DNA fragment rapid assembly method based on programmable nuclease MfAgo

Abstract

The invention discloses a DNA fragment rapid assembly method based on programmable nuclease MfAgo, and belongs to the field of molecular biology, the nuclease MfAgo from Methanocaldococcus services is utilized, site-specific cleavage of plasmids can be achieved, and rapid assembly and cloning of cleavage fragments are achieved through T5 exonuclease. Fragment cutting can be completed without adding polymerase and other complex components, the visualization effect is good, and the assembly result can be quickly seen.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and in particular relates to a method for rapid assembly of DNA fragments based on the programmable nuclease MfAgo. Background Art

[0002] In the field of synthetic biology, large DNA fragment assembly technology, as a key supporting technology, is playing an increasingly important role. Its core goal is to accurately and efficiently splice multiple smaller DNA fragments into larger DNA molecules, which has far-reaching significance for the construction of complex biosynthetic pathways, customized genomes, and even the study of artificial life. From the perspective of application scale, the ability to assemble large DNA fragments directly determines the upper limit of artificially synthesized genomes. For example, in viral genome research, the ability to successfully assemble viral genomes of several thousand bases can help to gain a deeper understanding of the genetic mechanism and pathogenic mechanism of the virus, providing a key basis for the development of antiviral drugs and vaccine design. At the yeast genome level, assembly attempts on the scale of megabases have opened up broad space for the study of gene function in eukaryotes and bioengineering transformation.

[0003] Over the past two decades, DNA synthesis and assembly technology has achieved rapid development, and numerous assembly technologies suitable for different scales have emerged. These technologies can be roughly divided into two categories: in vivo assembly and in vitro assembly. In vitro assembly technology has certain advantages when dealing with small DNA fragments, because its operation is relatively convenient, small DNA fragments are not easy to break, and the experimental controllability is relatively strong. However, when faced with the task of assembling DNA molecules of up to several hundred kb, in vitro assembly technology exposes many disadvantages, such as extremely cumbersome operation procedures, the need for precise control of experimental conditions and a large amount of manpower and material resources. More importantly, most DNA molecules assembled in vitro still need to be transferred into the host body for subsequent cloning and expression, which undoubtedly increases the complexity and uncertainty of the experiment.

[0004] Given the limitations of in vitro assembly techniques, in vivo assembly based on homologous recombination has gradually become the mainstream method for constructing large DNA fragments. Currently, the commonly used in vivo assembly chassis organisms include Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae.

[0005] As a classic model organism, Escherichia coli is widely used in molecular biology research. It has significant advantages such as clear genetic background, short reproduction cycle, and easy cultivation. In terms of large DNA fragment assembly, with the help of the Lambda-red system, researchers have developed a series of systems that have broken through the barriers to constructing megabase-sized DNA fragments. Among them, the REXER and CONEXER systems developed by Jason Chin's team innovatively combined CRISPR / Cas9 technology to successfully assemble a 1.1Mb human genome fragment in Escherichia coli. This achievement not only demonstrates the potential of Escherichia coli in large-fragment DNA assembly, but also provides a new technical path for human genome-related research. However, Escherichia coli is not perfect in large-fragment DNA assembly. Compared with Saccharomyces cerevisiae, it can only assemble medium-sized DNA fragments, and its own short homology arm recombination mechanism has not yet been fully understood, which to a certain extent limits its application in more complex assembly tasks.

[0006] Bacillus subtilis has an extremely high gene transformation ability and can efficiently integrate foreign DNA through RecA-mediated homologous recombination. In this chassis strain, the main strategies currently used to assemble large fragments of DNA are the inchworm elongation method (IWe) and the domino method (The dominomethod). These methods have been successfully applied to the assembly of 3.5Mb foreign DNA in photosynthetic bacteria and the mitochondrial and chloroplast genomes of eukaryotic organisms. However, they also have some limitations, such as the extremely high quality requirements of the DNA template, which increases the experimental difficulty in actual operation. Especially in the process of de novo assembly, it is not easy to obtain high-quality DNA templates, which limits the widespread application of these methods.

