A method for amplifying and detecting genes of long-fragment double-stranded circular nucleic acids

Abstract

The present invention discloses a method for amplifying double-stranded circular nucleic acids, comprising: mixing and incubating a CRISPR RNP complex with a double-stranded circular nucleic acid, wherein the double-stranded circular nucleic acid includes a first strand and a second strand, wherein the sgRNA can specifically bind to a target site on the first strand, and wherein the Cas9 nickase can cleave the second strand; adding a nuclease exonuclease to digest the second strand to obtain a nicked strand; separating the CRISPR RNP complex from the first strand; and using the nicked strand as a primer and the first strand as a template, using a polymerase to extend a nucleic acid complementary to the template from the primer. The double-stranded circular nucleic acid amplification method of the present invention has strong specificity and simple operation, can obtain a long single-stranded rolling circle amplification product, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a method for amplifying and detecting genes of long-fragment double-stranded circular nucleic acids. Background Art

[0002] Rolling circle amplification (RCA) is an isothermal amplification technique based on the circular structure of DNA, capable of generating single-stranded DNA amplification products. Since its introduction in the 1990s, this technique has become an important tool in molecular biology and biotechnology, with widespread applications in genomic analysis, DNA fingerprinting, pathogen detection, and biosensors. The basic principle of RCA is to utilize the continuous synthesis activity of DNA polymerase on a single-stranded DNA template to form a stable complex. This complex is continuously extended by the polymerase to form a long single-stranded DNA strand, known as the rolling circle amplification product. RCA typically uses a circular plasmid or other closed DNA molecule containing a single-stranded DNA template as the starting material. At the beginning of the reaction, the DNA polymerase recognizes and binds to a specific primer sequence and then begins synthesizing a new DNA strand along the template. Because the template is closed, the newly synthesized DNA strand displaces the existing strand on the template, forming a displacement loop. This process continues until the entire template is covered with newly synthesized DNA. A key feature of RCA is that it can be performed under isothermal conditions, typically between 30-40°C, making it simpler than PCR, which requires temperature cycling. Furthermore, RCA's relatively short reaction time makes it suitable for rapid detection and diagnostic applications. RCA technology has a wide range of applications. In genomic analysis, RCA can be used to amplify and analyze the entire genome of organisms with small genomes or to study the expression patterns of specific genes. In pathogen detection, RCA can be used to rapidly identify and quantify microbial infections, particularly in food safety and environmental monitoring. Furthermore, RCA can be combined with fluorescent labels, radioisotopes, or other reporter molecules to develop highly sensitive biosensors and diagnostic tools. Despite its many advantages, RCA also has some limitations. For example, RCA requires high template quality; any damage or contamination of the template DNA can affect amplification efficiency. Furthermore, because RCA generates single-stranded DNA, additional steps may be required to verify the accuracy and specificity of the amplified product. Nevertheless, RCA remains a very useful tool, and its high efficiency and isothermal nature give it unique advantages in many research and applications. With the continuous improvement and optimization of technology, RCA will continue to play an important role in future bioscience research and clinical diagnosis.

[0003] There are two main rolling circle methods for circular DNA. The first is for the amplification of single-stranded circular DNA. Common methods include using splint chains to convert short linear single-stranded DNA into single-stranded circular DNA, using the splint chains as primers for rolling circle amplification, or digesting double-stranded circular DNA with nucleases to convert it into single-stranded circular DNA, and then adding primers for amplification. The second method is to add random primers to the double-stranded circular DNA for rolling circle amplification. The random primers can randomly bind to the circular DNA and initiate rolling circle amplification using one strand of the double-stranded circular DNA as a template.

[0004] The disadvantage of the first rolling circle amplification method is that it has high requirements for the amplification template and can only amplify single-stranded DNA templates. The method of using a splint chain to convert linear single-stranded DNA into circular single-stranded DNA and then performing rolling circle amplification is only suitable for forming shorter single-stranded circular DNA, usually less than 200nt in length. The circular DNA present in organisms is usually double-stranded and has a wide range of length distribution. If restriction enzymes and nucleases are used to convert double-stranded circular DNA into single-stranded circular DNA before rolling circle amplification, on the one hand, the experimental operation is complicated, and on the other hand, the conversion process is limited by the restriction enzyme binding site, and the amplification specificity is poor.

[0005] The second method uses random primers to amplify double-stranded circular DNA and is often used for the preparation of sequencing libraries. Its disadvantage is that it has no specificity for the amplification of circular DNA. All double-stranded circular DNA in the random amplification system will be amplified, and the amplified products will continue to amplify with the random primers to form hyperbranched products. Debranching treatment is required before the amplified products are used, which is complicated.

