WO2025222410A1 - Method for synthesis and assembly of large fragment genomic dna in vitro - Google Patents

Abstract

Provided is a method for the synthesis and assembly of large fragment genomic DNA in vitro. A double-stranded DNA sticky-end generation system is used in the method. The generation system comprises DNA assembly sequences, single-stranded guide DNA, nicking endonuclease A, nicking endonuclease B, and nicking endonuclease C. The DNA assembly sequence comprises, from the 5' end to the 3' end: a first nicking endonuclease recognition site unit, a target DNA sequence, and a second nicking endonuclease recognition site unit. The method comprises: dividing a genomic sequence into n target DNA sequences, preparing DNA assembly sequences, designing and synthesizing single-stranded guide DNA, and adding nicking endonuclease A, nicking endonuclease B, and nicking endonuclease C to each DNA assembly sequence and the single-stranded guide DNA for enzyme digestion; and performing in vitro DNA assembly on the enzyme-digested DNA assembly sequences. The method overcomes the limitation of short overhangs generated by restriction endonucleases and improves the success rate of large DNA molecule assembly.

Description

A method for synthesizing and assembling large genomic DNA fragments in vitro Technical Field

This invention belongs to the field of synthetic biology technology, specifically relating to a method for synthesizing and assembling large fragments of genomic DNA in vitro.

Background Technology

The ability to synthesize DNA allows researchers to control the composition of DNA sequences, eliminating reliance on naturally occurring sequences isolated from organisms and providing a versatile tool for designing living systems. The demand for synthetic DNA, particularly large DNA fragments, is steadily increasing as the need to design complex metabolic pathways, genetic circuits, and even entire genomes grows. Various methods exist for linking small DNA fragments to large molecules using restriction endonucleases, such as BioBrick, Golden Gate, and YeastFab. However, restriction endonucleases typically produce cantilevers of only 0-4 nucleotides, which are too short to provide sufficient specificity and affinity for assembling multiple fragments. This limits most assembly techniques to producing DNA molecules smaller than 10 kbp. As the size and complexity of the desired DNA sequence increase, the success rate of assembly drops sharply, making it difficult to scale up to larger assemblies.

Several restriction endonuclease-independent methods, such as sequence and ligation-independent cloning (SLIC), Gibson assembly, or polymerase cycle assembly (PCA), have also been developed. Gibson assembly is widely used due to its simplicity, efficiency, and the absence of restriction sites or scar sequences in the final structure. However, Gibson assembly is only suitable for assembling DNA frameworks of a certain length, typically around 10-15 kb, and is not suitable for assembling entire chromosomes. Furthermore, by utilizing the homologous recombination ability of Saccharomyces cerevisiae, synthetic DNA structures ranging from tens to hundreds of kb in size, and even entire bacterial genomes, have been successfully assembled. In recent years, scientists have used a method called "swAP-In" (swap auxotrophic body progressive integration) to construct multiple synthetic yeast chromosomes in vivo, a method that iteratively replaces natural sequences with synthetic sequences. Although synthetic chromosomes can be successfully assembled and integrated into yeast cells, transferring the assembled chromosomes from yeast to other organisms (such as mammalian cells) remains very difficult.

Summary of the Invention

To address the shortcomings of existing technologies, the present invention aims to provide a method for synthesizing and assembling large fragments of genomic DNA in vitro.

The specific technical solution of the present invention is as follows:

This invention provides the use of a double-stranded DNA sticky end generation system in the in vitro synthesis and assembly of large genomic DNA fragments. The system includes a DNA assembly sequence, a single-stranded guide DNA, nicking endonuclease A, nicking endonuclease B, and nicking endonuclease C.

The DNA assembly sequence includes a first nicking endonuclease recognition site unit, a target DNA sequence, and a second nicking endonuclease recognition site unit from the 5' end to the 3' end. The first nicking endonuclease recognition site unit includes 2 or 3 nicking endonuclease A recognition sites and spacer bases located between the nicking endonuclease A recognition sites. The second nicking endonuclease recognition site unit includes 2 or 3 nicking endonuclease B recognition sites and spacer bases located between the nicking endonuclease B recognition sites. The nicking endonuclease A and nicking endonuclease B belong to the same type of nicking endonuclease. The cleavage sites of the nicking endonuclease A and nicking endonuclease B are located on the upper and lower strands of the DNA. After the DNA assembly sequence is digested by nicking endonuclease A and nicking endonuclease B, single-stranded regions are formed at both ends of the DNA assembly sequence.

The single-stranded guide DNA includes a first single-stranded guide DNA and a second single-stranded guide DNA. The single-stranded guide DNA consists of a recognition sequence for the target sequence and a short DNA sequence folded into a stem-loop structure. The target sequence is a single-stranded DNA sequence and includes a portion of the single-stranded region. The short DNA sequence comprises a single-stranded region and a partial sequence of its adjacent target DNA sequence. The partial sequence of this single-stranded region is referred to as the first target sequence, and the partial sequence of the adjacent target DNA sequence is referred to as the second target sequence. The stem-loop structure formed by the short DNA sequence has a nicking endonuclease C recognition site, but lacks a sequence that can be cleaved by the nicking endonuclease C. After the recognition sequence of the target sequence hybridizes with the first target sequence, the other DNA strand corresponding to the second target sequence is separated. The recognition sequence of the target sequence further hybridizes with the second target sequence. The target sequence and the recognition sequence hybridize to form a double-stranded structure. This double-stranded structure can be recognized by the nicking endonuclease C, and the predetermined position of the second target sequence is located at a position that can be cleaved by the nicking endonuclease C through the recognition of the recognition sequence on the single-stranded guide DNA by the nicking endonuclease C. The recognition sequences of the target sequences of the first single-stranded guide DNA and the second single-stranded guide DNA hybridize with the partial sequences of the single-stranded regions formed at both ends of the DNA assembly sequence after nicking endonuclease A and nicking endonuclease B digestion, as well as the partial sequences of their adjacent target DNA sequences, to form a double-stranded structure.

