WO2022242464A1 - 一种新型crispr相关转座酶 - Google Patents

一种新型crispr相关转座酶 Download PDF

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WO2022242464A1
WO2022242464A1 PCT/CN2022/091102 CN2022091102W WO2022242464A1 WO 2022242464 A1 WO2022242464 A1 WO 2022242464A1 CN 2022091102 W CN2022091102 W CN 2022091102W WO 2022242464 A1 WO2022242464 A1 WO 2022242464A1
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homology
seq
plasmid
gene
crispr
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杨晟
杨思琪
张译文
徐佳琪
张姣
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中国科学院分子植物科学卓越创新中心
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Definitions

  • the invention belongs to the technical field of gene editing, and in particular relates to a novel CRISPR-related transposase and its application in gene editing.
  • Zhang Feng's research group extracted a transposase from cyanobacteria and named it CAST, which stands for CRISPR-associated transposase (CRISPR-associated transposase).
  • CAST CRISPR-associated transposase
  • the Sternberg research group developed a gene called INTEGRATE (Insertion of transposable elements by guide RNA-assisted targeting, guide RNA-assisted targeted transposable element insertion) Editing tools that can insert large stretches of genes in the genome without introducing DNA breaks.
  • the new technology utilizes transposable genes to insert DNA sequences into the genome without cutting the DNA.
  • Both the CAST system and the INTEGRATE system can integrate DNA fragments into preset sites on the E. coli chromosome through transposition without relying on homologous recombination, without residual resistance markers and double-stranded DNA breaks.
  • MUCICAT multi-copy gene insertion system
  • MUCICAT based on a single CRISPR-related transposase can only explore the optimal dosage of a single cargo gene carrying a single or multiple genes, and cannot screen the optimal ratio of gene dosages for multiple genes or pathways. There are no reports of chromosomal copy number techniques for parallel amplification of multiple genes/pathways.
  • MUCICAT technology containing multiple CRISPR-related transposases that are orthogonal to each other is expected to achieve simultaneous and independent amplification of multiple genes or pathways in a single cell, forming a strain library with different gene dosage ratios to screen the optimal multiple genes or pathways dosage ratio.
  • CRISPR-related transposases ideally, if there are other new and efficient CRISPR-related transposases, or an orthogonal CRISPR-related transposase system can be formed with MUCICAT, it can be applied to complex metabolic engineering and synthesis in biological design.
  • the inventors have extensively screened CRISPR transposases derived from other microorganisms, and found a type I-F3 CRISPR transposition system derived from Pseudoalteromonas translucida KMM520 (Pseudoalteromonas translucida KMM520), which The insertion efficiency in Escherichia coli is no less than or even better than that of Vibrio cholerae Tn6677, and can be inserted into the eda-purT and lacZ sites without interfering with Vibrio cholerae Tn6677, respectively.
  • This novel CRISPR transposition system recognizes all 16 PAMs without PAM dependence.
  • the first aspect of the present invention is to provide a CRISPR-related transposase, which includes a polypeptide selected from the group consisting of the transposase protein tnsA derived from Pseudoalteromonas bacteria, the transposase protein tnsA derived from Pseudoalteromonas Transposase protein tnsB from bacteria of the genus Pseudoalteromonas, transposase protein tnsC from bacteria of the genus Pseudoalteromonas, transposase protein tniQ from bacteria of the genus Pseudoalteromonas, Bacterial nuclease protein Cas5/8, nuclease protein Cas6 derived from bacteria of the genus Pseudoalteromonas, nuclease protein Cas7 derived from bacteria of the genus Pseudoalteromon
  • the bacteria of the genus Pseudoalteromonas is Pseudoalteromonas translucidum. More preferably, the Pseudoalteromonas translucida is Pseudoalteromonas translucida KMM520 (Pseudoalteromonas translucida KMM520).
  • the above-mentioned CRISPR-related transposase preferably includes a polypeptide selected from the group consisting of:
  • tnsA it is a polypeptide having the amino acid sequence of SEQ ID NO: 1, or has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO: 1, and Functionally identical polypeptides;
  • tnsB it is a polypeptide having the amino acid sequence of SEQ ID NO: 2, or has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO: 2, and Functionally identical polypeptides;
  • tnsC it is a polypeptide having the amino acid sequence of SEQ ID NO:3, or has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO:3, and Functionally identical polypeptides;
  • tniQ it is a polypeptide having the amino acid sequence of SEQ ID NO: 4, or has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO: 4, and Functionally identical polypeptides;
  • Cas5/8 it is a polypeptide having the amino acid sequence of SEQ ID NO:5, or has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO:5 , and a polypeptide with the same function;
  • Cas6 it is a polypeptide having the amino acid sequence of SEQ ID NO:6, or has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO:6, and Functionally identical polypeptides;
  • Cas7 it is a polypeptide having the amino acid sequence of SEQ ID NO:7, or has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO:7, and functionally identical peptides.
  • nucleases Cas5/8, Cas7, and Cas6 are the Cascade complexes of the type I CRISPR system, which are associated with the transposases tnsABC and tniQ to become CRISPR-associated transposases. These polypeptides are derived from Pseudoalteromonas translucida KMM520 (Pseudoalteromonas translucida KMM520).
  • the second aspect of the present invention provides a gene encoding the above-mentioned polypeptide.
  • the gene encoding the polypeptide tnsA having the amino acid sequence of SEQ ID NO: 1 is the nucleotide sequence of SEQ ID NO: 8, or has more than 80% homology with SEQ ID NO: 8, preferably Polynucleotides with more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology;
  • the gene encoding the polypeptide tnsB having the amino acid sequence of SEQ ID NO: 2 is the nucleotide sequence SEQ ID NO: 9, or has more than 80% homology, preferably more than 85% homology, more Polynucleotides with preferably more than 90% homology, more preferably more than 95% homology;
  • the gene encoding the polypeptide tnsC having the amino acid sequence of SEQ ID NO:3 is the nucleotide sequence of SEQ ID NO:10, or has more than 80% homology, preferably more than 85% homology, more homology with SEQ ID NO:10 Polynucleotides with preferably more than 90% homology, more preferably more than 95% homology;
  • the gene encoding the polypeptide tniQ having the amino acid sequence of SEQ ID NO: 4 is the nucleotide sequence SEQ ID NO: 11, or has more than 80% homology, preferably more than 85% homology, more homology with SEQ ID NO: 11 Polynucleotides with preferably more than 90% homology, more preferably more than 95% homology;
  • the gene encoding the polypeptide Cas5/8 having the amino acid sequence of SEQ ID NO:5 is the nucleotide sequence SEQ ID NO:12, or has more than 80% homology, preferably more than 85% homology with SEQ ID NO:12 , more preferably a polynucleotide with more than 90% homology, more preferably more than 95% homology;
  • the gene encoding the polypeptide Cas6 having the amino acid sequence of SEQ ID NO: 6 is the nucleotide sequence SEQ ID NO: 13, or has more than 80% homology, preferably more than 85% homology, more homology with SEQ ID NO: 13 Polynucleotides with preferably more than 90% homology, more preferably more than 95% homology;
  • the gene encoding the polypeptide Cas7 having the amino acid sequence of SEQ ID NO: 7 is the nucleotide sequence SEQ ID NO: 14, or has more than 80% homology, preferably more than 85% homology, more homology with SEQ ID NO: 14 It is preferably a polynucleotide with a homology of more than 90%, more preferably a homology of more than 95%.
  • the third aspect of the present invention is to provide a CRISPR transposon system.
  • the CRISPR transposon system of the present invention also includes plasmid pQCascade, helper plasmid pTns, and helper plasmid pDonor carrying cargo genes. Any two of these three plasmids can even be combined into one plasmid, and even these three plasmids can be combined together to form one plasmid. in particular,
  • a plasmid pQCascade for the CRISPR transposon system which includes a gene segment selected from the group consisting of: the above-mentioned Cas5/8 coding gene; the above-mentioned Cas6 coding gene; the above-mentioned Cas7 coding gene; the above-mentioned tniQ coding gene.
  • the plasmid pQCascade is named pQCascadePtr herein.
  • the plasmids pTns and pDonor of the present invention may be denoted as pTnsPtr and pDonorPtr, respectively, hereinafter.
  • the above-mentioned plasmid pQCascadePtr may also include the following genes: crRNA sequence targeting the target site in the genome, CloDF13 replicon, promoter such as anhydrocycline-inducible promoter, streptomycin resistance gene.
  • the crRNA in the above CRISPR transposase can function in the form of an array.
  • the above-mentioned spacer of the crRNA series may be a spacer targeting a single site in the genome of the cell to be treated.
  • the above-mentioned crRNA series may be a crRNA array (also known as CRISPRarray, crRNA array or array) targeting multiple sites in the genome of the cell to be treated.
  • crRNA array also known as CRISPRarray, crRNA array or array
  • the above-mentioned crRNA sequence is a crRNA array targeting multiple sites in the genome, wherein the repeat sequence region repeat includes more than one sequence selected from the following group: the nucleotide sequence is repeat1, nucleotide sequence of SEQ ID NO:15
  • the sequence is repeat2 of SEQ ID NO:16
  • the nucleotide sequence is repeat3 of SEQ ID NO:17
  • the nucleotide sequence is repeat4 of SEQ ID NO:18
  • these nucleotide sequences of SEQ ID NOs:15-18 are any base A, T, G or C.
  • a helper plasmid pTnsPtr for the CRISPR transposon system which is used in conjunction with the above-mentioned plasmid pQCascadePtr, which includes a gene segment selected from the group consisting of the above-mentioned tnsA-encoding gene, the above-mentioned tnsB-encoding gene, and the above-mentioned tnsC-encoding gene .
  • CRISPR transposase gene sequence When the above-mentioned CRISPR transposase gene sequence is used for CRISPR transposition, it needs to be combined with the host (gram-negative bacteria such as Escherichia coli, natrivibrio, and Tatumella citronella; gram-positive bacteria such as glutamic acid rod Bacillus) available promoters, Left end (LE)-cargo-Right end (RE) work together, whether in the form of a plasmid or in an integrated form.
  • host gram-negative bacteria such as Escherichia coli, natrivibrio, and Tatumella citronella; gram-positive bacteria such as glutamic acid rod Bacillus
  • L Left end
  • RE Left end
  • helper plasmid pTnsPtr also includes the following genes: ColA replicon, promoter such as anhydrocycline-inducible promoter, and kanamycin resistance gene.
  • the present invention provides a plasmid pQCasTnsPtr for the CRISPR transposon system, which is formed by merging the above-mentioned plasmid pQCascadePtr and the helper plasmid pTnsPtr, including: the above-mentioned Cas5 /8, Cas6, Cas7, tniQ, tnsA, tnsB, and tnsC genes, crRNA sequences targeting genomic target sites, ColA replicons, promoters such as anhydrocycline-inducible promoters, kanamycin resistance genes.
  • helper plasmid pDonorPtr that is used for CRISPR transposon system, it is used in conjunction with above-mentioned plasmid pQCascadePtr and above-mentioned helper plasmid pTnsPtr, and it comprises the gene segment that is selected from the following group: sequence Left end (LE), its nucleotide The sequence is SEQ ID NO: 19, or has more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID NO: 19 sex and include the 3' end 33bp sequence of SEQ ID NO: 19; sequence Right end (RE), its nucleotide sequence is SEQ ID NO: 20, or there is more than 80% homology with SEQ ID NO: 20, preferably More than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology and include the 27bp sequence of the 5' end of SEQ ID NO:
  • the LE with the nucleotide sequence of SEQ ID NO: 19 and the RE with the nucleotide sequence of SEQ ID NO: 20 are derived from Pseudoalteromonas translucidus KMM520 ( P seudoalteromonas tr anslucida KMM520), the plasmid pDonor Name it pDonorPtr.
