Construction and optimization of MUSCULAR-CAST-based transcription inhibition tool and application of MUSCULAR-CAST-based transcription inhibition tool in escherichia coli

Abstract

The invention discloses construction and optimization of a transcription inhibition tool based on MUSCULAR-CAST and application of the transcription inhibition tool in escherichia coli, and belongs to the field of synthetic biology and genetic engineering. According to the invention, through the strategies of optimizing transcriptional regulation through promoter engineering, adding a spacer sequence at the upstream of crRNA, replacing a TniQ-Cascade promoter with a constitutive weak promoter J23109 and the like, the growth condition of a strain is improved while the inhibition rate on a gene is improved. The system is successfully applied to construction of a 3-fucosyllactose high-producing strain, 17 target genes of escherichia coli chassis cells can be inhibited, the yield of 3-FL is increased by 4.40 times, and the important value of 3-FL in metabolic engineering is proved. The Tn-CRISPRi system established by the invention solves the core problems of strict PAM limitation, low multiple inhibition efficiency, high cytotoxicity and the like in the prior art, and provides a new generation of gene regulation and control tool for synthetic biology.

Description

Technical Field

[0001] This invention relates to the construction and optimization of a transcriptional repression tool based on MUSCULAR-CAST and its application in Escherichia coli, belonging to the fields of synthetic biology and genetic engineering. Background Technology

[0002] Innovation in gene editing technology is a core driving force for the development of synthetic biology. CRISPR interference (CRISPRi), a gene transcription regulation tool based on the CRISPR-Cas system, achieves efficient and specific gene expression inhibition by designing guide RNAs (crRNA / sgRNA) to target specific DNA sequences. It is widely used in microbial gene function analysis, metabolic pathway optimization, and the construction of synthetic biology infrastructure. However, existing CRISPRi systems still have significant limitations: 1. Dependence on the classic Cas9 protein: CRISPRi systems based on dCas9 are strictly limited by the requirement that the prototype adjacent motif (PAM) be NGG, and the inhibition efficiency on the template strand is generally less than 30%. Figure 1 e) This greatly limits the flexibility of target site selection. 2. Insufficient efficiency of multiple gene repression: Traditional methods require multiple deliveries or complex vector assembly, which easily leads to decreased repression efficiency and increased cellular metabolic burden. 3. Poor adaptability of regulatory elements: Constitutive promoter-driven CRISPRi components often induce cytotoxicity, while inducible systems (such as tetracycline promoters) can alleviate growth inhibition, but have problems such as basal leakage expression and delayed induction kinetics. Therefore, it is necessary to develop new gene editing tools with high integration efficiency. Summary of the Invention

[0003] This invention provides a gene transcription repression system Tn-CRISPRi based on CRISPR-associated transposases (CAST), comprising: a CRISPR-Cas targeting module Cascade (Cas8, Cas7, Cas6), a transposase TniQ, and a crRNA expression cassette; The crRNA expression cassette contains the 32 bp base sequence required for the target sequence (referred to as N32). The CRISPR-Cas targeting module Cascade is constructed on plasmid pQ876 and is regulated by a constitutive weak promoter, J23109, as shown in SEQ ID NO.2, which regulates the TniQ-Cascade complex (TniQ / Cas8 / Cas7 / Cas6).

[0004] In an embodiment, the target position of the crRNA specifically recognizing the NCC / NCA type PAM can be selected as required, wherein the highest inhibition intensity is targeted at -10 to +15 bp relative to the transcription start site, different positions of the target gene nucleotide sequence can obtain different inhibition intensity, and the template strand is slightly better than the non-template strand.

[0005] In an embodiment, the promoter contains a nonsense spacer sequence between the crRNA expression cassette; the nonsense spacer sequence is shown in SEQ ID NO. 12.

[0006] The application also provides a construction method of the Tn-CRISPRi system, comprising the following steps: (1) cloning the TniQ-Cascade gene cluster (SEQ ID NO. 13) from Vibrio cholerae Tn6677 to the plasmid pSC101, replacing the original promoter with the J23109 promoter (SEQ ID NO. 2) to obtain the basic plasmid p119AQC; (2) inserting a nonsense spacer sequence (SEQ ID NO. 12) between the promoter J23119 of the crRNA expression cassette and the target sequence; (3) designing a crRNA array for the target gene, meeting: the PAM type is NCC or NCA (efficiency > 96%), and the template strand is targeted at a position about 10% of the total length of the gene sequence from the 5' end of the gene sequence (efficiency > 90%) (4) integrating the crRNA array of step (3) into the plasmid of step (2) to form the final plasmid p119A109QC-crRNA~target.

