Construction and optimization of gene editing tool based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated transsposases and application of gene editing tool in escherichia coli

Abstract

The invention discloses construction and optimization of a gene editing tool based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated transsposases and application of the gene editing tool in escherichia coli, and belongs to the field of synthetic biology and gene engineering. According to the invention, an efficient gene editing system is established in Escherichia coli, and the burden of a MUSCULAR-CAST system on strains is reduced through element optimization; by eliminating the self-targeting effect, large-fragment (> 10k bp) target gene expression cassettes can be efficiently integrated, and the editing degree of the gene editing system is improved. According to the invention, a MUSCULAR-CAST gene editing system is used for constructing the chassis strains for producing 3-fucosyllactose, L-tryptophan, 2 '-fucosyllactose and neotetraose, so that the chassis strains have equivalent or higher expression yield compared with plasmids, and the growth condition is better or the conversion rate is higher.

Description

Technical Field

[0001] The present invention relates to the construction and optimization of a gene editing tool based on CRISPR-associated transposases and its application in Escherichia coli, and belongs to the field of synthetic biology and genetic engineering. Background Art

[0002] Industrial chassis strains are becoming plasmid-free, which not only reduces the metabolic pressure of production strains, but also reduces the use of antibiotics and inducers. As the most widely used and most comprehensively studied industrial chassis strain, Escherichia coli is now becoming a research hotspot. However, due to the complex metabolic relationship between strain growth and product production, it takes a long time to screen the optimal copy number for genome integration, and the efficiency of genome integration of large fragments (>10k bp) expression frames is not high. The expression frames required for the integration of target metabolic pathways in metabolic engineering contain multiple genes, which generates high metabolic pressure and makes genome integration difficult. The currently widely used CRISPR-Cas editing system is difficult to achieve multi-site gene editing, and the homologous recombination efficiency is not high. In 2019, MIT Feng Zhang’s team (Jonathan Strecker et al., RNA-guided DNA insertion with CRISPR-associated transposases. Science 365, 48-53 (2019).) and Columbia University Samuel H. Sternberg’s team (Klompe, SE, et al., Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration. Nature, 2019. 571(7764): p. 219-225.) proposed using transposases from different sources for gene editing, referred to as the INTEGRATE (INsert Transposable Elements by Guide RNA-Assisted Targeting) system and the CAST (CRISPR-associated transposase) system, and successfully completed multi-site gene editing in Escherichia coli with extremely high efficiency.In 2021, Yang Sheng's team from the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, discovered a new CAST editing system derived from Pseudomonas transparenta KMM520, called the MUCICAT (multicopy chromosomal integration using CRISPR-associated transposases) system (Yang, S., et al., Orthogonal CRISPR-associated transposases for parallel and multiplexed chromosomal integration. Nucleic Acids Research, 2021. 49(17): p. 10192-10202.), and successfully achieved rapid multi-site insertion of glucose dehydrogenase (GDH) into the Escherichia coli genome, and screened out recombinant bacteria with enzyme activity higher than plasmid expression (Zhang, Y., et al., Multicopy Chromosomal Integration Using CRISPR-Associated Transposases. ACS Synthetic Biology, 2020. 9(8): p. 1998-2008.). The above research has brought new ideas and tools for the construction of synthetic biology chassis strains. However, due to the high complexity of the metabolic network, it is not limited to the enzymatic properties of a single enzyme, and the application scenarios are more complex and more difficult. Therefore, there is an urgent need to develop a gene editing system suitable for large fragments, multiple sites, low pressure, and easy programming. Summary of the invention

[0003] The present invention provides a gene editing system, comprising a plasmid combination of (a) or (b):

[0004] (a) plasmid pABC, plasmid pQ876, and plasmid pDonorA;

[0005] (b) plasmid pABCQ876 and plasmid pDonorA;

[0006] Among them, plasmid ABC is based on plasmid pTnsABC, but the promoter of transposase TnsABC is replaced with J23100 promoter as shown in SEQ ID NO.6; plasmid pQ876 is based on plasmid pQCascade, but the promoter of TniQ-Cascade is replaced with J23100 promoter as shown in SEQ ID NO.6; plasmid pABCQ876 has the nucleotide sequence shown in SEQ ID NO.4.

[0007] In one embodiment, the replicon in the plasmid pDonorA is replaced with a temperature-sensitive replicon as shown in SEQ ID NO.5.

[0008] The present invention also provides a method for using the gene editing system to perform gene editing on a strain, comprising the following steps:

[0009] (1) Based on the gene editing system, the cargo gene fragment for integration is constructed between the LE (Left end) and RE (Right end) of the pDonor plasmid, and the crRNA array for identifying the insertion site is constructed upstream of the LE of the pDonor plasmid;

[0010] (2) The gene editing system constructed in step (1) is transformed into the host strain and cultured at 30-37°C for 12-16 hours. All colonies on the plate are scraped off and resuspended in 1 mL LB. After gradient dilution, 100 μL is spread on the corresponding resistance plate and cultured at 37°C for 12-14 hours or 30°C for 14-16 hours. This process is called transfer plate.

[0011] (3) The copy number and integration site of the genome of the strain obtained by transfer plate were determined by colony PCR.

[0012] In one embodiment, the method further comprises cutting and eliminating the strain obtained by screening using the pCutamp plasmid of the MUCICAT system or the promoter of the commercial pCas targeting resistance gene.

[0013] In one embodiment, the pCutamp plasmid is eliminated using sucrose counter selection.

[0014] In one embodiment, the pCas plasmid is eliminated by culturing at 42° C. for 12 to 14 hours.