  • auxiliary plasmid pDonorPtr can also include the following genes: pMB1 replicon, ampicillin resistance gene, target cargo gene (Cargo gene); or the following genes: p15A replicon, chloramphenicol resistance gene, target cargo Gene (Cargo gene).
  • the present invention provides a plasmid pEffectorPtr for the CRISPR transposon system, which is formed by merging the above-mentioned plasmid pQCascadePtr, the above-mentioned helper plasmid pTnsPtr and the above-mentioned helper plasmid pDonorPtr Composition, including: the above-mentioned Cas5/8, Cas6, Cas7, tniQ, tnsA, tnsB and tnsC genes, Left end (LE) and Right end (RE) sequences, target cargo genes, crRNA sequences targeting genomic target sites, ColA replicon, promoters such as anhydrocycline-inducible promoters, kanamycin resistance genes.
  • a plasmid pEffectorPtr for the CRISPR transposon system which is formed by merging the above-mentioned plasmid pQCascadeP
  • the fourth aspect of the present invention provides a CRISPR transposon system, which includes: plasmids pQCascadePtr, pTnsPtr and pDonorPtr; or plasmids pQCasTnsPtr and pDonorPtr; or plasmid pEffectorPtr.
  • a fifth aspect of the present invention provides a combination of the above-mentioned CRISPR transposon system derived from Pseudomonas translucidus KMM520 and the prior art CRISPR transposon system derived from Vibrio cholerae (Vibrio cholerae) Tn6677 Combinations use, specifically,
  • a CRISPR transposon system which, in addition to including the above-mentioned CRISPR transposon system (including: plasmids pQCascadePtr, pTnsPtr and pDonorPtr; or plasmids pQCasTnsPtr and pDonorPtr; or plasmid pEffectorPtr ), also includes Vibrio cholerae (Vibrio ch olerae) Tn6677-derived CRISPR transposase-associated plasmid.
  • Vibrio cholerae Vibrio ch olerae
  • the gene sequence of the type I-F3 CRISPR transposase of Vibrio cholerae Tn6677 is NCBI: NZ_ALED01000027.1.
  • CRISPR transposase-related plasmids derived from Vibrio cholerae Tn6677 include plasmid pQCasTnsVch and helper plasmid pDonorVch, wherein
  • Plasmid pQCasTnsVch includes: Cas5/8, Cas6, Cas7, tniQ, tnsA, tnsB and tnsC genes derived from Vibrio cholerae Tn6677, CloDF13 replicator, promoter such as anhydrocycline-inducible promoter, streptomycin resistance gene;
  • Plasmid pDonorVch includes CRISPR array derived from Vibrio cholerae Tn6677, Left end (LE) and Right end (RE), pMB1 replicon, ampicillin resistance gene, target cargo gene (Cargo gene).
  • the above-mentioned plasmids pQCasTnsVch and pDonorVch can be combined into plasmid pEffectorVch.
  • the above-mentioned promoter adopts anhydrotetracycline-inducible promoter
  • the above-mentioned CRISPR transposon system derived from Pseudomonas translucidus KMM520 is used to transform Escherichia coli BL21 (DE3), BL21Star TM (DE3) or W3110 (DE3) and other strains can be induced with anhydrotetracycline.
  • anhydrocycline can also be used for induction.
  • plasmids pEffectorPtr and pEffectorVch or plasmids pQCasTnsVch, pDonorVch, pQCasTnsPtr, and pDonorPtr are used to transform strains such as Escherichia coli BL21 (DE3), BL21Star TM (DE3) or W3110 (DE3), anhydrotetracycline is used for induction.
  • the sixth aspect of the present invention provides the above-mentioned CRISPR-related transposase, the above-mentioned gene encoding a polypeptide, the above-mentioned plasmid pQCascadePtr, the above-mentioned plasmid pTnsPtr, the plasmid pQCasTnsPtr, the plasmid pDonorPtr, the plasmid pEffectorPtr, the above-mentioned CRISPR transposon system Applications in gene editing.
  • the gene editing can be gene editing in any cell, especially gene editing in microorganism (including fungi and bacteria) cells, especially gene editing in industrial microorganism cells.
  • the bacteria for gene editing include Gram-negative bacteria such as Escherichia coli, Narvibrio natrium, Tatumella citrum, Gram-positive bacteria such as Corynebacterium glutamicum, etc., but are not limited thereto.
  • the new CRISPR-associated transposase system developed by the present invention can insert cargo genes into 8 sites at one time, with an efficiency of 100%, which is higher than that of the CRISPR-associated transposase derived from Vibrio cholerae Tn6677; transposition 15.4kb
  • the efficiency of the cargo gene to the corresponding target is 100%; the novel CRISPR-associated transposase can recognize 16 PAMs.
  • two CRISPR-related transposase systems are used in the same Escherichia coli for the first time, and they can function without interfering with each other, thus providing an option for accelerating the construction of metabolic engineering strains.
  • Fig. 1 has shown the gel electrophoresis photograph of targeting crRNA3 (lacZ) site in embodiment 1.
  • Fig. 2 shows the gel electrophoresis of the cargo gene GFP insertion situation at 8 sites in the genome of the strain in Example 2.
  • NC is a negative control (Negative Control).
  • Fig. 3 is a statistical bar chart of the 8-site insertion status of each cloned genome verified by colony PCR and nucleic acid gel electrophoresis in Example 2.
  • the abscissa is the copy number of the cargo gene GFP, and the ordinate is the insertion rate of the cargo gene GFP.
  • Figure 4 shows the gel electrophoresis images of the insertion of the "terminator sequence" of the cargo gene GFP carried by Ptr and the cargo gene "terminator sequence” carried by Vch at 6 sites in the genome of the strain in Example 3. where NC is the negative control.
  • Fig. 5 is a schematic diagram of the structure of plasmid pQCascadePtr.
  • Fig. 6 is a schematic diagram of the structure of plasmid pDonorPtr.
  • Fig. 7 is a schematic diagram of the structure of plasmid pTnsPtr.
  • Fig. 8 is a schematic diagram of the structure of plasmid pQCasTnsPtr.
  • Fig. 9 is a schematic diagram of the structure of plasmid pQCasTnsVch.
  • Fig. 10 is a schematic diagram of the structure of plasmid pEffectorPtr.
  • Fig. 11 is a schematic diagram of the structure of plasmid pEffectorVch.
  • Fig. 12 is a schematic diagram of the structure of plasmid pVnQCasTnsPtr.
  • Fig. 13 is a schematic diagram of the structure of plasmid pCgQCasTnsVch.
  • Fig. 14 is a schematic diagram of the structure of plasmid pCgDonorPtr.
  • Fig. 15 shows the photograph of the gel electrophoresis image targeting the crtYf gene locus of the Corynebacterium glutamicum ATCC13032 strain in Example 5.
  • novel CRISPR-associated transposase system of the present invention is derived from Pseudoalteromonas translucenta KMM520, which is a further development and improvement of the gene editing tools CAST system, INTEGRATE system and MUCICAT system, especially the gene copy amount and cargo gene insertion efficiency improvement.
  • CRISPR-associated transposase system is sometimes abbreviated as “CRISPR-associated transposase”, “CRISPR transposase”, or “CRISPR transposase system”, etc., which mean the same can be used interchangeably.
  • tnsA when used to describe the function or class of transposase, it refers to the protein; when described as a gene, it refers to the gene encoding the transposase tnsA protein.
  • RNA such as crRNA and the name of its coding gene are mixed, and those skilled in the art should understand that they represent different substances in different description occasions. Their meanings are easily understood by those skilled in the art depending on the context and context.
  • Each of the plasmids pQCascadePtr, pTnsPtr, pDonorPtr, pQCasTnsPtr, and pEffectorPtr provided by the present invention contains multiple genetic elements, for example, the plasmid pQCascadePtr contains Cas5/8 gene, Cas6 gene, Cas7 gene, tniQ coding gene, crRNA gene, CloDF13 replication promoters, promoters such as anhydrocycline-inducible promoters, and streptomycin resistance genes, the sequence of these gene elements can be arbitrary, and those skilled in the art can arrange them according to their habits and easily prepare plasmids.
  • codon optimization is a technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of a gene of interest. Different organisms often show a particular preference for one of several codons encoding the same amino acid due to mutation propensity and natural selection. For example, in fast-growing microorganisms such as E. coli, codons are optimized to reflect the composition of their respective genomic tRNA pools. Thus, in fast-growing microorganisms, low-frequency codons for amino acids can be replaced by high-frequency codons for the same amino acids. Thus, expression of optimized DNA sequences is improved in fast growing microorganisms.
  • the temperature usually refers to room temperature (15-30° C.).
  • the molecular biology experiments in the examples include plasmid construction, enzyme digestion, connection, competent cell preparation, transformation, medium preparation, etc., mainly referring to "Molecular Cloning Experiment Guide” (third edition), J. Sambrook, Edited by D.W. Russell (US), translated by Huang Peitang et al., Science Press, Beijing, 2002).
  • the specific experimental conditions can be determined through simple experiments if necessary.
  • PCR amplification experiments were carried out according to the reaction conditions or kit instructions provided by the plasmid or DNA template suppliers. It can be adjusted by simple experiment if necessary.
  • LB medium 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH7.2, sterilized under high temperature and high pressure at 121°C for 20min. Add 20g/L agar powder to the solid medium.
  • LBv2 medium add v2 salt (21.7204g mmol/L NaCl, 0.34.2g mmol/L KCl, 4.723.14g mmol/L MgCl 2 ) to LB medium.
  • BHIS medium 37g/L BHI, 91g/L sorbitol. Add 20g/L agar powder to the solid medium.
  • Plasmids pQCascadePtr, pTnsPtr, and pCgQCasTnsPtr used in the examples were commissioned by Nanjing GenScript Biotechnology Co., Ltd. to construct and synthesize them. Any unit or individual can obtain these plasmids to verify the present invention, but they cannot be used for other purposes, including development and utilization, scientific research and teaching, without the permission of the Center for Excellence in Molecular Plant Science, Chinese Academy of Sciences. in
  • Plasmid pQCascadePtr comprises genes tniQ (SEQ ID NO:11), Cas5/8 (SEQ ID NO:12), Cas7 (SEQ ID NO:14) and Cas6 (SEQ ID NO:13) derived from Pseudoalteromonas translucida KMM520, targeting The crRNA sequence of the genomic target site, CloDF13 replicon, promoter such as anhydrocycline-inducible promoter, streptomycin resistance gene was commissioned to synthesize by Nanjing GenScript Biotechnology Co., Ltd., and its nucleotide sequence is SEQ ID NO: 21, its structure is shown in Figure 5.
  • Plasmid pDonorPtr contains gene LE (SEQ ID NO:19) and RE (SEQ ID NO:20) sequences derived from Pseudoalteromonas translucida KMM520, pMB1 replicon, ampicillin resistance gene, target cargo gene Cargo such as chloramphenicol without promoter
  • LE SEQ ID NO:19
  • RE SEQ ID NO:20
  • Plasmid pTnsPtr comprises the genes tnsA (SEQ ID NO:8), tnsB (SEQ ID NO:9) and tnsC (SEQ ID NO:10) derived from Pseudoalteromonas translucida KMM520, a ColA replicon, a promoter such as an anhydrocycline-inducible promoter , Kanamycin resistance gene, entrusted Nanjing GenScript Biotechnology Co., Ltd. to synthesize, its nucleotide sequence is SEQ ID NO: 22, and its structure is shown in Figure 7.