[0007] With its sophisticated genetic toolbox and robust recombination capabilities, Saccharomyces cerevisiae has become one of the most commonly used platforms for large DNA assembly. It can tolerate large chromosome fragments and demonstrates unique advantages through the support of a variety of recombination methods. For example, the transformation-associated recombination method (TAR), which utilizes only homology arm design; the chromosome assembly methods (SWAP-In and eSWAP-In) that utilize auxotrophic alternation; and the assembly method (MRA) based on the mating-meiosis process in Saccharomyces cerevisiae, have all played a significant role in large DNA assembly. Furthermore, the YLC assembly technique, which combines yeast mating-sporulation with the CRISPR / Cas system, has successfully assembled a 95 kb essential yeast gene fragment. In the field of plant and animal genome synthesis, Saccharomyces cerevisiae holds even greater potential and has been used in numerous synthetic artificial genome projects, such as the SynMoss genome synthesis project, which is being conducted using this platform. However, DNA breaks can sometimes occur when assembling DNA fragments as large as 300 kb, and fragment loss is also common when cloning repetitive sequence-rich fragments.

[0008] In practical application scenarios, such as the artificial synthesis of the Saccharomyces cerevisiae genome project (sc2.0), its important goal is to achieve the artificial full synthesis of the 12MB genome of Saccharomyces cerevisiae. So far, although 8.5 complete artificial synthetic chromosomes have been successfully designed and synthesized, because Saccharomyces cerevisiae is a eukaryotic organism, the genome fragments are long and assembly is extremely difficult. In the process of chromosome assembly, short fragments are mainly constructed in vitro and enriched by Escherichia coli self-replication using plasmids as vectors, while slightly longer fragments are assembled using the yeast's endogenous homologous recombination mechanism, and a hierarchical assembly method is adopted as a whole. This complex process highlights the urgency of developing a more convenient, fast and accurate method for assembling large-fragment DNA.

[0009] As a commonly used gene carrier, plasmids hold a unique position in the assembly of large DNA fragments. They are able to replicate autonomously within host cells, providing a stable environment for the amplification and preservation of exogenous DNA fragments. In the process of large DNA assembly, plasmids can serve as the basic unit for carrying multiple small DNA fragments, and these small fragments can be gradually connected into large fragments through a series of assembly strategies. For example, when constructing a complex biosynthetic pathway, gene fragments encoding different enzymes can be cloned separately into plasmids, and then the fragments on these plasmids can be integrated together using appropriate assembly techniques to form a complete biosynthetic pathway. At the same time, the development of large plasmid DNA assembly technology has an important driving force in the construction of efficient gene expression systems, the development of new biosensors, and the customized transformation of cell factories.

[0010] The field of synthetic genomics is rapidly developing, with successful de novo chromosome synthesis achieved for several single-cell organisms. However, this field also faces numerous challenges, such as the difficulty in synthesizing DNA with high GC content and multiple repeat sequences, as well as the challenges of precise assembly and delivery of very large DNA fragments. These challenges pose a severe challenge to the cost and accuracy of assembly. As a key component in addressing these challenges, further optimization and innovation of large plasmid DNA fragment assembly technology is crucial. Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide a method for rapid assembly of DNA fragments based on the programmable nuclease MfAgo.

[0012] The technical solution of the present invention is: a method for cutting DNA based on the programmable nuclease MfAgo, comprising the following steps:

[0013] (1) Designing gDNA targeting the site to be cleaved;

[0014] (2) incubating the nuclease MfAgo with the gDNA from step (1) in a reaction buffer at a temperature of 55-70° C. to form an MfAgo-gDNA complex;

[0015] (3) adding the target DNA containing the cleavage site to the MfAgo-gDNA complex obtained in step (2); reacting at 60-85°C for more than 5 minutes to cleave the target DNA;

[0016] The reaction buffer comprises: 10 mM HEPES, 150-750 mM NaCl, 5% glycerol, 0.2-0.4 mM Mn 2+ ;

[0017] The nuclease MfAgo is derived from Methanocaldococcus fervens, and its amino acid sequence is shown in SEQ ID No.1.

[0018] Furthermore, the molar ratio of the nuclease MfAgo, gDNA and target DNA is 3:2:1.

