Fluorescently-labeled composite sgRNA as well as preparation method and application thereof

Abstract

The invention provides a fluorescently-labeled composite sgRNA as well as a preparation method and application thereof. The fluorescently-labeled composite sgRNA sequentially comprises a guide RNA sequence, a skeleton sequence, a linker sequence and a Hulu sequence which are complementary with a target nucleic acid sequence from the 5 '-3' end, and the Hulu is DNA oligonucleotide labeled by at least one fluorophore. The fluorescently-labeled composite sgRNA can be used for genome editing, diagnosis and genome site imaging.

Description

A fluorescently labeled composite sgRNA and its preparation method and application Technical Field

[0001] The present invention belongs to the field of molecular biology, and specifically relates to a fluorescently labeled composite sgRNA and a preparation method and application thereof. Background Art

[0002] DNA FISH is a powerful technique for detecting chromosome structure and has been widely used in the study of chromosome structure and the clinical diagnosis of chromosomal abnormalities. However, DNA FISH technology requires that genomic DNA be opened by heat denaturation, and the target region to be detected must be long enough (usually greater than 150Kb) to generate hybridization signals for detection. DNA FISH probes based on BACs (bacterial artificial chromosomes) usually span dozens or even hundreds of kb of genomic DNA. In the study of fine chromosome structure, in some cases, genomic DNA cannot be heat denatured, or an overly long target region is selected. For example, in gene therapy or when introducing exogenous genes into the genome, the target region should be less than 10kb.

[0003] 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 that site or open the genome for hybridization. Due to the secondary structure and length of the sgRNA, chemically synthesized sgRNA labeling is inefficient, especially when the sgRNA length exceeds 100 bp. Chemical synthesis of long RNAs requires demanding conditions, resulting in yields below 10%, and the addition of fluorescent labels further reduces the yield. CRISPR-Cas9 / sgRNA-based FISH detection methods have the advantage of allowing for more precise detection of target regions compared to traditional FISH. In multiplexed CRISPR-Cas9 / sgRNA-based FISH detection, if a fluorescent group is labeled on the Cas9 protein, the dissociation and reassociation of the Cas9 protein and the sgRNA will result in different fluorescence signals for the same sgRNA, making it impossible to distinguish the fluorescent signal attributable to the corresponding sgRNA target under a microscope.

[0004] Summary of the Invention

[0005] In response to the problems existing in the prior art, the present invention provides a fluorescently labeled composite sgRNA and its preparation method and application.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a fluorescently labeled composite sgRNA, comprising a targeting sgRNA specific for a target nucleic acid sequence, a Hulu connected to the targeting sgRNA, and a linker sequence for connecting the targeting sgRNA and the Hulu, wherein the Hulu is a DNA oligonucleotide labeled with at least one fluorescent group;

[0008] The targeting sgRNA includes a guide RNA sequence complementary to the target nucleic acid sequence and a backbone sequence, wherein 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 via a linker sequence, wherein 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, and the Gibbs free energy of the secondary structure formed by the nucleotide sequence of the Hulu is greater than -10 kJ / mol (e.g., -5 kJ / mol). , 0 kJ / mol, 5 kJ / mol); 80-100% (e.g., 85%, 90%, 95%) of the nucleotide bases in the Hulu 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 genomic or transcriptomic sequence of the gene in which the target nucleotide sequence complementary to the guide RNA sequence is located does not contain a homologous sequence of the Hulu, and the homologous sequence of the Hulu refers to a sequence with a homology of more than 80% to the Hulu.

[0009] The Gibbs free energy (Gibbs free energy deltaG) of the Hulu nucleotide sequence to form a secondary structure in the present invention can be predicted using UNAfold software.

[0010] The 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.

[0011] In the above-mentioned fluorescently labeled composite sgRNA, as a preferred embodiment, the linker sequence is shown in the sequence listing SEQ ID NO: 12.

[0012] In the above-mentioned fluorescently labeled composite sgRNA, as a preferred embodiment, the nucleotide length of Hulu is 5 to 450 bp (e.g., 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp), more preferably 10 to 135 bp (e.g., 15 bp, 30 bp, 60 bp, 90 bp, 130 bp).

[0013] In the fluorescently labeled composite sgRNA, as a preferred embodiment, 80-100% of the nucleotide bases of Hulu are H, and the number ratio of adenine (A), cytosine (C), and thymine (T) in H is (0.1-10): (0.1-10): (0.1-10) (e.g., 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).

[0014] In the fluorescently labeled composite sgRNA, as a preferred embodiment, 80 to 100% of the nucleotides of Hulu are bases B, and the ratio of the number of cytosine (C), guanine (G), and thymine (T) in B is (0.1 to 10): (0.1 to 10): (0.1 to 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).

[0015] In the fluorescently labeled composite sgRNA, as a preferred embodiment, 80 to 100% of the nucleotide bases of Hulu are V, and the ratio of the number of adenine (A), cytosine (C), and guanine (G) in V is (0.1 to 10): (0.1 to 10): (0.1 to 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).

[0016] In the fluorescently labeled composite sgRNA, as a preferred embodiment, 80 to 100% of the nucleotide bases of Hulu are D, and the ratio of the number of adenine (A), guanine (G), and thymine (T) in D is (0.1 to 10): (0.1 to 10): (0.1 to 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).

[0017] In the above-mentioned fluorescently labeled composite sgRNA, as a preferred embodiment, the backbone sequence is shown in the sequence listing SEQ ID NO: 16.

[0018] In the above-mentioned fluorescently labeled composite sgRNA, as a preferred embodiment, the fluorescent group is selected from one or more of Atto series dyes, Alexa series dyes, Cy series dyes, CF series dyes, Dylight series dyes, Abberior series dyes, FAM, ROX, TAMRA, FITC, Rodamine, JF series dyes, etc.