[0015] In one embodiment, the method comprises the following steps:

[0016] (1) constructing a cargo gene fragment for integration into the genome and constructing it between the LE (Left end) and RE (Right end) of the pDonor plasmid in the gene editing system;

[0017] (2) Designing the crRNA targeting the gene, and constructing the crRNA array targeting the genome into the plasmid pDonor in step (1) to finally form the plasmid pDonorA;

[0018] (3) TnsABC and TniQ-Cascade were transcribed using the J23100 promoter and constructed into plasmids containing low-copy replicons. The replicons and resistance genes were based on the plasmid pTnsABC and plasmid pQCascade of INTEGRATE. The constructed plasmids were named plasmid pABC and plasmid pQ876, respectively.

[0019] (4) The three-plasmid editing system constructed in step (3) was transformed into the host strain and cultured at 37°C for 12 to 14 h or 30°C for 14 to 16 h. All colonies on the plate were scraped off and resuspended in 1 mL LB. After gradient dilution, 100 μL was spread on the corresponding resistance plate and cultured at 37°C for 12 to 14 h or 30°C for 14 to 16 h. This process is called transfer plate.

[0020] (5) After repeating the plate transfer 2 to 3 times, the copy number and integration site of the genome of different strains were determined by colony PCR.

[0021] (6) According to the needs of the user, the strain that has successfully integrated at a specific site in step (5) is further transferred to a plate. A single colony can be expanded and then streaked or transferred to a plate as shown in step (5). Finally, a series of strains with different copy numbers and the same copy number at different sites are screened, and the genome of the strain with insertion is sequenced to confirm the absence of base mutations and deletions;

[0022] (7) Cutting and eliminating the strain selected in step (6) using the pCutamp plasmid of the MUCICAT system or the promoter of the commercial pCas targeting resistance gene;

[0023] (8) Ferment the strain obtained in step (7) in a shake flask to screen for high-yield plasmid-free strains.

[0024] In one embodiment, in step (6), the pCutamp plasmid is eliminated by reverse screening using sucrose (10 g / L), and the pCas plasmid is eliminated by culturing at 42° C. for 12 to 14 h.

[0025] In one embodiment, the method comprises the following steps:

[0026] (1) constructing a cargo gene fragment for integration into the genome and constructing it between the LE (Left end) and RE (Right end) of the pDonor plasmid in the gene editing system;

[0027] (2) Designing the crRNA targeting the gene, and constructing the crRNA array targeting the genome into the plasmid pDonor in step (1) to finally form the plasmid pDonorA;

[0028] (3) The inducible Tac promoter, Tet promoter and constitutive promoter J23100 carrying the lactose operon or tetracycline operon were built into the plasmid of the low copy replicon to form plasmid pABCQ876, which formed a dual plasmid editing system with plasmid pDonorA;

[0029] (4) The dual-plasmid editing system successfully constructed in step (3) was transformed into the host strain and cultured at 37°C for 12-14 h or 30°C for 14-16 h. All colonies on the plate were scraped off and resuspended in 1 mL LB. After gradient dilution, 100 μL was spread on the corresponding resistance plate and cultured at 37°C for 12-14 h or 30°C for 14-16 h. This process is called transfer plate.

[0030] (5) Repeat step (4) for 2 to 3 times, and then determine the copy number and integration site of the genome of different strains by colony PCR;

[0031] (6) According to the needs of the user, the strain that has successfully integrated at a specific site in step (5) is further transferred to a plate. A single colony can be expanded and then streaked or transferred to a plate as shown in step (4). Finally, a series of strains with different copy numbers and the same copy number at different sites are screened, and the genome of the strain with insertion is sequenced to confirm the absence of base mutations and deletions;

[0032] (7) Cutting and eliminating the strain selected in step (6) using the pCutamp plasmid of the MUCICAT system or the promoter of the commercial pCas targeting resistance gene;

[0033] (8) Ferment the strain obtained in step (7) in a shake flask to screen for high-yield plasmid-free strains.

[0034] In one embodiment, for a gene editing system containing an inducible promoter, an inducer is also added to the plate culture medium during the plate transfer process in step (4).

[0035] In one embodiment, the nucleotide sequences of LE (Left end) and RE (Right end) are shown as SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0036] In one embodiment, the Escherichia coli includes Escherichia coli BL21, Escherichia coli K-12 MG1655, and Escherichia coli K-12 W3110.

[0037] In one embodiment, the crRNA is designed with its self-complementarity in mind, and a certain distance needs to be maintained between different crRNAs when targeting the genome.

[0038] In one embodiment, the method for constructing plasmid pDonorA is as follows: rationally screen different locations of the targeted genome, design a crRNA array that specifically targets the gene, and connect the crRNA array to the 5' end of the RE sequence of the plasmid pDonor; connect the target gene expression cassette between the RE sequence and the LE sequence of the plasmid pDonor to form a plasmid pDonorA carrying the target gene expression cassette and its targeted gene crRNA array.

[0039] In one embodiment, the plasmid pABCQ876 is constructed as follows: TniQ-Cascade is connected between the replicon and the promoter of the plasmid pABC by seamless cloning to form plasmid pABCQ876, the sequence of which is shown in SEQ ID NO.12.

[0040] In one embodiment, the sequencing method in step (9) is Sanger sequencing.

[0041] The present invention also provides the use of the method or the recombinant Escherichia coli in producing 3-fucosyllactose, 2'-fucosyllactose, L-tryptophan and neotetraose.

[0042] Beneficial effects:

[0043] (1) The present invention constructs a gene editing system named MUSCULAR-CAST. By optimizing the components, the growth pressure of the system on the gene editing strain is reduced while eliminating the self-targeting effect. The large fragment (>10k bp) target gene expression frame can be inserted into multiple sites of the E. coli genome at a very high efficiency. It is found that the remaining sites in the strains that have undergone genome integration are more likely to undergo genome integration. This feature makes it easier to screen high-copy production strains. Finally, high-yield and healthy chassis strains with different copy numbers or the same copy number at different sites can be screened out through simple screening, providing an efficient gene editing tool for the construction of high-yield chassis strains without plasmid systems.