[0019] Furthermore, the GC content of the site to be cleaved is not higher than 45%.

[0020] Furthermore, the gDNA has 18 nucleotides and is phosphorylated at the 5' end.

[0021] Furthermore, in the reaction buffer, the NaCl concentration is 250 mM.

[0022] Furthermore, the Mn 2+ The concentration is 0.2 mM.

[0023] Furthermore, the incubation temperature is 55°C.

[0024] Furthermore, the reaction temperature is 85° C. and the reaction time is more than 10 minutes.

[0025] A method for rapid assembly of large DNA fragments, comprising the following steps:

[0026] (1) Cutting multiple plasmids containing target fragments using the method described above;

[0027] (2) gel recovery and purification of the target fragment;

[0028] (3) digesting the purified target fragment with T5 exonuclease;

[0029] (4) The digested target fragment is transferred into Escherichia coli for homologous recombination to obtain a plasmid containing the target fragment.

[0030] Furthermore, the target fragment is connected to a fluorescent protein encoding gene or an antibiotic resistance gene.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] Fragment cutting can be completed without adding polymerase and other complex components. At the same time, because the assembled fragments are all cut fragments, no base mutation occurs. In addition, this method has good visualization effect and the assembly results can be quickly seen. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 To detect the activity of MfAgo in cleaving single-stranded nucleic acids, Urea-PAGE was used to identify and analyze the results. In the figure, 5'P-gDNA is a guide DNA with a phosphorylation modification at the 5' end; 5'OH-gDNA is a guide DNA with a hydroxylation modification at the 5' end; 5'P-gRNA is a guide RNA with a phosphorylation modification at the 5' end; 5'OH-gRNA is a guide RNA with a hydroxylation modification at the 5' end; M1 is a 34nt product chain, which serves as a product control for successful target cleavage by MfAgo (the target nucleic acid is 45nt, and after MfAgo cleavage at positions 10-11, the target is broken into two DNA segments of 11nt and 34nt); T-tDNA is a 45nt target chain; Product is a 34nt product control.

[0034] Figure 2 Figure 1 shows the results of targeted cleavage of MfAgo in plasmid pUC19 at different GC content regions, identified and analyzed by agarose gel electrophoresis. Figure A shows the cleavage profile after direct use of guide targeting plasmids such as 29-R and 29-F. The highest cleavage occurred in a region with a GC content of 45%, which was then linearized. Figure B shows the product from Figure A further cleaved with NdeI or ScaI, yielding two DNA fragments of the expected size. Expected fragment sizes were obtained at the 29%, 39%, and 45% regions.

[0035] Figure 3 This is an activity analysis of MfAgo cutting the 29% region of plasmid pUC19 under different NaCl concentrations; the results show that when the NaCl concentration is in the range of 100mM-750mM, MfAgo can completely cut the plasmid pUC19 and completely linearize it.

[0036] Figure 4 For different Mn 2+ The results showed that MfAgo could cut 29% of the plasmid pUC19 at the concentration of Mn. 2+The activity is observed in the concentration range of 0.1mM-1mM, and the plasmid pUC19 can be completely cut and linearized in the range of 0.2-0.4mM.

[0037] Figure 5 This is an activity analysis of MfAgo cutting the 29% region of plasmid pUC19 at different incubation temperatures; the results show that MfAgo is active in the incubation temperature and concentration range of 37-70°C, and can completely cut the plasmid pUC19 and completely linearize it at the range of 55-70°C.

[0038] Figure 6 This is an activity analysis of MfAgo cutting the 29% region of plasmid pUC19 at different reaction temperatures; the results show that MfAgo is active in the incubation temperature and concentration range of 37-93°C, and can completely cut plasmid pUC19 and completely linearize it within the range of 60-85°C.

[0039] Figure 7 Exploration of the optimal ratio of Protein:gDNA:Target; Figure A is the exploration of the ratio of Protein:gDNA, the Target concentration is fixed at 2pM, and the ratios of channels AF are Protein:gDNA:Target=0.4:2:2, 1:2:2; 2:2:2, 3:2:2, 4:2:2, and 5:2:2; Figure B is the exploration of the optimal ratio of Protein:gDNA:Target, and the ratios of channels 1-7 are Protein:gDNA:Target=:60:40:10, 30:20:10, 15:10:10, 7.5:5:10, 6:4:10, 5:3.4:10, and 3:2:10. When the ratio is 3:2:1, the cutting efficiency is highest in the 29% and 39% regions, and the most linearized plasmid is present; Figure C is the visualization of the results of Figure B, and the results of Figure B are represented in the form of a bar graph.