[0019] In the above-mentioned fluorescently labeled composite sgRNA, as a preferred embodiment, the fluorescent group is connected to the Hulu through the base in the Hulu nucleotide sequence; the number of nucleotides in the Hulu is determined according to the number of fluorescent groups. When the number of fluorescent groups increases, the length of the Hulu increases. Preferably, one fluorescent group is labeled in every 10 bp-20 bp of the Hulu; preferably, when multiple fluorescent groups are labeled on the Hulu, the fluorescent groups can be the same or different; preferably, the Hulu is a DNA oligonucleotide labeled with 1 to 9 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) fluorescent groups.

[0020] In the above-mentioned fluorescently labeled composite sgRNA, as a preferred embodiment, the composite sgRNA targets human telomeric DNA, the guide RNA sequence complementary to the target nucleic acid sequence in the composite sgRNA is shown in the sequence listing SEQ ID NO: 14, and the sequence of Hulu in the composite sgRNA is shown in the sequence listing SEQ ID NO: 13; preferably, the nucleotide sequence of the composite sgRNA is shown in the sequence listing SEQ ID NO: 10.

[0021] In the above-mentioned fluorescently labeled composite sgRNA, as a preferred embodiment, the composite sgRNA targets the first exon of the TRAC gene, the guide RNA sequence complementary to the target nucleic acid sequence in the composite sgRNA is shown in the sequence listing SEQ ID NO: 15, and the sequence of Hulu in the composite sgRNA is shown in the sequence listing SEQ ID NO: 13; preferably, the nucleotide sequence of the composite sgRNA is shown in the sequence listing SEQ ID NO: 11.

[0022] In a second aspect, the present invention provides a ribonucleoprotein complex, which includes: a Cas enzyme, and the fluorescently labeled composite sgRNA described in the first aspect complexed with the Cas enzyme.

[0023] In the above-mentioned ribonucleoprotein complex, as a preferred embodiment, the Cas enzyme is dCas9, Cas9 Nickase, or CasX; preferably, the CasX is Cas9, Cas12, or Cas13.

[0024] In a third aspect, the present invention provides a method for preparing the above-mentioned ribonucleoprotein complex, the method comprising the following steps in sequence:

[0025] S1. Synthesis of the fluorescently labeled composite sgRNA: The chemically synthesized composite sgRNA first fragment, the composite sgRNA second fragment, and the Hulu are mixed using T4 DNA ligase or T4 RNA ligase 2 to form an enzyme ligation system, and then an enzyme ligation reaction is performed. The enzyme ligation product is purified to obtain a fluorescently labeled composite sgRNA solution;

[0026] S2. Add the fluorescently labeled composite sgRNA solution to a binding buffer to prepare a first solution; add the Cas enzyme protein solution to the binding buffer to dissolve it to prepare a second solution; and mix the first solution and the second solution to react, thereby obtaining the ribonucleoprotein complex;

[0027] The sizes of the composite sgRNA first fragment and the composite sgRNA second fragment are both 40 to 70 bp (e.g., 45 bp, 50 bp, 55 bp, 60 bp, 65 bp), and the composite sgRNA first fragment includes the guide RNA sequence and part of the backbone sequence; wherein the guide RNA sequence is 18 to 24 (preferably 20) nucleotides of the composite sgRNA first fragment from the 5' end; the composite sgRNA second fragment includes the remaining backbone sequence and the linker sequence.

[0028] In the present invention, the targeting sgRNA is divided into a first composite sgRNA fragment and a second composite sgRNA fragment by selecting the tetraloop region (located in the 22nd to 51st nucleotides of the backbone sequence) or Stem1 (located in the 52nd to 61st nucleotides of the backbone sequence) in the secondary structure of the targeting sgRNA, so as to convert the long targeting sgRNA into short fragments for easy chemical synthesis. The two are then connected to a composite sgRNA by ligase and Hulu (HuluLINK technology) to overcome the problems of low yield and high mutation rate of chemically synthesized overly long RNA or fluorescently labeled composite sgRNA. The region with insignificant secondary structure in the targeting sgRNA is used as one of the connection points to promote connection efficiency.

[0029] In the above preparation method, as a preferred embodiment, in step S1, the enzyme ligation system further includes a first splint oligonucleotide for assisting in the ligation of the first fragment of the composite sgRNA and the second fragment of the composite sgRNA, and a second splint oligonucleotide for assisting in the ligation of the second fragment of the composite sgRNA and Hulu;

[0030] The length of the first splint oligonucleotide and the second splint oligonucleotide are both 20 to 100 nucleotides, wherein 30 to 70% (e.g., 40%, 50%, 60%) of the nucleotides of the first splint oligonucleotide from the 3' end are fully complementary to the sequence from the 3' end of the first segment of the composite sgRNA, and except for the nucleotides complementary to the sequence from the 3' end of the first segment of the composite sgRNA, the remaining nucleotides in the first splint oligonucleotide are fully complementary to the sequence from the 5' end of the second segment of the composite sgRNA;

[0031] 30 to 70% (e.g., 40%, 50%, 60%) of the nucleotides of the second splint oligonucleotide from the 3' end are completely complementary to the sequence from the 3' end of the second fragment of the composite sgRNA, and in addition to the nucleotides complementary to the sequence from the 3' end of the second fragment of the composite sgRNA, the remaining nucleotides in the second splint oligonucleotide are completely complementary to the sequence from the 5' end of Hulu.

[0032] In the above preparation method, as a preferred embodiment, the sequence of the first splint oligonucleotide is shown as SEQ ID NO: 9, and the sequence of the second splint oligonucleotide is shown as SEQ ID NO: 8.

[0033] In the above preparation method, as a preferred embodiment, the 3' ends of the composite sgRNA first fragment, the composite sgRNA second fragment and Hulu are all hydroxyl groups, and the 5' ends are all phosphorylated.