  • Plasmid pQCasTnsPtr comprises gene tnsA (SEQ ID NO:8), tnsB (SEQ ID NO:9), tnsC (SEQ ID NO:10), tniQ (SEQ ID NO:11), Cas5/8 ( SEQ ID NO: 12), Cas7 (SEQ ID NO: 14) and Cas6 (SEQ ID NO: 13), crRNA sequences targeting genomic target sites, ColA replicon, promoters such as anhydrocycline-inducible promoters, Cas The structure of the namycin resistance gene is shown in FIG. 8 .
  • Plasmid pQCasTnsVch contains genes Cas5/8, Cas7, Cas6, tniQ, tnsA, tnsB, tnsC and CRISPR array derived from Vibrio cholerae Tn6677, CloDF13 replicon, promoter such as anhydrocycline-inducible promoter, streptomycin resistance gene , whose structure is shown in Figure 9, constructed by Yang Sheng's research group of Center for Excellence in Molecular Plant Science, Chinese Academy of Sciences.
  • Plasmid pEffectorPtr comprises gene tnsA (SEQ ID NO: 8), tnsB (SEQ ID NO: 9), tnsC (SEQ ID NO: 10), tniQ (SEQ ID NO: 11), Cas5/8 ( SEQ ID NO:12), Cas7 (SEQ ID NO:14) and Cas6 (SEQ ID NO:13), crRNA sequences targeting genomic target sites, LE (SEQ ID NO:19) and RE (SEQ ID NO: 20) Sequence, target cargo gene, ColA replicon, promoter such as anhydrocycline-inducible promoter, kanamycin resistance gene, the structure of which is shown in FIG. 10 .
  • Plasmid pEffectorVch contains genes Cas5/8, Cas7, Cas6, tniQ, tnsA, tnsB, tnsC and CRISPR array, LE and RE sequences derived from Vibrio cholerae Tn6677, target cargo gene, CloDF13 replicon, promoter such as anhydrocycline-inducible
  • the structure of the promoter and streptomycin resistance gene is shown in Figure 11, which was constructed by Yang Sheng's research group of the Center for Excellence in Molecular Plant Science, Chinese Academy of Sciences.
  • a CRISPR-associated enzyme from Pseudomonas translucidus KMM520 was demonstrated to have programmable transposition activity by targeting the lacZ and T7 RNA polymerase pro-lacZ of the Escherichia coli BL21Star TM (DE3) genome.
  • the pQCascadePtr-cr3 plasmid targeting genomic lacZ and pre-lacZ of T7 RNA polymerase and the verified primers are listed in Table 1.
  • the suffix "-cr3" in the plasmid name pQCascadePtr-cr3 represents the construction of targeting genome lacZ.
  • the suffix F in the primer name in the table represents the forward primer, and R represents the reverse primer.
  • the pQCascadePtr plasmid was digested with NcoI and BamHI to obtain the backbone fragment of pQCascadePtr.
  • Enzyme digestion reaction conditions 37°C, 1h.
  • the digested plasmid fragments were separated by gel electrophoresis and gel recovered.
  • the restriction endonuclease kit was purchased from Thermofisher Company, and the DNA gel recovery kit was purchased from Shanghai Tolo Harbor Biotechnology Co., Ltd.
  • the primer pair PtrDR-F and PtrDR-R in Table 1 can be annealed and self-assembled to obtain a DR fragment containing a 4bp sticky end, which is complementary to the sticky end of the pQCascadePtr plasmid backbone after NcoI and BamHI digestion.
  • T4 ligase kit was purchased from TAKARA company.
  • the pQCascadePtr-DR plasmid was digested with BsaI to obtain the pQCascadePtr-DR backbone fragment.
  • Enzyme digestion reaction conditions 37°C, 1h.
  • the digested plasmid fragments were separated by gel electrophoresis and gel recovered.
  • the restriction endonuclease kit was purchased from Thermofisher Company, and the DNA gel recovery kit was purchased from Tolo Harbor Company.
  • the primer pair Ptrcr3-F and Ptrcr3-R in Table 1 can be annealed and self-assembled to obtain a DR fragment containing a 4bp sticky end, which is complementary to the sticky end of the pQCascadePtr-DR plasmid backbone after BsaI digestion.
  • T4 ligase kit was purchased from TAKARA company.
  • the positive clones were selected and made into competent cells for electroporation, and pQCascadePtr-cr3 was electrotransformed into the above-mentioned bacteria, and screened on LB solid plates containing ampicillin, kanamycin and streptomycin at 37°C to obtain cells containing E. coli BL21Star TM (DE3) strains of pDonorPtr, pTnsPtr and pQCascadePtr-cr3.
  • crRNA3-F/crRNA3-R and crRNA3-R/T7lacZcr3-R located upstream and downstream of the insertion site in Table 1, verify the efficiency of targeting the two sites of crRNA3 by colony PCR.
  • PCRMix was purchased from Novizan.
  • the donor insert on plasmid pDonorPtr includes LE (Left end), RE (Right end) and cargo gene CmR fragment (chloramphenicol resistance gene fragment without promoter), a total of 1433bp, positive band 1601/1759bp, negative Band 168/326bp. According to statistics, all 16 clones had insertions at two sites.
  • the gel electrophoresis picture is shown in Figure 1.
  • Figure 1 clearly shows the band of the CmR fragment of the cargo gene, which was confirmed to be derived from Pseudoalteromonas translucidus by targeted cloning of the CmR fragment into the lacZ of the Escherichia coli BL21Star TM (DE3) genome and the pre-lacZ of T7 RNA polymerase
  • the CRISPR-associated transposase KMM520 has programmable transposition activity.
  • Example 2 Using array to target 8 different sites in the genome to achieve multi-copy integration
  • Example 2 the crRNA targeting 8 different sites of the Escherichia coli BL21Star TM (DE3) genome was combined to form 9 fixed direct repeat sequences and 8 targeting different sites
  • the array sequences arranged at spacer intervals were inserted between the NcoI and BamHI sites of the pQCasTnsPtr plasmid to construct the plasmid pQCasTnsPtr-array8.
  • the array sequence synthesis and the above plasmid construction were completed by Nanjing GenScript Biotechnology Co., Ltd.
  • the cargo gene in the plasmid pDonorPtr is the green fluorescent protein GFP gene (about 1.29 kb).
  • the suffix F in the primer name in the table represents the forward primer, and R represents the reverse primer.
  • step 1.8 The PCR system and reaction conditions are the same as step 1.8.
  • Colony PCR and nucleic acid gel electrophoresis were used to verify the insertion of 8 loci in the genome of each clone.
  • all cargo genes at 8 loci can be inserted in the target colony at one time, and the specific distribution of cargo gene copy numbers is shown in the figure 3. It shows that using the novel CRISPR-associated transposase, the cargo gene insertion efficiency is 100%, which is higher than that of the CRISPR-associated transposase derived from Vibrio cholerae Tn6677.
  • the pQCasTnsVch-cr3 plasmid construction method is the same as steps 1.4, 1.5, and 1.6, and the plasmid pQCasTnsVch comes from our laboratory.
  • the cargo gene carried by pDonorPtr is the green fluorescent protein GFP gene (about 1.29kb), and the cargo gene carried by pDonorVch is the terminator sequence (about 0.64kb).
  • Genomic lacZ and T7 RNA polymerase pro-lacZ were targeted with a CRISPR-associated transposase derived from Vibrio cholerae Tn6677, with a cargo gene size of 0.64 kb.
  • the crRNA array of pQCasTnsPtr was designed to target the genomes nagB, nagE and manX, and the cargo gene green fluorescent protein GFP gene (about 1.29kb).
  • the positive band size should be 1.0kb and 1.17kb, and the negative band size should be 0.17kb and 0.33kb; use nagB, nagE and manX PCR detection of primers upstream and downstream of the upper target site, the positive band size is about 2.47kb, 2.70kb, 2.49kb and 2.36kb, and the negative band size should be 0.43kb, 0.66kb, 0.46kb and 0.33kb.
  • Figure 4 The results are shown in Figure 4.
  • Both the CRISPR-related transposase and pQCasTnsPtr derived from Vibrio cholerae Tn6677 target the corresponding sites, and the corresponding cargo genes are inserted (the cargo gene of pDonorPtr is the green fluorescent protein GFP gene, and the cargo gene carried by pDonorVch is terminator sequence) to obtain 2*4 copies of the strain with an efficiency of about 100%, which was verified by sequencing, see Figure 4.
  • Figure 4 shows the insertion of the cargo gene GFP carried by Ptr and the "terminator sequence" of the cargo gene carried by Vch at 6 sites in the strain genome, indicating that the two CRISPR-related transposases can be used in the same E. coli, and can Function without interfering with each other, thus providing an option for accelerating the construction of metabolic engineering strains.
  • Plasmid pVnQCasTnsPtr comprises gene tnsA (SEQ ID NO: 8), tnsB (SEQ ID NO: 9), tnsC (SEQ ID NO: 10), tniQ (SEQ ID NO: 11), Cas5/8 ( SEQ ID NO: 12), Cas7 (SEQ ID NO: 14) and Cas6 (SEQ ID NO: 13), crRNA sequences targeting genomic target sites, p15A replicon, promoters such as anhydrocycline-inducible promoters, chlorine Mycin resistance gene, the plasmid structure is shown in Figure 12.
  • Plasmid pDonorPtr-GFP contains gene LE (SEQ ID NO:19) and RE (SEQ ID NO:20) sequences derived from Pseudoalteromonas translucida KMM520, pMB1 replicon, ampicillin resistance gene, target cargo gene (Cargo) without promoter The green fluorescent protein gene fragment.
  • pVnQCasTnsPtr-wbfF plasmid targeting genome wbfF and the verified primers are listed in Table 4.
  • the suffix "-wbfF" in the plasmid name pVnQCasTnsPtr-wbfF represents the construction of targeted genome wbfF.
  • the pVnQCasTnsPtr-wbfF plasmid construction method is the same as steps 1.4, 1.5, and 1.6.
  • the suffix F in the primer name in the table represents the forward primer, and R represents the reverse primer.
  • the efficiency of targeting the two sites of wbfF was verified by colony PCR using the primer pair wbfF-test-F/wbfF-test-R located upstream and downstream of the insertion site in Table 4.
  • the colony PCR method is the same as step 1.8.
  • the donor insert on the plasmid pDonorPtr-GFP includes LE (Left end), RE (Right end) and cargo gene GFP fragment (green fluorescent protein gene fragment without promoter), a total of 720bp, positive band 1220bp, negative band 500bp. According to statistics, all 16 clones had insertions.
  • LE Left end
  • RE Right end
  • cargo gene GFP fragment green fluorescent protein gene fragment without promoter
  • Example 5 Verify the transposition activity of CRISPR-related transposase in Corynebacterium glutamicum
  • the plasmid pCgQCasTnsPtr used in the examples comprises genes tnsA (SEQ ID NO:8), tnsB (SEQ ID NO:9), tnsC (SEQ ID NO:10), tniQ (SEQ ID NO:11) derived from Pseudoalteromonas translucida KMM520 ), Cas5/8 (SEQ ID NO: 12), Cas7 (SEQ ID NO: 14) and Cas6 (SEQ ID NO: 13), the above-mentioned genes are all codon-optimized for Corynebacterium glutamicum, targeting genomic target sites
  • the crRNA sequence, ColA replicon, pBL1ts replicon, promoter such as anhydrocycline-inducible promoter, kanamycin resistance gene, its structure is shown in Figure 13.
  • Plasmid pCgDonorPtr contains gene LE (SEQ ID NO: 19) and RE (SEQ ID NO: 20) sequences derived from Pseudoalteromonas translucida KMM520, pMB1 replicon, pGA1 replicon, spectinomycin resistance gene, target cargo gene Cargo, for example, no
  • LE SEQ ID NO: 19
  • RE SEQ ID NO: 20 sequences derived from Pseudoalteromonas translucida KMM520, pMB1 replicon, pGA1 replicon, spectinomycin resistance gene, target cargo gene Cargo, for example, no
  • FIG. 14 The structure of the chloramphenicol-resistant CmR gene fragment of the promoter is shown in FIG. 14 .