[0044] (2) The MUSCULAR-CAST gene editing system constructed by the present invention was used to transform Escherichia coli K-12MG1655 to produce 3-fucosyllactose, and strains with a yield equivalent to or even higher than the plasmid expression level were screened, and the strains grew well.

[0045] (3) The MUSCULAR-CAST gene editing system constructed by the present invention was used to insert a polycistronic expression cassette of >10k bp at multiple sites to produce L-tryptophan. When the cassette was inserted at four sites in the genome, the conversion rate was increased by 30% compared with the control, and the yield was comparable to that of the control, at 5.2 g / L and 5.35 g / L, respectively.

[0046] (4) The MUSCULAR-CAST gene editing system constructed by the present invention was used to transform Escherichia coli BL21 to produce 2'-fucosyllactose. The production strain CBGW-4 with genome-integrated target gene had a yield of 17.43 g / L, which was higher than that of the production strain CBGW with plasmid-expressed target gene (15.59 g / L).

[0047] (5) The MUSCULAR-CAST gene editing system constructed by the present invention was used to transform Escherichia coli BL21 to produce neotetraose. The neotetraose yields of the production strains LgtB Cassette4, LgtB Cassette5, LgtBCassette6, and LgtB Cassette7 with the target gene integrated into the genome were 7.66 g / L, 8.38 g / L, 8.28 g / L, and 6.78 g / L, respectively, which were all higher than the production strain NC using a plasmid to express the target gene. In addition, the growth condition of the strain was better, and the stickiness during the fermentation process was significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The different component plasmids of the MUSCULAR-CAST system were constructed; A is a schematic diagram of the three-plasmid editing system; B is a schematic diagram of the two-plasmid editing system.

[0049] Figure 2 This is a gel image of the nucleic acid inserted into the genome of Escherichia coli K-12MG1655 by the MUSCULAR-CAST system; A. Preliminary verification of the insertion of the target gene at site 5 of the genome; B. After a few rounds of transfer plate screening, 6, 7, and 8 high-copy strains can be screened.

[0050] Figure 3 A nucleic acid gel image showing an increase in copy number after plasmid elimination for genome insertion using the MC1 system of the present invention.

[0051] Figure 4 To replace the temperature-sensitive replicon plasmid pABCQ876-Ts and plasmid pQ876-Ts.

[0052] Figure 5 The following are the nucleic acid gel images of the genome insertion of different fragments. A. The nucleic acid gel image of the genome insertion of the expression frame with a fragment size of 4000bp. B. The nucleic acid gel image of the genome insertion of the expression frame with a fragment size of 1400bp. C. The genome insertion of all sites of the strain with successful genome insertion at sites 1 and 2 after transfer.

[0053] Figure 6 This is the liquid phase detection diagram of 3-fucosyllactose.

[0054] Figure 7This is a nucleic acid gel image showing the insertion of L-tryptophan into the genome of Escherichia coli K-12W3110 using the MUSCULAR-CAST system.

[0055] Figure 8 The L-tryptophan yield and OD of the recombinant bacteria constructed in Example 5 600 , conversion rate chart.

[0056] Fig. 9 The 2'-fucosyllactose production and OD of the recombinant bacteria constructed in Example 6 600 picture.

[0057] Fig.10 The new tetrasaccharide yield and OD of the recombinant bacteria constructed in Example 7 600 picture. DETAILED DESCRIPTION

[0058] (I) The abbreviation and full name of the invention, as well as the Chinese and English translations:

[0059] INTEGRATE: INsert Transposable Elements by Guide RNA-AssistedTargEting;

[0060] CAST: CRISPR-associated transposase;

[0061] MUCICAT: multicopy chromosomal integration using CRISPR-associated transposases;

[0062] MUSCULAR-CAST(MUlti-Site CUt-burden,LARge-fragment genomic integration using CRISPR-ASsociated Transposases);

[0063] crRNA: CRISPR RNA, clustered regularly interspaced short palindromicrepeat sequences RNA;

[0064] PAM: Protospacer Adjacent Motif;

[0065] LB: Luria-Bertani medium, used for the culture of Escherichia coli, the specific composition is peptone 10g·L -1 , sodium chloride 10g·L-1 , yeast powder 5g·L -1 .

[0066] (II) Detection methods involved in specific embodiments of the present invention:

[0067] (1) Shake flask fermentation and sampling

[0068] 1) The BL21 bacterial suspension stored in the glycerol tube was streaked onto LB solid medium to which Amp with a final concentration of 100 μg / mL had been added, and the streaked plate was cultured overnight in a 37°C constant temperature incubator.

[0069] 2) Pick a single colony from the culture dish and inoculate it with Amp added at a final concentration of 100 μg / mL. Incubate the inoculated shake flask in LB liquid culture medium at 37°C and 200 r·min-1 for 8-9 hours.

[0070] 3) Transfer the culture medium to 50 ml of fermentation medium at a transfer volume of 2% and culture at 37°C and 200 rpm.

[0071] 4) When the bacterial concentration of the culture medium reaches 2-3, 0.1 mM IPTG is added to the culture medium and the two cultures are placed in a shaker at 25°C.

[0072] 5) Two hours after adding IPTG, add lactose to a final concentration of 10 g / L.

[0073] 6) Take the first sample at the time of adding IPTG on the second day (take the sample on day 5).

[0074] sampling:

[0075] 1) First, take 400 μL and place it in a 5 mL EP tube to measure OD600.

[0076] 2) Take out 800 μL and place it evenly in a 1.5EP tube, place in a metal bath at 100°C for 15 min, then at 12000 rpm for 10 min, take the supernatant and pass it through a membrane and place it in a liquid phase vial.

