized and purified by HPLC respectively.
  • Fluorescently labeled composite sgRNA is a chimera of DNA and RNA that can be used for a wider range of CRISPR applications, such as genome editing, diagnostics, and imaging of genomic loci.
  • CRISPR applications such as genome editing, diagnostics, and imaging of genomic loci.
  • fluorescently labeled sgRNA chimeras can be used to image human telomeres.
  • the connection described in the present invention can be a direct connection between the two or an indirect connection through other sequences.
  • the present invention synthesizes complex sgRNA (sgRNA-Hulu) through enzymatic ligation, and further synthesizes a ribonucleoprotein complex (dCas9-sgRNA-Hulu).
  • the method sequentially includes the following steps:
  • Step S1 synthesis of the fluorescently labeled composite sgRNA: combine the chemically synthesized first fragment of composite sgRNA, the second fragment of composite sgRNA (SEQ ID NO: 3), and the first splint oligonucleotide (SEQ ID NO: 9) , the second splint oligonucleotide (SEQ ID NO:8) and the Hulu (SEQ ID NO:13) are mixed through T4 DNA ligase or T4 RNA ligase 2 to form an enzyme ligation system;
  • the enzyme ligation system is as follows:
  • T4 DNA ligase buffer 50mM Tris-HCl, 10mM MgCl 2 , 1mM ATP, 10mM DTT (pH 7.5@25°C) contains the following reactants and enzymes (concentrations are final concentrations):
  • T4 RNA Ligase 2 buffer 50mM Tris-HCl, 2mM MgCl 2 , 1mM DTT, 400 ⁇ M ATP, (pH 7.5@25°C) contains the following reactants and enzymes (concentrations are final concentrations):
  • the temperature of the enzyme conjugation reaction is 35 to 38°C, and the enzyme conjugation time is 0.5 to 6 hours.
  • the enzyme conjugation product is purified by PAGE gel or HPLC to obtain a fluorescently labeled composite sgRNA solution.
  • Use Nanodrop to quantitatively adjust the concentration of the fluorescently labeled composite sgRNA solution to 50 ⁇ 300ng/ ⁇ L, when adjusting the concentration, add 1:100 diluted NEB RNase inhibitor (RNase inhibitor product model: M0314) to prevent RNase contamination.
  • the second fragment of the composite sgRNA, the first splint oligonucleotide, the second splint oligonucleotide and the nucleotide sequence of Hulu are all universal sequences.
  • sgRNA can be used repeatedly.
  • the nucleotide sequence of the second fragment of the composite sgRNA is as shown in the sequence list SEQ ID NO:3, and the nucleotide sequence of the first splint oligonucleotide is as shown in the sequence list SEQ ID NO:9
  • the nucleotide sequence of the second splint oligonucleotide is shown in the sequence list SEQ ID NO:8, and the nucleotide sequence of Hulu is shown in the sequence list SEQ ID NO:13.
  • the nucleotide sequence of the first fragment of the composite sgRNA used is as shown in the sequence list SEQ ID NO: 1; preparing the first fragment targeting the TRAC gene When compounding sgRNA of exons, the nucleotide sequence of the first fragment of the compound sgRNA used is shown in the sequence list SEQ ID NO:2.
  • Step S2 According to the volume ratio, mix the fluorescently labeled composite sgRNA solution:binding buffer at (0.8 ⁇ 1.2):4 to prepare the first solution, wherein the binding buffer includes: HEPES with a final concentration of 18 ⁇ 22mM.
  • the final concentration is 90-110 mM KCl, the final concentration is 4-6mM MgCl 2 , the final concentration is 4-6% glycerol by volume, the final concentration is 0.08-0.12% Tween-20 by volume, the final concentration is the mass-to-volume ratio 0.08 ⁇ 0.12mg/ml bovine serum albumin;
  • the ribonucleoprotein complex is obtained.
  • the binding buffer formula used in the embodiment of the present invention includes: HEPES with a final concentration of 20mM, KCl with a final concentration of 100mM, MgCl 2 with a final concentration of 5mM, glycerol with a final concentration of 5% by volume, and glycerol with a final concentration of 5% by volume. 0.1% Tween-20, the final concentration is bovine serum albumin with a mass to volume ratio of 0.1 mg/ml.
  • the pH of HEPES is 7.5.
  • sgRNA-Hulu a composite sgRNA
  • the Hulu sequence is divided into three segments, such as SEQ ID NO:5, SEQ ID NO:6 and SEQ ID As shown in NO:7, each of the three sequences is labeled with an Atto565 dye.
