CN118620975A - A universal method for preparing circular single-stranded DNA - Google Patents

Abstract

The invention discloses a method for preparing circular single-stranded DNA, comprising: providing a circular double-stranded DNA molecule as a template; mixing Cas9 nickase, sgRNA and the circular double-stranded DNA to obtain an initial reaction system, introducing a nick on any one chain of the double-stranded DNA molecule, and the sgRNA targets the cleavage site on any one chain of the circular double-stranded DNA molecule; allowing the Cas9 nickase and sgRNA complex to separate from the circular double-stranded DNA molecule; mixing a nuclease exonuclease with the circular double-stranded DNA molecule with a nick introduced on one chain to obtain a digestion reaction system, and reacting to obtain circular single-stranded DNA. The method for preparing the circular single-stranded DNA of the invention has universality, no sequence restriction, simple operation, and good application prospects.

Description

A universal method for preparing circular single-stranded DNA

Technical Field

The invention relates to the technical field of DNA synthesis, and in particular to a universal method for preparing circular single-stranded DNA.

Background Art

Circular single-strand DNA (CssDNA) is a type of nucleic acid molecule with a covalently closed loop topological structure. Compared with linear nucleic acids, it has higher resistance to exonuclease shearing and thermodynamic stability, and is often used as a carrier for drug delivery or gene editing donors. In addition, CssDNA also has abundant molecular recognition sites and is often used for the detection and imaging of biomarkers. At present, many methods for preparing CssDNA have been developed, such as splint chain-assisted cyclization, secondary structure-assisted cyclization, and phage biosynthesis. These methods have special requirements for DNA sequences, and the preparation process is complicated. In particular, as the length of the prepared sequence increases, the difficulty of preparation also increases, which in turn affects the biomedical applications based on CssDNA. Therefore, it is urgent to develop a method for preparing circular single-stranded DNA that is universal and easy to operate.

Summary of the invention

The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the object of the present invention is to provide a universal method for preparing circular single-stranded DNA.

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

The present invention provides a method for preparing circular single-stranded DNA, comprising:

Providing a circular double-stranded DNA molecule as a template;

Mixing Cas9 nickase, sgRNA and circular double-stranded DNA to obtain an initial reaction system reaction, introducing a nick on any one strand of the double-stranded DNA molecule, the sgRNA targeting the cleavage site on any one strand of the circular double-stranded DNA molecule, the number of the cleavage sites being N, where N is an integer not less than 1;

allowing the Cas9 nickase and sgRNA complex to detach from the circular double-stranded DNA molecule;

The exonuclease is mixed with a circular double-stranded DNA molecule with an incision introduced on one strand to obtain a digestion reaction system, and the reaction obtains a circular single-stranded DNA.

In one or more embodiments, the Cas9 nickase is selected from any one of Cas9 D10A, Cas9 H840A, Cas9N854A, and Cas9 N863A.

In one or more embodiments, the exonuclease is selected from at least one of the following: (a) T7 exonuclease; (b) a mixture of exonuclease I and exonuclease III.

In one or more embodiments, the step of separating the Cas9 nickase and sgRNA complex from the circular double-stranded DNA molecule comprises adding proteinase K to the initial reaction system after the reaction to carry out the reaction.

In one or more embodiments, the method further comprises the step of inactivating proteinase K.

In one or more embodiments, the number of cleavage sites N is at least 2, and the sgRNA is a sgRNA targeting different cleavage sites. When there are multiple cleavage sites, the Cas9 nickase can act to form multiple nicks, and the exonuclease can digest the single strand with a nick in the open ring structure from the multiple nicks, thereby improving the reaction efficiency.

In one or more embodiments, the concentration of the circular double-stranded DNA in the initial reaction system is 25-50 ng/μL.

In one or more embodiments, the A260/A280 of the circular double-stranded DNA is 1.7-1.9.

In one or more embodiments, the molar ratio of the sgRNA to the circular double-stranded DNA in the initial reaction system is (10N-30N): 1, and the molar concentration of the sgRNA targeting each cleavage site in the sgRNA is the same. It can be understood that when there is one cleavage site, the sgRNA is a sgRNA targeting a single cleavage site, and this case only includes one sgRNA. When there are multiple cleavage sites, the sgRNA includes all sgRNAs targeting each cleavage site, and the molar concentration of the sgRNA targeting each cleavage site in the sgRNA is the same. That is, the molar concentration of the sgRNA targeting each cleavage site is 10-30 times that of the circular double-stranded DNA. There are N cleavage sites in the reaction system, and the molar concentration of the sgRNA is 10N-30N times that of the circular double-stranded DNA. The molar ratio of the Cas9 nickase to the sgRNA is 1: (1-5).