[0006] Therefore, it is of great significance to provide a new rolling circle amplification method for double-stranded circular DNA. Summary of the Invention

[0007] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention aims to provide a method for amplifying a double-stranded circular nucleic acid.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A first aspect of the present invention provides a method for amplifying a double-stranded circular nucleic acid, comprising:

[0010] The CRISPR RNP complex is mixed and incubated with a double-stranded circular nucleic acid, wherein the double-stranded circular nucleic acid includes a first strand and a second strand, the CRISPR RNP complex includes a Cas9 nickase and an sgRNA, wherein the Cas9 nickase is a Cas9 protein with unilateral cleavage activity, the sgRNA can specifically bind to the target site of the first strand, and the Cas9 nickase can cleave the second strand;

[0011] adding exonuclease to digest the second strand to obtain a nicked strand;

[0012] causing the CRISPR RNP complex to dissociate from the first strand;

[0013] The gap strand is used as a primer and the first strand is used as a template, and a polymerase is used to extend a nucleic acid complementary to the template from the primer.

[0014] In the present invention, the target site can be selected and the sgRNA can be designed according to the sequence characteristics of the double-stranded circular nucleic acid to be amplified, so the sgRNA sequence is not particularly limited.

[0015] The Cas9 nickase used in the present invention has unilateral cleavage activity. After the CRISPR RNP complex binds to the double-stranded circular nucleic acid, it will cut the second strand without cutting the first strand.

[0016] In one or more embodiments, the Cas9 nickase is Cas9 H840A.

[0017] In one or more embodiments, the exonuclease is Exonuclease I.

[0018] In one or more embodiments, the polymerase is Klenow Large Fragmentase.

[0019] The Klenow large fragment enzyme used in the present invention is an N-terminal truncation of DNA polymerase I, which retains DNA polymerase activity but loses 5'→3' exonuclease activity. Meanwhile, the 3'→5' exonuclease activity is removed through mutation.

[0020] In one or more embodiments, the CRISPR RNP complex is detached from the first strand under the action of proteinase K.

[0021] In one or more embodiments, the amplification method further comprises a proteinase K inactivation step.

[0022] In one or more embodiments, the inactivation refers to high temperature treatment inactivation, such as incubating the entire reaction system at 95° C. for 5 minutes.

[0023] In one or more embodiments, the gap length of the gap chain is 10-30 nt.

[0024] In one or more embodiments, the extension of the nucleic acid complementary to the template from the primer using a polymerase is carried out at 30° C. to 37° C. for 12 to 48 hours.

[0025] In one or more embodiments, the method for amplifying a double-stranded circular nucleic acid comprises:

[0026] Mix Cas9 nickase and sgRNA for reaction;

[0027] Add double-stranded circular nucleic acid to react;

[0028] Add exonuclease I to react;

[0029] Proteinase K was added to react;

[0030] Add dNTP and Klenow large fragment enzyme to carry out amplification reaction.

[0031] In one or more embodiments, the final concentration of Cas9 nickase is 25-75 nM.

[0032] In one or more embodiments, the final concentration of the sgRNA is 25-75 nM.

[0033] In one or more embodiments, the final concentration of the double-stranded circular nucleic acid is 2.5-7.5 nM.

[0034] In one or more embodiments, the final concentration of Exonuclease I is 0.5-1.5 U / μL.

[0035] In one or more embodiments, the final concentration of Proteinase K is 1-3 mg / mL.

[0036] In one or more embodiments, the final concentration of dNTPs is 0.5-1.5 mM.

[0037] In one or more embodiments, the final concentration of Klenow large fragment enzyme is 0.125-0.375 U / μL.

[0038] The final concentration of the present invention = the amount of reagent / the final reaction system volume, the amount of reagent can be mass, amount of substance or enzyme activity, and the final reaction system volume refers to the volume of the system for the amplification reaction. Specifically, the final concentration of Cas9 nickase = the amount of substance of Cas9 nickase / the final reaction system volume, the final concentration of sgRNA = the amount of substance of sgRNA / the final reaction system volume; the final concentration of double-stranded circular nucleic acid = the amount of substance of double-stranded circular nucleic acid / the final reaction system volume; the final concentration of nuclease I = the enzyme activity of nuclease I / the final reaction system volume; the final concentration of proteinase K = the mass of proteinase K / the final reaction system volume; the final concentration of dNTP = the amount of substance of dNTP / the final reaction system volume; the final concentration of Klenow large fragment enzyme = the enzyme activity of Klenow large fragment enzyme / the final reaction system volume.

