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CN-121975792-A - High-flux long gene synthesis method and application thereof

CN121975792ACN 121975792 ACN121975792 ACN 121975792ACN-121975792-A

Abstract

High-flux long gene synthesis method and its application. The present invention provides methods for assembling (e.g., synthesizing) nucleic acid sequences of a variety of genes of interest, pools of oligonucleotide fragments, and uses thereof. The methods and pools of oligonucleotide fragments of the invention can be used to assemble the full-length nucleic acid sequences of multiple genes of interest simultaneously in a single or multiple reaction systems.

Inventors

  • ZHENG JIA
  • GUO NING
  • WANG CHEN
  • ZHUO WEIWEI
  • GUO XIANG

Assignees

  • 西湖大学

Dates

Publication Date
20260505
Application Date
20250430

Claims (20)

  1. 1. A method for synthesizing multiple genes of interest in a single reaction system, comprising: Respectively designing corresponding oligonucleotide fragments aiming at each of the multiple target genes, wherein the full-length sequence of each target gene is subjected to non-interval non-overlapping sequential splitting to obtain sequences of the multiple oligonucleotide fragments; synthesizing a pool of oligonucleotide fragments based on the sequences of the designed plurality of oligonucleotide fragments; phosphorylating the 5' -end of the oligonucleotide fragment, and Assembling the 5' -phosphorylated oligonucleotide fragment into a full-length nucleic acid sequence of the target gene by Ligase Chain Reaction (LCR); optionally, the oligonucleotide fragment is designed with a tag sequence at both ends, and the method comprises removing the tag sequence prior to LCR.
  2. 2. The method of claim 1, wherein the method of designing a corresponding pool of oligonucleotide fragments for the target gene comprises: Sequentially splitting the full-length sequence from two different splitting starting points in two splitting modes without interval and overlapping to obtain two groups of different oligonucleotide fragments; Wherein, the oligonucleotide fragments obtained by splitting the same splitting starting point have no interval and overlapping region, and the oligonucleotide fragments obtained by different splitting modes have overlapping or complementary regions of 6-5000 nt; optionally, the target gene is sequence optimized, e.g., codon optimized, by a computer program prior to resolution.
  3. 3. The method of claim 1 or 2, wherein the oligonucleotide fragments of the plurality of genes of interest comprise a pool of oligonucleotide fragments, optionally the pool of oligonucleotide fragments comprises two or more sub-pools of oligonucleotide fragments; optionally, wherein a single sub-pool comprises oligonucleotide fragments derived from 1-1000 genes of interest, preferably from 1-100 genes of interest.
  4. 4. A method according to any one of claims 1 to 3, wherein the method of designing the corresponding oligonucleotide fragment for the gene of interest comprises: For each target gene, the full-length sequence of the target gene is designed to have a first pair of tag sequences at both ends thereof, Optionally, the first pair of tag sequences comprises an enzyme recognition site that is an enzyme recognition site of a restriction enzyme and/or a barcode sequence that is genotype-specific, sub-library-specific or gene-specific.
  5. 5. The method of claim 1 to 4, wherein the method of designing a corresponding pool of oligonucleotide fragments for the target gene comprises designing each oligonucleotide fragment sequence to have a second pair of tag sequences at both ends thereof, Optionally, the second pair of tag sequences comprises a barcode sequence and one or more cleavage recognition sites, the barcode sequence being sub-library specific.
  6. 6. The method of claim 5, wherein the oligonucleotide fragment having the second pair of tag sequences is amplified by Polymerase Chain Reaction (PCR) using primers corresponding to the second pair of tag sequences prior to phosphorylating the 5' end of the oligonucleotide fragment.
  7. 7. The method of claim 6, wherein the number of cycles of the polymerase chain reaction is from 2 to 50 cycles, more preferably from 8 to 20 cycles; Preferably, the polymerase is a high-fidelity polymerase.
  8. 8. The method of claim 6, wherein the second pair of tag sequences is removed and 5 'end phosphorylated by contacting the oligonucleotide fragment having the second pair of tag sequences with an enzyme capable of hydrolyzing a phosphodiester bond and generating a 5' phosphate group.