In one or more embodiments, the initial reaction system reaction refers to incubation at 37° C. for 1-4 hours.

In one or more embodiments, the concentration of proteinase K in the reaction system after adding proteinase K is 0.2-1 mg/mL.

In one or more embodiments, the adding of proteinase K to react refers to incubation at 37° C.-50° C. for 1-4 hours.

In one or more embodiments, the step of inactivating proteinase K refers to inactivating proteinase K by heat treatment.

In one or more embodiments, the exonuclease is T7 exonuclease.

In one or more embodiments, in the digestion reaction system, the concentration of the T7 exonuclease is 300-700 U/mL.

In one or more embodiments, in the digestion reaction system, the concentration of the circular double-stranded DNA is 15-35 ng/μL.

In one or more embodiments, the reaction to obtain circular single-stranded DNA refers to incubating at 20-30° C. for 1-4 hours.

The beneficial effects of the present invention are as follows: the general method for preparing circular single-stranded DNA provided by the present invention, a gap is created on one of the single strands of the circular double-stranded DNA by Cas9 nickase to form an open-loop structure, and Cas9 nickase does not require a fixed recognition sequence, which improves the universality of the method, and then the Cas9 nickase and sgRNA complex are detached from the circular double-stranded DNA to prevent the residual Cas9 nickase from hindering the exonuclease from taking effect, and finally the single strand with a gap in the open-loop structure is digested by exonuclease, because the closed structure of the circular single strand can resist exonuclease, and finally the corresponding circular single-stranded DNA is obtained. In addition, the circular single-stranded DNA preparation method of the present invention is applicable to circular double-stranded DNA of any length and any sequence, and circular single-stranded DNA of any length and any sequence can be obtained. The circular single-stranded DNA preparation method of the present invention has universality, no sequence restriction, simple operation, and good application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram showing a general method for preparing circular single-stranded DNA provided by the present invention;

FIG2 shows the agarose gel electrophoresis characterization of the Cas9 nickase nicking four sites on the 15638 bp circular double-stranded DNA to form an open ring structure;

FIG3 shows the agarose gel electrophoresis characterization that Cas9 nickase recognizes 15638 bp circular double-stranded DNA and causes 1-4 nicks;

FIG. 4 shows the agarose gel electrophoresis characterization of the 15638 nt circular single-stranded DNA prepared by digesting the open circular structure with T7 exonuclease.

DETAILED DESCRIPTION

The present invention provides a universal method for preparing circular single-stranded DNA, comprising the following steps (schematic diagram as shown in FIG1 ):

1) Provide circular double-stranded DNA (such as plasmid) as a template;

2) Analyze the double-stranded DNA sequence and select a specific cleavage site on one of the single strands of the circular double-stranded DNA;

3) Synthesize the corresponding sgRNA according to the designed cleavage site;

4) Mix Cas9 nickase, sgRNA and circular double-stranded DNA for reaction;

5) adding an appropriate amount of proteinase K to digest the Cas9 nickase in the reaction of step 4) to remove the Cas9 nickase and sgRNA complex from the circular double-stranded DNA. After the reaction is completed, heat inactivate the proteinase K;

6) Add an appropriate amount of exonuclease to the solution after the reaction in step 5) for digestion to obtain the corresponding circular single-stranded DNA.

The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

All reagents of the present invention are commercially available. Cas9 nickase (D10A) protein was purchased from Suzhou Jinan Protein Technology Co., Ltd., T7 exonuclease was purchased from New England Biolands (NEB), 15638bp circular double-stranded DNA (pLenti-U6-gRNA-Cas9-P2A-mCherry-Hygro plasmid) was purchased from Beyotime Biotechnology Co., Ltd., proteinase K was purchased from Sangon Biotech (Shanghai) Co., Ltd., DL 15,000 DNA indicator strip was purchased from Takara Biotech (Beijing) Co., Ltd., and all DNA sequences were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

Example 1: Comparison of the effect of Cas9 nickase on nicking four different sites on a 15638 bp circular double-stranded DNA to form an open ring structure

According to the sequence information of the 15638bp circular double-stranded DNA, four specific cleavage sites were selected, and Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize the corresponding DNA sequences (SEQ ID NO.1-SEQ ID NO.5). The sequences were used to synthesize DNA templates and transcribe sgRNAs for the corresponding sites.