[0034] In the above preparation method, as a preferred embodiment, the composite sgRNA targets human telomeric DNA. In the composite sgRNA, the RNA sequence of the first fragment of the composite sgRNA is shown as SEQ ID NO: 1, and the RNA sequence of the second fragment of the composite sgRNA is shown as SEQ ID NO: 3; the complete nucleotide sequence of the composite sgRNA is shown in the sequence listing as SEQ ID NO: 10.

[0035] In the above preparation method, as a preferred embodiment, the composite sgRNA targets the first exon of the TRAC gene. In the composite sgRNA, the RNA sequence of the first fragment of the composite sgRNA is shown as SEQ ID NO: 2, and the RNA sequence of the second fragment of the composite sgRNA is shown as SEQ ID NO: 3; the complete nucleotide sequence of the composite sgRNA is shown in the sequence listing as SEQ ID NO: 11.

[0036] In the above preparation method, as a preferred embodiment, the binding buffer comprises: HEPES with a final concentration of 18-22 mM (e.g., 19 mM, 20 mM, 21 mM), KCl with a final concentration of 90-110 mM (e.g., 95 mM, 100 mM, 105 mM), MgCl2 with a final concentration of 4-6 mM (e.g., 4.5 mM, 5 mM, 5.5 mM), glycerol with a final concentration of 4-6% by volume (e.g., 4.5%, 5%, 5.5%), Tween-20 with a final concentration of 0.08-0.12% by volume (e.g., 0.09%, 0.1%, 0.11%), and Tween-20 with a final concentration of 0.08-0.12 mg / ml (e.g., 0.09 mg / ml, 0.1 mg / ml, 0.11 mg / ml) bovine serum albumin; preferably, the binding buffer comprises: HEPES with a final concentration of 20 mM, KCl with a final concentration of 100 mM, MgCl2 with a final concentration of 5% by volume of glycerol, Tween-20 with a final concentration of 0.1% by volume, and bovine serum albumin with a final concentration of 0.1 mg / ml by mass volume.

[0037] In the above preparation method, as a preferred embodiment, the pH of the HEPES buffer used to prepare the binding buffer is 7-8, preferably 7.5.

[0038] In the above preparation method, as a preferred embodiment, in step S1, the purification is performed by using PAGE gel or HPLC purification to obtain the fluorescently labeled composite sgRNA solution.

[0039] In the above preparation method, as a preferred embodiment, in step S1, the temperature of the enzymatic ligation reaction is 35-38°C (e.g., 36°C, 37°C), and the enzymatic ligation time is 0.5-6h (e.g., 1h, 2h, 3h, 4h, 5h).

[0040] In the above preparation method, as a preferred embodiment, in step S1, in the enzyme-linked system, the molar ratio of the total amount of the composite sgRNA first fragment and the composite sgRNA second fragment to T4 RNA ligase 2 is (5-100):1 (e.g., 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-linked system, the molar ratio of the total amount of the composite sgRNA first fragment and the composite sgRNA second fragment to T4 DNA ligase is (0.5-5):1 (e.g., 1:1, 1.5:1, 2:1, 3:1, 4:1, 4.5:1).

[0041] In the above preparation method, as a preferred embodiment, in the first solution, the volume ratio of the fluorescently labeled composite sgRNA solution to the binding buffer is (0.8-1.2):4 (e.g., 0.9:4, 1:4, 1.1:4); preferably, in the fluorescently labeled composite sgRNA solution, the concentration of the fluorescently labeled composite sgRNA is 50-300 ng / μL (e.g., 60 ng / μL, 70 ng / μL, 80 ng / μL, 90 ng / μL, 100 ng / μL, 150 ng / μL, 200 ng / μL, 250 ng / μL).

[0042] In the above preparation method, as a preferred embodiment, in the second solution, the volume ratio of the Cas enzyme protein solution to the binding buffer is (0.8~1.2):4 (for example, 0.9:4, 1:4, 1.1:4); preferably, the mass concentration of the Cas enzyme protein solution is 2~40nM.

[0043] In the above preparation method, as a preferred embodiment, in step S2, the molar ratio of the fluorescently labeled composite sgRNA to the Cas enzyme protein in the reaction system is (1 to 5): 1 (for example, 2:1, 3:1, 4:1).

[0044] Compared with the prior art, the present invention has at least the following beneficial effects:

[0045] Compared with the existing technology, the present invention has more than 10 times the advantages in the cost and scale of composite sgRNA preparation, which is specifically reflected in:

[0046] 1. The guide RNA sequence complementary to the target nucleic acid sequence is a region specific to each composite sgRNA. It is located in the first 18 to 24 nucleotides of the first fragment of the composite sgRNA (abbreviated as sgRNA P1) and is a variable item in the production of each customized composite sgRNA.

[0047] 2. The second segment of the composite sgRNA (sgRNA P2) is a universal sequence that can be synthesized in large quantities and reused repeatedly, reducing costs. All composite sgRNA molecules required by the Cas9 protein can be synthesized based on the same universal sgRNA P2.

[0048] 3. Hulu's nucleotide sequence is also a universal sequence, which can be synthesized in large quantities and reused repeatedly to reduce costs.

[0049] 4. For DNA FISH (fluorescently labeled composite sgRNA) applications, multiple different composite sgRNAs (with different guide RNA sequences and / or Hulu) can be synthesized and purified in the same reaction system. The preparation method of the present invention produces composite sgRNAs. The time and material costs of simultaneously producing multiple different sgRNAs in the same reaction system are similar to those of synthesizing a single composite sgRNA, thereby improving the synthesis efficiency of composite sgRNAs and saving costs.

[0050] 5. After the short fragments of sgRNA P1, sgRNA P2 and Hulu enzyme were used to synthesize the composite sgRNA, the mutation rate of each nucleic acid segment was greatly reduced due to the reduction in length. The mutation rate did not increase after the three were compositely spliced, so the overall mutation rate of the synthesized composite sgRNA was also lower than that of the directly chemically synthesized composite sgRNA.