  • the crRNA sequence targeting the genome crtYf gene is constructed as follows:
  • Electrotransformation of pCgDonorPtr and pCgQCasTnsPtr into Corynebacterium glutamicum ATCC13032 was performed at 30°C on a BHIS solid plate containing spectinomycin and kanamycin to obtain Corynebacterium glutamicum ATCC13032 strains containing pCgDonorPtr and pCgQCasTnsPtr. Scraped a part of the clones on the above plate, resuspended in liquid BHIS medium, and reapplied on the BHIS solid plate containing anhydrotetracycline, spectinomycin and kanamycin at a final concentration of 100ng/ml.
  • Anhydrotetracycline is responsible for inducing transformation. Expression of loci-associated enzymes. After culturing at 30°C for 24 hours, a layer of bacterial film may form, which is normal. Scrape a part of the clones on the plate containing 100ng/ml anhydrotetracycline and resuspend in liquid BHIS medium, adjust OD 600 to about 0.5, dilute 50 times with liquid BHIS medium, draw 100 ⁇ L and spread to the final concentration 1000ng/ml anhydrotetracycline, spectinomycin and kanamycin on the BHIS solid plate, cultured at 30°C for 48h.
  • the efficiency of targeting crtYf was verified by colony PCR using the primer pair F-crtYf (TGCTGTGGGAACTTTTCGGT) and R-crtYf (ACTACCACTCCCGAGGTTGA) located upstream and downstream of the insertion site.
  • the donor insert on plasmid pDonorPtr includes LE (Left end), RE (Right end) and cargo gene CmR fragment (chloramphenicol resistance gene fragment without promoter), a total of 1433bp, positive band 2110bp, negative band 677bp. According to statistics, all 6 clones were inserted successfully.
  • the gel electrophoresis image is shown in Figure 15, confirming the transposition activity of the CRISPR-associated transposase derived from Pseudoalteromonas translucenta KMM520 in Corynebacterium glutamicum.

Abstract

一种新型CRISPR相关转座酶，其来源于半透明假交替单胞菌KMM520，可识别16种PAM，可以一次将货物基因插入至8个位点，效率100％，高于来源于霍乱弧菌Tn6677的CRISPR相关转座酶；转座15.4kb货物基因至对应靶点的效率为100％，能够与来源于霍乱弧菌Tn6677的CRISPR相关转座酶在同一大肠杆菌内使用，且互不干扰地发挥功能，应用前景广阔。

Description

一种新型CRISPR相关转座酶 技术领域
本发明属于基因编辑技术领域，具体地说，涉及一种新型CRISPR相关转座酶及其在基因编辑中的应用。
背景技术
在代谢工程中，可以通过调整酶基因的剂量与比值使代谢途径不同酶或不同代谢途径之间协调一致最大化总体反应速度。在细菌中提高基因表达盒剂量的最常用方式是质粒，但会遇到遗传稳定性问题。将基因表达盒逐个整合到细菌染色体上则耗时长，难以快速测试诸多构建方案。CRISPR-Cas虽然可以切割基因组多拷贝序列或以crRNA阵列同时靶向多个不同序列，但受限于双链断裂的修复效率，难以一次性插入3个拷贝以上的基因表达盒。因此，需要一种更好的方法获得不同基因剂量的组合。
2019年，Broad研究所张锋研究组和哥伦比亚大学Sam Sternberg研究组公布了两项相似的研究成果：利用细菌转座基因，将DNA序列精确地插入基因组而不切割DNA。其中张锋研究组从蓝藻中抽提了一种转座酶，将其命名为CAST，即CRISPR相关转座酶(CRISPR-associated transposase)。Sternberg研究组在霍乱弧菌中发现了一个独特的转座基因后，开发了一种名为INTEGRATE(Insertion of transposable elements by guide RNA-assisted targeting，引导RNA辅助靶向的转座元件插入)的基因编辑工具，可以在基因组中插入大片段基因而不引入DNA断裂。该新技术INTEGRATE利用转座基因将DNA序列插入基因组而不切割DNA。CAST系统和INTEGRATE系统都可不依赖于同源重组将DNA片段通过转座整合到大肠杆菌染色体预设的位点，无抗性标记残留，无双链DNA断口。
发明人以CAST系统和INTEGRATE系统为基础，开发出了一种的多拷贝基因插入系统MUCICAT(参见专利文献CN202010083919.7)可以在5天得到染色体插入10拷贝货物基因(Cargo gene)的菌株，具有可编辑、快速、无marker、定点等优势，目前MUCICAT已被成功应用于酶工程菌株与代谢工程菌株的构建(Zhang,Y.,Multicopy chromosomal integration using CRISPR-associated transposases.Acs Synthetic Biology,2020.)。然而，代谢 工程与合成生物学通常涉及多酶或多条途径表达水平的优化。在一轮转座中，基于单一CRISPR相关转座酶的MUCICAT只能探索负载单或多基因的单一货物基因最优剂量，不能筛选多基因或途径的基因剂量的最优配比。目前还没有平行扩增多基因/途径的染色体拷贝数技术的报道。包含多个彼此正交的CRISPR相关转座酶的MUCICAT技术则有希望实现单一细胞中多基因或途径的同时独立扩增，形成含不同基因剂量配比的菌株文库从而筛选最优多基因或途径的剂量配比。
已知活性的CRISPR转座酶中，仅来源于霍乱弧菌Tn6677的I-F3型CRISPR转座酶插入效率和中靶率均高(Klompe,S.E.,et al.,Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration.Nature,2019.571(7764):p.219-225.)，来源于霍夫曼尼斯藻和柱状鱼腥藻的V-K型(Jonathan Strecker et al.,RNA-guided DNA insertion with CRISPR-associated transposases.Science，2019)、杀鲑气单胞菌S44的I-F3型(Michael T.Petassi et al.,Guide RNA Categorization Enables Target Site Choice in Tn7-CRISPR-Cas Transposons.Cell,2020)、多变鱼腥藻和Peltigera membranacea cyanobiont 210A的I-B型均效率低或/和脱靶率高(Makoto Saito,A.L.and Jonathan Strecker,Han Altae-Tran,Rhiannon K.Macrae,Feng Zhang,Dual modes of CRISPR-associated transposon homing.Cell,2021.)。
发明内容
根据现有技术的CRISPR转座酶的特点，理想的是，若有其他新型高效的CRISPR相关转座酶，或可与MUCICAT组成正交的CRISPR相关转座系统，应用于复杂的代谢工程与合成生物学设计中。为了实现这一目的，发明人对于其他微生物来源的CRISPR转座酶进行了广泛筛选，发现了来源于半透明假交替单胞菌KMM520(Pseudoalteromonas translucida KMM520)的I-F3型CRISPR转座系统，其在大肠杆菌中具有不亚于、甚至更优于霍乱弧菌Tn6677的插入效率，且能与霍乱弧菌Tn6677互不干扰地分别靶向插入eda-purT间和lacZ位点。该新型CRISPR转座系统可识别所有16种PAM，没有PAM依赖性。
因此，本发明的第一个方面在于提供一种CRISPR相关转座酶，其包括选自下组的多肽：来源于假交替单胞菌属细菌的转座酶蛋白tnsA、来源于假交替单胞菌属细菌的转座酶蛋白tnsB、来源于假交替单胞菌属细菌的转座酶蛋白tnsC、来源于假交替单胞菌属细菌的转座酶蛋白tniQ、来源于假交替单胞菌属细菌的核酸酶蛋白Cas5/8、来源于假交替单胞菌属细菌的核酸酶蛋白Cas6、来源于假交替单胞菌属细菌的核酸酶蛋白Cas7。
优选地，所述假交替单胞菌属细菌是半透明假交替单胞菌。更优选所述半透明假交替单胞菌是半透明假交替单胞菌KMM520(Pseudoalteromonas translucida KMM520)。
在一种具体的实施方式中，上述CRISPR相关转座酶优选包括选自下组的多肽：
tnsA：其为具有SEQ ID NO:1氨基酸序列的多肽，或者与SEQ ID NO:1有95％以上同源性、优选地98％以上同源性、更优地99％以上同源性、且功能相同的多肽；
tnsB：其为具有SEQ ID NO:2氨基酸序列的多肽，或者与SEQ ID NO:2有95％以上同源性、优选地98％以上同源性、更优地99％以上同源性、且功能相同的多肽；
tnsC：其为具有SEQ ID NO:3氨基酸序列的多肽，或者与SEQ ID NO:3有95％以上同源性、优选地98％以上同源性、更优地99％以上同源性、且功能相同的多肽；
tniQ：其为具有SEQ ID NO:4氨基酸序列的多肽，或者与SEQ ID NO:4有95％以上同源性、优选地98％以上同源性、更优地99％以上同源性、且功能相同的多肽；
Cas5/8：其为具有SEQ ID NO:5氨基酸序列的多肽，或者与SEQ ID NO:5有95％以上同源性、优选地98％以上同源性、更优地99％以上同源性、且功能相同的多肽；
Cas6：其为具有SEQ ID NO:6氨基酸序列的多肽，或者与SEQ ID NO:6有95％以上同源性、优选地98％以上同源性、更优地99％以上同源性、且功能相同的多肽；
Cas7：其为具有SEQ ID NO:7氨基酸序列的多肽，或者与SEQ ID NO:7有95％以上同源性、优选地98％以上同源性、更优地99％以上同源性、且功能相同的多肽。
上述核酸酶Cas5/8、Cas7、Cas6是I型CRISPR系统的Cascade复合物，与转座酶tnsABC和tniQ关联变成CRISPR相关转座酶。这些多肽均来源于半透明假交替单胞菌KMM 520(Pseudoalteromonas translucida KMM520)。
本发明的第二个方面提供了编码上述多肽的基因。