[0040] Figure 8 The reaction time for MfAgo cleavage at the 29% and 39% regions was explored. Based on the optimized conditions described above, the time required for MfAgo to cleave a plasmid was explored, using the 29% and 39% regions as an example. As shown in Figure A, cleavage of the pUC29% region required 20 minutes of reaction time for complete plasmid cleavage; as shown in Figure B, cleavage of the pUC39% region required 5 minutes of reaction time for complete plasmid cleavage (i.e., complete conversion of the plasmid to the Lin state).

[0041] Figure 9The figure shows the principle of DNA assembly. Plasmid B and plasmid C are used as the starting plasmids. A single guide can target and cut a single target DNA. Four guides, gDNA-1-F, gDNA-1-R, gDNA-2-F, and gDNA-2-R, are used to target and cut plasmid B. Four guides, gDNA-3-F, gDNA-3-R, gDNA-4-F, and gDNA-4-R, are used to target and cut plasmid C. Four double-stranded DNA fragments with blunt ends are obtained. The fragments sfGFP-MfAgo and pET28a(+) vector are treated with T5 exonuclease to digest their ends into sticky ends with 10bp homologous complementary sequences. The two double-stranded DNA fragments are mixed and added to BL21 competent cells. The sticky ends are connected using the homologous complementary repair ability of E. coli to finally obtain recombinant plasmid D.

[0042] Figure 10 This is the cutting result detection of starting plasmid B and starting plasmid C. It can be seen from the figure that the two plasmids were cut into two fragments by MfAgo, and the fragments are consistent with the expected size.

[0043] Figure 11 The results of DNA assembly are displayed in the form of a plate; as shown in the figure, the green fluorescent colonies are colonies grown from the successfully assembled recombinant plasmid.

[0044] Figure 12 To verify the recombinant plasmid sequencing results, the recombinant plasmid D with green fluorescence was sequenced, and the sequencing results were consistent with expectations, proving that the recombination was successful. DETAILED DESCRIPTION

[0045] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0046] Example 1 Preparation of experimental materials

[0047] 1. Plasmid Construction

[0048] MfAgo from Methanocaldococcus fervens was retrieved from NCBI (protein sequence as SEQ ID No. 1, coding gene sequence as SEQ ID No. 2), and the nucleotide sequence of MfAgo was synthesized by Wuhan Jinkairui Company after codon optimization. A 6x His tag was added to its N-terminus and then constructed on the pET28a(+) vector to obtain plasmid A (pET28a-MfAgo).

[0049] The protein MfAgo was constructed on the pET23a(+) vector to obtain the plasmid pET23a-MfAgo. Similarly, the sfGFP sequence was constructed on the plasmid pET23a-MfAgo to obtain the starting plasmid B (pET23a-sfGFP-MfAgo). Protein P2 was constructed on the pET28a(+) vector by PCR to obtain the starting plasmid C (pET28a-P2).

[0050] 2. Heterologous Expression and Purification of MfAgo Protein

[0051] Plasmid A was transformed into Escherichia coli BL21 (DE3), cultured at 37°C for 16 hours, and single colonies were picked and transferred to LB liquid medium containing kanamycin at a final concentration of 50 μg / ml. The culture was continued at 37°C in a shaker at 220 rpm until the OD 600 The pH value was between 0.6 and 0.8, the temperature was lowered to 18°C, IPTG was added for induction, and the cells were harvested after 17 hours at 6000 rpm. The cells were resuspended in Buffer A (50 mM Tris, 500 mM NaCl, 5% Glycerol, 5 mM MgCl2) and washed by centrifugation at 6000 rpm. The cells were then resuspended in Buffer A and PMSF was added to a final concentration of 1 mM before high-pressure lysis. After centrifugation at 17000 rpm for 30 minutes, the supernatant was incubated with Ni-NTA at 4°C with a silent mixer for 50 minutes. The cells were then eluted with Buffer A containing 20 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM imidazole, respectively. At each step, samples were taken for SDS-PAGE detection, and the elution group containing high-purity target protein was collected and purified in two steps using Heparin Focurose 6FF. Then, three steps of purification were performed using Superdex 200Increase 10 / 300GL. Samples were taken for SDS-PAGE detection, and the elution group containing high-purity target protein was frozen in a -80℃ refrigerator for later use.