[0051] 6. The preparation efficiency is high. The yield of fluorescently labeled composite sgRNA prepared by the method of the present invention can reach 50%, while the yield of fluorescently labeled composite sgRNA synthesized purely chemically is less than 1%. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 is a schematic diagram of the enzymatic synthesis of fluorescently labeled composite sgRNA (abbreviated as sgRNA-Hulu), wherein sgRNA P1 (the first fragment of composite sgRNA) and sgRNA P2 (the second fragment of composite sgRNA) are chemically synthesized RNA fragments, each of which is no more than 70 bp in length. Hulu is a fluorescently labeled DNA oligonucleotide, which is connected by T4 DNA ligase or T4 RNA ligase 2 with the help of splint molecules (the first and second splint oligonucleotides). sgRNA P1 and sgRNA P2 can form a double-stranded structure with the first splint oligonucleotide, and sgRNAP2 and Hulu can form a double-stranded structure with the second splint oligonucleotide. The splint molecules can promote the connection of the connection sites.

[0053] Figure 2 is a schematic diagram of CRISPR FISH with dCas9 and fluorescently labeled composite sgRNA, i.e., the binding of the ribonucleoprotein complex dCas9-sgRNA-Hulu to the target sequence.

[0054] Figure 3 shows the CRISPR FISH (ribonucleoprotein complex dCas9-sgRNA) prepared in Example 1 Telomere -Hulu) Human telomere imaging results, the cell nucleus is counterstained by DA PI (blue), and the human telomere target sequence is stained by dCas9-sgRNA Telomere -Hulu tinting.

[0055] Figure 4 shows the test results of the biological activity (gene editing activity) of the ribonucleoprotein complex prepared by the method of the present invention in Example 2. The test used T7EI to analyze mutations. In the figure, the leftmost lane is a 1 kb marker (ThermoScientific's GeneRuler 1 Kbplus), lane 1 is a positive control (proving that the kit is working properly), lane 2 is a negative control (no gene editing), and lane 3 is a 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. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the specific embodiments or embodiments of the present invention will be clearly and completely described below in conjunction with the specific embodiments or examples of the present invention. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention.

[0057] The embodiments of the present invention are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and processes are given. However, the protection scope of the present invention is not limited to the following embodiments. The process parameters in the following embodiments that do not specify specific conditions are generally based on conventional conditions.

[0058] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to form one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed in the present invention.

[0059] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies. The quantitative tests in the following examples were performed in triplicate, and the results were averaged.

[0060] Unless otherwise defined, all technical terms, symbols and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those skilled in the art to which this application belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and such definitions contained herein are not necessarily to be construed as representing a substantial difference from what is generally understood in the art.

[0061] The sgRNA used alone in the following examples refers to the targeting sgRNA not linked to Hulu, including or excluding the linker sequence. For fluorescently labeled composite sgRNA (referred to as composite sgRNA), the full name is used in the following content, or it is represented by sgRNA-Hulu.

[0062] "Hulu" is a DNA oligonucleotide labeled with at least one fluorescent group.

[0063] In the present invention, the Hulu nucleotide sequence was meticulously designed and modeled using the UNAfold (http: / / www.unafold.org / ) and RNAfold (http: / / rna.tbi.univie.ac.at / / cgi-bin / RNAWebSuite / RNAfold.cgi) web servers. The design principle is to use bases H (H for A, C, and T), B (H for C, G, and T), V (H for A, C, and G), or D (H for A, G, and T) in 80-100% of Hulu's nucleotides to avoid forming strong secondary structures with the Hulu sequence itself or with the sgRNA P1 or sgRNA P2 sequences. A large number of Hulu sequences of the desired length were randomly generated by computer. These sequences generally lacked one of the bases A, T, G, or C. Secondary structures were predicted using the UNAfold software, and sequences were screened for those with a Gibbs free energy of secondary structure formation greater than -10 kJ / mol, preferably close to or greater than 0 kJ / mol. Finally, Blast software was used to confirm that the Hulu sequence had no obvious homologous sequences with the genome and transcriptome sequences of the target species, and the homology was less than 80%.

[0064] 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 by conventional methods in the art.

[0065] "Targeting sgRNA" is an sgRNA that does not include a linker sequence and Hulu, and includes a guide RNA sequence complementary to the target nucleic acid sequence and a backbone sequence, wherein the 3' end of the guide RNA sequence is connected to the 5' end of the backbone sequence. In the present invention, for the purpose of distinguishing between targeting sgRNAs targeting different genes, the name of the gene targeted is used as a subscript and marked on the sgRNA, for example, sgRNA TRAC represents the targeting sgRNA targeting the TRAC gene.

[0066] "Compound sgRNA" is a fluorescently labeled sgRNA, also known as DNA FISH, which includes a targeting sgRNA, Hulu, and a linker sequence. The linker sequence is used to connect the targeting sgRNA and Hulu. In the present invention, the compound sgRNAs targeting different genes are involved. In order to distinguish them, the names of the genes they target are marked as subscripts on the sgRNAs, for example, sgRNA TRAC -Hulu indicates the composite sgRNA targeting the TRAC gene.

[0067] The "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.

[0068] "Ribonucleoprotein complex", also known as CRISPR FISH, is obtained by combining the Cas enzyme with a fluorescently labeled composite sgRNA. For example, the ribonucleoprotein complex formed by combining dCas9 with the composite sgRNA is represented by dCas9-sgRNA-Hulu.

[0069] A specific embodiment of the present invention proposes a novel method for connecting sgRNA to multiple fluorescent dyes, wherein the sgRNA is divided into two segments, each of which is chemically synthesized, and a linker sequence for connection to Hulu is added to the 3' end of the sgRNA. The first composite sgRNA segment (abbreviated as sgRNA P1), the second composite sgRNA segment (abbreviated as sgRNA P2), and a Hulu oligonucleotide labeled with a fluorescent group are then ligated using T4 DNA ligase or T4 RNA ligase 2 to synthesize a fluorescently labeled sgRNA. sgRNA P1 and sgRNA P2 are chemically synthesized and purified by HPLC.