[0007] In an embodiment, the nucleotide sequence of the J23109 promoter is shown in SEQ ID NO. 2.

[0008] In an embodiment, the replicon in the plasmid p119A109QC is replaced with a temperature-sensitive replicon shown in SEQ ID NO. 5.

[0009] The application also provides a method for single gene or multi-gene inhibition of a strain using the gene inhibition system, which comprises constructing the target sequence of the gene site to be inhibited to the crRNA region of the plasmid p119A109QC on the basis of the gene inhibition system.

[0010] In an embodiment, for multi-gene inhibition, the crRNAs of the genes to be inhibited are connected in series.

[0011] In an embodiment, the method comprises the following steps: (1) Construct the required gene crRNAx array for inhibition, and construct into the plasmid p119A109QC in the gene editing system; (2) Sequence the constructed plasmid p119A109QC-crRNAx to confirm that the sequence is correct; (3) Prepare the competent cells of the fermentation strain to be transformed, and transform the successfully constructed plasmid p119A109QC-crRNAx into the competent cells; (4) Perform shake flask fermentation on the positive clone strain successfully constructed in step (3), and screen the inhibition sites of high-yield strains.

[0012] In an embodiment, the E. coli includes E. coli BL21, E. coli K-12 MG1655, and E. coli K-12 W3110.

[0013] The application also provides a recombinant E. coli constructed using the Tn-CRISPRi system.

[0014] In an embodiment, the recombinant E. coli inhibits the expression of genes in a 3-fucosyllactose synthesis competitive pathway or genes that are not essential for cell growth; the genes are selected from one or more of the following: lacA, fucI, mdoH, clpYQ, fucK, lon, iclR, poxB, ybeQ, ydeU, motA, caiB, intQ, yjiV, yihS, sgcC adhE , lacA, fucI, mdoH, clpYQ, fucK, lon, iclR, poxB .

[0015] The application also provides the use of the method or the recombinant E. coli in the production of 3-fucosyllactose.

[0016] In an embodiment, the TniQ-Cascade is regulated by a J23109 promoter (SEQ ID NO. 2), and the crRNA is regulated by a J23119 promoter (SEQ ID NO. 1).

[0017] In an embodiment, the E. coli includes but is not limited to E. coli BL21 (DE3), E. coli K-12 MG1655, and E. coli W3110.

[0018] The application also provides the use of the Tn-CRISPRi system in the field of metabolic engineering.

[0019] In an embodiment, the use includes inhibiting E. coli competitive pathway genes to improve the yield of 3-fucosyllactose (3-FL).

[0020] In an embodiment, the competitive pathway genes include ybeQ, ydeU, motA, caiB, intQ, yjiV, yihS, sgcC adhE The genes are competitive pathway genes for 3-fucosyllactose synthesis, and the genes are Figure 2 Figure 1 ,Figure 1 Genes that are not essential for cell growth.

[0021] Beneficial effects: The Tn-CRISPRi system constructed by the application solves the core problems of the prior art, such as strict PAM restriction, low multiplexing inhibition efficiency and high cell toxicity, and has the following improvements compared with the prior art: (1) The Tn-CRISPRi transcription inhibition system is constructed, and after promoter engineering optimization (TniQ-Cascade is driven by promoter J23109), the growth rate of the strain is increased by 25% (compared with the initial tetracycline induction system, Figure 1 ), and the metabolic burden is significantly reduced. And the inhibition rate is maintained at 92.5%.

[0022] (2) Mechanism innovation and universality: using TniQ-Cascade steric hindrance to block transcription initiation ( Figure 1 a), avoiding the off-target cutting risk of dCas9. Spacing sequence optimization: adding nonsense spacer region makes the inhibition rate jump from 45.02% to 83.58% ( Figure 1 d), breaking through the bottleneck of RNA polymerase binding efficiency Low PAM dependence broadens target selection: the inhibition rate of non-canonical PAM (such as NCC / NCA) is >96% ( Figure 2 f), compared with the dCas9 system (dependent on NGG), the target range is expanded by 5.3 times (based on the number of PAM combinations). Template strand efficient inhibition: crRNA GFP2 targeting the template strand has an inhibition rate of 87.19% ( Figure 2 h), solving the defect that the dCas9 system has less than 30% inhibition of the template strand ( motA e).

[0023] (3) Rapid construction and precise regulation: system optimization only needs 2 rounds of promoter replacement, and the strain construction cycle is shortened by 50% (compared with the traditional CRISPRi iterative process). The transcription level of crRNA is positively correlated with the inhibition rate, supporting the quantitative fine-tuning of metabolic flux.