[0052] 3. Sequence Design

[0053] 3.1. Oligonucleotide sequences required for single-stranded nucleic acid cleavage experiments

[0054] The present invention designed an 18nt guide DNA (gDNA) with phosphorylation modification at the 5' end. A 45nt target was also designed and modified with a FAM fluorescent group at the 5' end. The gDNA and target were complementary sequences and synthesized by Shanghai Sangon Biotechnology Co., Ltd. The specific sequence is as follows:

[0055] gDNA: 5'P-TGAGGTAGTAGGTTGTAT

[0056] T-tDNA: FAM-AAACGACGGCCAGTGCCAAGCTTACTATACAACCTACTACCTCAT

[0057] M1: FAM-AAACGACGGCCAGTGCCAAGCTTACTATACAACC

[0058] 3.2. Oligonucleotide sequences required for plasmid cleavage experiments

[0059] The present invention cuts the plasmid pUC19 with reference to the literature, designs and synthesizes forward and reverse gDNAs targeting five different GC content positions of 29%, 39%, 45%, 53%, and 65% in the pUC19 plasmid, which are synthesized by Shanghai Bioengineering Co., Ltd. The specific sequences are as follows:

[0060] 29-R:TAGGTGAAGATCCTTTTT

[0061] 29-F:TCAAAAGGATCTTCACC

[0062] 39-R:CATGATAATAATGGTTTC

[0063] 39-F: AAGAAACCATTATTATCA

[0064] 45-R:TTCCAATGATGAGCACTT

[0065] 45-F:AAAAGTGCTCATCATTGG

[0066] 53-R:TCGTGACTGGGAAAACCC

[0067] 53-F:CAGGGTTTTCCCAGTCAC

[0068] 65-R:CGGAACAGGAGAGCGCAC

[0069] 65-F:TCGTGCGCTCTCCTGTTC

[0070] The present invention designed four gDNAs for cutting the starting plasmid B, namely gDNA-1-F, gDNA-1-R, gDNA-2-F, and gDNA-2-R, and four gDNAs for cutting the starting plasmid C, namely gDNA-3-F, gDNA-3-R, gDNA-4-F, and gDNA-4-R. All eight gDNAs were phosphorylated at their 5' ends and synthesized by Shanghai Shenggong Company. The specific sequences are as follows:

[0071] gDNA-1-F:5'P-ctctagaaataattttgt

[0072] gDNA-1-R: 5'P-aaacaaaattatttctag

[0073] gDNA-2-F: 5'P-tttacaatttcaggtggc

[0074] gDNA-2-R: 5'P-gtgccacctgaaattgta

[0075] gDNA-3-F: 5'P-tgtttaactttaagaagg

[0076] gDNA-3-R: 5'P-ctccttcttaaagttaaa

[0077] gDNA-4-F: 5'P-aacaaaatattaacgttt

[0078] gDNA-4-R: 5'P-gtaaacgttaatattttg

[0079] Example 2 Single-stranded nucleic acid cleavage experiment

[0080] Before the plasmid cleavage experiment, the activity of protein cleavage of single-stranded nucleic acid should be determined. The molar ratio of each component in the reaction system is: MfAgo: gDNA: T-tDNA = 10:2:1. The final concentration of MfAgo is 2μM, gDNA is 0.4μM, and 5mM Mn is added. 2+ The cells were incubated at 37°C for 10 min with Buffer B (10 mM HEPES, 250 mM NaCl, 5% Glycerol), and then 0.2 μM T-tDNA was added. The cells were reacted at 85°C for 30 min, and then Buffer C (95% formamide, 18 mM EDTA, 0.025% SDS, 0.025% bromophenol blue) was added to terminate the reaction. The samples were heated at 95°C for 5 min, and the cleavage results were detected by 20% Urea-PAGE. The cells were then analyzed using Gel Doc. TM The results were observed using XR+ (Bio-Rad gel imaging system).