[0070] Fluorescently labeled composite sgRNAs are chimeras of DNA and RNA that can be used for a wider range of CRISPR applications, such as genome editing, diagnostics, and genomic locus imaging. In a specific example, we demonstrated that fluorescently labeled sgRNA chimeras can be used to image human telomeres. The linkage described in the present invention can be direct or indirect via other sequences.

[0071] The method of the present invention for synthesizing a composite sgRNA (sgRNA-Hulu) by enzyme ligation and further synthesizing a ribonucleoprotein complex (dCas9-sgRNA-Hulu) comprises the following steps in sequence:

[0072] Step S1, synthesis of the fluorescently labeled composite sgRNA: the chemically synthesized composite sgRNA first fragment, the composite sgRNA second fragment (SEQ ID NO: 3), the first splint oligonucleotide (SEQ ID NO: 9), the second splint oligonucleotide (SEQ ID NO: 8) and the Hulu (SEQ ID NO: 13) were mixed using T4 DNA ligase or T4 RNA ligase 2 to form an enzyme ligation system;

[0073] When using T4 DNA ligase, the enzyme ligation system is as follows:

[0074] T4 DNA ligase buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT (pH 7.5 @ 25°C)) contains the following reactants and enzymes (all concentrations are final):

[0075] 1.5 μM composite sgRNA first fragment (sgRNA P1),

[0076] 1.5 μM composite sgRNA first fragment (sgRNA P2),

[0077] 1.5 μM Hulu,

[0078] 1.5 μM first splint oligonucleotide,

[0079] 1.5 μM second splint oligonucleotide,

[0080] 1.5 μM T4 DNA ligase;

[0081] When using T4 RNA ligase 2, the enzyme ligation system is as follows:

[0082] T4 RNA Ligase 2 buffer (50 mM Tris-HCl, 2 mM MgCl2, 1 mM DTT, 400 μM ATP, (pH 7.5 @ 25°C)) contains the following reactants and enzymes (all concentrations are final):

[0083] 1.5 μM composite sgRNA first fragment (sgRNA P1),

[0084] 1.5 μM composite sgRNA first fragment (sgRNA P2),

[0085] 1.5 μM Hulu,

[0086] 1.5 μM first splint oligonucleotide,

[0087] 1.5 μM second splint oligonucleotide,

[0088] 0.15 μM T4 RNA ligase 2;

[0089] The enzyme ligation reaction temperature is 35-38°C and the enzyme ligation time is 0.5-6 h. The enzyme ligation product is purified by PAGE gel or HPLC to obtain a fluorescently labeled composite sgRNA solution. The concentration of the fluorescently labeled composite sgRNA solution is quantitatively adjusted to 50-300 ng / μL using Nanodrop. When adjusting the concentration, a 1:100 diluted NEB RNase inhibitor (RNase inhibitor product model: M0314) is added to prevent RNase contamination.

[0090] In the reactants used in step S1, the nucleotide sequences of the second fragment of the composite sgRNA, the first splint oligonucleotide, the second splint oligonucleotide and Hulu are all universal sequences and can be repeatedly used when preparing composite sgRNAs targeting different genes. Preferably, the nucleotide sequence of the second fragment of the composite sgRNA is shown in the sequence listing SEQ ID NO: 3, the nucleotide sequence of the first splint oligonucleotide is shown in the sequence listing SEQ ID NO: 9, the nucleotide sequence of the second splint oligonucleotide is shown in the sequence listing SEQ ID NO: 8, and the nucleotide sequence of Hulu is shown in the sequence listing SEQ ID NO: 13.

[0091] In a specific embodiment of the present invention, when preparing a composite sgRNA targeting human telomeric DNA, the nucleotide sequence of the first fragment of the composite sgRNA used is shown in the sequence listing SEQ ID NO: 1; when preparing a composite sgRNA targeting the first exon of the TRAC gene, the nucleotide sequence of the first fragment of the composite sgRNA used is shown in the sequence listing SEQ ID NO: 2.

[0092] Step S2, according to a volume ratio, the fluorescently labeled composite sgRNA solution: binding buffer is mixed at a ratio of (0.8-1.2):4 to prepare a first solution, wherein the binding buffer comprises: HEPES with a final concentration of 18-22 mM, mMKCl with a final concentration of 90-110 mM, MgCl2 with a final concentration of 4-6 mM, glycerol with a final concentration of 4-6% by volume, Tween-20 with a final concentration of 0.08-0.12% by volume, and bovine serum albumin with a final concentration of 0.08-0.12 mg / ml by mass volume ratio;

[0093] A Cas enzyme protein solution with a mass concentration of 2 to 40 nM is mixed with a volume ratio of Cas enzyme protein solution to binding buffer of (0.8 to 1.2):4 to prepare a second solution;

[0094] After the first solution and the second solution are mixed and reacted (the molar ratio of the composite sgRNA to the Cas enzyme protein in the reaction system is 1 to 5:1), the ribonucleoprotein complex is obtained.

[0095] Table 1

[0096] The binding buffer used in the present invention comprises: HEPES at a final concentration of 20 mM, KCl at a final concentration of 100 mM, MgCl2 at a final concentration of 5% by volume, glycerol at a final concentration of 5% by volume, Tween-20 at a final concentration of 0.1% by volume, and bovine serum albumin at a final concentration of 0.1 mg / ml by mass / volume. The pH of HEPES is 7.5.