[0024] (4) Double gene synchronous near complete inhibition: synchronous inhibition of GFP / mCherry double fluorescent proteins is realized, and the inhibition rates are 98.6% and 99.8% respectively ( mdhA b), which is one of the most efficient multi-target regulation records in the field of synthetic biology at present. Key mechanism: crRNA mCherry1 targets the shared promoter region ( Figure 1 a), breaking through the competitive inhibition bottleneck of traditional CRISPRi array.

[0025] (5) The Tn-CRISPRi system constructed by the application is successfully used for 3-fucosyllactose (3-FL) high-yield strain construction. Inhibition Figure 2 With Figure 3 the gene makes the 3-FL yield increase by 4.4 times and 2.78 times respectively, and the pH stability of the strain is enhanced (the acetic acid yield is reduced). BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 4 is the construction and test of the Tn-CRISPRi system. (a) Schematic diagram of the CRISPRi system based on CAST. (b) Schematic diagram of the CRISPRi system based on dCas9. (c) Relative fluorescence and OD 600 values when using plasmid p119A100QC, and relative fluorescence and OD 00 values when using plasmid pTA100QC and p119ATQC under gradient concentrations of the inducer. (d) Relative fluorescence and OD 600 values when using plasmid p119A100QC and p119A100QC2, and relative fluorescence and OD 600 values when using plasmid p119ATQC and p119ATQC2 under gradient concentrations of the inducer. (e) Relative fluorescence of the CRISPRi system based on dCas9 under gradient concentrations of the inducer. (f) Schematic diagram of the Tn-CRISPRi system based on Tn6677 and Tn7016, using different crRNAs for inhibition. (g) (h) Unit OD 600 fluorescence intensity and OD 600 values of the Tn-CRISPRi system based on Tn6677 and Tn7016 under gradient concentrations of the inducer. In the column chart, all comparison data are from the same batch, and the data are represented by mean value ± standard deviation (n = 3 biological replicates).

[0027] iclR::gfp is the modular transcription level optimization and identification of PAM preference bases in the Tn-CRISPRi system; wherein (a) schematic diagram of batch replacement of different promoters, TniQ-Cascade and crRNA; (b)~(c) are unit OD600 fluorescence intensity and OD 600 values of TniQ-Cascade transcribed by using arabinose promoter and T7 promoter respectively. (d)~(e) are relative fluorescence intensity and OD 600 values of TniQ-Cascade and crRNA transcribed by using different intensity constitutive promoters respectively.

[0028] iclR is the result of the three-factor four-level orthogonal experiment of PAM.

[0029] Figure 1 Optimization of Tn-CRISPRi system; wherein (a) is a schematic diagram of optimizing Tn-CRISPRi system to simultaneously suppress two fluorescent proteins in the genome. (b) is the relative fluorescence intensity of two fluorescent proteins simultaneously inhibited by crRNA GFP2 and different crRNA mCherry1, which can simultaneously target the same promoter sequence of two fluorescent proteins; (c) is TS1 ~ TS17: the influence of single competitive pathway gene on strain 3-FL production, OD600 and pH value. TS18 ~ TS19: the influence of simultaneous inhibition of two sites (single gene inhibition significantly improves production, two tandem configurations are used) on strain 3-FL production, OD600 and pH value. CherryX 600 DETAILED DESCRIPTION

[0030] (I) Reagents and culture medium All antibiotics were prepared with sterile ddH2O, and then sterilized with a filter after preparation.

[0031] Ampicillin (100 μg / mL), kanamycin (30 μg / mL), spectinomycin (50 μg / mL) All culture media were prepared with ddH2O, and then sterilized at 121℃ for 20 min after preparation.

[0032] LB liquid medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L.

[0033] Fermentation medium (FB): 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, pork bone peptone 2.5 g / L, glycerol 30 g / L, trace metal solution 10 mL.

[0034] Trace metal solution (g / L): 5 mol / L HCl as mother liquor, CaCl2 2.0 g / L, (NH4)6Mo7O 24 0.1 g / L, ZnSO4·7H2O 2.25 g / L, MnSO4·4H2O 0.5 g / L, FeSO4·7H2O 10.0 g / L, CuSO4·5H2O 3.0 g / L, Na2B4O7·10H2O 0.23 g / L, CoCl2·6H2O 0.18 g / L.

[0035] ​​(ii) The method for detecting 3-FL in the embodiment of the present application is described as follows: (1) Sampling: 1) First, 400 μL was taken and placed in a 5 mL EP tube to measure OD600.