[0077] (2) Liquid phase detection:

[0078] Parameters: use differential; column oven temperature 50°C; differential 45°C; flow rate 0.5min / mL;

[0079] Elution time: 25 min; Liquid column: Aminex HPX-87H column; Mobile phase: 5 mM sulfuric acid;

[0080] Specific operation: Take 1mL of fermentation liquid, centrifuge, take the supernatant, put it in a metal bath, 100℃, 15min. After the time is up, centrifuge, take the supernatant, pass it through a 0.22μm aqueous membrane, insert the inner cannula, put it in a liquid phase bottle, and then put it in an Agilent liquid phase detector for detection.

[0081] (3) Preparation and transformation of electroporation cells:

[0082] Competent cell preparation:

[0083] 1) Inoculate the bacteria from the glycerol tube into a 10 mL LB medium shake flask with an inoculum size of 0.2% and culture at 37°C, 200 rpm for 10 h.

[0084] 2) Inoculate the bacteria from the shake flask into a 50 mL LB medium shake flask (or a 50 mL 2YT medium shake flask) with an inoculum size of 1%. Cultivate at 37°C, 200 rpm for about 2-3 hours until the OD value reaches 0.5-0.8. Place the shake flask on ice for 30 minutes.

[0085] 3) Centrifuge the bacterial solution at 4000 rpm and 4°C for 10 min, and slowly resuspend the bacteria in sterile water.

[0086] 4) Repeat step 3).

[0087] 5) Centrifuge the bacterial solution at 4000 rpm and 4°C for 10 min, remove the supernatant, and slowly resuspend the bacteria in 10% glycerol on ice.

[0088] 6) Repeat step 5).

[0089] 7) Centrifuge the bacterial solution at 4000 rpm and 4°C for 10 min, remove the supernatant, slowly resuspend the bacteria in 0.6-1 mL of 10% glycerol on ice (depending on the amount of bacteria), dispense 100 μL each into clean 1.5 mL EP tubes, and store at -80°C for later use.

[0090] Conversion:

[0091] 1) Place the -80℃ refrigerator or the newly prepared competent cells on ice, and take out the plasmid to be transformed and the 1mm electroporation cup and place them on ice for 10 minutes to pre-cool them.

[0092] 2) Pipette 10uL of plasmid into competent cells, blow gently to mix, and place on ice for 15-20 minutes.

[0093] 3) Transfer to an electric shock cup (with no water at the bottom), shock once at 2500 V, quickly add 1 mL of LB, and transfer the bacteria to a clean and sterilized EP tube, seal it with a sealing film, and resuscitate and culture in a shaking incubator at 37°C and 200 rpm for 1.5 h.

[0094] 4) Centrifuge at 3000 rpm for 5 min, open the tube in a clean bench, discard the supernatant and keep about 200uL, resuspend the bacterial solution, and spread it on the corresponding resistance plate.

[0095] 5) Culture in a 30°C incubator for 10-12 hours.

[0096] (III) Reagents involved in the specific embodiments of the present invention:

[0097] Antibiotics were prepared using sterile ddH2O and then sterilized using a filter.

[0098] Ampicillin (100 μg / mL), kanamycin (30 μg / mL), spectinomycin (50 μg / mL), apramycin (50 μg / mL)

[0099] The culture medium was prepared with ddH2O and sterilized at 121°C for 20 min.

[0100] LB liquid culture medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L.

[0101] Fermentation medium: KH2PO4 13.5 g / L, MgSO4·7H2O 1.4 g / L, (NH4)2HPO4 4.0 g / L, C6H8O7·H2O 1.7 g / L, industrial yeast powder 5 g / L, pig bone peptone 2.5 g / L, glycerol 30 g / L, trace metal liquid 10 mL.

[0102] Trace metal liquid (g / L): 5 mol·L -1 HCl as mother liquor, CaCl2 2.0g / L, (NH4)6Mo7O 24 0.1g / L, ZnSO4·7H2O 2.25g / L, MnSO4·4H2O 0.5g / L, FeSO4·7H2O 10.0g / L, CuSO4·5H2O 3.0g / L, Na2B4O7·10H2O 0.23g / L, CoCl2·6H2O0.18g / L.

[0103] Example 1: Construction of the E. coli Muscular-CAST system

[0104] (I) Construction of the three-plasmid editing system (MCI)

[0105] A three-plasmid editing system (MCI) was constructed based on the three plasmids of the INTEGRATE system: pQCascade plasmid, pTnsABC plasmid and pDonor plasmid. The specific steps are as follows:

[0106] The promoter of TniQ-Cascade in the INTEGRATE system pQCascade plasmid and the promoter of transposase TnsABC in the pTnsABC plasmid were replaced with J23100 promoter (nucleotide sequence as shown in SEQ ID NO.6), and the plasmids after the replacement of the promoters were named plasmid pABC and plasmid pQ876, respectively.

[0107] The crRNA sequence transcribed by the constitutive promoter J23100 (also called the targeting sequence) is constructed in front of the RE of the plasmid pDonor to form the plasmid pDonorA. The targeting sequence and the cargo gene can be replaced according to the needs of the user.

[0108] Based on MCI-1, the promoter of plasmid pABC was replaced with Tac promoter Ptac-LacO carrying LacO (shown in SEQ ID NO.3). The obtained plasmid was named pABC-Tac, which formed a three-plasmid inducible editing system with plasmid pQ876 and plasmid pDonorA, referred to as MCI-2.

[0109] (II) Construction of the dual-plasmid editing system (MCII)

[0110] Based on the first part, the transposases TnsABC, TniQ, and Cascade regulated by the J23100 promoter were used in a polycistronic form to replace the lactose operon and P on the pTnsABC plasmid of the INTEGRATE system. T7 -TnsABC part, forming plasmid pABCQ876 (nucleotide sequence is shown in SEQ ID NO.4). Plasmid pABCQ876 and plasmid pDonorA constitute a double plasmid constitutive editing system, referred to as MCII-1.

[0111] Based on MCII-1, the J23100 promoter of TnsABC in plasmid pABCQ876 was replaced with the Tac promoter carrying LacO (shown in SEQ ID NO.3), thus forming a dual-plasmid inducible editing system, referred to as MCII-2.