[0081] The results are as follows Figure 1 As shown in the figure, MfAgo can specifically cut single-stranded DNA under the guidance of one of the guides: 5'P-gDNA, 5'OH-gDNA, and 5'OH-gRNA within 5 minutes.

[0082] Example 3 Cutting process optimization

[0083] (1) Cutting positions of different GC contents in pUC19

[0084] First, the pUC19 plasmid was cut with MfAgo. MfAgo with a final concentration of 2 μM was incubated with the corresponding forward and reverse primers with different GC contents in Buffer B for 10 minutes to obtain an Ago-gDNA binary complex. 1600 ng of pUC19 was added and reacted at 85°C for 20 minutes. Buffer D (30 mM EDTA, 36% glycerol, 0.05% xylene cyanol FF, 0.05% bromophenol blue) was added to stop the reaction. The cutting results were detected on a 1% agarose gel and the gel Doc was used. TM XR+ (Bio-Rad) observation results.

[0085] The results are as follows Figure 2 As shown in Figure A, MfAgo can cut double-stranded DNA with a GC content of 29%-45%. When the GC content is 29% and 39%, it can completely cut the double-stranded DNA and convert the pUC19 plasmid into a linear state. When the GC content is 45%, it can partially convert the pUC19 plasmid into a linear state. Figure 4 In Figure B, the linearized pUC19 plasmid obtained in the previous step was digested with the restriction endonuclease NdeI at the 29% and 45% cleavage products, and with the restriction endonuclease ScaI at the 39% cleavage product. Two fragments of the expected sizes were obtained, confirming that the pUC19 plasmid was cleaved by MfAgo. Therefore, the GC content of the selected cleavage fragments in the assembly experiment was kept below 45%.

[0086] (2) Effect of NaCl concentration in Buffer B on cleavage activity

[0087] The concentration of NaCl is crucial to the activity of nucleases, so the present invention explored the cleavage activity of MfAgo at different concentrations, taking the cleavage activity of the 29% GC region of the pUC19 plasmid as an example.

[0088] like Figure 3 As shown, when the NaCl concentration is less than 150mM, the MfAgo cleavage efficiency is low. When the NaCl concentration is ≥150mM, the MfAgo cleavage efficiency reaches 100%, and the pUC19 plasmid is completely converted to a linear state. Therefore, the present invention continued the subsequent experiments with a 250mM NaCl buffer.

[0089] (3)Mn 2+ Effect of concentration on cleavage activity

[0090] Metal ions are auxiliary factors for the activity of nucleases. The present invention determines the activity of different concentrations of Mn 2+Under the condition of 40 ℃, MfAgo has the activity of cutting the 29% GC region of pUC19 plasmid.

[0091] The results are as follows Figure 4 As shown, in Mn 2+ When the concentration is 0.2mM-0.4mM, MfAgo can completely cut the pUC19 plasmid into linearized plasmid, and the cutting activity is optimal. Subsequent reactions are all carried out at 0.2mM Mn 2+ Conditions are carried out.

[0092] (4) Effect of incubation temperature on cleavage activity

[0093] Incubating Ago protein with gDNA before adding the target can obtain a more firmly bound binary complex. Different incubation temperatures may affect its thermal stability and binding. Therefore, the present invention explored the activity of MfAgo in cutting the 29% GC region of the pUC19 plasmid at different incubation temperatures.

[0094] The results are as follows Figure 5 As shown, the activity is best when the incubation temperature is 55-70℃ for 10 minutes. MfAgo can cut all the plasmids of pUC19 into a linear state. Therefore, the incubation temperature of the subsequent reaction is 55℃.

[0095] (5) Effect of reaction temperature on cleavage activity

[0096] MfAgo is derived from thermophilic bacteria and can cut single-stranded nucleic acids at higher temperatures. Therefore, the present invention also explores the activity of MfAgo in cutting plasmid DNA at different temperatures.