[0097] Example 1

[0098] We tested CRISPR-FISH on human telomeres in Hela cells. A targeting sgRNA targeting human telomeric DNA was linked to Hulu containing three Atto565 dyes to form a composite sgRNA (sgRNA-Hulu). The Hulu sequence was divided into three segments, as shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, each labeled with an Atto565 dye. In Figure 3, a guide RNA sequence targeting human telomeric DNA was designed based on 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 to the telomeric region, achieving capture of the target sequence.

[0099] 1. Synthesis of fluorescently labeled composite sgRNA (sgRNA-Hulu):

[0100] In this example, chemically synthesized sgRNA P1 was 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: 13; the first splint oligonucleotide, whose nucleotide sequence is shown in SEQ ID NO: 9; and the second splint oligonucleotide, whose nucleotide sequence is shown in SEQ ID NO: 8.

[0101] Enzyme-linked system: 1.5 μM sgRNA P1 (sgRNA first fragment)

[0102] 1.5 μM sgRNA P2 (sgRNA second fragment)

[0103] 1.5μM Hulu,

[0104] 1.5 μM first splint oligonucleotide

[0105] 1.5 μM second splint oligonucleotide

[0106] 0.15 μM T4 RNA ligase 2 (commercially available from NEB)

[0107] The enzyme was ligated 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 400μM, (pH7.5@25℃)) and reacted at 37℃ for 30 minutes. The enzyme ligation reaction product was then purified by urea PAGE gel to obtain the fluorescently labeled composite sgRNA after ligation at a concentration of 150-300ng / μL. Fluorescently labeled composite sgRNA (sgRNA Telomere -Hulu) was 50% (yield = product mass / sum of raw material mass × 100%).

[0108] 2. Ribonucleoprotein complex dCas9-sgRNA Telomere -Hulu Preparation

[0109] The sgRNA prepared in this example Telomere -Hulu follows sgRNA Telomere -Hulu was mixed with binding buffer in a volume ratio of 1:4 to prepare a first solution; dCas9 protein solution (10 nM, commercially available from IDTDNA) was mixed with binding buffer in a volume ratio of 1:4 to prepare a second solution.

[0110] The first solution and the second solution were mixed and incubated at room temperature for 10 minutes. The molar ratio of sgRNA to dCas9 protein in the reaction system was 1:1.

[0111] dCas9-sgRNA prepared in this example Telomere -Hulu is shown in Figure 2. Endonuclease-inactivated Cas9 (dCas9) is combined with a composite sgRNA labeled with a fluorescent dye.

[0112] 3. Staining of Human Telomeres

[0113] (1) HeLa cells were fixed on coverslips with cold fixative (methanol:acetic acid = 3:1 (v / v), stored at -20°C) for 10 minutes and stored at -20°C or used immediately.

[0114] (2) The fixed HeLa cells were washed with 1× PBS containing 0.5% Triton X-100 at room temperature (25°C) for 15 minutes, and then washed again with binding buffer, 1 mM dithiothreitol (DTT), and 0.4 U / μL mouse RNase inhibitor (NEB, M0314).

[0115] (3) At room temperature, dCas9-sgRNA containing 20 nM ribonucleoprotein complex was added. Telomere -Hulu(dCas9:sgRNA Telomere -Hulu=1:1) binding buffer was added to the Hela cells washed with the binding buffer in step (2) and reconstituted for 30 minutes.

[0116] (4) After the recombination is completed, the Hela cells are washed with the binding buffer 3 times, each time for 5 minutes, and then washed with 1×PBS 2 times, each time for 5 minutes, to remove the ribonucleoprotein complex dCas9-sgRNA that has not been recombined with the cells. Telomere The cells were washed with PBS containing 5 μg / ml DAPI for 5 min (for nucleus staining). The stained cells were mounted in VectorShield HardSet (Vector Laboratories, H-1400) and imaged under a Leica TCS SP8 confocal microscope. The imaging results are shown in Figure 3 below. Telomere The Hulu ribonucleoprotein complex binds to its specific target region in genomic DNA. Under a microscope, these bound complexes appear as a bright spot due to their close proximity.

[0117] Example 2

[0118] This example tests the gene editing activity of the composite sgRNA synthesized by enzyme ligation in vivo in Hela cells. Based on the designed sgRNA sequence for the human TRAC gene, a composite sgRNA containing Hulu (sgRNA TRAC -Hulu) and targeting sgRNA without Hulu (sgRNA TRAC ) sequences (two identical parallel control groups were set up in each group), and enzyme ligation was performed in the same manner as in Example 1. The fluorescent dye connected to each group of Hulu was also the same as in Example 1.

[0119] 1. Synthesis of sgRNA targeting human TRAC gene (targeting the first exon of TRAC gene):

[0120] In this example, two groups of sgRNAs targeting human TRAC gene were synthesized, namely sgRNA TRAC (nucleotide sequence shown in SEQ ID NO: 19) and sgRNA TRAC -Hulu (nucleotide sequence shown in SEQ ID NO: 11).

[0121] Among them, synthetic sgRNA TRAC sgRNA TRAC The nucleotide sequence of sgRNA P1 used by Hulu is shown in SEQ ID NO: 2;

[0122] Synthetic sgRNA TRAC The nucleotide sequence of the sgRNA P2 used is shown in SEQ ID NO: 4. TRAC The nucleotide sequence of sgRNAP2 used by Hulu is shown in SEQ ID NO: 3;

[0123] Synthetic sgRNA TRAC - The nucleotide sequence of the Hulu segment of Hulu is shown in SEQ ID NO: 13;

[0124] Synthetic sgRNA TRAC and sgRNA TRAC - the first splint oligonucleotide used by Hulu, the nucleotide sequence of which is shown in SEQ ID NO: 9;

[0125] Synthetic sgRNA TRAC Synthesize sgRNA without the need for a second splint oligonucleotide TRAC - The second splint oligonucleotide used by Hulu, the nucleotide sequence of which is shown in SEQ ID NO: 8;

[0126] The enzyme ligation was performed according to the enzyme ligation system and method in Example 1 to obtain sgRNA. TRAC (SEQ ID NO: 19), sgRNA TRAC -Hulu (SEQ ID NO: 11).