[0039] In one or more embodiments, mixing the Cas9 nickase and the sgRNA for reaction refers to incubating at 30-37° C. for 15-25 minutes.

[0040] In one or more embodiments, adding the double-stranded circular nucleic acid to react refers to incubating at 30-37° C. for 25-35 minutes.

[0041] In one or more embodiments, adding exonuclease I to carry out the reaction means incubating at 30-37° C. for 15-25 minutes.

[0042] In one or more embodiments, adding proteinase K to react means incubating at 48-55° C. for 40-80 min.

[0043] In one or more embodiments, adding dNTPs and Klenow large fragment enzyme to perform amplification reaction refers to amplification at 30-37° C. for 12-48 hours.

[0044] A second aspect of the present invention provides a double-stranded circular nucleic acid amplification kit comprising:

[0045] Cas9 nickase, sgRNA, exonuclease, proteinase K, dNTPs, Klenow large fragment enzyme;

[0046] The sgRNA can specifically bind to the target site on one strand of the double-stranded circular nucleic acid.

[0047] In one or more embodiments, the Cas9 nickase is Cas9 H840A.

[0048] In one or more embodiments, the exonuclease is Exonuclease I.

[0049] The third aspect of the present invention provides a gene detection method, comprising amplifying a target double-stranded circular nucleic acid using the amplification method provided in the first aspect of the present invention. The gene detection method is not a diagnostic gene detection method.

[0050] The beneficial effects of the present invention are:

[0051] In the double-stranded circular nucleic acid amplification method provided by the present invention, the CRISPR-Cas9 nickase system targets circular DNA containing the target sequence, and after modification at the target site, rolling circle amplification is initiated with the target site as the starting point. Other circular DNAs that do not contain the target site will not undergo rolling circle amplification, and the site specificity is strong.

[0052] The double-stranded circular nucleic acid amplification method provided in the present invention is simple to operate and does not require the additional design of rolling circle amplification primers. After the CRISPR-Cas9 nickase system binds to the target site, the present invention uses one strand of the double-stranded circular nucleic acid as a template and the other strand as a primer to initiate rolling circle amplification, eliminating the need to design synthetic rolling circle amplification primers.

[0053] The double-stranded circular nucleic acid amplification method provided by the present invention can amplify larger circular DNA fragments and produce long single-stranded DNA products. This solves the problem that artificially synthesized circular DNA is small in size, while longer DNA circles existing in organisms are difficult to convert into single-stranded circles, making it difficult to produce long single-stranded rolling circle amplification products.

[0054] The double-stranded circular nucleic acid amplification method provided in the present invention can perform site-specific rolling circle amplification on long double-stranded circular DNA fragments including plasmids, eccDNA, mitochondrial and chloroplast DNA, and has application potential in circular DNA detection, gene editing, gene sequencing, gene transfection and drug delivery, DNA storage and information materials, DNA nanotechnology, SELEX and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Schematic diagram of the process of exonuclease I-mediated rolling circle amplification method.

[0056] Figure 2 Gel electrophoresis characterization of rolling circle amplification of a 2.7 kb double-stranded circular DNA using the exonuclease I-mediated rolling circle amplification method. Lane 1 is the 2.7 kb double-stranded circular DNA without Cas9n-sgRNA, lane 2 is the 2.7 kb double-stranded circular DNA with Cas9n-sgRNA, and lanes 3 and 4 are the rolling circle amplification products of the 2.7 kb double-stranded circular DNA. Among them, superhelix is ​​the circular DNA without Cas9 and has supercoiling, and loose circle is the circular DNA with Cas9 and has no supercoiling. Compared with the DNA with supercoiling, the position of the band has changed, proving that Cas9 successfully binds to the target site under the action of sgRNA and cuts the second chain.

[0057] Figure 3Gel electrophoresis characterization of rolling circle amplification of a 5.4 kb double-stranded circular DNA using the exonuclease I-mediated rolling circle amplification method. Lane 1 is the 5.4 kb double-stranded circular DNA without Cas9n-sgRNA, lane 2 is the 5.4 kb double-stranded circular DNA with Cas9n-sgRNA, and lane 3 is the rolling circle amplification product of the 5.4 kb double-stranded circular DNA. The superhelix is ​​the circular DNA without Cas9 and has supercoiling, and the loose circle is the circular DNA with Cas9 and has no supercoiling. Compared with the DNA with supercoiling, the position of the band has changed, proving that Cas9 successfully binds to the target site under the action of sgRNA and cuts the second chain.