[0097] The results are as follows Figure 6 As shown, MfAgo can cut the plasmid at room temperature of 37°C. As the reaction temperature increases, the activity is significantly enhanced. The optimal temperature range is between 60-85°C. Therefore, the reaction temperature in the present invention is set to 85°C.

[0098] (6) Optimal ratio of protein:gDNA:target

[0099] Excessive nucleases can degrade the plasmid. The right ratio maximizes MfAgo's cleavage efficiency. To verify MfAgo's cleavage efficiency, we explored different ratios of protein:guide:target, using cleavage activity at the 29% GC region and the 39% GC region of the pUC19 plasmid as an example. We first explored the protein:gDNA ratio, then the protein:gDNA:TargetD ratio.

[0100] The results are as follows Figure 7As shown, the cleavage efficiency is highest when Protein:gDNA:Target=3:2:1.

[0101] (7) Reaction time exploration

[0102] Furthermore, based on the optimal conditions obtained above, the reaction time of MfAgo cleavage plasmid was further explored. Taking the activity of cleavage of 29% GC and 39% GC regions of pUC19 plasmid as an example

[0103] The results are as follows Figure 8 As shown, the pUC19 plasmid can be substantially linearized in 5 minutes when cutting the 29% GC region and the 39% GC region. To ensure complete plasmid cutting, the reaction time was controlled within 10 minutes.

[0104] Example 4 DNA assembly

[0105] (1) Cutting the starting plasmid B and starting plasmid C

[0106] The optimized conditions in Example 3 (Protein 0.375 μM, gDNA 0.25 μM, plasmid 0.23 μM, incubation temperature 55°C, reaction temperature 85°C, 0.2 mM Mn 2+ , 250 mM NaCl) to cut the starting plasmid B and the starting plasmid C.

[0107] Principle Figure 9 As shown: MfAgo is first incubated with different gDNAs to form a binary complex, and then specifically recognizes the complementary target chain under the guidance of gDNA. The cutting activity of MfAgo is activated, and the phosphodiester bond between the 10th and 11th nucleotides of the complementary plasmid is targeted for cutting. After the cutting is completed, the circular plasmid is cut into two linear DNA fragments with blunt ends.

[0108] The specific operations are as follows:

[0109] First, 2 μM MfAgo was mixed with 1 μM gDNA-1-F, gDNA-1-R, gDNA-2-F, gDNA-2-R, gDNA-3-F, gDNA-3-R, gDNA-4-F, gDNA-4-R in a 0.2 mM Mn 2+ Incubate with Buffer B at 55°C for 10 min.

[0110] After incubation, mix the four tubes of samples, gDNA-1-F, gDNA-1-R, gDNA-2-F, and gDNA-2-R, evenly, take 2 μL of the mixture, add 8 μL of the starting plasmid B, and react at 85°C for 10 min.

[0111] Mix the four tubes of samples, gDNA-3-F, gDNA-3-R, gDNA-4-F, and gDNA-4-R, evenly, take 2 μL of the mixture, add 8 μL of the starting plasmid C, and react at 85°C for 10 min.

[0112] The cleavage results were checked using 1% agarose gel. Figure 10 As shown, the starting plasmid B was cut into two linear DNA segments with blunt ends, namely pET23a(+) vector and sfGFP-MfAgo; the starting plasmid C was cut into two linear DNA segments with blunt ends, namely pET28a(+) vector and P2.

[0113] The fragment sfGFP-MfAgo and the fragment pET28a(+) vector were subjected to gel recovery treatment to obtain cleaner DNA fragments.

[0114] (2) Assembly plasmid D—Visualization method

[0115] T5 exonuclease can degrade DNA in the 5'→3' direction, starting from the 5' end of linear double-stranded DNA. Based on this, the two fragments recovered were treated with T5 exonuclease to obtain two fragments with 10bp complementary sequences at the 5' end. Then, using the homologous recombination repair mechanism of E. coli, the two fragments were constructed into a recombinant plasmid. The steps are as follows:

[0116] 1. Place 1 μL T5 exonuclease (purchased from NEB), 4.5 μL sterile water, and 5 μL of buffer 4 in a 1.5 mL centrifuge tube, mix thoroughly, and store at 4°C.