[0036] 2) 800 μL was taken and placed in a 1.5 EP tube, and a metal bath was used at 100°C for 15 min, followed by centrifugation at 12000 rpm for 10 min, and then the supernatant was taken, filtered, and placed in a liquid chromatography vial.

[0037] (2) Liquid chromatography detection: A differential detector was used; the column oven temperature was 50°C; the differential temperature was 45°C; and the flow rate was 0.5 min / mL. The elution time was 25 min; the liquid chromatography column was an Aminex HPX-87H chromatography column; and the mobile phase was 5 mM sulfuric acid. Specific operation: 1 mL of the fermentation broth was centrifuged, the supernatant was taken, and the mixture was placed in a metal bath at 100°C for 15 min, centrifuged to precipitate impurities such as proteins, the supernatant was taken, filtered through a 0.22 μm water phase membrane, inserted into a syringe, placed in a liquid chromatography vial, and then placed in an Agilent liquid chromatography detector for detection.

[0038] (3) Preparation of electroshock competent cells: 1) The bacteria were inoculated from a glycerol tube into a 10 mL LB medium shake flask at an inoculation amount of 0.2%, and cultured at 37°C and 200 rpm for 10 h.

[0039] 2) The bacteria were inoculated from the shake flask into a 50 mL LB medium shake flask (or a 50 mL 2YT medium shake flask) at an inoculation amount of 1%, and cultured at 37°C and 200 rpm for 2-3 h until the OD value reached 0.5-0.8, and then the shake flask was placed in an ice bath for 30 min.

[0040] 3) The bacterial solution was centrifuged at 4000 rpm and 4°C for 10 min, and the bacterial cells were slowly resuspended with sterile water.

[0041] 4) Step 3 was repeated.

[0042] 5) The bacterial solution was centrifuged at 4000 rpm and 4°C for 10 min, and the supernatant was removed, and the bacterial cells were slowly resuspended with 10% glycerol on ice.

[0043] 6) Step 5 was repeated.

[0044] 7) The bacterial solution was centrifuged at 4000 rpm and 4°C for 10 min, and the supernatant was removed, and the bacterial cells were slowly resuspended with 0.6-1 mL of 10% glycerol on ice (depending on the amount of bacterial cells), and 100 μL was aliquoted into a clean 1.5 mL EP tube, and stored at -80°C for standby use.

[0045] (4) Transformation: 1) -80℃ refrigerator or just prepared competent cells are placed on ice, and the standby plasmid, 1 mm electric shock cup is placed on ice for 10 min to precool.

[0046] 2) 10 uL of plasmid is aspirated and added to the competent cells, mixed gently and then ice-bathed for 15-20 min.

[0047] 3) Transfer to the electric shock cup (the bottom needs to be anhydrous), shock once at a voltage of 2500 V, quickly add 1 mL of LB, and transfer the bacteria to a clean sterilized EP tube, seal with a sealing film, and recover and culture in a 37℃, 200 rpm, shaking bed for 1.5 h.

[0048] 4) Centrifuge at 3000 rpm for 5 min, open in the clean bench, discard the supernatant, resuspend the bacterial liquid, and then spread on the corresponding resistance plate.

[0049] 5) Incubate in a 30℃ incubator for 10-12 h.

[0050] (Three) Sequences involved in the specific embodiments Table 1 Nucleotide sequences of crRNA involved in the specific embodiments

[0051] Table 2 Nucleotide sequences of crRNA involved in the specific embodiments

[0052] Table 3 Nucleotide sequences of sgRNA involved in the specific embodiments

[0053] Example 1: Construction and identification of E. coli Tn-CRISPRi system The targeting module of MUSCULAR-CAST is used to regulate gene transcription. First, the TniQ-Cascade (containing TniQ, Cas6, Cas7, Cas8) in the plasmid pQ876 (disclosed in the patent application with publication number CN119842771A) of the MUSCULAR-CAST system is expressed in series with the crRNA to construct a CRISPRi system, named Tn-CRISPRi. The test strain is the BLG (BL21(DE3) Figure 1 ) strain derived from E. coli BL21(DE3), which is a strain in which the Figure 1The site (Genbank: NZ_CP053602.1) is inserted with a green fluorescent protein (GFP) expression cassette (the nucleotide sequence is shown in SEQ ID NO. 14). The promoter J23119 (the nucleotide sequence is shown in SEQ ID NO. 1) transcribes crRNAx (the nucleotide sequence of crRNA is shown in Table 1, x represents the crRNA lacA crRNA AdhE Different genes represented by the lower right corner are integrated into the pQ876 plasmid to form p119A100QC-crRNA GFP0 Plasmid. Similarly, the crRNA transcribed by the tetracycline-induced Ptet promoter (the nucleotide sequence is shown in SEQ ID NO. 4) GFP0 is also integrated into the pQ876 plasmid to produce the plasmid pTA100QC-crRNA GFP0 .