[0058] Figure 4 Gel electrophoresis characterization of rolling circle amplification of a 6.4 kb double-stranded circular DNA using the exonuclease I-mediated rolling circle amplification method. Lane 1 is the 6.4 kb double-stranded circular DNA without Cas9n-sgRNA, lane 2 is the 6.4 kb double-stranded circular DNA with Cas9n-sgRNA, and lane 3 is the rolling circle amplification product of the 6.4 kb double-stranded circular DNA. The superhelix is ​​the circular DNA without Cas9 and has supercoiling, and the loose circle is the circular DNA with Cas9 and has no supercoiling. Compared with the DNA with supercoiling, the position of the band has changed, proving that Cas9 successfully binds to the target site under the action of sgRNA and cuts the second chain.

[0059] Figure 5 Gel electrophoresis characterization of rolling circle amplification of a 15kb double-stranded circular DNA using the exonuclease I-mediated rolling circle amplification method. Lane 1 is the 15kb double-stranded circular DNA without Cas9n-sgRNA, lane 2 is the 15kb double-stranded circular DNA with Cas9n-sgRNA, and lane 3 is the rolling circle amplification product of the 15kb double-stranded circular DNA. The superhelix is ​​the circular DNA without Cas9 and has supercoiling, and the loose circle is the circular DNA with Cas9 and has no supercoiling. Compared with the DNA with supercoiling, the position of the band has changed, proving that Cas9 successfully binds to the target site under the action of sgRNA and cuts the second strand.

[0060] Figure 6Gel electrophoresis characterization of rolling circle amplification of 5.4 kb double-stranded circular DNA using Phi29 DNA polymerase. Lane 1 is the 5.4 kb double-stranded circular DNA without Cas9n-sgRNA, lane 2 is the 5.4 kb double-stranded circular DNA with Cas9n-sgRNA, and lanes 4, 5, 6, and 7 are the double-stranded circular DNA with Phi29. The results of DNA polymerase rolling circle amplification at four sites on the 5.4kb double-stranded circular DNA. The starting site of the rolling circle amplification in lane 8 is the same as that in lane 7, but Klenow large fragmentase is used for rolling circle amplification. The starting site of the rolling circle amplification in lane 3 is the same as that in lane 4, but nuclease I is not used to digest the non-complementary chain of the sgRNA. Among them, superhelix is ​​the circular DNA without binding to Cas9 and has supercoiling. Loosecircle is the circular DNA with binding to Cas9 and has no supercoiling. Compared with the DNA with supercoiling, the position of the band has changed, proving that Cas9 successfully binds to the target site under the action of sgRNA and cuts the second chain. DETAILED DESCRIPTION

[0061] The present invention provides a method for site-specific rolling circle amplification of double-stranded circular DNA. The main process is to target and bind the Cas9 nickase with unilateral cutting activity to the double-stranded circular DNA, then release the non-complementary chain of the sgRNA outside the Cas9 nickase, and then use nuclease exonuclease to digest the non-complementary chain of the sgRNA. At this time, one chain of the double-stranded circular DNA has a gap of about 20 nt in length. Using the other complete circular single chain as a template and the chain that produces the gap as a primer, a polymerase can be used to start rolling circle amplification of the original double-stranded circular DNA. Figure 1 This is a schematic flow chart of the rolling circle amplification method according to an embodiment of the present invention. The specific steps of the rolling circle amplification method are as follows:

[0062] Step 1: Select the target site on the target circular DNA and design and synthesize the sgRNA required for Cas9 nickase binding based on the target site sequence information.

[0063] Step 2: Cas9 nickase and sgRNA are mixed and incubated to allow Cas9 nickase to combine with sgRNA to obtain CRISPRRNP complex (also known as Cas9n-sgRNA).

[0064] Step 3: Add the target double-stranded circular DNA to the reaction system and incubate to allow the CRISPR RNP complex to bind to the target site of the target double-stranded circular DNA.

[0065] The Cas9 nickase used in the present invention is a Cas9 protein variant (Cas9n, Cas9 H840A) in which the H840A mutation is introduced into the HNH domain. It only cuts one of the single strands in the DNA double strand. When Cas9n binds to the target site on the DNA, the non-complementary strand of the sgRNA is exposed outside the package of the Cas9n protein. The non-complementary strand of the sgRNA can be digested with a nuclease, creating a nick in the non-complementary strand of the sgRNA.

[0066] Step 4: Add exonuclease to the reaction system, incubate, and digest the non-complementary strand of sgRNA ( Figure 1 medium red chain).