Furthermore, the nicking endonuclease A and nicking endonuclease B are selected from one of Nt.BbvCI, Nt.AlwI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPII, or one of Nb.BbvCI, Nb.BsmI, Nb.BsrDI, and Nb.BtsI, respectively;

Preferably, the first nicking endonuclease recognition site unit includes three nicking endonuclease A recognition sites, and the second nicking endonuclease recognition site unit includes three nicking endonuclease B recognition sites;

Preferably, the nicking endonuclease A and nicking endonuclease B are the same nicking endonuclease;

Preferably, the nicking endonuclease A and nicking endonuclease B are Nt.BspQI;

Preferably, the first nicking endonuclease recognition site unit includes three nicking endonuclease A recognition sites, and the second nicking endonuclease recognition site unit includes three nicking endonuclease B recognition sites.

Furthermore, the spacer bases between the nicking endonuclease A recognition sites satisfy the following condition: the cleavage site of the nicking endonuclease A is not located in other nicking endonuclease A recognition sequences;

The spacer bases between the nicking endonuclease B recognition sites satisfy the following condition: the nicking endonuclease B cleavage site is not located in other nicking endonuclease B recognition sequences;

Preferably, the spacer bases between the nicking endonuclease A recognition sites and the spacer bases between the nicking endonuclease B recognition sites are 4 to 10 randomly arranged A, C, G or T bases, and the spacer bases do not form nicking endonuclease recognition sites.

Furthermore, the nicking endonuclease C is selected from one of Nt.BbvCI, Nt.AlwI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, Nb.BbvCI, Nb.BsmI, Nb.BsrDI, and Nb.BtsI;

Preferably, the nicking endonuclease C is selected from Nt.BstNBI.

Furthermore, the 5' end of the first nicking endonuclease recognition site unit and the 3' end of the second nicking endonuclease recognition site unit of the DNA assembly sequence each include an auxiliary sequence of 500-1500 bp in size; or, the DNA assembly sequence is cloned into a plasmid.

Furthermore, the length of the single-chain region is 5-100 bases.

Furthermore, the length of the second target sequence is between 5 and 100 bases, preferably between 5 and 30 bases, and more preferably between 10 and 30 bases.

Furthermore, the length of the sticky end is between 5 and 30 bases, preferably between 5 and 20 bases.

Furthermore, the large fragment of genomic DNA is a yeast chromosome or a T7 phage genome.

This invention also provides a method for synthesizing and assembling large genomic DNA fragments in vitro using the aforementioned system, comprising the following steps:

(1) Divide the genome sequence into n target DNA sequences, where n is a positive integer, prepare n DNA assembly sequences, and design and synthesize single-stranded guide DNA for each DNA assembly sequence;

(2) Add nicking endonuclease A, nicking endonuclease B and nicking endonuclease C to each DNA assembly sequence and its corresponding single-stranded guide DNA for enzyme digestion. The sticky ends generated by the enzyme digestion of adjacent target DNA sequences are complementary.

(3) The DNA assembly sequence after enzyme digestion in step (2) is assembled into DNA in vitro.

Furthermore, the target DNA sequence described in step (1) is 300bp-50kb in size;

Preferably, the method for preparing the DNA assembly sequence in step (1) is as follows: cloning the target DNA sequence into a plasmid containing a first nicking endonuclease recognition site unit and a second nicking endonuclease recognition site unit, wherein the target DNA sequence is located between the first nicking endonuclease recognition site unit and the second nicking endonuclease recognition site unit; or, inserting the first nicking endonuclease recognition site unit and the second nicking endonuclease recognition site unit at both ends of each target DNA sequence, and amplifying to obtain the DNA assembly sequence;

Preferably, the plasmid is pCCE, and its nucleotide sequence is shown in SEQ ID NO.13;

Preferably, the 5' end of the first nicking endonuclease recognition site unit and the 3' end of the second nicking endonuclease recognition site unit of the DNA assembly sequence each include an auxiliary sequence of 500-1500 bp in size, and the DNA assembly sequence is obtained by amplification.

Furthermore, the enzymatic digestion process described in step (2) is carried out at 30-75°C, preferably at 45-65°C, and more preferably at 50-60°C;

Preferably, the enzyme digestion system in step (2) is: 4 μg DNA assembly sequence, 1x cutsmart buffer, 1 mM DTT, 8% PEG4000 (v/v), 3 μM single-stranded guide DNA, 12 U nicking endonuclease A, 12 U nicking endonuclease B and 12 U nicking endonuclease C; the enzyme digestion conditions are incubation at 50°C for 2 h.

Furthermore, the DNA in vitro assembly described in step (3) involves multiple rounds of assembly based on the size of the genomic DNA fragment, thereby obtaining a large fragment of genomic DNA;

Preferably, the DNA in vitro assembly in step (3) includes two steps: a hybridization reaction and an assembly reaction performed sequentially.

1) Hybridization reaction: Add adjacent enzyme-digested DNA assembly sequences to the hybridization buffer and perform the hybridization reaction; the hybridization reaction conditions are: 55±5℃, 5±3min; 45±5℃, 10±5min; 40±5℃, 10±5min; 35±5℃, 10±5min; 20±5℃, 10±5min; the hybridization buffer composition is 100mM NaCl, 20mM Tris-Ac, 1mM EDTA, PEG4000 with a volume percentage concentration of 8%-16%, pH 7.5;

2) Assembly reaction: After the hybridization reaction, add assembly buffer and DNA ligase to the hybridization reaction system; the assembly reaction conditions are: 30±5℃, 10±5min; 50-100 cycles × (30±5℃, 1±0.5min; 42±5℃, 1±0.5min); the assembly buffer components are 30mM Tris-HCl, 4mM MgCl2, _26μM NAD, 1mM DTT, 50μg/mL BSA;

Preferably, the volume percentage concentration of PEG4000 in the hybridization buffer component is 8%;

Preferably, Escherichia coli DNA ligase or T7 DNA ligase is added to the hybridization reaction system, and more preferably Escherichia coli DNA ligase is added.