15k-sg-F1: 5’-TAATACGACT CACTATAGGG CAATAGCCCT CAGCAAATTG GTTTT AGAGCTAGAAATAGC AAGTT AAA-3’ (SEQ ID NO. 1);

15k-sg-F2: 5’-TAATA CGACT CACTA TAGGG TGTAG GTGGT CTTGA CCTCA GTTTTAGAGC TAGAA ATAGC AAGTT AAA-3’ (SEQ ID NO. 2);

15k-sg-F3: 5’-TAATA CGACT CACTA TAGGG GGTCG AAGTT GCTCT TGAAG GTTTTAGAGC TAGAA ATAGC AAGTT AAA-3’ (SEQ ID NO. 3);

15k-sg-F4: 5’-TAATA CGACT CACTA TAGGG CAACT AGAAT GCAGT GAAAA GTTTTAGAGC TAGAA ATAGC AAGTT AAA-3’ (SEQ ID NO. 4);

15k-sg-R: 5'-GCACC GACTC GGTGC CACTT TTTCA AGTTG ATAAC GGACT AGCCTTATTT TAACT TGCTA TTTCT AGC-3' (SEQ ID NO. 5).

Cas9 nickase, sgRNA and 15638 bp linear double-stranded DNA solution (600 ng) were mixed to a molar ratio of 10:10:1. The reaction system was 20 μL and placed at 37°C for 4 hours.

1 μL of proteinase K (20 mg/mL) was added and the mixture was reacted at 37° C. for 1 hour. After the reaction was completed, proteinase K was inactivated at 90° C. for 10 minutes.

The mixed solution after the reaction was immediately subjected to 0.8% agarose gel electrophoresis at 80 V for 1 hour.

As shown in Figure 2, circular double-stranded DNA molecules generally have three conformations: open loop, linear, and supercoiled. Theoretically, in agarose gel electrophoresis, supercoiled structures migrate the fastest, followed by linear structures, and open loops migrate the slowest. From left to right are the DL 15,000 DNA indicator band, 15638bp circular double-stranded DNA, open loop structure formed by Cas9 nickase cleavage site 1, open loop structure formed by Cas9 nickase cleavage site 2, open loop structure formed by Cas9 nickase cleavage site 3, and open loop structure formed by Cas9 nickase cleavage site 4.

Example 2: Cas9 nickase nicks 15638 bp circular double-stranded DNA, creating 1, 2, 3, and 4 nicks respectively

According to the sequence information of the 15638bp circular double-stranded DNA, four specific cleavage sites were selected, and Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize the corresponding DNA sequences (SEQ ID NO.1-SEQ ID NO.5). The sequences were used to synthesize DNA templates and transcribe sgRNAs for the corresponding sites.

Cas9 nickase, sgRNA and 15638bp linear double-stranded DNA solution (600ng) were mixed. For one cleavage site, the molar ratio of the three was 10:10:1. It should be noted that the reaction system that caused multiple cleavages contained sgRNAs corresponding to multiple sites, and the molar concentration of sgRNAs at each site was the same, that is, for two cleavage sites, the molar ratio of the three was 20:20:1, and the sgRNAs were sgRNAs corresponding to the two cleavage sites, and the two sgRNAs had the same molar concentration; for three cleavage sites, the molar ratio of the three was 30:30:1, and the sgRNAs were sgRNAs corresponding to the three cleavage sites, and the three sgRNAs had the same molar concentration; for four cleavage sites, the molar ratio of the three was 40:40:1, and the sgRNAs were sgRNAs corresponding to the four cleavage sites, and the four sgRNAs had the same molar concentration. The reaction system was 20μL and placed at 37℃ for 4 hours.

1 μL of proteinase K (20 mg/mL) was added and the mixture was reacted at 37° C. for 1 hour. After the reaction was completed, proteinase K was inactivated at 90° C. for 10 minutes.

The reaction mixture was immediately subjected to 1% agarose gel electrophoresis at 70 V for 1 hour.

As shown in Figure 3, circular double-stranded DNA molecules generally have three conformations, open loop, linear and supercoiled structures. Theoretically, in agarose gel electrophoresis, the supercoil has the fastest migration rate, followed by the linear type, and the open loop structure migrates the slowest. From left to right are the DL 15,000DNA indicator band, 15638bp circular double-stranded DNA, an open loop structure formed by 1 incision caused by Cas9 nickase, an open loop structure formed by 2 incisions caused by Cas9 nickase, an open loop structure formed by 3 incisions caused by Cas9 nickase, and an open loop structure formed by 4 incisions caused by Cas9 nickase. The results show that Cas9 nickase can introduce multiple incisions at one time on one strand of 15638bp circular double-stranded DNA. Compared with the nickase that requires a fixed sequence, this method is more universal and can cause multiple incisions at the same time, which is conducive to subsequent exonuclease digestion.