[0067] Step 5: Add proteinase K to the reaction system and incubate to allow the CRISPR RNP complex to detach from the target site.

[0068] Step 6: Inactivate Proteinase K.

[0069] Step 7: After the reaction system is cooled to room temperature, dNTPs, Klenow large fragment enzyme and corresponding reaction buffer are added to start rolling circle amplification.

[0070] The polymerase used in the present invention is Klenow Large Fragmentase. Unlike the Phi29 DNA polymerase used in traditional rolling circle amplification (RCA) methods, Klenow Large Fragmentase is an N-terminal truncation of DNA polymerase I. It retains DNA polymerase activity but loses 5'→3' exonuclease activity. Furthermore, a mutation has eliminated 3'→5' exonuclease activity. Klenow Large Fragmentase is crucial to the RCA method of the present invention; using Phi29 DNA polymerase cannot achieve the same results.

[0071] The present invention is further described in detail below using specific examples. Unless otherwise specified, the raw materials, reagents, and apparatus used in the examples and comparative examples are available from conventional commercial sources or by conventional methods. Unless otherwise specified, all experimental or testing methods are conventional in the art.

[0072] The Cas9n protein used in the examples of the present invention was purchased from Jinan Protein, catalog number E367-01A; pUC19 plasmid was purchased from Sangon Biotechnology, catalog number B610005-0050; exonuclease I (ExoI) was purchased from Sangon Biotechnology, catalog number C610019-0001; proteinase K was purchased from Sangon Biotechnology, catalog number B600452-0001; Klenow large fragment enzyme was purchased from Sangon Biotechnology, catalog number B110065-0500; PhiX174 phage was purchased from NEB, catalog number N3021S; M13 phage was purchased from NEB, catalog number N4018S; pLen-ti-U6-gRNA-Cas9-P2A-mCherry-Hygro plasmid was purchased from Biyuntian, catalog number D8310.

[0073] Example 1

[0074] A 2.7 kb double-stranded circular DNA (pUC19 plasmid, purchased from Sangon Biotechnology, catalog number B610005-0050) was subjected to rolling circle amplification using the exonuclease I-mediated method. The specific steps are as follows:

[0075] Step 1: After selecting the target site based on the sequence information of the 2.7 kb double-stranded circular DNA (pUC19 plasmid), the sgRNA primers (Sg-F and Sg-R) for the Cas9n protein corresponding to the target site were synthesized from Sangon Biotech Co., Ltd., and the sgRNA for the Cas9n protein at the target site was synthesized and transcribed using the sgRNA primers.

[0076] Sg-F: 5'-TAATA CGACT CACTA TAGGG ACAGA ATCAG GGGAT AACGC GTTTT AGAGCTAGAA ATAGC AAGTT AAA-3' (SEQ ID No. 1).

[0077] Sg-R: 5'-GCACC GACTC GGTGC CACTT TTTCA AGTTG ATAAC GGACT AGCCT TATTTTAACT TGCTA TTTCT AGC-3' (SEQ ID No. 2).

[0078] Step 2: Take a 1.5mL Eppendorf tube, add Cas9n protein and sgRNA corresponding to the pUC19 double-stranded circular DNA target site (final concentration of both is 50nM), add 10× reaction buffer (the buffer provided with the Cas9n (H840A) of the nearshore protein) 2μL, and incubate at 37°C for 20min to allow the Cas9n protein to combine with the sgRNA to obtain Cas9n-sgRNA.

[0079] Step 3: Add pUC19 double-stranded circular DNA (final concentration of 5 nM) to the reaction system and incubate at 37°C for 30 min to allow Cas9n-sgRNA to bind to the target site of the target double-stranded circular DNA.

[0080] Step 4: Add exonuclease I (ExoI) (final concentration of 1 U / μL) and 10X ExoⅠBuffer (the amount added refers to the instructions for exonuclease I) to the reaction system and incubate at 37°C for 20 minutes to digest the non-complementary strand of sgRNA.

[0081] Step 5: Add proteinase K (final concentration of 2 mg / mL) to the reaction system and incubate at 52°C for 60 min to allow Cas9n-sgRNA to fall off from the target site.

[0082] Step 6: Incubate the entire reaction system at 95°C for 5 min to inactivate proteinase K.

[0083] Step 7: After the reaction system is cooled to room temperature, dNTPs (final concentration of 1 mM), Klenow large fragment enzyme (final concentration of 0.25 U / μL) and corresponding reaction buffer (the matching buffer provided with the purchased Klenow large fragment enzyme, the amount added is referred to the reagent instructions) are added to start rolling circle amplification and incubate at 30°C for 48 hours.