[0127] 2. Preparation of Ribonucleoprotein Complex

[0128] Take the sgRNA prepared in this example TRAC sgRNA TRAC -Hulu, and Cas9 protein (10nM, commercial brand: IDTDNA), according to the method in Example 1, respectively complexed to Cas9-sgRNA TRAC , Cas9-sgRNA TRAC -Hulu.

[0129] 3. Detection of gene editing activity of ribonucleoprotein complexes

[0130] Cas9-sgRNA prepared in this example TRAC , Cas9-sgRNA TRAC -Hulu tested its TRAC gene editing activity in Hela cells according to the method in the literature (CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells[J]. Cell Research: English Edition, 2017, 27(1):4.).

[0131] Three days after transfection of the ribonucleoprotein complex into HeLa cells, genomic DNA was collected and subjected to T7EI mutation analysis targeting the TRAC edited region. Genomic DNA was extracted and amplified using a primer pair specific for the first exon of TRAC (SEQ ID NO:17, SEQ ID NO:18), followed by T7EI analysis. The results are shown in Figure 4.

[0132] In Figure 4, lane 1 is the positive control of the kit (proving that the kit works properly); lane 2 is the negative control, Hela cell genomic DNA control 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, lane 6 is Cas9-sgRNA TRAC -Hulu group 2. As can be seen from Figure 4, there are cut bands in lanes 3-6, and the results show that the Hulu marker (Cas9-sgRNATRAC -Hulu) and sgRNA without Hulu label (Cas9-sgRNA TRAC ) can edit the TRAC gene and induce mutations in Hela cells, demonstrating that the sgRNAs with or without Hulu produced by our enzymatic synthesis method are both biologically active.

[0133] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent replacements for some of the technical features therein. However, any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A fluorescently labeled composite sgRNA, characterized in that The fluorescently labeled composite sgRNA includes a targeting sgRNA specific to a target nucleic acid sequence, a Hulu connected to the targeting sgRNA, and a linker sequence for connecting the targeting sgRNA and the Hulu, wherein the Hulu is a DNA oligonucleotide labeled with at least one fluorescent group; The targeting sgRNA includes a guide RNA sequence complementary to the target nucleic acid sequence and a backbone sequence, wherein 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 via a linker sequence, wherein 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, and the gibbs free energy of the secondary structure formed by the nucleotide sequence of the Hulu is greater than -10 kJ / mol; and 80-100% of the nucleotide bases in the Hulu are H, B, V, or D; Base H is selected from adenine, cytosine and thymine; base B is selected from cytosine, guanine and thymine; base V is selected from adenine, cytosine and guanine; base D is selected from adenine, guanine and thymine; The target nucleotide sequence complementary to the guide RNA sequence is located in a gene where the homologous sequence of Hulu does not exist in the genome or transcriptome sequence of the gene. The homologous sequence of Hulu refers to a sequence with a homology of more than 80% with Hulu.

2. The fluorescently labeled composite sgRNA according to claim 1, wherein The linker sequence is shown in SEQ ID NO: 12 in the sequence listing; and / or, the nucleotide length of Hulu is 5 to 450 bp, preferably 10 to 135 bp; And / or, the backbone sequence is shown in SEQ ID NO: 16; And / or, the fluorescent group is selected from one or more of Atto series dyes, Alexa series dyes, Cy series dyes, CF series dyes, Dylight series dyes, Abberior series dyes, FAM, ROX, TAMRA, FITC, Rodamine, JF series dyes, etc.

3. The fluorescently labeled composite sgRNA according to claim 1 or 2, wherein 80-100% of the nucleotides in Hulu have bases of H, and the ratio of the number of adenine, cytosine, and thymine in H is (0.1-10):(0.1-10):(0.1-10); Alternatively, 80-100% of the nucleotides in Hulu have bases B, and the ratio of cytosine, guanine, and thymine in B is (0.1-10):(0.1-10):(0.1-10); Alternatively, 80-100% of the nucleotides in Hulu have bases of V, and the ratio of the number of adenine, cytosine, and guanine in V is (0.1-10):(0.1-10):(0.1-10); Alternatively, 80-100% of the nucleotides in Hulu have bases of D, and the ratio of the numbers of adenine, guanine, and thymine in D is (0.1-10):(0.1-10):(0.1-10).

4. The fluorescently labeled composite sgRNA according to any one of claims 1 to 3, wherein The fluorescent group is connected to the Hulu through the base in the Hulu nucleotide sequence; the number of nucleotides in the Hulu is determined according to the number of fluorescent groups, and when the number of fluorescent groups increases, the length of the Hulu increases; preferably, one fluorescent group is marked in every 10bp-20bp in the Hulu.

5. The fluorescently labeled composite sgRNA according to any one of claims 1 to 4, wherein The composite sgRNA targets human telomeric DNA, the guide RNA sequence complementary to the target nucleic acid sequence in the composite sgRNA is shown in the sequence listing SEQ ID NO: 14, and the sequence of Hulu in the composite sgRNA is shown in the sequence listing SEQ ID NO: 13; preferably, the nucleotide sequence of the composite sgRNA is shown in the sequence listing SEQ ID NO:

10.

6. The fluorescently labeled composite sgRNA according to any one of claims 1 to 4, wherein The composite sgRNA targets the first exon of the TRAC gene, the guide RNA sequence complementary to the target nucleic acid sequence in the composite sgRNA is shown in the sequence listing SEQ ID NO: 15, and the sequence of Hulu in the composite sgRNA is shown in the sequence listing SEQ ID NO: 13; preferably, the nucleotide sequence of the composite sgRNA is shown in the sequence listing SEQ ID NO:

11.