Preferably, the number of adjacent DNA assembly sequences after enzyme digestion added to the hybridization buffer is 3-12, rounded to the nearest integer.

Preferably, the number of adjacent post-digestion DNA assembly sequences added to the hybridization buffer is 6 or 7.

The beneficial effects of this invention are as follows:

This invention provides a system for generating sticky ends of double-stranded DNA, which can generate ultra-long and precise cantilevers at both ends of double-stranded DNA fragments. The cantilevers are generated by programmable DNA-guided strand cutting (DSC) technology, which can be designed autonomously without length or sequence preference.

This invention also provides an in vitro iterative assembly strategy that uses DNA guide strand cutting (DSC) technology to process small DNA fragments, generating ultra-long and precise cantilevers at both ends of the small DNA fragments. These small fragments with ultra-long cantilevers are further used for in vitro assembly. A series of tests are used to optimize the assembly efficiency of DNA fragments containing target cantilevers, thereby achieving the in vitro synthesis and assembly of large genomic DNA fragments. This invention overcomes the limitation of restriction endonucleases (REs) in generating short cantilevers and improves the success rate of assembling large DNA molecules. Taking T7 bacteriophage as an example, this invention assembles a complete T7 bacteriophage genome from PCR amplicon, which can generate infectious viral particles. Finally, using 62 synthesized DNA fragments, a telomere-telomere synthetic yeast chromosome was constructed in vitro, isolated, and transferred into yeast cells. This technique is commonly used for the seamless construction of large DNA molecules and can accelerate future genome synthesis.

Attached Figure Description

Figure 1 illustrates the in vitro iterative DNA assembly strategy based on DNA guide strand cutting technology. (A) Schematic diagram of DSC recognition and cleavage of ssDNA. Gray in the ssDNA strand represents the target ssDNA. The scissors represent the cleavage sites of the endonuclease in the target ssDNA. (B) Schematic diagram of generating designed sticky ends by cutting dsDNA using DSC. The scissors represent the cleavage sites of the endonuclease, and the gray in the dsDNA strand where the scissors are located represents the target dsDNA and the desired 5' cantilever. (C) Schematic diagram of syn I assembly. DNA fragments of ~5kb in length treated by DSC have the desired cantilever at both ends. Adjacent fragments are ligated to obtain DNA fragments of approximately 40kb in length. This ligation process is repeated to generate DNA fragments of approximately 200kb in length in the next stage of assembly, ultimately assembling a linear chromosome of 1Mb size. (D) PAGE analysis of the target DNA after DSC treatment. Lane 1 represents the target ssDNA before DSC treatment. Lanes 2 to 12 are target ssDNAs treated with DSC using different sgDNAs (sgDNA-1 to sgDNA-11). An asterisk indicates the full-length target ssDNA. (E) Direct Sanger sequencing analysis was performed on the resulting cantilever. TO indicates the target cantilever. Sequencing signals are shown on the right, with the corresponding nucleotides indicated by dashed boxes, perfectly matching the designed cantilever. The additional A peak at the end originates from the termination transferase activity of the sequencing enzyme.

Figure 2 shows the SnapGene map of the test plasmid.

Figure 3 shows the effect of different reaction conditions on DNA assembly efficiency. (A) Effect of different ligases on DNA assembly efficiency. Asterisks indicate the target product. (B) Effect of PEG4000 dosage on DNA assembly efficiency. Asterisks indicate the target product. (C) Effect of fragment number on DNA assembly efficiency. Two parallel experiments were performed for each reaction condition.

Figure 4 shows the assembly and characterization of the T7 genome and synI. (A) Electrophoretic analysis of PCR-amplified T7 DNA fragment TP15a. (B) Electrophoretic analysis of DSC-treated PCR fragment TP6a. (C) Electrophoretic analysis after the first stage of T7 DNA assembly. (D) Electrophoretic analysis after the second stage of T7 DNA assembly. (E) Phage plaque assay of *E. coli* transformed with the assembled T7 genome. (F) Electrophoretic analysis of homologous DNA fragments with desired cantilevers generated by DSC. Synthesized DNA was provided in 2-4 kb fragments in plasmids and digested using DSC (top). The released DNA fragments were purified and validated (bottom). (G) Electrophoretic analysis after the first stage of synI DNA assembly. Target products are indicated by colored asterisks. +/- indicates the presence or absence of DNA ligase. (H) Electrophoretic analysis after the second stage of synI DNA assembly. Colored asterisks point to assembled synI. +/- indicates the presence or absence of DNA ligase. (1) Pulsed-field gel electrophoresis analysis (left image) and Southern blotting analysis of four yeast clones containing synI (middle image: YAL003C specific probe, right image: URA3 specific probe). BY4742 was used as a control. P1 and P2 were transformed with purified synI. Clones. M1 and M2 are clones transformed from the mixed ligation product after secondary assembly. (J) Nanopore sequencing analysis of chromosome isoforms of four yeast clones. Each circle represents a genome fragment. Open, filled, and half-filled circles represent wild-type, synthetic, and hybrid types, respectively. P1 and P2 are clones transformed with purified synI. M1 and M2 are clones transformed from the mixed ligation product after secondary assembly.

Figure 5 shows a schematic diagram of the structure and assembly of synI. (A) The two markers of synI, HIS3 and URA3, are located near the two ends of the telomeres. (B) SynI is divided into 9 chunks, each containing 6-7 fragments.

Figure 6 shows the PCR tag analysis of four yeast clones containing synI. M1(A) and M2(B) are clones transformed from the mixed ligation product of the second assembly. P1(C) and P2(D) are clones transformed with purified synI. SYN: synthetic PCR tags, WT: wild-type PCR tags.