[0084] The reaction system for rolling circle amplification in step 7 is the final reaction system, and the final reaction system is 20 μL.

[0085] like Figure 2 Agarose gel electrophoresis was used to characterize the rolling circle amplification results of 2.7 kb double-stranded circular DNA mediated by nuclease I. Lane 1 was the 2.7 kb double-stranded circular DNA without Cas9n-sgRNA binding, lane 2 was the 2.7 kb double-stranded circular DNA bound to Cas9n-sgRNA, and lanes 3 and 4 were the rolling circle amplification products of 2.7 kb double-stranded circular DNA, indicating that the rolling circle amplification proceeded smoothly.

[0086] Example 2

[0087] Rolling circle amplification (RCA) of 5.4 kb double-stranded circular DNA (PhiX174 bacteriophage, purchased from NEB, Cat. No. N3021S) was performed using an exonuclease I-mediated method. The specific steps are as follows:

[0088] Step 1: After selecting the target site based on the sequence information of the 5.4 kb double-stranded circular DNA (PhiX174), the sgRNA primers (Sg-F and Sg-R) for the Cas9n protein corresponding to the target site were synthesized from Sangon Biotech Co., Ltd., and the sgRNA for the Cas9n protein at the target site was synthesized and transcribed using the sgRNA primers.

[0089] Sg-F: 5'-TAATA CGACT CACTA TAGGG AAGGT CATGC GGCAT ACGCT GTTTT AGAGCTAGAAATAGC AAGTT AAA-3' (SEQ ID No. 3).

[0090] Sg-R: 5'-GCACC GACTC GGTGC CACTT TTTCAAGTTG ATAAC GGACT AGCCT TATTTTAACT TGCTATTTCT AGC-3' (SEQ ID No. 4).

[0091] Step 2: Take a 1.5mL Eppendorf tube, add Cas9n protein and sgRNA corresponding to the PhiX174 double-stranded circular DNA target site (final concentration of both is 50nM), add 2μL of 10× reaction buffer, and incubate at 37°C for 20min to allow Cas9n protein to combine with sgRNA to obtain Cas9n-sgRNA.

[0092] Step 3: Add PhiX174 double-stranded circular DNA (final concentration of 5 nM) to the reaction system and incubate at 37°C for 30 minutes to allow Cas9n-sgRNA to bind to the target site of the target double-stranded circular DNA.

[0093] Step 4: Add exonuclease I (ExoI) (final concentration of 1 U / μL) and 10X ExoⅠBuffer (the amount added is referred to the reagent instructions) to the reaction system and incubate at 37°C for 20 minutes to digest the non-complementary chain of sgRNA.

[0094] Step 5: Add proteinase K (final concentration of 2 mg / mL) to the reaction system and incubate at 52°C for 60 min to allow Cas9n-sgRNA to fall off from the target site.

[0095] Step 6: Incubate the entire reaction system at 95°C for 5 min to inactivate proteinase K.

[0096] Step 7: After the reaction system is cooled to room temperature, dNTPs (final concentration of 1 mM), 1 μL Klenow large fragment enzyme (final concentration of 0.25 U / μL), and the corresponding reaction buffer (the matching buffer provided with the purchased Klenow large fragment enzyme, the amount added is referred to the reagent instructions) are added to start rolling circle amplification and incubate at 30°C for 48 hours.

[0097] The reaction system for rolling circle amplification in step 7 is the final reaction system, and the final reaction system is 20 μL.

[0098] like Figure 3 Agarose gel electrophoresis was used to characterize the rolling circle amplification results of 5.4 kb double-stranded circular DNA mediated by nuclease I. Lane 1 was the 5.4 kb double-stranded circular DNA without Cas9n-sgRNA binding, lane 2 was the 5.4 kb double-stranded circular DNA bound to Cas9n-sgRNA, and lane 3 was the rolling circle amplification product of the 5.4 kb double-stranded circular DNA, indicating that the rolling circle amplification proceeded smoothly.

[0099] Example 3

[0100] Rolling circle amplification (RCA) of 6.4 kb double-stranded circular DNA (M13 bacteriophage, purchased from NEB, Cat. No. N4018S) was performed using an exonuclease I-mediated method. The specific steps are as follows:

[0101] Step 1: After selecting the target site based on the sequence information of the 6.4 kb double-stranded circular DNA (M13 phage), the sgRNA primers (Sg-F and Sg-R) of the Cas9n protein corresponding to the target site were synthesized from Sangon Biotech Co., Ltd., and the sgRNA of the target site Cas9n protein was synthesized and transcribed using the sgRNA primers.