7. A ribonucleoprotein complex, characterized in that The ribonucleoprotein complex includes: a Cas enzyme, and a fluorescently labeled composite sgRNA according to any one of claims 1 to 6, which is complexed with the Cas enzyme.

8. The ribonucleoprotein complex according to claim 7, wherein The Cas enzyme is dCas9, Cas9 Nickase or CasX; preferably, the CasX is Cas9, Cas12, or Cas13.

9. The method for preparing the ribonucleoprotein complex according to claim 7 or 8, comprising the following steps in sequence: S1. Synthesis of the fluorescently labeled composite sgRNA: The chemically synthesized composite sgRNA first fragment, the composite sgRNA second fragment, and the Hulu are mixed using T4 DNA ligase or T4 RNA ligase 2 to form an enzyme ligation system, and then an enzyme ligation reaction is performed. The enzyme ligation product is purified to obtain a fluorescently labeled composite sgRNA solution; S2. Add the fluorescently labeled composite sgRNA solution to a binding buffer to prepare a first solution; The Cas enzyme protein solution is added to the binding buffer to dissolve and prepare a second solution; After the first solution and the second solution are mixed and reacted, the ribonucleoprotein complex is obtained; The sizes of the composite sgRNA first fragment and the composite sgRNA second fragment are both 40 to 70 bp, and the composite sgRNA first fragment includes the guide RNA sequence and part of the backbone sequence; wherein the guide RNA sequence is 18 to 24 nucleotides from the 5' end of the composite sgRNA first fragment; the composite sgRNA second fragment includes the remaining backbone sequence and the linker sequence.

10. The preparation method according to claim 9, characterized in that In step S1, the enzyme ligation system further includes a first splint oligonucleotide for assisting in the ligation of the first segment of the composite sgRNA and the second segment of the composite sgRNA, and a second splint oligonucleotide for assisting in the ligation of the second segment of the composite sgRNA and Hulu; The length of the first splint oligonucleotide and the second splint oligonucleotide are both 20 to 100 nucleotides, wherein 30 to 70% of the nucleotides from the 3' end of the first splint oligonucleotide are completely complementary to the sequence from the 3' end of the first segment of the composite sgRNA, and except for the nucleotides complementary to the sequence from the 3' end of the first segment of the composite sgRNA, the remaining nucleotides in the first splint oligonucleotide are completely complementary to the sequence from the 5' end of the second segment of the composite sgRNA; 30-70% of the nucleotides from the 3' end of the second splint oligonucleotide are completely complementary to the sequence from the 3' end of the second segment of the composite sgRNA, and except for the nucleotides complementary to the sequence 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 from the 5' end of Hulu; Preferably, the sequence of the first splint oligonucleotide is shown in SEQ ID NO: 9, and the sequence of the second splint oligonucleotide is shown in SEQ ID NO: 8; Preferably, the first segment of the composite sgRNA, the second segment of the composite sgRNA and the 3' end of Hulu All are hydroxyl groups, and the 5' ends are phosphorylated; Preferably, the composite sgRNA targets human telomeric DNA, and in the composite sgRNA, the RNA sequence of the first fragment of the composite sgRNA is shown as SEQ ID NO: 1, and the RNA sequence of the second fragment of the composite sgRNA is shown as SEQ ID NO: 3; the complete nucleotide sequence of the composite sgRNA is shown as SEQ ID NO: 10 in the sequence listing; Preferably, the composite sgRNA targets the first exon of the TRAC gene. In the composite sgRNA, the RNA sequence of the first fragment of the composite sgRNA is shown as SEQ ID NO: 2, and the RNA sequence of the second fragment of the composite sgRNA is shown as SEQ ID NO: 3; the complete nucleotide sequence of the composite sgRNA is shown as SEQ ID NO: 11 in the sequence listing; Preferably, the binding buffer comprises: HEPES with a final concentration of 18 to 22 mM, KCl with a final concentration of 90 to 110 mM, MgCl2 with a final concentration of 4 to 6 mM, glycerol with a final concentration of 4 to 6% by volume, Tween-20 with a final concentration of 0.08 to 0.12% by volume, and bovine serum albumin with a final concentration of 0.08 to 0.12 mg / ml by mass volume; Preferably, the binding buffer comprises: HEPES with a final concentration of 20 mM, KCl with a final concentration of 100 mM, MgCl2 with a final concentration of 5% by volume of glycerol, Tween-20 with a final concentration of 0.1% by volume, and bovine serum albumin with a final concentration of 0.1 mg / ml by mass volume ratio; Preferably, the pH of the HEPES buffer used to prepare the binding buffer is 7 to 8, more preferably 7.5; Preferably, in step S1, the purification is performed by using PAGE gel or HPLC to obtain the fluorescently labeled composite sgRNA solution; Preferably, in step S1, the temperature of the enzymatic ligation reaction is 35-38°C, and the enzymatic ligation time is 0.5-6h; Preferably, in step S1, in the enzyme ligation system, the molar ratio of the total amount of the composite sgRNA first fragment and the composite sgRNA second fragment to T4 RNA ligase 2 is (5-100):1, or in the enzyme ligation system, the molar ratio of the total amount of the composite sgRNA first fragment and the composite sgRNA second fragment to T4 DNA ligase is (0.5-5):1; Preferably, in the first solution, the volume ratio of the fluorescently labeled composite sgRNA solution to the binding buffer is (0.8-1.2):4; Preferably, in the fluorescently labeled composite sgRNA solution, the concentration of the fluorescently labeled composite sgRNA is 50 to 300 ng / μL; Preferably, in the second solution, the volume ratio of Cas enzyme protein solution to binding buffer is (0.8～1.2)：4； Preferably, the mass concentration of the Cas enzyme protein solution is 2 to 40 nM; Preferably, in step S2, the molar ratio of the fluorescently labeled composite sgRNA to the Cas enzyme protein in the reaction system is (1-5):1.

Images