[0112] Example 2 Application of the Muscular-CAST System

[0113] In order to test the practicality of the gene editing system constructed by the present invention, the three-plasmid gene editing system MCI-1 (containing plasmid pABC, plasmid pQ876 and plasmid pDonorA1) constructed in Example 1 was used. The difference was that the targeting sequence in the plasmid pDonorA referred to the crRNA sequence targeting eight sites in the plasmid pQCascade-array1 in the MUCICAT system, namely crRNA1 between the targeted genes yadD and panC, crRNA2 between tktB and ypfG, crRNA3 between aroG and gpmA, crRNA4 between yghA and rxbD, crRNA5 between ompW and yciE, crRNA6 between cspA and hokA, crRNA7 between purT and eda, and crRNA8 between rluF and yjbD, and the crRNA sequence was disclosed in the paper "Multicopy Chromosomal Integration Using CRISPR-Associated Transposases"; and the cargo gene was also transcribed using the glucose dehydrogenase expression cassette P of the T7 promoter. T7 -GDH (the relevant plasmids have been disclosed in the table in Appendix S3 of the paper), and the expression cassette was inserted between LE (Left end) and RE (Right end) of the pDonor plasmid. The constructed plasmid was named pDonorA1.

[0114] The successfully constructed three-plasmid editing system was transformed into Escherichia coli K-12MG1655 and cultured at 37°C for 12-14 h. All colonies on the plate were scraped off and resuspended in 1 mL LB. The gradient dilution was about 10 -5 Take 100 μL and spread it on the corresponding resistance plate and culture it at 37℃ for 12-14h or 30℃ for 14-16h. This process is called transfer. Repeat the transfer 2-3 times and determine the copy number and integration site of the genome of different strains by colony PCR. The strains that have successfully integrated at a specific site will continue to be transferred to the plate, and the single colony will be expanded and streaked. Finally, a series of strains with different copy numbers and the same copy number at different sites will be screened, and the strains with genome insertion will be sequenced to determine the absence of base mutations and deletions.

[0115] like Figure 2 As shown in A, after transferring the plate once, the insertion of the fifth site (crRNA5) was tested, and most strains could achieve accurate genome insertion. After transferring the plate three times, strains with different copy numbers and different sites of system copy numbers can be screened, such as Figure 2 As shown in B.

[0116] Example 3: Optimizing the Muscular-CAST system to control, turn on, turn off, and regulate gene editing

[0117] (1) Debugging the expression of transposase elements to construct the MCIII system to achieve timely shutdown of gene editing

[0118] To compare different systems, the MUCICAT system was first used for genome insertion. In this process, the copy number of the cargo gene was increased, and the application scenarios of synthetic biology were broadened, and the target genes required for the 3-fucosyllactose production strain were introduced. The specific steps are as follows: the phosphomannose mutase ManB, mannose-1-phosphate guanyl transferase ManC, GDP-mannose 4,6-dehydratase Gmd, and GDP-L-fucose synthase WcaG regulated by the T7 promoter, a total of 5711 bp, were inserted into the P T7 -manC-manB-P T7 -gmd-wcaG-T7 terminator polycistronic expression cassette (nucleotide sequence is shown in SEQ ID NO.7). T7 -GDH expression cassette was replaced with the one shown in SEQ ID NO.7 T7 -manC-manB-P T7 -gmd-wcaG-T7 terminator polycistronic expression cassette to form plasmid pDonor1. Plasmid pQCascade and plasmid pTnsABC in the MUCICAT system were transformed into Escherichia coli K-12MG1655 strain, spread on kanamycin and spectinomycin double resistance plates and cultured at 37°C for 12h. After obtaining positive clone strains, plasmid pDonor1 was transformed and finally spread on kanamycin, spectinomycin and ampicillin triple resistance plates with 1mol / L IPTG added. After culture at 37°C for 12-15h, the plates were transferred. After 7 transfers, no strains with successful genome insertion were found. It is speculated that the transposition-related elements and cargo are also transcribed by the strong promoter T7. When the inducer is added, multiple genes are expressed in large quantities, which makes the metabolic pressure of the strain too large and reduces the genome insertion efficiency. At the same time, it was verified that the high expression of transposable elements is not conducive to genome insertion.

[0119] The MCI-1 system (containing plasmid pABC, plasmid pQ876 and plasmid pDonorA1) constructed in Example 1 was used for genome insertion, except that P T7 -manC-manB-P T7 -gmd-wcaG-T7 terminator polycistronic expression cassette (shown in SEQ ID NO.7) replaced the P in plasmid pDonorA1 T7-GDH expression cassette to form plasmid pDonorA3. First, plasmids pABC and pQ876 were transformed into Escherichia coli K-12MG1655 strain, spread on plates with three resistances of kanamycin and spectinomycin, cultured in a 37°C incubator for 14 hours, and then single colonies were selected and cultured in 10mL LB for 12 hours, and then transferred to 50mL LB at a 1% inoculation amount to prepare competent cells, and plasmid pDonorA3 was transformed into competent cells by electroporation, and finally spread on plates with three resistances of kanamycin, spectinomycin and ampicillin, and cultured in a 37°C incubator for 14 hours. After two plate transfers, strains with different copy numbers were successfully screened. However, after plasmid elimination, the copy number of individual strains previously screened was found to increase. The screened strains were cut and eliminated using the pCutamp plasmid or the commercial pCas targeted resistance gene promoter; the pCutamp plasmid was reversely screened and eliminated using 10 g / L sucrose, and the pCas plasmid was eliminated by culturing at 42°C for 12 to 14 hours, and finally a plasmid-free genome multi-site integration strain was obtained; Figure 3 As shown, site 4 also completed genomic integration after plasmid elimination, e.g. Figure 3 shown.