[0102] Sg-F: 5'-TAATACGACT CACTATAGGG CGCGATATTT GAAGT CTTTC GTTTT AGAGCTAGAAATAGC AAGTT AAA-3' (SEQ ID No. 5).

[0103] Sg-R: 5'-GCACC GACTC GGTGC CACTT TTTCAAGTTG ATAAC GGACT AGCCT TATTTTAACT TGCTATTTCT AGC-3' (SEQ ID No. 6).

[0104] Step 2: Take a 1.5mL Eppendorf tube, add Cas9n protein and sgRNA corresponding to the M13 phage double-stranded circular DNA target site (final concentration of both is 50nM), add 2μL of 10× reaction buffer, and incubate at 37°C for 20min to allow Cas9n protein to combine with sgRNA to obtain Cas9n-sgRNA.

[0105] Step 3: Add M13 phage double-stranded circular DNA (final concentration of 5 nM) to the reaction system and incubate at 37°C for 30 min to allow Cas9n-sgRNA to bind to the target site of the target double-stranded circular DNA.

[0106] Step 4: Add exonuclease I (ExoI) (final concentration of 1 U / μL) and 10X ExoⅠBuffer (the amount added is referred to the reagent instructions) to the reaction system and incubate at 37°C for 20 minutes to digest the non-complementary chain of sgRNA.

[0107] Step 5: Add proteinase K (final concentration of 2 mg / mL) to the reaction system and incubate at 52°C for 60 min to allow Cas9n-sgRNA to fall off from the target site.

[0108] Step 6: Incubate the entire reaction system at 95°C for 5 min to inactivate proteinase K.

[0109] Step 7: After the reaction system is cooled to room temperature, dNTPs (final concentration of 1 mM), Klenow large fragment enzyme (final concentration of 0.25 U / μL) and corresponding reaction buffer (the matching buffer provided with the purchased Klenow large fragment enzyme, the amount added is referred to the reagent instructions) are added to start rolling circle amplification and incubate at 30°C for 48 hours.

[0110] The reaction system for rolling circle amplification in step 7 is the final reaction system, and the final reaction system is 20 μL.

[0111] like Figure 4 Agarose gel electrophoresis was used to characterize the rolling circle amplification results of 6.4 kb double-stranded circular DNA mediated by nuclease I. Lane 1 was the 6.4 kb double-stranded circular DNA without Cas9n-sgRNA binding, lane 2 was the 6.4 kb double-stranded circular DNA bound to Cas9n-sgRNA, and lane 3 was the rolling circle amplification product of 6.4 kb double-stranded circular DNA, indicating that the rolling circle amplification proceeded smoothly.

[0112] Example 4

[0113] Rolling circle amplification (RCA) of 15 kb double-stranded circular DNA (pLen-ti-U6-gRNA-Cas9-P2A-mCherry-Hygro plasmid, purchased from Beyotime, Cat. No. D8310) was performed using an exonuclease I-mediated method. The specific steps are as follows:

[0114] Step 1: After selecting the target site based on the sequence information of the 15kb double-stranded circular DNA, the sgRNA primers (Sg-F and Sg-R) of the Cas9n protein corresponding to the target site were synthesized from Sangon Biotech Co., Ltd., and the sgRNA of the Cas9n protein of the target site was synthesized and transcribed using the sgRNA primers.

[0115] Sg-F: 5'-TAATACGACT CACTATAGGG GAGGC TAGAAGGAGAGAGAT GTTTT AGAGCTAGAAATAGC AAGTT AAA-3' (SEQ ID No. 7).

[0116] Sg-R: 5'-GCACC GACTC GGTGC CACTT TTTCA AGTTG ATAAC GGACT AGCCT TATTTTAACT TGCTA TTTCT AGC-3' (SEQ ID No. 8).

[0117] Step 2: Take a 1.5mL Eppendorf tube, add Cas9n protein and sgRNA corresponding to the 15kb double-stranded circular DNA target site (final concentration of both is 50nM), add 2μL of 10× reaction buffer, and incubate at 37°C for 20min to allow Cas9n protein to combine with sgRNA to obtain Cas9n-sgRNA.

[0118] Step 3: Add 15 kb double-stranded circular DNA (final concentration 5 nM) to the reaction system and incubate at 37°C for 30 min to allow Cas9n-sgRNA to bind to the target site of the target double-stranded circular DNA.