[0117] 2. Add the fragment sfGFP-MfAgo and the fragment pET28a(+) vector at a molar ratio of 1:3 to the new 1.5

[0118] mL centrifuge tube, add 1 μL of diluted T5 exonuclease to the centrifuge tube and place in ice water for 5 minutes;

[0119] 3. Add 100 μL of E. coli BL21 competent cells to the above 1.5 mL centrifuge tube and place on ice for 30 minutes;

[0120] Incubate in a 42°C water bath for 45 seconds, then quickly place on ice. Add 100 μL of LB liquid medium and incubate in a shaker at 37°C for 60 minutes.

[0121] 5. Heat the LB solid medium to fully melt it. When it cools to about 40°C, add Kana antibiotics at a final concentration of 50 μg / mL and pour it into a disposable plate. After solidification, spread the revived bacterial liquid on the plate and incubate it upside down at 37°C incubator for 18 hours.

[0122] 6. Use a blue light analyzer to observe the growth of colonies on the plate.

[0123] The results are as follows Figure 11 As shown: Through illumination with a blue light instrument, colonies with green fluorescence can be directly observed with the naked eye on the plate. These are recombinant plasmid colonies with successful fragment ligation, and colonies without green fluorescence are non-target colonies.

[0124] (3) Assembly of plasmid D—plate-free method

[0125] Because the successfully assembled recombinant plasmid carries a resistance gene, only the successfully assembled target colony can grow after the corresponding antibiotic is added. Therefore, this method can be directly cultured overnight in a liquid culture medium containing the corresponding antibiotic, without the need for plate culture. The specific operation is the same as steps 1-5 of method (2). In step 6, the revived bacterial liquid is transferred to LB liquid culture medium containing 50μg / mL Kana antibiotic and cultured in a shaking incubator at 37℃ for 18 hours.

[0126] The cultured colonies can be directly extracted to obtain the assembled recombinant plasmid D (28a-sfGFP-MfAgo). The colonies and plasmids obtained by method (2) and method (3) were sequenced. Figure 12 , the sequencing results were all correct, indicating that the target plasmid was successfully assembled.

Claims

1. A method for cutting DNA based on the programmable nuclease MfAgo, characterized in that: The steps include: (1) Designing gDNA targeting the site to be cleaved; (2) incubating the nuclease MfAgo with the gDNA from step (1) in a reaction buffer at a temperature of 55-70° C. to form an MfAgo-gDNA complex; (3) adding the target DNA containing the cleavage site to the MfAgo-gDNA complex obtained in step (2); reacting at 60-85°C for more than 5 minutes to cleave the target DNA; The reaction buffer consists of 10 mM HEPES, 150-750 mM NaCl, 5% glycerol, 0.2-0.4 mM Mn 2+ ; The nuclease MfAgo is derived from Methanocaldococcus fervens, and its amino acid sequence is shown in SEQ ID No.

1.

2. The method according to claim 1, characterized in that The molar ratio of the nuclease MfAgo, gDNA and target DNA is 3:2:

1.

3. The method according to claim 1, characterized in that The GC content of the site to be cleaved is not higher than 45%.

4. The method according to claim 1, wherein The gDNA has 18 nucleotides and is phosphorylated at the 5' end.

5. The method according to claim 1, wherein The NaCl concentration in the reaction buffer was 250 mM.

6. The method according to claim 1, characterized in that The Mn 2+ The concentration is 0.2 mM.

7. The method according to claim 1, characterized in that The incubation temperature is 55°C.

8. The method according to claim 1, characterized in that The reaction temperature is 85° C. and the reaction time is more than 10 minutes.

9. A method for rapid assembly of large DNA fragments, characterized in that: The steps include: (1) Cutting multiple plasmids containing target fragments using the method described in any one of claims 1 to 8; (2) gel recovery and purification of the target fragment; (3) digesting the purified target fragment with T5 exonuclease; (4) The digested target fragment is transferred into Escherichia coli for homologous recombination to obtain a plasmid containing the target fragment.

10. The method according to claim 9, characterized in that The target fragment is connected to a fluorescent protein encoding gene or an antibiotic resistance gene.

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