[0120] When all transposable elements are transcribed using constitutive promoters, the efficiency of transposition editing is high, the number of genomic insertions is difficult to control, it is difficult to screen for strains with low copy numbers, and after the target copy number is screened, the genome integration copy number will become higher after plasmid elimination. The use of an inducible promoter will solve this problem to a certain extent. In this regard, further optimization was performed, and the replicon of the plasmid pDonorA was replaced with a temperature-sensitive replicon (sequence shown in SEQ ID NO.5) based on the above-constructed MCI-1, to obtain the plasmid pDonorA-Ts (as shown in Figure 4 As shown). Together with pABC and pQ876, it forms the three-plasmid editing system MCIII. When preparing competent cells for plasmid elimination, pick a single colony, culture 10mL system at 37℃ for 12-14h and then transfer, so that the plasmid pDonorA-Ts is eliminated in advance, thereby reducing the genome insertion phenomenon during the plasmid elimination process, and then use the plasmid pCutamp in the MUCICAT system to cut and eliminate the remaining plasmid.

[0121] Plasmids pDonorA-Ts, pABC, and pQ876 were transformed into Escherichia coli K-12MG1655, and spread on plates with three resistances added with ampicillin (100 μg / mL), kanamycin (30 μg / mL), and spectinomycin (50 μg / mL). After culturing at 30°C for 16 hours, the plates were transferred. After transferring the plates 4 times, colonies were randomly selected for colony PCR to determine the gene copy number. Finally, one strain with 8 copies, one strain with 7 copies, two strains with 6 copies, three strains with 5 copies, and one strain with 4 copies were screened, among which strains with the same copy number inserted at different sites in the genome were selected, as shown in Table 1.

[0122] Table 1. Gene copy number of E. coli K-12MG1655

[0123] Stain S1 S2 S3 S4 S5 S6 S7 S8 Copy number 1 √ √ √ √ √ × √ √ 7 2 √ √ √ √ √ √ √ √ 8 3 √ √ × × √ √ √ √ 6 4 × √ × × √ √ √ √ 5 5 × √ √ × × × √ √ 4 6 × √ √ √ √ × √ √ 6 7 × √ × √ √ × √ √ 5 8 × √ √ × √ × √ √ 5

[0124] (2) The strains with successful genome insertion at some sites have higher genome insertion efficiency at the remaining sites

[0125] In order to simultaneously achieve the inactivation of the competitive metabolic pathway genes and the insertion of the target metabolic pathway genes, the crRNA array was replaced with a crRNA sequence targeting five sites of the Escherichia coli K-12MG1655 genome (shown in SEQ ID NO.8) designed with NCC as PAM (Protospacer Adjacent Motif), targeting galactoside O-acetyltransferase LacA, L-fucose isomerase FucI, glucan biosynthesis glucosyltransferase MdoH, HslVU protease ATPase ClpY, UDP-glucose: undecenyl phosphate glucose-1-phosphotransferase WcaJ, to form plasmid pDonorA2. On the basis of plasmid pDonorA2, the cargo gene was replaced with P T7 _gsk-P T7 _futA-T7terminator expression cassette (nucleotide sequence as shown in SEQ ID NO.9), construct plasmid pDonorA4. On the basis of plasmid pDonorA2, the cargo gene was replaced with P T7 _futA-T7 terminator expression cassette (nucleotide sequence is shown in SEQ ID NO.10) to construct pDonorA5.

[0126] Using the dual plasmid editing system, plasmid pABCQ876 was used for gene editing of Escherichia coli K-12MG1655 together with pDonorA4 and pDonorA5. First, plasmid pABCQ876 was transformed into Escherichia coli K-12MG1655, spread on kanamycin plates and cultured at 37°C for 14 hours, then single colonies were selected and cultured in 10mL LB for 12 hours, and then transferred to 50mL LB at a 1% inoculation amount to prepare competent cells, pDonorA4 and pDonorA5 were transformed into competent cells by electroporation, and finally spread on kanamycin and ampicillin double-resistant plates and placed in a 37°C incubator for 14 hours for transfer. After one transfer, the genome integration efficiency of larger fragments is lower than that of small fragments, such as Figure 5 As shown in A~5B. T7 _gsk-P T7 When the _futA-T7terminator expression cassette was integrated, only one strain with successful genome integration was screened after five plate transfers, and in P T7 When the _futA-T7 terminator expression cassette is integrated, strains with different copy numbers can be screened after transferring the plate once.

[0127] On this basis, P T7 _gsk-P T7 The single bacterium with the successful integration of the _futA-T7 terminator expression cassette genome was used as the starting strain for transfer to a plate and mixed with the plate colonies. That is, all the strains growing on the plate were picked into a 1.5 mL centrifuge tube and resuspended in 500 μL LB solution and diluted to an appropriate concentration (usually 10 -5 ) Take 100 μL to plate and test the efficiency of subsequent genome integration. The results show that the genome integration efficiency of other sites will be greatly improved if the strain with successful genome insertion is used as the starting strain and then transferred to the plate. Figure 5 As shown in C, after transferring the plate once, multiple single colonies were randomly selected for verification, and it was found that genome insertion was achieved at almost all sites. Figure 5 After the colonies on the plate containing the strain in A were mixed and transferred, most of the strains still failed to achieve P T7 _gsk-P T7 The _futA-T7 terminator expression cassette is inserted into the genome. Therefore, using the above method for strains that require high-copy genome insertion can greatly shorten the time for screening high-copy.

[0128] Example 4: Muscular-CAST system transforms Escherichia coli K-12MG1655 to produce 3-fucosyllactose

[0129] According to the method of Example 2, the MCI-1, MCII-1, and MCIII editing systems were used to integrate the P of the target metabolic pathway for producing 3-fucosyllactose in Escherichia coli K-12MG1655. T7 -manC-manB-P T7 -gmd-wcaG-T7terminator polycistronic expression cassette (shown in SEQ ID NO.7), the difference being that the plasmid pDonorA in the MCI-1 and MCII-1 systems is replaced by pDonorA-Ts containing a temperature-sensitive replicon.