Figure 7 shows the structures of the synI and chrI isoforms. Sequencing evidence for the synI and chrI subtype structures in strains M1(A), M2(B), P1(C), and P2(D) is plotted in the arrow diagram and stacked histogram. Breakpoints are identified by hybridization reads containing both synthetic and wild-type PCR tags. All reads spanning breakpoints longer than 10 kb are plotted as arrows. The stacked histogram shows the sequencing depth of the PCR tag sites. SYN: synthetic PCR tags are light gray; WT: wild-type PCR tags are dark gray.

Detailed Implementation

To better understand the present invention, it is now further described with reference to the following embodiments and accompanying drawings. The embodiments are for illustrative purposes only and do not limit the invention in any way. In the embodiments, all original reagents and materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

Example 1: Development of an in vitro iterative DNA assembly strategy based on DNA guide strand cutting technology

DNA-guided strand cleavage, abbreviated as DSC, is a technique that creates single-strand breaks in target single-stranded DNA using a DSC system. The DSC system includes a nicking endonuclease and a single-stranded guide DNA (sgDNA). The sgDNA interacts with the nicking endonuclease to anchor the target sequence and precisely cleaves at specific sites within the target single-stranded DNA, creating a notch. This invention, based on DSC technology, generates ultra-long and precise cantilever arms at both ends of double-stranded DNA fragments for subsequent DNA assembly. The following section details the in vitro iterative DNA assembly strategy based on DNA-guided strand cleavage technology.

1. Programmed DSC is used for precise cutting of single-stranded DNA.

Programmable DSC technology enables precise cleavage of single-stranded DNA based on a DSC system. The DSC system comprises a nicking endonuclease and a single-stranded guide DNA (sgDNA). The nicking endonuclease creates single-strand breaks in the target DNA (Figure 1A). The key component of DSC is the single-stranded guide DNA (sgDNA), which consists of a 5' target recognition sequence (TRS) and a short DNA (shDNA) sequence folded into a stem-loop structure at the 3' end. The TRS contains approximately 20 nucleotides complementary to the target single-stranded DNA (ssDNA) and binds to it via DNA-DNA base pairing. The stem-loop structure provides a recognition site for the nicking endonuclease, forming a DNA-enzyme complex that creates a gap within the target ssDNA. Therefore, DSC can be programmed to cleave any sequence in the target sequence that lacks a recognition site, allowing for seamless DNA assembly and avoiding the introduction of additional sequences.

In this embodiment, the system and reaction conditions for DSC cleavage of the target ssDNA were as follows: 4 μg of the target DNA molecule to be cleaved was digested in a mixed solution of 1x Cutsmart Buffer (NEB), 1 mM DTT, 8% PEG4000 (Thermo), 3 μM sgDNA, and 12 U Nt. BstNBI (NEB). The digestion was carried out at 50°C for 2 hours.

To verify that DSC can accurately cleave the target ssDNA, a series of sgDNAs (as shown in Table 1) were synthesized in this embodiment to guide the cleavage. Nt.BstNBI nicking endonuclease cleaves 50nt ssDNA of the ampicillin resistance gene (its nucleotide sequence is shown in SEQ ID NO.12). Each sgDNA contains 10–14nt TRS, depending on their calculated melting temperature, and pairs with the target ssDNA at 4nt intervals starting from the 5' end. sgDNA-1, meaning its TRS binds to nts 1–10, cleaves the target ssDNA with the nicking endonuclease Nt.BstNBI after the 4th nucleotide, producing two nucleotide segments of 4 and 46 nt. Similarly, sgDNA-11 binds 10 nts to the 3' end of the target ssDNA, producing oligonucleotides of 44 and 6 nt. These sgDNAs bind to Nt.BstNBI, cleave the target ssDNA, and are analyzed by PAGE (Figure 1D). As expected, lane 1 serves as a negative control, containing only the full-length target gene ssDNA and Nt.BstNBI, but no sgDNA. In lanes 2-11, for each sgDNA, the target ssDNA was digested into two fragments of the expected size. In lanes 2 and 11, the smaller squares were 4 nt and 6 nt, respectively, which were too short to be observed.

Table 1

2. DSC-assisted generation of the required sticky ends of dsDNA

Based on the strategy of using programmed DSC for precise single-stranded DNA cleavage, DSC is used to cleave double-stranded DNA (dsDNA) to generate double-strand breaks (DSBs). However, unlike ssDNA, the target sequence in dsDNA hybridizes with its complementary strand, preventing TRS binding and blocking enzyme cleavage. Therefore, to overcome this limitation, this invention designs a special vector pCCE (nucleotide sequence shown in SEQ ID NO. 13). The target DNA fragment is ligated into the vector pCCE, and three adjacent recognition sites of Nt.BspQI are designed on the sides of the target DNA fragment, exposing the first nucleotide of the desired 5' cantilever after enzyme digestion, and releasing short oligonucleotides between these gaps to form a single-stranded DNA (ssDNA) region (Figure 1B). Next, in the presence of DSC, the TRS sequence pairs with the ssDNA region, separating the designed 5' cantilever, and Nt.BstNBI cleaves the target strand. The other end of the DNA fragment is similarly treated, generating a designed cantilever at the other end for subsequent assembly.

In this embodiment, the system and reaction conditions for DSC-assisted generation of the required dsDNA sticky ends are as follows: 4 μg of the target DNA molecule to be cleaved is digested in a mixed solution of 1x cutsmart buffer (NEB), 1 mM DTT, 8% PEG4000 (Thermo), 3 μM sgDNA, 12 U Nt.BspQI (NEB), and 12 U Nt.BstNBI (NEB). The mixture is incubated at 50°C for 2 hours.

This embodiment uses DSC to cut the two strands at their relative positions, obtaining 5' cantilevers at both ends of the dsDNA. Since most DNA polymerases possess exonuclease activity, the generation of specific 5' cantilevers minimizes potential environmental contamination. It allows us to directly examine the integrity and accuracy of the cantilevers via Sanger sequencing, a prerequisite for error-free and efficient assembly. Sanger sequencing introduces dideoxynucleotide triphosphates (ddNTPs) during DNA synthesis to prepare DNA fragments of varying lengths. These fragments are separated according to their size in gel electrophoresis to obtain the complete DNA sequence. In the presence of 5' cantilevers, ddNTPs are used to extend the short strands in the dsDNA to obtain the desired signal.