在一种具体的实施方式中，编码具有SEQ ID NO:1氨基酸序列的多肽tnsA的基因是核苷酸序列SEQ ID NO:8，或者与SEQ ID NO:8有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性的多核苷酸；
编码具有SEQ ID NO:2氨基酸序列的多肽tnsB的基因是核苷酸序列SEQ ID NO:9，或者与SEQ ID NO:9有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性的多核苷酸；
编码具有SEQ ID NO:3氨基酸序列的多肽tnsC的基因是核苷酸序列SEQ ID NO:10，或者与SEQ ID NO:10有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性的多核苷酸；
编码具有SEQ ID NO:4氨基酸序列的多肽tniQ的基因是核苷酸序列SEQ ID NO:11，或者与SEQ ID NO:11有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性的多核苷酸；
编码具有SEQ ID NO:5氨基酸序列的多肽Cas5/8的基因是核苷酸序列SEQ ID NO:12，或者与SEQ ID NO:12有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性的多核苷酸；
编码具有SEQ ID NO:6氨基酸序列的多肽Cas6的基因是核苷酸序列SEQ ID NO:13，或者与SEQ ID NO:13有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性的多核苷酸；
编码具有SEQ ID NO:7氨基酸序列的多肽Cas7的基因是核苷酸序列SEQ ID NO:14，或者与SEQ ID NO:14有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性的多核苷酸。
本发明的第三个方面在于提供一种CRISPR转座子系统。如同转座系统INTEGRATE系统或者CAST系统，本发明的CRISPR转座子系统也包括质粒pQCascade、辅助质粒pTns、和携带货物基因的辅助质粒pDonor。这三种质粒中的任意两种甚至可以合并成一种质粒、甚至这三种质粒可以合并一起形成一种质粒。具体而言，
一种用于CRISPR转座子系统的质粒pQCascade，其包括选自下组的基因片段：上述的Cas5/8编码基因；上述的Cas6编码基因；上述的Cas7编码基因；上述的tniQ编码基因。
由于上述这些多肽均来源于半透明假交替单胞菌KMM520( Pseudoalteromonas  translucida KMM520)，本文中将该质粒pQCascade命名为pQCascadePtr。类似地，下文中可以将本发明的质粒pTns和pDonor分别表示为pTnsPtr和pDonorPtr。
优选地，上述的质粒pQCascadePtr还可以包括下述基因：靶向基因组目标位点的crRNA序列，CloDF13复制子，启动子例如脱水四环素诱导型启动子，链霉素抗性基因。
上述CRISPR转座酶中的crRNA可以呈array的形式发挥功能。
在一种实施方式中，上述crRNA系列的间隔序列spacer可以是靶向待处理细胞基因组中单个位点的spacer。
在另一种实施方式中，上述crRNA系列可以是靶向待处理细胞基因组中多个位点的crRNA阵列(又称CRISPRarray、crRNA array或者array)。
优选上述crRNA序列是靶向基因组中多个位点的crRNA阵列，其中重复序列区repeat包括选自下组序列中的一种以上：核苷酸序列为SEQ ID NO:15的repeat1、核苷酸序列为SEQ ID NO:16的repeat2、核苷酸序列为SEQ ID NO:17的repeat3、核苷酸序列为SEQ ID NO:18的repeat4，这些核苷酸序列SEQ ID NOs:15-18中的32个N(即[N32])是任意的碱基A、T、G或C。
一种用于CRISPR转座子系统的辅助质粒pTnsPtr，其与上述的质粒pQCascadePtr配合使用，其包括选自下组的基因片段：上述的tnsA编码基因，上述的tnsB编码基因，上述的tnsC编码基因。
上述CRISPR转座酶基因序列用于CRISPR转座时，需与宿主内(革兰氏阴性菌如大肠杆菌、需钠弧菌、柠檬塔特姆氏菌；革兰氏阳性菌如谷氨酸棒杆菌)可用的启动子，Left end(LE)-cargo-Right end(RE)一起发挥作用，不论是以质粒的形式或是整合的形式。
优选地，上述的辅助质粒pTnsPtr还包括下述基因：ColA复制子、启动子例如脱水四环素诱导型启动子、卡那霉素抗性基因。
作为两种质粒合并成一种质粒的实施方式，本发明提供了一种用于CRISPR转座子系统的质粒pQCasTnsPtr，其由上所述的质粒pQCascadePtr和辅助质粒pTnsPtr合并而成，包括：上述的Cas5/8、Cas6、Cas7、tniQ、tnsA、tnsB和tnsC基因，靶向基因组目标位点的crRNA序列，ColA复制子，启动子例如脱水四环素诱导型启动子，卡那霉素抗性基因。
一种用于CRISPR转座子系统的辅助质粒pDonorPtr，其与上述的质粒pQCascadePtr和上述的辅助质粒pTnsPtr配合使用，其包括选自下组的基因片段：序列Left end(LE)，其核苷酸序列为SEQ ID NO:19，或者与SEQ ID NO:19有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性且包含SEQ ID NO:19的3’端33bp序列；序列Right end(RE)，其核苷酸序列为SEQ ID NO:20，或者与SEQ ID NO:20有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性且包含SEQ ID NO:20的5’端27bp序列；目标货物基因(Cargo gene)。
由于核苷酸序列为SEQ ID NO:19的LE和核苷酸序列为SEQ ID NO:20的RE来源于半透明假交替单胞菌KMM520( Pseudoalteromonas  translucida KMM520)，本文中将该质粒pDonor命名为pDonorPtr。
优选地，上述辅助质粒pDonorPtr还可以包括下述基因：pMB1复制子、氨苄青霉素抗性基因、目标货物基因(Cargo gene)；或者下述基因：p15A复制子、氯霉素抗性基因、 目标货物基因(Cargo gene)。
作为上述三种质粒合并成一种质粒的实施方式，本发明提供了一种用于CRISPR转座子系统的质粒pEffectorPtr，其由上述的质粒pQCascadePtr、上述的辅助质粒pTnsPtr和上述的辅助质粒pDonorPtr合并而成，包括：上述的Cas5/8、Cas6、Cas7、tniQ、tnsA、tnsB和tnsC基因、Left end(LE)和Right end(RE)序列，目标货物基因，靶向基因组目标位点的crRNA序列、ColA复制子、启动子例如脱水四环素诱导型启动子、卡那霉素抗性基因。
基于上述的质粒，本发明的第四个方面提供了一种CRISPR转座子系统，其包括：质粒pQCascadePtr、pTnsPtr和pDonorPtr；或者质粒pQCasTnsPtr和pDonorPtr；或者质粒pEffectorPtr。
本发明的第五个方面提供了一种上述来源于半透明假交替单胞菌KMM520的CRISPR转座子系统与现有技术的来源于霍乱弧菌(Vibrio cholerae)Tn6677的CRISPR转座子系统的组合使用方式，具体而言，
一种CRISPR转座子系统，其除了包括上述的CRISPR转座子系统(包括：质粒pQCascadePtr、pTnsPtr和pDonorPtr；或者质粒pQCasTnsPtr和pDonorPtr；或者质粒pEffectorPtr)之外，还包括霍乱弧菌( Vibrio  cholerae)Tn6677来源的CRISPR转座酶相关质粒。
霍乱弧菌Tn6677的I-F3型CRISPR转座酶的基因序列是NCBI：NZ_ALED01000027.1。
上述霍乱弧菌Tn6677来源的CRISPR转座酶相关质粒包括质粒pQCasTnsVch和辅助质粒pDonorVch，其中
质粒pQCasTnsVch包括：霍乱弧菌Tn6677来源的Cas5/8、Cas6、Cas7、tniQ、tnsA、tnsB和tnsC基因，CloDF13复制子，启动子例如脱水四环素诱导型启动子，链霉素抗性基因；
质粒pDonorVch包括霍乱弧菌Tn6677来源的CRISPR阵列、Left end(LE)和Right end(RE)，pMB1复制子，氨苄青霉素抗性基因，目标货物基因(Cargo gene)。
在一种实施方式中，与本发明的两种或三种质粒合并成一种质粒的组合方式pQCasTnsPtr和pEffectorPtr相类似，上述的质粒pQCasTnsVch和pDonorVch可以合并为质粒pEffectorVch。
在上述启动子采用脱水四环素诱导型启动子的情况下，当上述来源于半透明假交替单胞菌KMM520的CRISPR转座子系统用于转化大肠杆菌BL21(DE3)、BL21Star TM(DE3) 或W3110(DE3)等菌株时，可以使用脱水四环素进行诱导。即，将质粒pQCasTnsPtr和pDonorPtr用于转化大肠杆菌BL21(DE3)、BL21Star TM(DE3)或W3110(DE3)等菌株时，使用脱水四环素进行诱导。
类似地，在上述启动子采用脱水四环素诱导型启动子的情况下，当上述来源于半透明假交替单胞菌KMM520的CRISPR转座子系统与来源于霍乱弧菌Tn6677的CRISPR转座子系统组合一起使用时，也可以使用脱水四环素进行诱导。即，将质粒pEffectorPtr和pEffectorVch，或将质粒pQCasTnsVch、pDonorVch、pQCasTnsPtr和pDonorPtr用于转化大肠杆菌BL21(DE3)、BL21Star TM(DE3)或W3110(DE3)等菌株时，使用脱水四环素进行诱导。
这两种微生物来源的CRISPR转座子系统能够在同一大肠杆菌内使用，且可以互不干扰的发挥功能，两者正交发挥作用。
本发明的第六个方面提供了上述的CRISPR相关转座酶、上述的编码多肽的基因、上述的质粒pQCascadePtr、上述的质粒pTnsPtr、质粒pQCasTnsPtr、质粒pDonorPtr、质粒pEffectorPtr、上述的CRISPR转座子系统在基因编辑中的应用。
所述的基因编辑可以是任何细胞内的基因编辑，尤其是在微生物(包括真菌和细菌)细胞内的基因编辑，特别是工业微生物细胞内的基因编辑。
优选地，进行基因编辑的细菌包括革兰氏阴性菌例如大肠杆菌、需钠弧菌、柠檬塔特姆氏菌、革兰氏阳性菌例如谷氨酸棒杆菌等，但不限于此。
实验证明，本发明开发的新型CRISPR相关转座酶系统可以一次将货物基因插入至8个位点，效率100％，效率高于来源于霍乱弧菌Tn6677的CRISPR相关转座酶；转座15.4kb货物基因至对应靶点的效率为100％；该新型CRISPR相关转座酶可识别16种PAM。本发明首次将两种CRISPR相关转座酶系统在同一大肠杆菌内使用，且可以互不干扰的发挥功能，因此为加速代谢工程菌株构建提供了一种选择。
附图说明
图1显示了实施例1中靶向crRNA3(lacZ)位点的凝胶电泳图照片。
图2显示了实施例2中菌株基因组中8个位点的货物基因GFP插入情况的凝胶电泳图。其中NC是阴性对照(Negative Control)。
图3是实施例2中通过菌落PCR与核酸凝胶电泳验证各克隆基因组8个位点插入情况的统计柱形图。其中横坐标为货物基因GFP的拷贝数，纵坐标是货物基因GFP插入率。
图4显示了实施例3中菌株基因组中6个位点的Ptr携带的货物基因GFP和Vch携带的货物基因“终止子序列”插入情况的凝胶电泳图。其中NC是阴性对照。
图5是质粒pQCascadePtr的结构示意图。
图6是质粒pDonorPtr的结构示意图。
图7是质粒pTnsPtr的结构示意图。
图8是质粒pQCasTnsPtr的结构示意图。
图9是质粒pQCasTnsVch的结构示意图。
图10是质粒pEffectorPtr的结构示意图。
图11是质粒pEffectorVch的结构示意图。
图12是质粒pVnQCasTnsPtr的结构示意图。
图13是质粒pCgQCasTnsVch的结构示意图。