  9. 9. The method of claim 8, wherein the enzyme is a restriction enzyme and the second pair of tag sequences comprises an enzyme recognition site for the restriction enzyme, preferably the restriction enzyme is a restriction enzyme capable of cleaving a double strand and producing a blunt end, more preferably an MlyI enzyme.
  10. 10. The method of claim 8, wherein the enzymes are a restriction enzyme and an S1 nuclease and the second pair of tag sequences comprises an enzyme recognition site for the restriction enzyme, preferably the restriction enzyme is a type IIS restriction enzyme.
  11. 11. The method of claim 8, wherein the enzymes are different nicking enzymes and the second pair of tag sequences comprises enzyme recognition sites for the different nicking enzymes.
  12. 12. The method of claim 8, wherein the enzymes are a restriction enzyme and a nicking enzyme, and the second pair of tag sequences comprises enzyme recognition sites for the restriction enzyme and the nicking enzyme, preferably the restriction enzyme is a type IIS restriction enzyme.
  13. 13. The method of claim 6, wherein the primers corresponding to the second pair of tag sequences comprise a forward primer comprising a 5 'phosphorothioate modification and optionally a 3' uracil modification and a reverse primer comprising a tag sequence reverse complement, the enzyme recognition site being selected from the group consisting of restriction endonucleases, nicking enzymes and enzyme recognition sites of the USER enzymes, or a combination thereof; optionally, the reverse primer comprises a 5' phosphorylation modification.
  14. 14. The method of claim 13, further comprising the step of preparing a 5 'phosphorylated single stranded oligonucleotide fragment after amplification for a reverse primer that does not comprise a 5' phosphorylation modification, wherein: Contacting the amplified oligonucleotide fragment with phosphokinase to phosphorylate one 5 'end thereof, or contacting the amplified oligonucleotide fragment with an enzyme corresponding to the enzyme recognition site, removing one tag from the second pair of tag sequences and phosphorylating one 5' end thereof, Treating the single-sided 5' -end phosphorylated oligonucleotide fragment with an exonuclease (e.g., lambda exonuclease) to obtain a single-stranded oligonucleotide fragment, The second pair of tag sequences is removed and the 5' end of the single stranded oligonucleotide fragment is phosphorylated.
  15. 15. The method of claim 13, further comprising the step of preparing a 5 'phosphorylated single stranded oligonucleotide fragment after amplification for a reverse primer comprising a 5' phosphorylation modification, wherein: Contacting the oligonucleotide fragment with an exonuclease (e.g., lambda exonuclease) to obtain a single stranded oligonucleotide fragment, The second pair of tag sequences is removed and the 5' end of the single stranded oligonucleotide fragment is phosphorylated.
  16. 16. The method of claim 14 or 15, wherein the second pair of tag sequences is removed by contacting the single stranded oligonucleotide fragment with an enzyme corresponding to the enzyme recognition site and optionally a primer comprising the second pair of tag sequences or the reverse complement thereof.
  17. 17. The method of any one of claims 11-16, wherein the nicking enzyme is selected from the group consisting of nt.bspqi, nt.cvipi, nt.bstnbi, nb.bsrdi, nb.btsi, nt.alwi, nb.bbvci, nt.bbvci, nb.bsmci, nb.bsssi, nt.bsmai, and Cas9 nicking enzymes, or a combination thereof.
  18. 18. The method of any one of claims 4-17, wherein the length of the first pair of tag sequences is 4nt to 60nt, e.g., 10nt to 60nt, 10nt to 50nt, 20nt to 40nt, 30nt to 50nt, 30nt to 40nt; preferably, the length of the first pair of labels is from 10nt to 30nt.
  19. 19. The method of any one of claims 5-18, wherein the length of the second pair of tag sequences is 4nt to 60nt, e.g., 10nt to 60nt, 10nt to 50nt, 20nt to 40nt, 30nt to 50nt, 30nt to 40nt; Preferably, the length of the second pair of labels is from 10nt to 30nt.
  20. 20. The method of any one of claims 1-4, wherein the 5' end phosphorylation is performed by a phosphokinase-mediated reaction or a chemical phosphorylation process (e.g., phosphoramidite or chemical modification reagent); preferably, the phosphokinase is a T4 polynucleotide kinase.