A 761 bp DNA fragment was inserted into pMV to construct a test plasmid (Figure 2, the nucleotide sequence of the test plasmid is shown in SEQ ID NO. 14). After DSC treatment, fragments with 5' cantilevers at both ends were released. For sequencing of both ends, this fragment was further digested into two smaller fragments by adding another restriction endonuclease, BtsI. As expected, fluorescence signals were detected between 276 nt–290 nt and 487 nt–501 nt (Figure 1E). DNA sequences corresponding to the 5' cantilevers were obtained from the fluorescence signals, and each 5' cantilever perfectly matched the design. The results indicate that DSC can generate the designed sticky ends with high fidelity, and the cantilevers remain intact during DNA extraction, ensuring high-quality DNA assembly in subsequent processes.

The nucleotide sequence of the vector pCCE (SEQ ID NO.13):

Nucleotide sequence of the test plasmid (SEQ ID NO.14):

3. DNA assembly

Utilizing the generated DNA fragments containing the desired overhangs, this invention develops a stepwise chromosome assembly strategy called DSC-assisted iterative (Dai) assembly for the in vitro construction of chromosome-sized linear DNA molecules (Figure 1C). In the specific assembly process, the linear chromosome is broken down into multiple DNA fragments obtained from a DNA synthesis company and cloned into a designed vector pCCE, or by inserting three adjacent Nt.BspQI sites at both ends of a synthesized DNA fragment. During assembly, these fragments are first treated with DSC to generate the desired overhangs at each end, and then purified by agarose gel electrophoresis. Next, multiple adjacent fragments are mixed and ligated to obtain a DNA fragment approximately 40 kb in length, which is then purified again by gel electrophoresis to remove unligated fragments. This process is repeated in the next round of assembly to generate larger DNA fragments (~200 kb), ultimately yielding the entire chromosome (Figure 1C). For longer chromosomes, more assemblies can be performed; theoretically, this assembly process can be repeated as needed.

Example 2: Construction of the T7 genome using Dai assembly

This embodiment utilizes Dai assembly to construct the T7 phage genome (approximately 30 kb in length). The entire genome was randomly divided into 15 fragments, each approximately 2 kb in length. Because some DNA fragments are toxic to *E. coli*, they could not be cloned into the designed pCCE vector. Therefore, three adjacent Nt.BspQI sites were inserted at both ends of the amplified DNA fragments, and the required 5' cantilever was directly generated using DSC. For subsequent assembly. This invention uses the "quasi-plasmid method" for the splicing and transformation of the T7 genome. The entire process does not require cloning the sequence into a plasmid, and specifically includes the following steps:

1. Primers were designed for three-round splicing PCR. Quasi-plasmids were obtained from the three rounds of PCR. Quasi-plasmids are PCR products with extended sequences at both ends. Their enzyme digestion products can be distinguished by electrophoresis.

(1) The T7 genome was divided into 15 segments (denoted as seg_3k), each segment being 2k-3k in length, as shown in Table 2 below. In addition to the vector pTEF, there were a total of 16 segments.

Table 2

Nucleotide sequence of vector pTEF (SEQ ID NO.15):

(2) Each seg_3k requires the addition of approximately 1k of auxiliary sequences at both ends. The advantage of this design is that after enzyme digestion, there is a length difference of 1k or more between the incompletely digested PCR product and the fully digested PCR product, which can be easily distinguished by electrophoresis. This ensures that the recovered fragment ends contain a high proportion of the expected pattern. In this invention, this PCR product with added long auxiliary sequences is referred to as "quasi-..." "Plasmids" are fragments obtained by digesting plasmids with enzymes, which have a ligation efficiency close to that of plasmid digestion fragments during splicing.

(3) To obtain the quasi-plasmid, three rounds of PCR are required (the polymerase used in this invention is Phantamax, which is to ensure the accuracy of the sequence as much as possible). The PCR primers for the 16 fragments are shown in Table 3.

Each seg_3k fragment corresponds to 3 PCR products, therefore 3 pairs of primers need to be designed. Taking the amplification of the TP1a fragment as an example, the three rounds of PCR are explained in detail:

The first round of PCR used three primer pairs: CCE1671F and TX1vR, TX1c24F and TX1c2224R, and TX1vF and CCE4357R. Primers CCE1671F and TX1vR, and TX1vF and CCE4357R, amplified sequences on the pCCE vector, containing the left and right sides of the Nt.BspQI sequence, respectively. Primers CCE1671F and CCE4357R were identical across all fragments, while the four middle primers had different seg_3k values for each fragment. Primers TX1c24F and TX1c2224R amplified the T7 genome as a template. The shaded regions of TX1vR and TX1vF were reverse complementary to TX1c24F and TX1c2224R, respectively, allowing for PCA splicing after the three fragments were obtained by PCR.

Second round PCR: The three recovered fragments from the first round PCR were mixed and used as templates for three-segment PCA amplification. The primers were CCE1671F and CCE4357R.

The third round of PCR: Using the recovered products from the second round of PCR as templates, amplification was performed using primers CCE1722F and CCE6819R. These primers are more internal than CCE1671F and CCE4357R, so the third round is essentially nested PCR. Nested PCR is used to obtain cleaner target fragments. The third round PCR products need to be methylated with M.MlyI methyltransferase before use. Methylation can be performed before or after gel recovery of the PCR products; in this invention, methylation is performed before recovery. These fragments are easily obtained through PCR amplification. Figure 4A shows the electrophoretic analysis results of the PCR amplified fragment TP15a.

Table 3

2. The recovered third-round PCR products were digested with designed sgDNA. The designed sgDNA sequences for the 16 fragments are shown in Table 4. The digestion reaction system and conditions were as follows: 4 μg of the target DNA molecule to be digested was digested in a mixed solution of 1x Cutsmart buffer (NEB), 1 mM DTT, 8% PEG4000 (Thermo), 3 μM sgDNA, 12 U Nt. BspQI (NEB), and 12 U Nt. BstNBI (NEB). The digestion was carried out at 50°C for 2 hours.