图14是质粒pCgDonorPtr的结构示意图。
图15显示了实施例5中靶向谷氨酸棒杆菌ATCC13032菌株crtYf基因位点的凝胶电泳图照片。
具体实施方式
本发明的新型CRISPR相关转座酶系统来源于半透明假交替单胞菌KMM520，是对基因编辑工具CAST系统和INTEGRATE系统以及MUCICAT系统的进一步发展和完善，尤其是基因拷贝量和货物基因插入效率的提高。
在本文中，为了描述简便，有时会将术语“CRISPR相关转座酶系统”简称为“CRISPR相关转座酶”、“CRISPR转座酶”、或者“CRISPR转座子系统”等，它们表示相同的含义，可以互换使用。
在本文中，为了描述简便，有时会将某种蛋白比如Cas6与其编码基因(DNA)名称混用，本领域技术人员应能理解它们在不同描述场合表示不同的物质。本领域技术人员根据语境和上下文容易理解它们的含义。例如，对于tnsA，用于描述转座酶功能或类别时，指的是蛋白质；在作为一种基因描述时，指的是编码该转座酶tnsA蛋白的基因。
类似地，为了描述简便，有时会将RNA比如crRNA与其编码基因名称混用，本领域技术人员应能理解它们在不同描述场合表示不同的物质。本领域技术人员根据语境和上下文容易理解它们的含义。
本发明提供的每种质粒pQCascadePtr、pTnsPtr、pDonorPtr、pQCasTnsPtr和pEffectorPtr 等都分别包含多个基因元件，例如质粒pQCascadePtr中包含Cas5/8基因、Cas6基因、Cas7基因、tniQ编码基因、crRNA基因、CloDF13复制子、启动子例如脱水四环素诱导型启动子、链霉素抗性基因，这些基因元件的排列顺序可以是任意的，本领域技术人员可以根据习惯进行安排、并且容易地制备出质粒。
应理解，对于本发明的多肽Cas5/8、Cas7、Cas6、tnsABC(即tnsA、tnsB和tnsC)和tniQ的编码基因，本领域技术人员可以根据待处理细胞的具体种类比如大肠杆菌进行密码子优化，而仅仅不限于上述的核苷酸序列SEQ ID NOs:8-14。
密码子优化的目的在于，使得这些多肽能够在待处理细胞中实现最佳表达。密码子优化是可用于通过增加感兴趣基因的翻译效率使生物体中蛋白质表达最大化的一种技术。不同的生物体由于突变倾向和天然选择而通常示出对于编码相同氨基酸的一些密码子之一的特殊偏好性。例如，在生长快速的微生物如大肠杆菌中，优化密码子反映出其各自的基因组tRNA库的组成。因此，在生长快速的微生物中，氨基酸的低频率密码子可以被用于相同氨基酸的但高频率的密码子置换。因此，优化的DNA序列的表达在快速生长的微生物中得以改良。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于举例说明目的，而不是对本发明的限制。此外应理解，在阅读了本发明的构思之后，本领域技术人员对其作出的各种改变或调整，均应落入本发明的保护范围内，这些等价形式同样属于本申请所附权利要求书限定的范围。
本文中涉及到多种物质的添加量、含量及浓度，其中所述的百分含量，除特别说明外，皆指质量百分含量。
本文的实施例中，如果对于反应温度或操作温度没有做出具体说明，则该温度通常指室温(15-30℃)。
实施例
材料和方法
实施例中的全基因合成由南京金斯瑞生物科技有限公司完成，引物合成由铂尚生物技术(上海)有限公司完成，测序由擎科生物有限公司完成。
实施例中的分子生物学实验包括质粒构建、酶切、连接、感受态细胞制备、转化、培养基配制等等，主要参照《分子克隆实验指南》(第三版)，J.萨姆布鲁克，D.W.拉塞尔(美)编著，黄培堂等译，科学出版社，北京，2002)进行。必要时可以通过简单试验 确定具体实验条件。
PCR扩增实验根据质粒或DNA模板供应商提供的反应条件或试剂盒说明书进行。必要时可以通过简单试验予以调整。
LB培养基：10g/L胰蛋白胨、5g/L酵母提取物、10g/L氯化钠，pH7.2，121℃高温高压灭菌20min。固体培养基另加20g/L琼脂粉。
LBv2培养基：LB培养基中添加v2盐(21.7204g mmol/L NaCl，0.3 4.2g mmol/L KCl，4.723.14g mmol/L MgCl 2)。
BHIS培养基：37g/L BHI，91g/L山梨醇。固体培养基另加20g/L琼脂粉。
实施例中所使用的质粒pQCascadePtr、pTnsPtr和pCgQCasTnsPtr委托南京金斯瑞生物科技有限公司构建合成，质粒pDonorPtr、pQCasTnsPtr、pEffectorPtr、pCgDonorPtr、pQCasTnsVch、pDonorVch和pEffectorVch由中国科学院分子植物科学卓越创新中心杨晟课题组构建，任何单位和个人都可以获得这些质粒用于验证本发明，但未经中国科学院分子植物科学卓越创新中心允许不得用作其他用途，包括开发利用、科学研究和教学。其中
质粒pQCascadePtr包含来源于Pseudoalteromonas translucida KMM520的基因tniQ(SEQ ID NO:11)、Cas5/8(SEQ ID NO:12)、Cas7(SEQ ID NO:14)和Cas6(SEQ ID NO:13)、靶向基因组目标位点的crRNA序列、CloDF13复制子、启动子例如脱水四环素诱导型启动子、链霉素抗性基因，委托南京金斯瑞生物科技有限公司合成，其核苷酸序列为SEQ ID NO:21，其结构如图5所示。
质粒pDonorPtr包含来源于Pseudoalteromonas translucida KMM520的基因LE(SEQ ID NO:19)和RE(SEQ ID NO:20)序列，pMB1复制子、氨苄青霉素抗性基因、目标货物基因Cargo例如无启动子的氯霉素抗性CmR基因片段，其结构如图6所示。
质粒pTnsPtr包含来源于Pseudoalteromonas translucida KMM520的基因tnsA(SEQ ID NO:8)、tnsB(SEQ ID NO:9)和tnsC(SEQ ID NO:10)、ColA复制子、启动子例如脱水四环素诱导型启动子、卡那霉素抗性基因，委托南京金斯瑞生物科技有限公司合成，其核苷酸序列为SEQ ID NO:22，其结构如图7所示。
质粒pQCasTnsPtr包含来源于Pseudoalteromonas translucida KMM520的基因tnsA(SEQ ID NO:8)、tnsB(SEQ ID NO:9)、tnsC(SEQ ID NO:10)、tniQ(SEQ ID NO:11)、Cas5/8(SEQ ID NO:12)、Cas7(SEQ ID NO:14)和Cas6(SEQ ID NO:13)、靶向基因组目标位点的crRNA序列、ColA复制子、启动子例如脱水四环素诱导型启动子、 卡那霉素抗性基因，其结构如图8所示。
质粒pQCasTnsVch包含来源于霍乱弧菌Tn6677的基因Cas5/8、Cas7、Cas6、tniQ、tnsA、tnsB、tnsC及CRISPR array、CloDF13复制子、启动子例如脱水四环素诱导型启动子、链霉素抗性基因，其结构如图9所示，由中国科学院分子植物科学卓越创新中心杨晟课题组构建。
质粒pEffectorPtr包含来源于Pseudoalteromonas translucida KMM520的基因tnsA(SEQ ID NO:8)、tnsB(SEQ ID NO:9)、tnsC(SEQ ID NO:10)、tniQ(SEQ ID NO:11)、Cas5/8(SEQ ID NO:12)、Cas7(SEQ ID NO:14)和Cas6(SEQ ID NO:13)、靶向基因组目标位点的crRNA序列、LE(SEQ ID NO:19)和RE(SEQ ID NO:20)序列、目标货物基因、ColA复制子、启动子例如脱水四环素诱导型启动子、卡那霉素抗性基因，其结构如图10所示。
质粒pEffectorVch包含来源于霍乱弧菌Tn6677的基因Cas5/8、Cas7、Cas6、tniQ、tnsA、tnsB、tnsC及CRISPR array、LE和RE序列、目标货物基因、CloDF13复制子、启动子例如脱水四环素诱导型启动子、链霉素抗性基因，其结构如图11所示，由中国科学院分子植物科学卓越创新中心杨晟课题组构建。
实施例1 验证CRISPR相关转座酶的转座活性
通过靶向大肠杆菌BL21Star TM(DE3)基因组lacZ和T7RNA聚合酶前lacZ，证实来源于半透明假交替单胞菌KMM520的CRISPR相关酶具有可编程的转座活性。
构建靶向基因组lacZ和T7RNA聚合酶前lacZ的pQCascadePtr-cr3质粒及验证引物列于表1。其中质粒名称pQCascadePtr-cr3中的后缀“-cr3”代表构建靶向基因组lacZ。
表1：引物序列
Figure PCTCN2022091102-appb-000001
注：表中引物名称后缀F代表正向引物，R代表反向引物。
1.1酶切获得pQCascadePtr骨架片段
NcoI和BamHI酶切pQCascadePtr质粒，获得pQCascadePtr骨架片段。
酶切反应体系(50μL)
Figure PCTCN2022091102-appb-000002
酶切反应条件：37℃，1h。通过凝胶电泳分离酶切后质粒片段并胶回收。
限制性内切酶试剂盒购自Thermofisher公司，DNA凝胶回收试剂盒购自上海吐露港生物科技有限公司。
1.2引物退火自搭
表1中的引物对PtrDR-F和PtrDR-R通过退火自搭，可获得含有4bp粘性末端的DR片段，该粘性末端与pQCascadePtr质粒骨架NcoI和BamHI酶切后的粘性末端互补。
退火自搭体系(50μL)
Figure PCTCN2022091102-appb-000003
95℃保温5min，每分钟减低5-10℃，16℃保温10min，用ddH 2O稀释20倍，备用。
1.3连接构建质粒pQCascadePtr-DR
T4连接体系(10μL)
Figure PCTCN2022091102-appb-000004
16℃连接1h。T4连接酶试剂盒购自TAKARA公司。
将上述连接产物全部转化至DH5α感受态细胞中(购自深圳康体生命科技有限公司)，在含有链霉素的LB固体平板上进行筛选，获得质粒pQCascadePtr-DR。用引物8测序验证正确。
1.4酶切获得pQCascadePtr-DR骨架片段
BsaI酶切pQCascadePtr-DR质粒，获得pQCascadePtr-DR骨架片段。
酶切反应体系(50μL)
Figure PCTCN2022091102-appb-000005
酶切反应条件：37℃，1h。通过凝胶电泳分离酶切后质粒片段并胶回收。
限制性内切酶试剂盒购自Thermofisher公司，DNA凝胶回收试剂盒购自吐露港公司。
1.5引物退火自搭
表1中的引物对Ptrcr3-F和Ptrcr3-R通过退火自搭可获得含有4bp粘性末端的DR片段，该粘性末端与pQCascadePtr-DR质粒骨架BsaI酶切后的粘性末端互补。
退火自搭体系(50μL)
Figure PCTCN2022091102-appb-000006
95℃保温5min，每分钟减低5-10℃，16℃保温10min，用ddH 2O稀释20倍，备用。
1.6连接构建质粒pQCascadePtr-cr3
T4连接体系(10μL)
Figure PCTCN2022091102-appb-000007
16℃连接1h。T4连接酶试剂盒购自TAKARA公司。
将上述连接产物全部转化至DH5α感受态细胞中(购自深圳康体生命科技有限公司)，在含有链霉素的LB固体平板上进行筛选，获得质粒pQCascadePtr-cr3。用引物8测序验证正确。
1.7转化转座工具质粒与诱导转座
电转化pDonorPtr与pTnsPtr至大肠杆菌BL21Star TM(DE3)，37℃条件下在含氨苄青霉素与卡那霉素的LB固体平板上进行筛选。挑选阳性克隆子，制备成电转感受态细胞后，电转化pQCascadePtr-cr3至上述菌中，37℃条件下在含氨苄青霉素、卡那霉素与链霉素的LB固体平板上进行筛选，获得含有pDonorPtr、pTnsPtr与pQCascadePtr-cr3的大肠杆菌BL21Star TM(DE3)菌株。将上述平板上的克隆刮取一部分重悬于液体LB培养基中，重新涂布于含有终浓度100ng/ml脱水四环素、氨苄青霉素、卡那霉素与链霉素的LB固体平板上，脱水四环素负责诱导转座相关酶的表达。37℃条件下培养16h，可能会形成一层菌膜，属正常情况。将上述含有100ng/ml脱水四环素平板上的克隆刮取一部分重悬于液体LB培养基中，调整OD 600至约0.5后，用液体LB培养基稀释50倍，吸取100μL涂布于添加了终浓度1000ng/ml脱水四环素、氨苄青霉素、卡那霉素与链霉素的LB固体平板上，37℃条件下培养24h。