[0119] Step 4: Add exonuclease I (ExoI) (final concentration of 1 U / μL) and 10X ExoⅠBuffer (the amount added is referred to the reagent instructions) to the reaction system and incubate at 37°C for 20 minutes to digest the non-complementary chain of sgRNA.

[0120] Step 5: Add proteinase K (final concentration of 2 mg / mL) to the reaction system and incubate at 52°C for 60 min to allow Cas9n-sgRNA to fall off from the target site.

[0121] Step 6: Incubate the entire reaction system at 95°C for 5 min to inactivate proteinase K.

[0122] Step 7: After the reaction system is cooled to room temperature, dNTPs (final concentration of 1 mM), Klenow large fragment enzyme (final concentration of 0.25 U / μL) and corresponding reaction buffer (the matching buffer provided with the purchased Klenow large fragment enzyme, the amount added is referred to the reagent instructions) are added to start rolling circle amplification and incubate at 30°C for 48 hours.

[0123] The reaction system for rolling circle amplification in step 7 is the final reaction system, and the final reaction system is 20 μL.

[0124] like Figure 5Agarose gel electrophoresis was used to characterize the rolling circle amplification results of 15kb double-stranded circular DNA mediated by nuclease I. Lane 1 was the 15kb double-stranded circular DNA without Cas9n-sgRNA binding, lane 2 was the 15kb double-stranded circular DNA bound to Cas9n-sgRNA, and lane 3 was the rolling circle amplification product of the 15kb double-stranded circular DNA, indicating that the rolling circle amplification proceeded smoothly.

[0125] From the above four examples, it can be seen that the rolling circle amplification method introduced in the present invention has a good amplification effect on double-stranded circular DNA of different lengths, which proves the wide applicability of the present invention to double-stranded circular DNA amplification.

[0126] Comparative Example 1

[0127] In this comparative example, Phi29 DNA polymerase was used as the polymerase for the amplification reaction, and the reaction system and reaction conditions were the same as those in Example 2.

[0128] like Figure 6 Agarose gel electrophoresis was used to characterize the rolling circle amplification (RCA) of a 5.4 kb double-stranded circular DNA. Lanes 4, 5, 6, and 7 show the results of RCA performed using Phi29 DNA polymerase at four sites on the 5.4 kb double-stranded circular DNA. Compared to the control group, no RCA product was produced. Lane 8, using the same RCA initiation site as lane 7, but using Klenow large fragmentase, produced a RCA product. This indicates that Phi29 DNA polymerase cannot achieve the same effect as Klenow large fragmentase.

[0129] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for amplifying a double-stranded circular nucleic acid, comprising: The Cas9 nickase and sgRNA were mixed to react, wherein the Cas9 nickase was Cas9 H840A; Adding a double-stranded circular nucleic acid to react, the double-stranded circular nucleic acid includes a first strand and a second strand, the sgRNA can specifically bind to the target site of the first strand, and the Cas9 nickase can cleave the second strand; Add exonuclease I to react; Proteinase K was added to react; Add dNTP and Klenow large fragment enzyme to carry out amplification reaction; The final concentration of Cas9 nickase is 25-75 nM; The final concentration of sgRNA is 25-75nM; The final concentration of double-stranded circular nucleic acid is 2.5-7.5 nM; The final concentration of exonuclease I was 0.5-1.5 U / μL; The final concentration of proteinase K is 1-3 mg / mL; The final concentration of dNTP is 0.5-1.5 mM; The final concentration of Klenow large fragment enzyme was 0.125-0.375 U / μL; Mixing Cas9 nickase and sgRNA for reaction means incubating at 30-37°C for 15-25 minutes; Adding double-stranded circular nucleic acid to the reaction means incubating at 30-37°C for 25-35 minutes; Adding exonuclease I to the reaction means incubating at 30-37°C for 15-25 minutes; Adding proteinase K to the reaction means incubating at 48-55°C for 40-80 minutes; Adding dNTPs and Klenow large fragment enzyme to perform amplification reaction means amplifying at 30-37°C for 12-48 hours.

2. The amplification method according to claim 1, characterized in that The amplification method further comprises a proteinase K inactivation step.

3. A double-stranded circular nucleic acid amplification kit comprising: Cas9 nickase, sgRNA, exonuclease, proteinase K, dNTP, Klenow large fragmentase, wherein the Cas9 nickase is Cas9 H840A, and the exonuclease is exonuclease I; in, The sgRNA can specifically bind to a target site on one strand of the double-stranded circular nucleic acid.

4. A gene detection method, comprising amplifying a target double-stranded circular nucleic acid using the amplification method according to any one of claims 1 to 2.

Images