  • a guide RNA sequence targeting human telomeric DNA was designed for the TAAGGG repeat in human telomeres (see SEQ ID NO: 14 in Table 1 above).
  • the dCas9-sgRNA-Hulu complex prepared in this example can effectively hybridize with the telomere region to achieve the capture of the target sequence.
  • sgRNA P1 chemically synthesized sgRNA P1 is used, whose nucleotide sequence is shown in SEQ ID NO:1; sgRNA P2, whose nucleotide sequence is shown in SEQ ID NO:3; Hulu, whose nucleotide sequence is shown in SEQ ID NO:3 SEQ ID NO:13; the first splint oligonucleotide, the nucleotide sequence of which is SEQ ID NO:9; the second splint oligonucleotide Plate oligonucleotide, the nucleotide sequence of which is shown in SEQ ID NO:8.
  • Enzyme conjugation system 1.5 ⁇ M sgRNA P1 (sgRNA first fragment)
  • the enzyme is connected in T4 RNA Ligase 2 buffer (which includes: Tris-HCl with a final concentration of 50mM, MgCl2 with a final concentration of 2mM, DTT with a final concentration of 1mM, ATP with a final concentration of 400 ⁇ M, (pH7. 5@25°C)), react at 37°C for 30 minutes, and then purify the enzyme ligation reaction product through urea PAGE gel to obtain the ligated fluorescently labeled composite sgRNA at a concentration of 150-300ng/ ⁇ L.
  • sgRNA Telomere- Hulu prepared in this example with the binding buffer at a volume ratio of 1:4 to prepare the first solution; mix the dCas9 protein solution (10 nM, commercial source: IDTDNA) according to the dCas9 protein solution and Mix the binding buffer with a volume ratio of 1:4 to prepare a second solution.
  • the molar ratio of sgRNA to dCas9 protein in the reaction system is 1:1.
  • dCas9-sgRNA Telomere -Hulu prepared in this example is shown in Figure 2.
  • Endonuclease-inactivated Cas9 (dCas9) binds to fluorescent dye-labeled complex sgRNA.
  • the in vivo gene editing activity of the composite sgRNA synthesized through enzymatic conjugation was tested in HeLa cells. Based on the designed sgRNA sequence for the human TRAC gene, two sets of sgRNA containing Hulu's composite sgRNA (sgRNA TRAC -Hulu) and a targeting sgRNA without Hulu (sgRNA TRAC ) sequence were constructed (two identical parallel sets were set in each group). (control group), enzyme conjugation was carried out according to the method of Example 1, and the fluorescent dyes connected to Hulu in each group were also the same as Example 1.
  • sgRNA TRAC the nucleotide sequence is as shown in SEQ ID NO: 19
  • sgRNA TRAC -Hulu the nucleotide sequence is as shown in SEQ ID NO: 11
  • nucleotide sequence of sgRNA P1 used to synthesize sgRNA TRAC and sgRNA TRAC -Hulu is shown in SEQ ID NO: 2;
  • nucleotide sequence of sgRNA P2 used to synthesize sgRNA TRAC is shown in SEQ ID NO: 4, and the nucleotide sequence of sgRNA P2 used to synthesize sgRNA TRAC -Hulu is shown in SEQ ID NO: 3;
  • the nucleotide sequence of the Hulu fragment of synthetic sgRNA TRAC -Hulu is shown in SEQ ID NO: 13;
  • the first splint oligonucleotide used to synthesize sgRNA TRAC and sgRNA TRAC -Hulu its nucleotide sequence is shown in SEQ ID NO: 9;
  • the second splint oligonucleotide used to synthesize sgRNA TRAC -Hulu has a nucleotide sequence as shown in SEQ ID NO: 8;
  • Enzyme conjugation was performed according to the enzyme conjugation system and method in Example 1 to obtain sgRNA TRAC (SEQ ID NO:19), sgRNA TRAC -Hulu (SEQ ID NO:11).
  • the sgRNA TRAC and sgRNA TRAC -Hulu prepared in this example were combined with the Cas9 protein (10 nM, commercial brand: IDTDNA) according to the method in Example 1 to form Cas9-sgRNA TRAC and Cas9-sgRNA TRAC -Hulu respectively.
  • Cas9-sgRNA TRAC and Cas9-sgRNA TRAC -Hulu prepared in this example were prepared according to the literature (CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells [J]. Cell Research: English Edition, 2017, 27(1): The method in 4.) was used to detect its activity on TRAC gene editing of HeLa cells.
  • lane 1 is the positive control of the kit (to prove that the kit is working properly);
  • lane 2 is the negative control, the genomic DNA control of Hela cells without transfection of ribonucleoprotein complex;
  • lane 3 is Cas9-sgRNA TRAC group 1 ;
  • Lane 4 is Cas9-sgRNA TRAC group 2 ;
  • lane 5 is Cas9-sgRNA TRAC -Hulu group 1, and
  • lane 6 is Cas9-sgRNA TRAC -Hulu group 2.