1.8菌落PCR鉴定靶向crRNA3的效率
使用表1中位于插入位点上下游的引物对crRNA3-F/crRNA3-R和crRNA3-R/T7lacZcr3-R，通过菌落PCR，验证靶向crRNA3的两个位点的效率。
菌落PCR反应体系(10μL)：
Figure PCTCN2022091102-appb-000008
PCRMix购自诺维赞公司。
PCR反应条件：
Figure PCTCN2022091102-appb-000009
质粒pDonorPtr上的供体插入片段包括LE(Left end)、RE(Right end)和货物基因CmR片段(无启动子的氯霉素抗性基因片段)，共1433bp，阳性条带1601/1759bp， 阴性条带168/326bp。经统计，16个克隆均在两位点有插入。凝胶电泳图如图1所示。
图1清楚地显示了货物基因CmR片段的条带，通过将该CmR片段靶向克隆于大肠杆菌BL21Star TM(DE3)基因组lacZ和T7RNA聚合酶前lacZ，证实了来源于半透明假交替单胞菌KMM520的CRISPR相关转座酶具有可编程的转座活性。
实施例2 用array靶向基因组8个不同位点实现多拷贝整合
2.1质粒pQCasTnsPtr-array8的构建
参照专利文献CN202010083919.7中实施例2的方法，将靶向大肠杆菌BL21Star TM(DE3)基因组8个不同位点的crRNA组合，形成由9个固定正向重复序列与8个靶向不同位点的spacer间隔排列的array序列，插入至pQCasTnsPtr质粒的NcoI和BamHI位点之间，构建质粒pQCasTnsPtr-array8。array序列合成以及上述质粒构建由南京金斯瑞生物科技有限公司完成。
2.2转化转座工具质粒与诱导转座
电转化质粒pDonorPtr、pQCasTnsPtr-array8至大肠杆菌BL21Star TM(DE3)，转化操作同步骤1.7。平板菌落诱导转座与传代操作同步骤1.7。质粒pDonorPtr中的货物基因是绿色荧光蛋白GFP基因(约1.29kb)。
2.3菌落PCR鉴定基因组8位点插入情况
在基因组8个位点的上下游处分别设计正向与反向引物，即验证所需的引物，序列见表2。
表2、验证基因组中8个位点插入所需引物序列
编号 引物 序列(5'→3')
1 site1-F GGACGTTGAATTTGCAGGAT
2 site1-R CGGCATAAACTACAGCTGGCA
3 site2-F CCGGATAATGTGCCGATGG
4 site2-R ATTCCGTATCGTCATGACCG
5 site3-F CTAACCTCGACGCATGTATTACC
6 site3-R TCTCTTCACCCGCTACCAG
7 site4-F TCTGACCACCTTGCAGGAC
8 site4-R TCCGGCGTTTCAGATTGTTG
9 site5-F GACAGTTTTGGTGCTGACGA
10 site5-R ACTCTGGATGCTAAAACCGTG
11 site6-F ACTATTGTGAAAGATCCGGCG
12 site6-R CCGATACCTGATTAAACGGGTC
13 site7-F GCTGATAGATTTGCCTGTCTAGTT
14 site7-R CAACCTGTGTGCGTTAGCAA
15 site8-F GCATGGCATATGCCGTTTG
16 site8-R TGGCGATTGTTGAAACCGC
注：表中引物名称后缀F代表正向引物，R代表反向引物。
PCR体系与反应条件同步骤1.8。
通过菌落PCR与核酸凝胶电泳验证各克隆基因组8位点的插入情况，如图2所示，目的菌落中一次即可完成8个位点的货物基因全部插入，货物基因拷贝数具体分布如图3所示。表明利用新型CRISPR相关转座酶，货物基因插入效率为100％，高于来源于霍乱弧菌Tn6677的CRISPR相关转座酶。
实施例3 两种CRISPR相关转座酶的正交实验
考察来源于Pseudoalteromonas translucida KMM520的新型CRISPR相关转座酶与和来源于霍乱弧菌Tn6677的CRISPR相关转座酶的正交性。
3.1质粒pQCasTnsPtr-nagman和pQCasTnsVch-cr3质粒构建
将靶向大肠杆菌BL21Star TM(DE3)基因组nagB、nagE和manX的crRNA组合，形成由5个固定正向重复序列与4个靶向不同位点的spacer间隔排列的array序列，插入至pQCasTnsPtr质粒的NcoI和BamHI位点之间，构建质粒pQCasTnsPtr-nagman。array序列合成以及上述质粒构建由南京金斯瑞生物科技有限公司完成。
pQCasTnsVch-cr3质粒构建方法同步骤1.4、1.5、1.6，质粒pQCasTnsVch来源于本实验室。
质粒构建及鉴定所用引物列于表3。
表3、质粒pQCasTnsPtr-nagman和pQCasTnsVch-cr3构建及鉴定所用引物
序号 引物 序列(5'→3')
1 Vchcr3-F ATAACTATCCCATTACGGTCAATCCGCCGTTTGTTCCG
2 Vchcr3-R TTCACGGAACAAACGGCGGATTGACCGTAATGGGATAG
3 tetR-seq AGTGAGTATGGTGCCTATCT
4 crRNA3-F CACCACAGATGAAACGCCG
5 crRNA3-R CATCTACACCAACGTGACCTATCC
6 T7lacZcr3-R CACCCATCTCGTAAGACTCATG
7 nagEF AACCTCGCATTAATCTTCGC
8 nagER GCAGTAACAGCAACAGAAAGCA
9 nagBF TGCAATATGACCGTCGTTACC
10 nagBR TGAGACTGATCCCCCTGACT
11 manXF GAAATGCTGTTAGGCGAGCA
12 manXR TGAATCAGACGGTCGTCGAT
13 manXF2 GATGAAGTGGCTGCGGATAC
14 manXR2 CCTTACGGACTTCCAGCTCA
3.2转化转座工具质粒与诱导转座
电转化质粒pDonorPtr、pQCasTnsPtr-nagman、pDonorVch和pQCasTnsVch-cr3至大肠杆菌BL21Star TM(DE3)，转化操作同步骤1.7。平板菌落诱导转座与传代操作同步骤1.7。其中pDonorPtr携带的货物基因是绿色荧光蛋白GFP基因(约1.29kb)，pDonorVch携带的货物基因是终止子序列(约0.64kb)。
3.3菌落PCR鉴定基因组插入情况
用来源于霍乱弧菌Tn6677的CRISPR相关转座酶靶向基因组lacZ和T7RNA聚合酶前lacZ，货物基因大小0.64kb。设计pQCasTnsPtr的crRNA阵列靶向基因组nagB、nagE和manX，货物基因绿色荧光蛋白GFP基因(约1.29kb)。经过大肠杆菌转座实验后，用lacZ上靶位点上下游引物PCR检测，阳性条带大小应为1.0kb和1.17kb，阴性条带大小应为0.17kb和0.33kb；用nagB、nagE和manX上靶位点上下游引物PCR检测，阳性条带大小约为2.47kb、2.70kb、2.49kb和2.36kb阴性条带大小应为0.43kb、0.66kb、0.46kb和0.33kb。结果如图4所示，霍乱弧菌Tn6677来源的CRISPR相关转座酶与pQCasTnsPtr均分别靶向对应位点，插入对应货物基因(pDonorPtr的货物基因是绿色荧光蛋白GFP基因，pDonorVch携带的货物基因是终止子序列)，获得2*4拷贝的菌株，效率约100％，该结果经过测序验证，参见图4。
图4显示了菌株基因组中6个位点的Ptr携带的货物基因GFP和Vch携带的货物基因“终止子序列”插入情况，表明两种CRISPR相关转座酶可以在同一大肠杆菌内使用，且可以互不干扰的发挥功能，从而为加速代谢工程菌株构建提供了一种选择方案。
实施例4 验证CRISPR相关转座酶在需钠弧菌的转座活性
通过靶向需钠弧菌ATCC14048基因组wbfF基因，证实来源于半透明假交替单胞菌KMM520的CRISPR相关酶在需钠弧菌具有可编程的转座活性。
质粒pVnQCasTnsPtr包含来源于Pseudoalteromonas translucida KMM520的基因tnsA(SEQ ID NO:8)、tnsB(SEQ ID NO:9)、tnsC(SEQ ID NO:10)、tniQ(SEQ ID NO:11)、Cas5/8(SEQ ID NO:12)、Cas7(SEQ ID NO:14)和Cas6(SEQ ID NO:13)、 靶向基因组目标位点的crRNA序列、p15A复制子、启动子例如脱水四环素诱导型启动子、氯霉素抗性基因，质粒结构如图12所示。
质粒pDonorPtr-GFP包含来源于Pseudoalteromonas translucida KMM520的基因LE(SEQ ID NO:19)和RE(SEQ ID NO:20)序列，pMB1复制子、氨苄青霉素抗性基因、目标货物基因(Cargo)无启动子的绿色荧光蛋白基因片段。
构建靶向基因组wbfF的pVnQCasTnsPtr-wbfF质粒及验证引物列于表4。其中质粒名称pVnQCasTnsPtr-wbfF中的后缀“-wbfF”代表构建靶向基因组wbfF。pVnQCasTnsPtr-wbfF质粒构建方法同步骤1.4、1.5、1.6。
表4：引物序列
编号 引物 序列(5'→3')
1 wbfF-F GAAATCTCTCTTTGGTATCTCTATCAGTCTACTCTAAG
2 wbfF-R TTCACTTAGAGTAGACTGATAGAGATACCAAAGAGAGA
3 wbfF-test-F TCAGCAGAACAACGACACTCAG
4 wbfF-test-R TATTGCCTTGGTTATCTACCGTGAC
5 tetR-seq AGTGAGTATGGTGCCTATCT
注：表中引物名称后缀F代表正向引物，R代表反向引物。
4.1转化转座工具质粒与诱导转座
电转化pDonorPtr-GFP至需钠弧菌ATCC14048，30℃条件下在含氨苄青霉素的LBv2固体平板上进行筛选。挑选阳性克隆子，制备成电转感受态细胞后，电转化pVnQCasTnsPtr-wbfF至上述菌中，30℃条件下在含氨苄青霉素、氯霉素的LBv2固体平板上进行筛选，获得含有pDonorPtr-GFP与pVnQCasTnsPtr-wbfF的需钠弧菌ATCC14048菌株。将上述平板上的克隆刮取一部分重悬于液体LBv2培养基中，重新涂布于含有终浓度100ng/ml脱水四环素、氨苄青霉素与氯霉素的LBv2固体平板上，脱水四环素负责诱导转座相关酶的表达。30℃条件下培养12h，可能会形成一层菌膜，属正常情况。将上述含有100ng/ml脱水四环素平板上的克隆刮取一部分重悬于液体LBv2培养基中，调整OD 600至约0.5后，用液体LBv2培养基稀释50倍，吸取100μL涂布于添加了终浓度1000ng/ml脱水四环素、氨苄青霉素与氯霉素的LBv2固体平板上，30℃条件下培养12h。
4.2菌落PCR鉴定靶向wbfF基因的效率
使用表4中位于插入位点上下游的引物对wbfF-test-F/wbfF-test-R，通过菌落PCR，验证靶向wbfF的两个位点的效率。菌落PCR方法同步骤1.8。
质粒pDonorPtr-GFP上的供体插入片段包括LE(Left end)、RE(Right end)和 货物基因GFP片段(无启动子的绿色荧光蛋白基因片段)，共720bp，阳性条带1220bp，阴性条带500bp。经统计，16个克隆均有插入。证实了来源于半透明假交替单胞菌KMM520的CRISPR相关转座酶在需钠弧菌中也具有可编程的转座活性。
实施例5 验证CRISPR相关转座酶在谷氨酸棒杆菌中的转座活性
实施例中所使用的质粒pCgQCasTnsPtr包含来源于Pseudoalteromonas translucida KMM520的基因tnsA(SEQ ID NO:8)、tnsB(SEQ ID NO:9)、tnsC(SEQ ID NO:10)、tniQ(SEQ ID NO:11)、Cas5/8(SEQ ID NO:12)、Cas7(SEQ ID NO:14)和Cas6(SEQ ID NO:13)、上述基因全部进行谷氨酸棒杆菌密码子优化，靶向基因组目标位点的crRNA序列、ColA复制子、pBL1ts复制子、启动子例如脱水四环素诱导型启动子、卡那霉素抗性基因，其结构如图13所示。