[0054] The plasmid pTA100QC-crRNA GFP0 is transformed into the strain BLG, cultured in LB medium at 37°C for 12-16h, and the fluorescence and OD are detected using a fluorescence enzyme label instrument. The plasmid pQ876 without a targeting sequence is transformed into the strain BLG to construct BLG-NC as a negative control. The inhibition rate is calculated according to the relative fluorescence (fluorescence / OD) of the negative control. The inhibition rate of the plasmid p119A100QC-crRNA GFP0 is only 45.02%, and the inhibition rate of the plasmid pTA100QC-crRNA GFP0 is 71.45%. Although the inhibition rate of the plasmid pTA100QC-crRNA GFP0 is higher, compared with the control strain BLG-NC and the plasmid p119A100QC-crRNA GFP0 , the growth of the strain is poor Figure 2 a、1c).

[0055] Further, the plasmid p119ATQC-crRNA GFP0 is constructed on the basis of the plasmid p119A100QC-crRNA GFP0 , and the J23100 promoter (SEQ ID NO. 6) of TniQ-Cascade is replaced with the tetracycline-induced promoter Ptet. The constructed plasmid p119ATQC-crRNA GFP0The plasmid was transformed into strain BLG, which was cultured in LB medium at 37°C for 12 h, and the inducer was added at the time of inoculation (shown in Fig. 1c). The fluorescence value was detected using a fluorescence microplate reader. The plasmid without targeting sequence was used as a negative control, and the inhibition rate was calculated according to the proportion of the negative control. The experimental results showed that the inhibition rate of plasmid p119ATQC-crRNA GFP0 was equivalent to that of pTA100QC-crRNA GFP0 , and the growth condition was improved.

[0056] To further improve the inhibition rate, the plasmid backbone structure was analyzed, and it was found that there was a lack of a spacer sequence between the promoter J23119 and the crRNA GFP0 , which might affect the binding of the transcription enzyme in the spatial structure. Therefore, a nonsense sequence (the sequence is shown in SEQ ID NO. 12) was added between the promoter J23119 and the crRNA GFP0 in the backbone of plasmids p119ATQC-crRNA GFP0 and p119A100QC-crRNA GFP0 , respectively, to construct plasmids p119ATQC2-crRNA GFP0 and p119A100QC2-crRNA GFP0 , in order to test whether the inhibition effect would be enhanced. The results showed that the inhibition rate was significantly improved after adding the nonsense sequence, among which the inhibition rate using p119ATQC2-crRNA GFP0 was 83.58% (Fig. 1d), so plasmid p119ATQC2-crRNA GFP0 was used for further research subsequently.

[0057] Therefore, based on plasmid p119ATQC2-crRNA GFP0 , different crRNAs GFPX（X代表1~5） were selected from 5' to 3' to about every 150 bp interval (see Table 1 for crRNA GFP1~ crRNA GFP5 ) to replace crRNA GFP0 in order to determine their effects on the inhibition rate. Five crRNAs were selected for further analysis (Fig. 1f). Among them, crRNA GFP1 targeted the non-template strand near the middle of the gene, and the PAM was a typical NCC; crRNA GFP2 targeted the template strand near the middle of the gene, and the PAM was a typical NCC. crRNA GFP3 moved 3 bp to the 5' end based on crRNA GFP2 , and the PAM was a non-typical NAT; crRNAGFP4 Targeting the non-template strand near the gene terminus, PAM is a typical NCC; crRNA GFP5 In crRNA GFP4 The plasmid was shifted 2 bp to the 5' end, and PAM was atypical NGT. The constructed plasmid was p119ATQC2-crRNA. GFP1 p119ATQC2-crRNA GFP2 p119ATQC2-crRNA GFP3 p119ATQC2-crRNA GFP4 and p119ATQC2-crRNA GFP5 Incubate at 37°C for 12 h, adding an inducer at inoculation ( Figure 2 (as shown in f-1h). The results showed that at a tetracycline concentration of 200 ng / mL, all five crRNAs exhibited the highest inhibition rates, with plasmid p119ATQC2-crRNA showing the highest inhibition rate. GFP1 The highest inhibition rate was 70.29%; plasmid p119ATQC2-crRNA GFP5 The highest inhibition rate was 87.19% for plasmid p119ATQC2-crRNA. GFP5 The highest inhibition rate was 69.68% for plasmid p119ATQC2-crRNA. GFP3 and plasmid p119ATQC2-crRNA GFP4 The inhibition rates were 47.87% and 48.79%, respectively.