Description

High-flux long gene synthesis method and application thereof Technical Field The present invention belongs to the field of gene synthesis. In particular, the invention relates to a method for synthesizing multiple genes in a single or multiple reaction systems, including the design of oligonucleotide fragments, and uses thereof. Background Gene synthesis is a widely used technique in life science research. Although the cost of 1kb DNA synthesis by commercial gene synthesis has fallen to $100, large quantities of DNA synthesis remain quite expensive for many research and applications. In 2010 Sriram Kosuri a method was developed to achieve high throughput parallel gene synthesis using an oligonucleotide library, the strategy of which was to split an independent one gene into overlapping fragments and distribute them in an independent one assembled sub-library, then assemble into full length genes by Golden Gate based on IIS restriction endonuclease (BtsI, bspQI, bsrDI, earI, bsaI, bsmBI, sapI, bbsI) or assemble by dpnli/USER/lambda exonuclease to generate single stranded DNA. Sriram Kosuri the synthetic gene approach is still to subject the genes to single gene single well synthesis, each gene being split in one reaction. This method involves many and complicated procedures for the synthesis of gene libraries containing thousands of genes. The Sriram Kosuri group developed a method DropSynth for multiplex gene synthesis in emulsion droplets (emulgens) in 2018 and 2020, as well as an upgrade DropSynth 2.0.0. The method divides each gene into a plurality of oligonucleotides, wherein each oligonucleotide contains a corresponding IIS restriction enzyme site and flanking sequences of a barcode tag (barcode), and uses different magnetic beads to adsorb the oligonucleotides of different genes to different droplet compartments, with the oligonucleotides of the same gene in each droplet. Then, by overlapping PCR with high-fidelity polymerase, a full-length gene library was obtained. The DropSynth method for preparing the liquid beads has complicated operation, is only suitable for synthesizing genes with the length of <800bp, and has the fidelity of only about 20 percent when assembled by high-fidelity polymerase. A method of assembling oligonucleotides into polynucleotides that rely on Zip sequences is disclosed in WO/2023/096890. It divides each gene into n fragments, cuts the Zip region into cohesive ends using methods such as exonuclease, and assembles fragments 1 and 2 of multiple genes by ligation, cyclization, and cleavage of the Zip region, followed by assembly of fragments 3, 4, and n. The oligonucleotide assembly method based on Zip sequence is used for synthesizing long genes, and needs to repeatedly carry out the steps of enzyme digestion and cyclization, has complex and time-consuming operation, and has the accuracy of only 4% when synthesizing hundreds of 500bp genes. The single gene synthesis method used in the current gene synthesis is mostly high in cost. There are few reports of large-scale polygene synthesis methods, but the problems of complex synthesis reaction system, short synthesis length, high error rate, low yield and difficult assembly of complex systems still face. Therefore, there is a need to develop a new high-throughput gene synthesis method to provide a gene synthesis method that is simpler to operate, higher in accuracy, and lower in cost. Disclosure of Invention The method of the invention can realize simultaneous synthesis of one or more genes in a single reaction system, reduce the reaction steps of synthesis of multiple gene libraries, and reduce reagent consumption. For example, the three-step method for synthesizing the gene is simple to operate, has fewer steps, is easier to realize without a special instrument, and adopts ligase-mediated melting-annealing for seamless splicing, thereby reducing mutation and error caused by splicing in the traditional overlap PCR method to the greatest extent. In some aspects, the invention provides methods for synthesizing multiple target genes in a single reaction system, comprising designing respective oligonucleotide fragments for each of the multiple target genes, wherein the full-length sequence of each target gene is sequentially split without gaps and overlapping to obtain sequences of multiple oligonucleotide fragments, synthesizing a pool of oligonucleotide fragments based on the designed sequences of the multiple oligonucleotide fragments, phosphorylating the 5 'ends of the oligonucleotide fragments, and assembling the 5' end-phosphorylated oligonucleotide fragments into the full-length nucleic acid sequence of the target gene by Ligase Chain Reaction (LCR). Optionally, the oligonucleotide fragment is designed with a tag sequence at both ends, and the method comprises removing the tag sequence prior to LCR. In some embodiments, a method of designing a pool of corresponding oligonucleotide fragments for a target gene comprises sequen