[0130] Gene editing was performed according to the method of Example 1, and the effect was verified by colony PCR. The results showed that the above editing systems can achieve genome insertion. Finally, strains 1 to 8 with different copy numbers were selected.

[0131] Then P T7 _gsk-P T7 The _futA-T7 terminator expression cassette (nucleotide sequence as shown in SEQ ID NO.9) was expressed in the plasmid pETDuet, wherein the nucleotide sequence of futA is shown in SEQ ID NO.6. pETDuet-P was electroporated into strains 1 to 8 with different copy numbers of the target 3-fucosyllactose metabolic pathway gene. T7 _gsk-P T7 _futA, the constructed strains were named strains MG1-8. T7 _gsk-P T7 The plasmid pETDuet-P was transformed into E. coli K-12MG1655 containing the _futA-T7 terminator expression cassette T7 _gsk-P T7 _futA, and P T7 -manC-manB-P T7 The gmd-wcaG-T7 terminator polycistronic expression cassette was expressed using plasmid pRSFDuet (nucleotide sequence shown in SEQ ID NO.7) and electroporated into plasmid pETDuet-P T7 _gsk-P T7 coli K-12 MG1655 expressing _futA, named strain MG9, was used as a negative control strain.

[0132] After strains MG1-MG9 were cultured at 37°C for 2 h in fermentation medium, 0.1 mM IPTG was added and then cultured at 25°C. After fermentation for 72 h, 3-fucosyllactose was detected in the liquid phase. The peaks of different substances in the liquid phase and the time are shown in the figure below. Figure 6As shown, the peak of 3-fucosyllactose is around 7 min. The image shows that the yield of No. 8 strain with genome integration is 1 g / L, and the yield of No. 9 strain with plasmid expression is 0.6 g / L. The OD of strains MG1 to MG9 at 72 h 600 The sequence is 8.40, 8.34, 10.60, 8.38, 8.40, 9.70, 8.28, 8.40, and 8.02. The strains with the target gene inserted into the genome are growing well. The results show that after genome insertion by the Muscular-CAST system, strains with an output equivalent to or even higher than the plasmid expression level can be screened, and the strains are growing better. This operation is conducive to reducing the metabolic burden of the strains, and the yield of the target product of the chassis strains with the key genes of the target metabolic pathway integrated into the genome can reach or even exceed the plasmid level, which greatly promotes the industrial application of plasmid-free chassis strains.

[0133] Example 5: Production of L-tryptophan by inserting a polycistronic expression cassette of >10 k bp at multiple sites using the Muscular-CAST system

[0134] In the laboratory, the L-tryptophan-producing Escherichia coli K-12W3110 FB-04 (Escherichia coli K-12W3110 FB-04 was modified in the early stage and disclosed in the paper "Modulating the direction of carbon flow in Escherichia coli to improve L-tryptophan production by inactivating the global regulator FruR.") used the above-mentioned MCI-1 system to integrate the target genes required for the production of L-tryptophan, a total of 7 enzymes, and the expression cassette sequence is shown in SEQ ID NO.8. First, the crRNA and the target gene expression frame targeting three different positions of crRNA1 between yadD and panC, crRNA5 between ompW and yciE, and crRNA8 between rluF and yjbD in the genome of Escherichia coli K-12W3110 were used to construct the plasmid pDonorA-Trp. Three strains with copy numbers of 1, 2, and 3 were screened in a very short time, as shown in FIG. Figure 7As shown, the experimental results show that the Muscular-CAST system modified by the present invention can integrate multiple copies of large fragments (>10k bp) complex expression cassettes into the genome with extremely high efficiency. On the basis of the above experimental results, the number of crRNA was increased, and finally the Muscular-CAST system was used to screen four bacteria with copy numbers of 1, 2, 3, and 4, named ZH-1, ZH-2, ZH-3, and ZH-4, all of which were plasmid-free strains. Shake flask fermentation was used to test the growth of the strain, L-tryptophan production and conversion rate. Ts-1 was used as a control and was a chassis strain for plasmid expression of the target gene. Analysis found that when the number of copies of the genome insertion was less than or equal to three, the strain growth OD 600 The OD value was significantly higher than that of the strain expressing plasmid, while the OD value was higher when the four sites of the genome were inserted. 600 The conversion rate was slightly decreased, but it was 30% higher than that of the control, and the yield was comparable to that of Ts-1, which were 5.2 g / L and 5.35 g / L, respectively. This once again proved that the Muscular-CAST system can be used to efficiently construct high-yield plasmid-free chassis strains, and the screening method is simple and convenient.

[0135] Example 6: Muscular-CAST system transforms Escherichia coli BL21 to produce 2'-fucosyllactose

[0136] The target gene required for producing 2'-fucosyllactose was integrated into the Escherichia coli BL21 star (DE3) F9 (the strain was disclosed in the paper "Combinatorial metabolic engineering of Escherichia coli for the high-yield production of 2'-fucosyllactose from glucose and glycerol") using the MCI-1 system (containing plasmid pABC, plasmid pQ876 and plasmid pDonorA3). According to the same method as in Example 2, 8 chassis strains with different copy numbers or the same copy number and different sites were finally screened. On this basis, plasmid pETDuet-Gsk-SUMO_FutC (nucleotide sequence as shown in SEQ ID NO.15) and plasmid pCDFDuet-RcsAB-SetA-Zwf-LacY (nucleotide sequence as shown in SEQ ID NO.16) were transformed to construct the obtained 2'-fucosyllactose chassis strains CBGW-1 to CBGW-8 containing genes with different copy numbers. The control strain CBGW was transformed with plasmid pRSFDuet-P using a strain without gene integration. T7 -manC-manB-P T7-gmd-wcaG (shown in SEQ ID NO.13), plasmid pETDuet-Gsk-SUMO_FutC (shown in SEQ ID NO.15), and plasmid pCDFDuet-RcsAB-SetA-Zwf-LacY (shown in SEQ ID NO.16) to produce 2'-fucosyllactose.