Figure 4B shows the electrophoresis image of the third-round PCR product of fragment TP6a after enzyme digestion. The main band is the target enzyme digestion fragment, the secondary band above it is the PCR product that was not completely digested, and you can see that there is a large distinction in the electrophoresis image. The band below is the auxiliary sequence of about 1k that was cut out.

Table 4

3. T7 genome assembly

(1) Assembly conditions optimization

The required dsDNA sticky ends were generated by DSC-assisted enzyme digestion, and the desired cantilever arms were obtained at both ends of 15 DNA fragments for subsequent assembly. In vitro DNA assembly includes two steps: hybridization and assembly. This example investigated the effects of different ligases, PEG4000 dosage, and fragment number on the efficiency of the assembly reaction through the following experiments. The hybridization reaction system was as follows: adjacent enzyme-digested DNA fragments were assembled in 50 μL of hybridization buffer (100 mM NaCl, 20 mM Tris-Ac, 1 mM EDTA, 8% PEG4000, pH 7.5), with 15 ng of each fragment. The hybridization reaction conditions were: 55℃, 5 min; 45℃, 10 min; 40℃, 10 min; 35℃, 10 min; 20℃, 10 min. After the hybridization reaction, the assembly reaction system was added to the hybridization reaction system to carry out the assembly reaction. The assembly reaction system was as follows: 0.1 U DNA was added to 5 μL of assembly buffer (30 mM Tris-HCl, 4 mM _MgCl₂ , 26 μM NAD, 1 mM DTT, 50 μg/mL BSA). Ligase. Assembly reaction conditions: 30℃, 10 min; 30℃, 1 min; 42℃, 1 min; cycle time: 3 h.

1) Study on the effect of different ligases on DNA assembly efficiency

Seven adjacent, enzyme-digested DNA fragments (TP1a-TP7a), 15 ng each, were collected and assembled using the in vitro DNA assembly conditions described above. The enzymes used in this experiment were Taq DNA ligase (NEB), E. coli DNA ligase (NEB), T4 DNA ligase (NEB), T7 DNA ligase (NEB), and 9°N ^™ DNA ligase (NEB), with each enzyme used in duplicate.

2) Effect of PEG4000 dosage on DNA assembly efficiency

Seven adjacent, enzyme-digested DNA fragments (TP1a-TP7a), 15 ng each, were collected and assembled under the aforementioned in vitro DNA assembly conditions. The DNA ligase used in this experiment was *E. coli* DNA ligase. Assembly experiments were conducted with PEG4000 volume percentage concentrations of 0%, 4%, 8%, 12%, and 16% to test the effect of PEG4000 volume percentage concentrations on assembly efficiency. Each concentration was tested in duplicate.

3) The effect of fragment number on DNA assembly efficiency

Assemble several adjacent, enzyme-digested DNA fragments, 15 ng each, using the in vitro DNA assembly conditions described above. The DNA ligase used in this experiment was E. coli DNA ligase. The assembly efficiency of 3 (TP1a-TP3a), 6 (TP1a-TP6a), 9 (TP1a-TP9a), 12 (TP1a-TP12a), and 15 (TP1a-TP15a) adjacent DNA fragments was tested, with each condition performed twice in parallel.

Figure 3 shows the experimental results of the effect of different reaction conditions on the efficiency of DNA assembly reaction.

After optimizing the above conditions, the optimal system and conditions for in vitro DNA assembly were determined. The hybridization reaction system consisted of assembling 6 or 7 adjacent enzyme-digested DNA fragments (90 ng each) in 50 μL of hybridization buffer (100 mM NaCl, 20 mM Tris-Ac, 1 mM EDTA, 8% PEG4000, pH 7.5). The hybridization reaction conditions were: 55℃ for 5 min; 45℃ for 10 min; 40℃ for 10 min; 35℃ for 10 min; 20℃ for 10 min. After the hybridization reaction, the assembly reaction system was added to the hybridization reaction system for assembly. The assembly reaction system consisted of 10 U of *E. coli* DNA ligase added to 5 μL of assembly buffer (30 mM Tris-HCl, 4 mM _MgCl₂ , 26 μM NAD, 1 mM DTT, 50 μg/mL BSA). The assembly reaction conditions were: 30℃ for 10 min; 30℃ for 1 min; 42℃ for 1 min; and the cycle time was 3 h.

(2) Full-length splicing

To generate a complete T7 genome, this embodiment performed two rounds of assembly. The first round involved assembling the 15 DSC-processed DNA fragments and the pTEF vector into four groups: TP3a, TP4a, TP5a, and TP6a; TP7a, TP8a, TP9a, and TP10a; TP11a, TP12a, TP13a, and TP14a; and TP1a, TP2a, TP15a, and pTEF, resulting in larger DNA fragments (Figure 4C). The second round of assembly used the fragments recovered in the first round (Figure 4D).

(3) Chemical transformation of splicing

The assembled product was directly transformed into E. coli for phage patch experiments (Figure 4E). Genomic DNA was isolated from the patches and sequenced, indicating that the T7 genome was successfully assembled.