[0058] Besides the transposase of Tn6677, Tn7016 is another CAST system characterized in recent years as having strong integration capabilities, and it has the potential to be developed into a highly efficient repression tool. The construction of the Tn7016-based Tn-CRISPRi system involves using a Tn7016-derived related enzyme (sequence shown in SEQ ID NO.13) and transcribing crRNA with a tetracycline promoter to create the system. The related plasmid was named pTA100QC-7016. The plasmid pTA100QC-7016-crRNA was constructed. GFP1 pTA100QC-7016-crRNA GFP2 pTA100QC-7016-crRNA GFP3 and pTA100QC-7016-crRNA GFP5 The above crRNA was tested GFP1 crRNA GFP2 crRNA GFP3 and crRNA GFP5inhibition rate. After 12 h incubation at 37℃, the inducer was added at the time of inoculation (shown in Fig. 1f-1h). The results showed that the inhibition rate of pTA100QC-7016-crRNA GFP1 was the highest, reaching 67.46%; the inhibition rate of pTA100QC-7016-crRNA GFP2 was the highest, reaching 51.95%; the inhibition rate of pTA100QC-7016-crRNA GFP3 and pTA100QC-7016-crRNA GFP5 was the highest, reaching 33% and 31%, respectively. The inhibition rates of the inhibition systems based on Tn7016 were lower than those based on Tn6677, so we subsequently optimized the Tn-CRISPRi system based on Tn6677.

[0059] To make a full comparison with the typical CRISPRi system based on dCas9, four sgRNAs (sgRNA1-sgRNA4, nucleotide sequences shown in Table 3) were designed to test the inhibition rate using dCas9. sgRNA1 targets the non-template strand at the 5' end of the green fluorescent protein GFP gene, sgRNA2 targets the non-template strand in the middle of the gene, sgRNA3 targets the non-template strand at the 3' end of the gene, and sgRNA4 targets the template strand at the 5' end of the gene. It is worth noting that all four sgRNAs use the typical NGG as the PAM. The inhibition rate of dCas9 targeting the non-template strand is as high as 96.6%, and there is little difference between different positions of the non-template strand, while the inhibition rate of targeting the template strand is lower, less than 30% (Fig. 1a-1d). Figure 2 b、1e)。

[0060] The above results show that, compared with the typical dCas9, the Tn-CRISPRi system based on Tn6677 initially exhibits lower PAM dependence and more target strand selection.

[0061] Example 2 Optimization of the Tn-CRISPRi system To further improve the inhibition efficiency and improve the growth condition of the strain, the transcription level of TniQ-Cascade and crRNA was optimized based on the plasmid p119ATQC2-crRNA GFP2 constructed in Example 1. The specific steps are as follows: the tetracycline promoter of TniQ-Cascade was replaced with the arabinose promoter P BAD, T7 promoter, J23101 (nucleotide sequence as shown in SEQ ID NO. 7), J23105 (nucleotide sequence as shown in SEQ ID NO. 8), J23106 (nucleotide sequence as shown in SEQ ID NO. 9) and J23109 (nucleotide sequence as shown in SEQ ID NO. 2), respectively, to obtain plasmids p119ABADQC, p119AT7QC, p119A101QC, p119A105QC, p119A106QC and p119A109QC (Figure 2a). Incubate at 37°C for 12 h. Respectively use tetracycline with a concentration of 100-300 ng / mL, 5-15 mM arabinose, 0.05-0.15 mM IPTG for induction, and add the corresponding inducers at the time of inoculation. The results show that when using the arabinose promoter, the significant increase in OD600 may be due to the metabolic utilization of part of the arabinose by the strain, and the maximum inhibition rate is 75.89%; while using the T7 promoter, the growth of the strain is most affected, and the maximum inhibition rate is 70.86% Figure 2 b- Figure 4 c). Using plasmid p119AQC results in an inhibition rate of 87.16% (Figure 2d), while using the constitutive promoter, the reduction of TniQ-Cascade transcription level has little effect on the inhibition rate, using the constitutive promoters J23101, J23105, J23106 and J23109 results in an inhibition rate of more than 90%, and has more favorable growth (Figures 2d-2e), among them, the plasmid p119A109QC containing the promoter J23109 has the highest inhibition rate, reaching 92.50%.