[0137] The constructed genetically engineered bacteria were cultured in a fermentation medium at 37°C for 2 h, 0.1 mM IPTG was added, and then cultured at 25°C. After 72 h of fermentation, the 2'-fucosyllactose production was detected in the liquid phase, and the OD of the fermentation liquid was detected. 600 .like Fig. 9 As shown, the 2'-fucosyllactose production of the production strain CBGW-4 with the target gene integrated into the genome is 17.43 g / L, and the production strain CBGW with the target gene expressed by the plasmid has a production yield of 15.59 g / L. After simple screening, production strains with higher yields than those expressed by the plasmid can be selected, which greatly promotes the construction of plasmid-free strains and lays the foundation for industrial applications.

[0138] Example 7: Muscular-CAST system to transform Escherichia coli BL21 to produce neotetrasaccharide (LNnT)

[0139] The target genes required for the production of neotetraose were integrated into the neotetraose-producing Escherichia coli BL21 star (DE3) BL21–16 (published in “High-Level Production of Lacto-N-neotetraose in Escherichia coli by Stepwise Optimization of the Biosynthetic Pathway”) using the MCI-1 system (containing plasmids pABC, pQ876, and pDonorA6), wherein the genes glmUMS and lgtA for the target metabolic pathway of LNnT were synthesized with P T7 -glmUMS-P T7 -lgtA-T7 terminator polycistronic expression cassette (shown in SEQ ID NO.17) replaced the P in plasmid pDonorA1 T7 -GDH expression cassette to form plasmid pDonorA6. Try to screen chassis strains with less metabolic pressure and a yield equivalent to or even higher than the level of the target gene expressed by the plasmid to solve the problem of stickiness of high-yield strains.

[0140] Finally, 6 chassis strains with different copy numbers were screened. The strain transformed with the target gene plasmid required for producing the new tetraose was used as the control. The strains LgtB Cassette3~LgtB Cassette8 and chassis strain NC for producing the new tetraose were constructed, totaling 7 strains. The copy numbers of LgtB Cassette3~LgtB Cassette8 integrated into the genome were 3 to 8, respectively.

[0141] The strain was cultured in fermentation medium at 37°C for 2 h, 0.1 mM IPTG was added, and then cultured at 25°C for 72 h. The content of neotetraose in the fermentation broth after shake flask fermentation was detected by liquid phase (the detection method refers to the paper "High-Level Production of Lacto-N-neotetraose in Escherichia coli by StepwiseOptimization of the Biosynthetic Pathway"). The yield after 72 h of fermentation was compared with the OD 600 like Fig.10 The results showed that the yield of strain NC was 6.42 g / L, and the yields of new tetrasaccharides of the production strains LgtB Cassette4, LgtBCassette5, LgtB Cassette6, and LgtB Cassette7 with the target gene integrated into the genome were 7.66 g / L, 8.38 g / L, 8.28 g / L, and 6.78 g / L, respectively, which were all higher than the production strain NC using plasmid to express the target gene. In addition, the growth condition of the strain was better, and the stickiness during the fermentation process was significantly improved.

[0142] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A gene editing system, characterized in that: A plasmid combination comprising (a) or (b): (a) plasmid pABC, plasmid pQ876, and plasmid pDonorA; (b) plasmid pABCQ876 and plasmid pDonorA; Among them, plasmid ABC is based on plasmid pTnsABC, but the promoter of transposase TnsABC is replaced with J23100 promoter as shown in SEQ ID NO.6; plasmid pQ876 is based on plasmid pQCascade, but the promoter of TniQ-Cascade is replaced with J23100 promoter as shown in SEQ ID NO.6; The plasmid pABCQ876 has the nucleotide sequence shown in SEQ ID NO.

4.

2. The gene editing system according to claim 1, characterized in that: The replicon in the plasmid pDonorA is replaced with a temperature-sensitive replicon as shown in SEQ ID NO.

5.

3. A gene editing method, characterized in that: Using the gene editing system of claim 1 or 2 to edit the gene of a strain comprises: (1) constructing the cargo gene fragment for integration between the LE and RE of the pDonor plasmid in the gene editing system, and constructing the crRNA array of the targeted gene upstream of the LE of the pDonor plasmid; (2) The gene editing system constructed in step (1) is transformed into the host strain and cultured at 30-37°C for 12-16 hours. All colonies on the plate are scraped off and resuspended in 1 mL LB. After gradient dilution, 100 μL is spread on the corresponding resistance plate and cultured at 37°C for 12-14 hours or 30°C for 14-16 hours. This process is called transfer plate. (3) Determine the copy number and integration site of the genome of the strain obtained by transfer plate by colony PCR, and screen the desired gene editing strain.

4. The method according to claim 3, characterized in that The method also includes eliminating the plasmid in the strain obtained by screening in step (3).

5. The method according to claim 4, characterized in that The elimination includes but is not limited to cutting and eliminating by using the pCutamp plasmid of the MUCICAT system or the promoter of the pCas targeting resistance gene.

6. The method according to claim 3, characterized in that The nucleotide sequences of LE and RE are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively.

7. The method according to claim 3, characterized in that Add an inducer to the plate culture medium in step (2).

8. The method according to claim 3, characterized in that The strains include but are not limited to Escherichia coli BL21, Escherichia coli K-12 MG1655, and Escherichia coli K-12 W3110.

9. Use of the gene editing system according to claim 1 or 2, or the method according to any one of claims 3 to 8 in constructing gene-edited microorganisms.

10. The use according to claim 9, characterized in that: Including but not limited to constructing a genetically engineered microorganism that produces 3-fucosyllactose, or produces L-tryptophan, or produces 2'-fucosyllactose, or produces neotetraose.

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