Example 3: Construction of a complete yeast chromosome in vitro using the Dai assembly method

This embodiment uses Dai assembly to test whether a complete linear chromosome can be reassembled in vitro. For this purpose, chromosome 1 (synI) synthesized by budding yeast is used as an example, with a length of approximately 180 kb. In Sc2.0, synI is synthesized as a fusion with synthesized chromosome 13 (synVIII). SynI, as an independent chromosome, had never been tested. Here, the inventors restored synI to an independent chromosome by placing the native centromere of chromosome I at its original site and adding UTC (universal telomere cap) sequences to both ends. To facilitate the selection of synI in yeast, two markers, HIS3 and URA3, were inserted near the left and right telomeres of yeast (Figure 5A), with the remaining sequences consistent with reports. The designed synI sequence was divided into 62 fragments, each approximately 3kb in length (YST1-YST62). The sequences of YST1-YST62, the corresponding sgDNA, and the generated cantilever are shown in Tables 5 and 6. These fragments were chemically synthesized and cloned into pCCE for subsequent assembly. SynI assembly consisted of two stages (Figure 5B). In the first stage, each plasmid was treated with DSC to release the DNA fragment, which had 5' cantilever of 8-17 nts at both ends (Figure 4F and Table 5). Nine parallel assembly reactions were established, each with six to seven adjacent fragments, which were ligated together by annealing to produce nine large DNA fragments of ~20 kb in length (Figure 4G). As a control, the same reaction was performed without ligase. In the next stage, the nine fragments were assembled together to form the full-length synI, as indicated by the asterisk in Figure 4H.

Table 5

Table 6

Example 4: Transformation and Characterization of synI

The assembled SynI was transformed into BY4742 via yeast protoplast transformation and cultured on synthetic intact medium (SC-His-Ura) without histidine and uracil. Clones M1/M2 (transformed with the mixed ligation product after the second assembly) and P1/P2 (transformed with purified synI) were isolated and confirmed using all synthetic and wild-type PCR tags (Figure 6 and Table 7). Next, pulsed-field gel electrophoresis (PFGE) was performed on the four strains to assess the presence of synI and native chromosome I (chrI). As shown in the left panel of Figure 4I, chrI is the smallest chromosome that migrated to the front of the PFGE in BY4742, indicated by a colored asterisk. The chromosomes of all four clones are smaller than chrI, indicating the presence of synI. Multiple bands of different sizes inferred the presence of different isoforms of synI. Considering the high sequence similarity between synI and chrI, we speculate that these bands may be recombinants of the two chromosomes.

Table 7

To differentiate between multiple chrI/synI subtypes, Southern blotting was employed. The inventors used two different probes: one specific to the YAL003C gene in both chrI and synI, and the other specific to the URA3 gene in synI (Table 8). As shown in the middle and right of Figure 4I, URA3 was present in four clones, P1, P2, and M1, exhibiting the expected size, indicating the presence of assembled synI or its recombinants. In P2, M1, and M2, the YAL003C probe detected an intermediate band between synI and chrI, indicating they were recombinants. In M1 and M2, the URA3 probe did not detect an intermediate band, indicating the absence of the URA3 gene in the recombinants. Interestingly, in M2, the URA3 probe observed a band the same size as chrI, possibly due to a small region, including URA3, being recombined into native chrI. In clone P1, the inventors observed that both chrI and synI were intact. The inventors further verified the presence of synI and chrI using Oxford nanopore technology (ONT) sequencing. Consistent with the Southern blot results, intact synI and chrI were found only in the P1 clone, while other clones showed more chimeric regions of synI and chrI recombination (Figures 4J and 7). In summary, the above data indicate that synI can be completely delivered into yeast cells and exist independently.

Table 8

Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims (16)