[0062] Further, on the basis of plasmid p119A109QC using the weaker promoter J23109 for Tni-QCascade, further replace the promoter J23119 of crRNA with J23111 (SEQ ID NO. 10), J23105 (SEQ ID NO. 8) and J23117 (SEQ ID NO. 11) to obtain plasmids p111A109QC-crRNA GFP2 , p105A109QC-crRNA GFP2 and p117A109QC-crRNA GFP2 (Figure 2a). With the decrease of promoter strength, the inhibition rate shows a trend from 90% to 0% (Figures 2d-2e), which indicates that the transcription level of crRNA is positively correlated with the inhibition rate of Tn-CRISPRi system.

[0063] To confirm whether using multiple crRNAs targeting the same gene would enhance the inhibition, we added crRNAs targeting GFP in plasmid p119A109QC GFP1 The transcription cassette (containing tandem crRNAGFP1 and crRNAGFP2), plasmid p119A109QC-2crRNAs was constructed Figure 4 Contrary to expectation, the inhibition rate using plasmid p119A109QC-2crRNAs was 87.16% (d-2e), slightly lower than when only a single crRNAGFP2 was used, which could be related to the competition effect between the two crRNAs. lacA, fucI, mdoH, clpYQ, fucK, lon, iclR, poxB, ybeQ, ydeU, motA, caiB, intQ, yjiV, yihS, sgcC

[0064] On the basis of the preliminary verification of PAM and strand preference, we designed a three-factor four-level orthogonal experiment, four bases (A, T, C, G) were designed at the-1 and-2 positions of PAM species and four target positions on GFP to systematically characterize the dependence of Tn-CRISPRi on PAM. The results showed that the inhibition rate of most PAMs was 50-70%, and the inhibition rate was higher when the PAM was NCC and NCA, which was 96.76% and 96.65% respectively (Figure 3). This result is consistent with previous studies that when the PAM is NCA or NGT, the integration efficiency of the Tn6677 CAST system is higher.

[0065] Example 3: Inhibition of Tn-CRISPRi system on double genes On the basis of the crRNA targeting GFP constructed in Example 1 GFP2 , by adding crRNA targeting red fluorescent protein mCherry mCherry (shown in Table 1 of nucleotide sequences), to achieve the effect of inhibiting double genes. From crRNA mCherry1 to crRNA mCherry6 , a total of six crRNAs targeting mCherry (crRNA mCherry1 ~crRNA mCherry6 ) were selected, each of which was sequentially targeted to the 5' end to the 3' end of mCherry. All PAMs were NCC, and the template strand was selected for targeting. On the basis of plasmid p119A109QC-crRNA GFP2 , plasmids p119A109QC-crRNA GFP2 -crRNA mCherry1 to p119A109QC-crRNA GFP2 -crRNA mCherry6 ​To suppress two genes (Figure 4a), mCherry was inserted into the wcaC locus of strain BLG to construct strain BLGR. Strain BLGR was cultured at 37℃ for 12 h, and the efficiency of simultaneously suppressing GFP and mCherry was tested. The results showed that crRNA… mCherry1 The inhibition rate against mCherry reached 99.8%, and crRNA... mCherry2 94.8%, crRNA mCherry4 It is 97.8% ( adhE b). Observations showed that when the inhibition rate of mCherry reached 99.8%, the inhibition rate of GFP also reached 98.6%. It is noteworthy that GPF and mCherry share the same promoter sequence. crRNA mCherry1 It can target two fluorescent proteins downstream of their respective promoters ( ldhA:: (a) This characteristic may be key to achieving near 100% inhibition of two fluorescent proteins simultaneously. Furthermore, multiple crRNA arrays are easier to assemble when performing simultaneous multi-gene inhibition.