质粒pCgDonorPtr包含来源于Pseudoalteromonas translucida KMM520的基因LE(SEQ ID NO:19)和RE(SEQ ID NO:20)序列，pMB1复制子、pGA1复制子、壮观霉素抗性基因、目标货物基因Cargo例如无启动子的氯霉素抗性CmR基因片段，其结构如图14所示。
通过靶向谷氨酸棒杆菌ATCC13032基因组crtYf基因，证实来源于半透明假交替单胞菌KMM520的CRISPR相关酶在谷氨酸棒杆菌中的转座活性。
构建靶向基因组crtYf基因的crRNA序列为：
AGGCAACCATAGGGCAGGAATCAGAAGTACTG。
5.1转化转座工具质粒与诱导转座
电转化pCgDonorPtr与pCgQCasTnsPtr至谷氨酸棒杆菌ATCC13032，30℃条件下在含壮观霉素与卡那霉素的BHIS固体平板上进行筛选，获得含有pCgDonorPtr与pCgQCasTnsPtr的谷氨酸棒杆菌ATCC13032菌株。将上述平板上的克隆刮取一部分重悬于液体BHIS培养基中，重新涂布于含有终浓度100ng/ml脱水四环素、壮观霉素与卡那霉素的BHIS固体平板上，脱水四环素负责诱导转座相关酶的表达。30℃条件下培养24h，可能会形成一层菌膜，属正常情况。将上述含有100ng/ml脱水四环素平板上的克隆刮取一部分重悬于液体BHIS培养基中，调整OD 600至约0.5后，用液体BHIS培养基稀释50倍，吸取100μL涂布于添加了终浓度1000ng/ml脱水四环素、壮观霉素与卡那霉素的BHIS固体平板上，30℃条件下培养48h。
5.2菌落PCR鉴定靶向crtYf的效率
使用位于插入位点上下游的引物对F-crtYf(TGCTGTGGGAACTTTTCGGT)和 R-crtYf(ACTACCACTCCCGAGGTTGA)，通过菌落PCR，验证靶向crtYf的效率。
质粒pDonorPtr上的供体插入片段包括LE(Left end)、RE(Right end)和货物基因CmR片段(无启动子的氯霉素抗性基因片段)，共1433bp，阳性条带2110bp，阴性条带677bp。经统计，6个克隆均成功插入。凝胶电泳图如图15所示，证实了来源于半透明假交替单胞菌KMM520的CRISPR相关转座酶在谷氨酸棒杆菌中的转座活性。

Claims (16)

  1. 一种CRISPR相关转座酶，其包括选自下组的多肽：来源于假交替单胞菌属细菌的转座酶蛋白tnsA、来源于假交替单胞菌属细菌的转座酶蛋白tnsB、来源于假交替单胞菌属细菌的转座酶蛋白tnsC、来源于假交替单胞菌属细菌的转座酶蛋白tniQ、来源于假交替单胞菌属细菌的核酸酶蛋白Cas5/8、来源于假交替单胞菌属细菌的核酸酶蛋白Cas6、来源于假交替单胞菌属细菌的核酸酶蛋白Cas7。
  2. 如权利要求1所述的CRISPR相关转座酶，其特征在于，所述假交替单胞菌属细菌是半透明假交替单胞菌，优选所述半透明假交替单胞菌是半透明假交替单胞菌KMM520(Pseudoalteromonas translucida KMM520)。
  3. 如权利要求2所述的CRISPR相关转座酶，其特征在于，
    所述tnsA是具有SEQ ID NO:1氨基酸序列的多肽，或者与SEQ ID NO:1有95％以上同源性、优选地98％以上同源性、更优地99％以上同源性、且功能相同的多肽；
    所述tnsB是具有SEQ ID NO:2氨基酸序列的多肽，或者与SEQ ID NO:2有95％以上同源性、优选地98％以上同源性、更优地99％以上同源性、且功能相同的多肽；
    所述tnsC是具有SEQ ID NO:3氨基酸序列的多肽，或者与SEQ ID NO:3有95％以上同源性、优选地98％以上同源性、更优地99％以上同源性、且功能相同的多肽；
    所述tniQ是具有SEQ ID NO:4氨基酸序列的多肽，或者与SEQ ID NO:4有95％以上同源性、优选地98％以上同源性、更优地99％以上同源性、且功能相同的多肽；
    所述Cas5/8是具有SEQ ID NO:5氨基酸序列的多肽，或者与SEQ ID NO:5有95％以上同源性、优选地98％以上同源性、更优地99％以上同源性、且功能相同的多肽；
    所述Cas6是具有SEQ ID NO:6氨基酸序列的多肽，或者与SEQ ID NO:6有95％以上同源性、优选地98％以上同源性、更优地99％以上同源性、且功能相同的多肽；
    所述Cas7是具有SEQ ID NO:7氨基酸序列的多肽，或者与SEQ ID NO:7有95％以上同源性、优选地98％以上同源性、更优地99％以上同源性、且功能相同的多肽。
  4. 编码如权利要求1-3中任一项中所述的多肽的基因。
  5. 如权利要求4所述的基因，其特征在于，
    编码具有SEQ ID NO:1氨基酸序列的多肽tnsA的基因是核苷酸序列SEQ ID NO:8，或者与SEQ ID NO:8有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性的多核苷酸；
    编码具有SEQ ID NO:2氨基酸序列的多肽tnsB的基因是核苷酸序列SEQ ID NO:9，或者与SEQ ID NO:9有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性的多核苷酸；
    编码具有SEQ ID NO:3氨基酸序列的多肽tnsC的基因是核苷酸序列SEQ ID NO:10，或者与SEQ ID NO:10有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性的多核苷酸；
    编码具有SEQ ID NO:4氨基酸序列的多肽tniQ的基因是核苷酸序列SEQ ID NO:11，或者与SEQ ID NO:11有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性的多核苷酸；
    编码具有SEQ ID NO:5氨基酸序列的多肽Cas5/8的基因是核苷酸序列SEQ ID NO:12，或者与SEQ ID NO:12有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性的多核苷酸；
    编码具有SEQ ID NO:6氨基酸序列的多肽Cas6的基因是核苷酸序列SEQ ID NO:13，或者与SEQ ID NO:13有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性的多核苷酸；
    编码具有SEQ ID NO:7氨基酸序列的多肽Cas7的基因是核苷酸序列SEQ ID NO:14，或者与SEQ ID NO:14有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性的多核苷酸。
  6. 一种用于CRISPR转座子系统的质粒pQCascade或称pQCascadePtr，其特征在于，包括选自下组的基因片段：如权利要求4或5中所述的Cas5/8编码基因；如权利要求4或5中所述的Cas6编码基因；如权利要求4或5中所述的Cas7编码基因；如权利要求4或5中所述的tniQ编码基因。
  7. 如权利要求6所述的质粒pQCascadePtr，其特征在于，所述crRNA序列的间隔序列spacer是靶向基因组中单个位点的spacer，或者是靶向基因组中多个位点的crRNA阵列。
  8. 如权利要求7所述的质粒pQCascadePtr，其特征在于，所述crRNA序列是靶向基因组多位点的crRNA阵列，其中重复序列区repeat包括选自下组序列中的一种以上：核苷酸序列为SEQ ID NO:15的repeat1、核苷酸序列为SEQ ID NO:16的repeat2、核苷酸序列为SEQ ID NO:17的repeat3、核苷酸序列为SEQ ID NO:18的repeat4，这些核苷酸序列SEQ ID NOs:15-18中的32个N即[N32]是任意的碱基A、T、G或C。
  9. 一种用于CRISPR转座子系统的辅助质粒pTns或称pTnsPtr，其与如权利要求6-8中任一项所述的质粒pQCascadePtr配合使用，其特征在于，包括选自下组的基因片段：如权利要求4或5中所述的tnsA编码基因；如权利要求4或5中所述的tnsB编码基因；如权利要求4或5中所述的tnsC编码基因。
  10. 一种用于CRISPR转座子系统的质粒pQCasTnsPtr，其特征在于，由如权利要求6-8中任一项所述的质粒pQCascadePtr和如权利要求9所述的辅助质粒pTnsPtr合并而成，包括：上述的Cas5/8、Cas6、Cas7、tniQ、tnsA、tnsB和tnsC基因，靶向基因组目标位点的crRNA序列，ColA复制子，启动子，卡那霉素抗性基因。
  11. 一种用于CRISPR转座子系统的辅助质粒pDonorPtr，其与如权利要求6-8中任一项所述的质粒pQCascadePtr和如权利要求9所述的辅助质粒pTnsPtr配合使用，其特征在于，包括选自下组的基因片段：序列Left end(LE)，其核苷酸序列为SEQ ID NO:19，或者与SEQ ID NO:19有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性且包含SEQ ID NO:19的3’端33bp序列；序列Right end(RE)，其核苷酸序列为SEQ ID NO:20，或者与SEQ ID NO:20有80％以上同源性、优选地85％以上同源性、更优地90％以上同源性、更优地95％以上同源性且包含SEQ ID NO:20的5’端27bp序列；目标货物基因(Cargo gene)。
  12. 一种用于CRISPR转座子系统的质粒pEffectorPtr，其特征在于，由如权利要求6-8中任一项所述的质粒pQCascadePtr、如权利要求9所述的辅助质粒pTnsPtr和如权利要求11所述的辅助质粒pDonorPtr合并而成，包括：上述的Cas5/8、Cas6、Cas7、tniQ、tnsA、tnsB和tnsC基因、Left end(LE)和Right end(RE)序列，靶向基因组目标位点的crRNA序列，ColA复制子，启动子，卡那霉素抗性基因。
  13. 一种CRISPR转座子系统，其特征在于，包括：如权利要求6-8中任一项所述的质粒pQCascadePtr，如权利要求9所述的辅助质粒pTnsPtr，如权利要求11所述的辅助质粒pDonorPtr；或者如权利要求10所述的质粒pQCasTnsPtr，如权利要求11所述的辅助质粒pDonorPtr；或者如权利要求12所述的质粒pEffectorPtr。
  14. 如权利要求13所述的CRISPR转座子系统，其特征在于，还包括霍乱弧菌(Vibrio cholerae)Tn6677来源的CRISPR转座酶相关质粒，所述霍乱弧菌Tn6677来源的CRISPR转座酶相关质粒包括质粒pQCasTnsVch和辅助质粒pDonorVch，
    其中质粒pQCasTnsVch包括：霍乱弧菌Tn6677来源的Cas5/8、Cas6、Cas7、tniQ、tnsA、tnsB和tnsC基因，CloDF13复制子，启动子，链霉素抗性基因；
    质粒pDonorVch包括霍乱弧菌Tn6677来源的Left end(LE)和Right end(RE)，以及目标货物基因(Cargo gene)。
  15. 如权利要求1-3中任一项所述的CRISPR相关转座酶、如权利要求4或5所述的基因、如权利要求6-8中任一项所述的质粒pQCascadePtr、如权利要求9所述的质粒pTnsPtr、如权利要求10所述的质粒pQCasTnsPtr、如权利要求11所述的质粒pDonorPtr、如权利要求12所述的质粒pEffectorPtr、如权利要求13或14所述的CRISPR转座子系统在基因编辑中的应用。
  16. 如权利要求13或14所述的CRISPR转座子系统在基因编辑中的应用，其特征在于，用于革兰氏阴性菌例如大肠杆菌、需钠弧菌、柠檬塔特姆氏菌、革兰氏阳性菌例如谷氨酸棒杆菌的基因编辑。
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