The use of a double-stranded DNA sticky end generation system in the in vitro synthesis and assembly of large genomic DNA fragments, characterized in that the system comprises a DNA assembly sequence, a single-stranded guide DNA, nicking endonuclease A, nicking endonuclease B, and nicking endonuclease C; The DNA assembly sequence includes a first nicking endonuclease recognition site unit, a target DNA sequence, and a second nicking endonuclease recognition site unit from the 5' end to the 3' end. The first nicking endonuclease recognition site unit includes 2 or 3 nicking endonuclease A recognition sites and spacer bases located between the nicking endonuclease A recognition sites. The second nicking endonuclease recognition site unit includes 2 or 3 nicking endonuclease B recognition sites and spacer bases located between the nicking endonuclease B recognition sites. The nicking endonuclease A and nicking endonuclease B belong to the same type of nicking endonuclease. The cleavage sites of the nicking endonuclease A and nicking endonuclease B are located on the upper and lower strands of the DNA. After the DNA assembly sequence is digested by nicking endonuclease A and nicking endonuclease B, single-stranded regions are formed at both ends of the DNA assembly sequence. The single-stranded guide DNA includes a first single-stranded guide DNA and a second single-stranded guide DNA. The single-stranded guide DNA consists of a recognition sequence of the target sequence and a short DNA sequence folded into a stem-loop structure. The target sequence is a single-stranded DNA sequence including a portion of the single-stranded region and a portion of the adjacent target DNA sequence. This portion of the single-stranded region is referred to as the first target sequence, and the portion of the adjacent target DNA sequence is referred to as the second target sequence. The stem-loop structure folded into the short DNA sequence has a nicking endonuclease C recognition site but lacks a sequence that can be cleaved by the nicking endonuclease C. After the recognition sequence of the target sequence hybridizes with the first target sequence, the second target sequence... The corresponding other DNA strand is separated, and the recognition sequence of the target sequence further hybridizes with the second target sequence. The target sequence and the recognition sequence hybridize to form a double-stranded structure, which can be recognized by the nicking endonuclease C. This places the predetermined position of the second target sequence at a position that can be cleaved by the nicking endonuclease C through the recognition of the recognition sequence on the single-stranded guide DNA. The recognition sequences of the target sequences of the first single-stranded guide DNA and the second single-stranded guide DNA hybridize with partial sequences of the single-stranded regions formed at both ends of the DNA assembly sequence after digestion by nicking endonuclease A and nicking endonuclease B, as well as partial sequences of their adjacent target DNA sequences, to form a double-stranded structure. According to the use described in claim 1, the nicking endonuclease A and nicking endonuclease B are respectively selected from one of Nt.BbvCI, Nt.AlwI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPII, or one of Nb.BbvCI, Nb.BsmI, Nb.BsrDI, and Nb.BtsI; Preferably, the first nicking endonuclease recognition site unit includes three nicking endonuclease A recognition sites, and the second nicking endonuclease recognition site unit includes three nicking endonuclease B recognition sites; Preferably, the nicking endonuclease A and nicking endonuclease B are the same nicking endonuclease; Preferably, the nicking endonuclease A and nicking endonuclease B are Nt.BspQI. According to the use described in claim 1, the first nicking endonuclease recognition site unit includes three nicking endonuclease A recognition sites, and the second nicking endonuclease recognition site unit includes three nicking endonuclease B recognition sites. According to the use of claim 1, the spacer bases between the nicking endonuclease A recognition sites satisfy the following condition: the nicking endonuclease A cleavage site is not located in other nicking endonuclease A recognition sequences; The spacer bases between the recognition sites of the nicking endonuclease B satisfy the following condition: the cleavage site of the nicking endonuclease B is not located in other... The cleavage sequence is recognized by endonuclease B. Preferably, the spacer bases between the nicking endonuclease A recognition sites and the spacer bases between the nicking endonuclease B recognition sites are 4 to 10 randomly arranged A, C, G or T bases, and the spacer bases do not form nicking endonuclease recognition sites. According to the use described in claim 1, the nicking endonuclease C is selected from one of Nt.BbvCI, Nt.AlwI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, Nb.BbvCI, Nb.BsmI, Nb.BsrDI, and Nb.BtsI; Preferably, the nicking endonuclease C is selected from Nt.BstNBI. According to the use described in claim 1, the 5' end of the first nicking endonuclease recognition site unit and the 3' end of the second nicking endonuclease recognition site unit of the DNA assembly sequence each include an auxiliary sequence of 500-1500 bp in size; or, the DNA assembly sequence is cloned into a plasmid. According to the use described in claim 1, the single-chain region has a length of 5-100 bases. According to the use of claim 1, the second target sequence is characterized in that its length is between 5 and 100 bases, preferably between 5 and 30 bases, and more preferably between 10 and 30 bases. According to the use described in claim 1, the sticky end has a length of 5-30 bases, preferably 5-20 bases. According to the use described in claim 1, the large fragment of genomic DNA is a yeast chromosome or a T7 phage genome. A method for synthesizing and assembling large genomic DNA fragments in vitro, characterized by employing the double-stranded DNA sticky end generation system described in claim 1, comprising the following steps: (1) Divide the genome sequence into n target DNA sequences, where n is a positive integer, prepare n DNA assembly sequences, and design and synthesize single-stranded guide DNA for each DNA assembly sequence; (2) Add nicking endonuclease A, nicking endonuclease B and nicking endonuclease C to each DNA assembly sequence and its corresponding single-stranded guide DNA for enzyme digestion. The sticky ends generated by the enzyme digestion of adjacent target DNA sequences are complementary. (3) The DNA assembly sequence after enzyme digestion in step (2) is assembled into DNA in vitro. According to the synthesis and assembly method of claim 11, the target DNA sequence in step (1) is 300bp-50kb in size; Preferably, the method for preparing the DNA assembly sequence in step (1) is as follows: cloning the target DNA sequence into a plasmid containing a first nicking endonuclease recognition site unit and a second nicking endonuclease recognition site unit, wherein the target DNA sequence is located between the first nicking endonuclease recognition site unit and the second nicking endonuclease recognition site unit; or, inserting the first nicking endonuclease recognition site unit and the second nicking endonuclease recognition site unit at both ends of each target DNA sequence, and amplifying to obtain the DNA assembly sequence. The synthesis and assembly method according to claim 12, characterized in that the plasmid is pCCE, and its nucleotide sequence is as shown in SEQ ID NO. Shown in NO.13; The DNA assembly sequence is obtained by amplifying the 5' end of the first nicking endonuclease recognition site unit and the 3' end of the second nicking endonuclease recognition site unit, which each include an auxiliary sequence of 500-1500 bp in size. According to the synthesis and assembly method of claim 11, the enzymatic digestion in step (2) is carried out at 30-75°C, preferably at 45-65°C, and more preferably at 50-60°C. Preferably, the enzyme digestion system in step (2) is: 4 μg DNA assembly sequence, 1x cutsmart buffer, 1 mM DTT, 8% PEG4000 (v/v), 3 μM single-stranded guide DNA, 12 U nicking endonuclease A, 12 U nicking endonuclease B and 12 U nicking endonuclease C; the enzyme digestion conditions are incubation at 50°C for 2 h. According to the synthesis and assembly method of claim 11, the DNA in vitro assembly in step (3) is performed in multiple rounds according to the size of the genomic DNA fragment, thereby obtaining a large fragment of genomic DNA; Preferably, the DNA in vitro assembly in step (3) includes two steps: a hybridization reaction and an assembly reaction performed sequentially. 1) Hybridization reaction: Adjacent DNA assembly sequences after enzyme digestion were added to the hybridization buffer for hybridization reaction; the hybridization reaction conditions were: 55±5℃, 5±3min; 45±5℃, 10±5min; 40±5℃, 10±5min; 35±5℃, 10±5min; 20±5℃, 10±5min; the hybridization buffer composition was 100mM NaCl, 20mM Tris-Ac, 1mM EDTA, PEG4000 with a volume percentage concentration of 8%-16%, and pH 7.5. 2) Assembly reaction: After the hybridization reaction, add assembly buffer and DNA ligase to the hybridization reaction system; the assembly reaction conditions are: 30±5℃, 10±5min; 50-100 cycles × (30±5℃, 1±0.5min; 42±5℃, 1±0.5min); the assembly buffer components are 30mM Tris-HCl, 4mM MgCl2, _26μM NAD, 1mM DTT, and 50μg/mL BSA. The synthesis and assembly method according to claim 15 is characterized in that the volume percentage concentration of PEG4000 in the hybridization buffer component is 8%; Preferably, Escherichia coli DNA ligase or T7 DNA ligase is added to the hybridization reaction system, and more preferably Escherichia coli DNA ligase is added. Preferably, the number of adjacent DNA assembly sequences after enzyme digestion added to the hybridization buffer is 3-12, rounded to the nearest integer. Preferably, the number of adjacent post-digestion DNA assembly sequences added to the hybridization buffer is 6 or 7.