[0066] Example 4: Application of the Tn-CRISPRi system Based on the Tn-CRISPRi system constructed and optimized in the aforementioned embodiments, its application effect in constructing synthetic biology matrix strains was further tested. 3-FL was used as the test subject, and 17 fucoidan-synthetic strains with gene knockout or silence were selected, such as... ΔlacZΔwcaJΔnudD mdoH and motA The genes were then inhibited individually to examine their effects on 3-FL production. The plasmid p119A109QC-crRNA was replaced with the sequences shown in Table 2. GFP2 crRNA GFP2 A plasmid p119A109QC-crRNA that suppresses 17 genes was constructed. LacA ~ p119A109QC-crRNA AdhE The chassis strain MG3 (Escherichia coli K-12 MG1655) was constructed earlier. Figure 4 DE3 ​ The negative control strain NC1 (disclosed in patent application CN119842771A) was used to transform plasmid p119A109QC-crRNA in strain MG3. GFP2 The negative control strain NC2 was used. Based on strain NC1, it was transformed with plasmid p119A109QC-crRNA. LacA ~p119A109QC-crRNA AdhEConstruction of test strains TS1~TS17. Single colonies were inoculated into LB liquid medium with a final concentration of 100 μg / mL Amp, and cultured at 37°C, 200 r / min for 8-9 h. Then, 2% of the culture was transferred into 50 ml of fermentation medium, and cultured at 37°C, 200 rpm until the OD reached 1.5-2.5. Then, 0.1 mM IPTG was added, and the culture was incubated at 25°C. Two hours after the addition of IPTG, 10 g / L of lactose was added, and the fermentation was continued for 72 h. The effect of the lactose on the production of 3-FL was detected. The results showed that, compared with the NC2 strain, the production of 3-FL of the TS3 strain inhibiting the expression of the gmd gene and the TS11 strain inhibiting the expression of the wcaG gene increased by 2.79 times and 4.40 times, respectively. ​ ​ ​ Compared with the strain NC2, the strains TS3 and TS11 did not produce acetic acid, and the pH value at the end of fermentation was higher, which was more conducive to the production of neutral fucose. In addition, the growth of the strain TS11 was better. Compared with the strain NC1, the decrease in the production of the strain NC2 might be due to the increase in the metabolic burden of the plasmid p119A109QC-crRNA GFP2

[0067] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application. Therefore, the protection scope of the present application should be defined by the claims.​​​

Claims

1. A gene transcription repression system Tn-CRISPRi based on CRISPR-related transposases, characterized in that, include: The CRISPR-Cas targeting module Cascade, the transposase TniQ-Cascade complex, and the crRNA expression cassette; the crRNA expression cassette contains the base sequence required for the targeting sequence. The CRISPR-Cas targeting module Cascade is constructed on plasmid pQ876 and is regulated by a constitutive weak promoter, J23109, as shown in SEQ ID NO.2, which regulates the TniQ-Cascade complex.

2. The method for constructing the gene transcription repression system Tn-CRISPRi, characterized in that, Includes the following steps: (1) The TniQ-Cascade gene cluster from Vibrio cholerae Tn6677 was cloned into plasmid pSC101, and the original promoter was replaced with J23109 promoter to obtain the basic plasmid p119AQC. (2) Insert the nonsense spacer sequence shown in SEQ ID NO.12 between the promoter J23119 of the crRNA expression cassette and the target sequence; (3) Design crRNA arrays for the target gene, satisfying the following conditions: PAM type is NCC or NCA, targeting the template strand and located at 300 bp from the 5' end of the target gene sequence; (4) Integrate the crRNA array from step (3) into the plasmid from step (2) to form the final plasmid p119A109QC-crRNA-target.

3. A method for suppressing single or multiple genes in a strain using the Tn-CRISPRi gene transcription repression system of claim 1, characterized in that, Includes the following steps: (1) Construct the desired gene crRNAx array for repression and insert it into the plasmid p119A109QC in the gene editing system; (2) The constructed plasmid p119A109QC-crRNAx was sequenced to confirm that the sequence was correct; (3) Prepare competent cells of the fermentation strain to be transformed, and transform the successfully constructed plasmid p119A109QC-crRNAx into competent cells; (4) The positive clone strain successfully constructed in step (3) is subjected to shake-flask fermentation.

4. The method according to claim 3, characterized in that, The strain is Escherichia coli, including Escherichia coli BL21, Escherichia coli K-12 MG1655 or Escherichia coli K-12 W3110.

5. Recombinant Escherichia coli constructed using the gene transcription repression system Tn-CRISPRi described in claim 1.

6. Recombinant Escherichia coli, characterized in that, The gene transcription repression system Tn-CRISPRi described in claim 1 represses one or more of the following genes: lacA, fucI, mdoH, clpYQ, fucK, lon, iclR, poxB, ybeQ, ydeU, motA, caiB, intQ, yjiV, yihS, sgcC , adhE .

7. The recombinant Escherichia coli according to claim 6, characterized in that, The Escherichia coli includes Escherichia coli BL21, Escherichia coli K-12 MG1655, or Escherichia coli K-12 W3110.

8. The use of the recombinant Escherichia coli according to claim 7 in the production of 3-fucosylated lactose.

9. The application of the gene transcription repression system Tn-CRISPRi as described in claim 1 in the field of metabolic engineering.

10. The application according to claim 9, characterized in that, The applications include, but are not limited to, constructing recombinant microorganisms with gene repression.

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