Example 3: Digestion of 15638 bp circular double-stranded DNA by T7 exonuclease to prepare 15638 nt circular single-stranded DNA and characterize it by agarose gel electrophoresis

Based on the sequence information of the 15638bp circular double-stranded DNA, Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize the corresponding DNA sequence (SEQ ID NO.3, SEQ ID NO.5), which was used to synthesize the DNA template and transcribe the sgRNA of the corresponding site.

Cas9 nickase, sgRNA and 15638 bp linear double-stranded DNA solution (600 ng) were mixed to a molar ratio of 10:10:1. The reaction system was 20 μL and placed at 37°C for 4 hours.

After the above reaction was completed, three different treatments were performed: ① Cas9 nickase was not inactivated; ② Cas9 nickase was thermally inactivated by reacting at 70°C for 15 minutes; ③ 1 μL proteinase K (20 mg/mL) was added and reacted at 37°C for 1 hour. After the reaction was completed, proteinase K was inactivated at 90°C for 10 minutes.

2 μL of T7 exonuclease (10,000 U/mL) was added to digest the open-circular structure. The reaction system was 30 μL and incubated at 25° C. for 4 hours to obtain the corresponding circular single-stranded DNA.

The reaction mixture was immediately subjected to 0.8% agarose gel electrophoresis at 85 V for 1 hour.

As shown in Figure 4, from left to right are the DL 15,000 DNA indicator band, 15638 bp circular double-stranded DNA, 15638 bp circular double-stranded DNA in an open-circular conformation, a sample digested with T7 exonuclease after not inactivating Cas9 nickase, a sample digested with T7 exonuclease after heat inactivation of Cas9 nickase, and a sample digested with T7 exonuclease after inactivation of Cas9 nickase by proteinase K. The results show that inactivation of Cas9 nickase by proteinase K is a key step of the technical solution, and a 15638 nt circular single-stranded DNA with high purity can be obtained according to the technical solution.

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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims (10)

1. A method of preparing circular single stranded DNA comprising:

Providing a circular double-stranded DNA molecule as a template;

Mixing Cas9 nickase, sgRNA and circular double-stranded DNA to obtain an initial reaction system, introducing a nick on any one strand of the double-stranded DNA molecule, wherein the sgRNA targets a cutting site on any one strand of the circular double-stranded DNA molecule, the number of the cutting sites is N, and N is an integer not less than 1;

Detaching the Cas9 nickase and sgRNA complex from the circular double stranded DNA molecule;

and mixing the exonuclease with a circular double-stranded DNA molecule with a notch introduced into one strand to obtain a digestion reaction system, and reacting to obtain circular single-stranded DNA.

2. The method of claim 1, wherein the Cas9 nickase is selected from any one of Cas 9D 10A, cas H840A, cas N854A, cas N863A.

3. The method of claim 1, wherein the exonuclease is selected from at least one of the following: (a) T7 exonuclease; (b) a mixture of exonuclease I and exonuclease III.

4. The method of claim 1, wherein the detaching the Cas9 nickase and sgRNA complex from the circular double-stranded DNA molecule comprises adding proteinase K to the initial reaction system after the reaction to perform the reaction.

5. The method of claim 4, further comprising the step of inactivating proteinase K.

6. The method of claim 1, wherein the number of cleavage sites N is at least 2 and the sgrnas are sgrnas targeting different cleavage sites.

7. The method according to claim 1, wherein the concentration of the circular double-stranded DNA in the initial reaction system is 25-50 ng/. Mu.l;

preferably, the A260/A280 of the circular double-stranded DNA is 1.7-1.9;

preferably, the molar ratio of the sgRNA to the circular double-stranded DNA in the initial reaction system is (10N-30N): 1, the molar concentration of the sgrnas in the sgrnas targeting each cleavage site is the same, the molar ratio of Cas9 nickase to the sgrnas is 1: (1-5);

Preferably, the initial reaction system reaction means incubation at 37 ℃ for 1-4 hours.

8. The method according to claim 4, wherein the concentration of proteinase K in the reaction system after proteinase K is 0.2-1mg/mL;

preferably, the reaction by adding proteinase K is performed by incubating at 37-50 ℃ for 1-4 hours.

9. The method of claim 5, wherein the step of inactivating proteinase K is heat treating the inactivated proteinase K.

10. The method of claim 1, wherein the exonuclease is a T7 exonuclease;

preferably, in the digestion reaction system, the concentration of the T7 exonuclease is 300-700U/mL;

preferably, the concentration of the circular double-stranded DNA in the digestion reaction system is 15-35 ng/. Mu.L;

Preferably, the reaction to obtain circular single stranded DNA means incubation at 20-30℃for 1-4 hours.