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Abstract

L'invention concerne un ARNsg composite marqué par fluorescence, son procédé de préparation et son utilisation. L'ARNsg composite marqué par fluorescence est constitué séquentiellement d'une séquence d'ARN guide complémentaire d'une séquence d'acide nucléique cible, d'une séquence de squelette, d'une séquence de lieur et d'une séquence de Hulu à partir de l'extrémité 5'-3'. Hulu est un oligonucléotide d'ADN marqué avec au moins un groupe fluorescent. L'ARNsg composite marqué par fluorescence peut être utilisé pour l'édition, le diagnostic et l'imagerie du génome de loci génomiques.
PCT/CN2023/072846 2023-01-18 2023-01-18 Arnsg composite marqué par fluorescence, son procédé de préparation et son utilisation Ceased WO2023241051A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150232915A1 (en) * 2014-02-19 2015-08-20 Personal Genomics, Inc. Method for Real-Time Single Molecule Sequencing
CN105755040A (zh) * 2016-01-21 2016-07-13 北京大学 一种改造的sgRNA及染色体标记应用
CN112384621A (zh) * 2018-04-13 2021-02-19 西格马-奥尔德里奇有限责任公司 使用成对的crispr切口酶核糖核蛋白修饰免疫相关基因组基因座

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US20150232915A1 (en) * 2014-02-19 2015-08-20 Personal Genomics, Inc. Method for Real-Time Single Molecule Sequencing
CN105755040A (zh) * 2016-01-21 2016-07-13 北京大学 一种改造的sgRNA及染色体标记应用
CN112384621A (zh) * 2018-04-13 2021-02-19 西格马-奥尔德里奇有限责任公司 使用成对的crispr切口酶核糖核蛋白修饰免疫相关基因组基因座

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Title
DONG CHANG; GOU YUANWEI; LIAN JIAZHANG: "SgRNA engineering for improved genome editing and expanded functional assays", CURRENT OPINION IN BIOTECHNOLOGY, LONDON, GB, vol. 75, 23 February 2022 (2022-02-23), GB , XP087088156, ISSN: 0958-1669, DOI: 10.1016/j.copbio.2022.102697 *
PARK HANSOL, OSMAN EIMAN A., CROMWELL CHRISTOPHER R., ST LAURENT CHRIS D., LIU YUNING, KITOVA ELENA N., KLASSEN JOHN S., HUBBARD B: "CRISPR-Click Enables Dual-Gene Editing with Modular Synthetic sgRNAs", BIOCONJUGATE CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 33, no. 5, 18 May 2022 (2022-05-18), US , pages 858 - 868, XP093117883, ISSN: 1043-1802, DOI: 10.1021/acs.bioconjchem.2c00106 *

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