Gene editing-transcriptional regulation system suitable for pseudomonas
By designing the CRISPR-AIE system, the precise and general regulation of Pseudomonas genes can be achieved by replacing sgRNA, which solves the problems of single function and off-target effects in existing technologies and realizes efficient gene editing and transcriptional regulation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANDONG UNIV
- Filing Date
- 2024-08-20
- Publication Date
- 2026-06-09
Smart Images
Figure CN118813663B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a gene editing-transcriptional regulation system suitable for Pseudomonas, belonging to the field of genetic engineering technology. Background Technology
[0002] The CRISPR / Cas system is an acquired immune system in prokaryotes, used to resist the invasion of foreign genetic elements present in bacteriophages or plasmids. It is a defense mechanism present in most bacteria and all archaea, used to destroy foreign plasmid or bacteriophage DNA. It is now widely used in genetic engineering. However, current CRISPR transcriptional regulatory systems often only achieve a single function, targeting only a specific single promoter, and cannot achieve high broad-spectrum regulation, let alone fine-tuning.
[0003] Pseudomonas putida is a saprophytic Gram-negative pseudomonad with multiple functions, including degrading harmful aromatic and aliphatic compounds in the environment, promoting chemical element cycling, biocatalysis, bio-pollution, and synthesizing bioplastics. It is a model strain for gene cloning and protein expression, and its genome was sequenced and analyzed in 2002. Research into gene regulation or editing systems suitable for Pseudomonas is of great significance. Summary of the Invention
[0004] In view of the above-mentioned prior art, the present invention provides a gene editing-transcriptional regulation system suitable for Pseudomonas, which can achieve fine and general regulation of gene transcription.
[0005] This invention is achieved through the following technical solution:
[0006] A gene editing-transcriptional regulation system suitable for Pseudomonas, named the CRISPR-AIE system, includes a regulatory vector containing the following elements: DNA corresponding to sgRNA, the gene encoding Cas9 protein, the gene encoding N4, the gene encoding sc, the gene encoding an activator, the Ptrc promoter, the xylR-pxylA promoter, and the ph5 promoter.
[0007] Furthermore, the sgRNA is designed based on the gene to be regulated or the gene to be edited.
[0008] Furthermore, the Cas9 protein is selected from Cas9n, dcas9, Xdcas9 and other Cas9 protein mutants. The nucleotide sequence of the encoding gene of Cas9n is shown in SEQ ID NO.1, the nucleotide sequence of the encoding gene of dcas9 is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene of Xdcas9 is shown in SEQ ID NO.3.
[0009] Furthermore, the nucleotide sequence of the gene encoding N4 is shown in SEQ ID NO.4; N4 is a tag that can be fused with the cas9 protein for expression and can specifically bind to sc.
[0010] Furthermore, the nucleotide sequence of the gene encoding sc is shown in SEQ ID NO.5; sc is a linker fused with the activator, which can specifically bind to N4.
[0011] Furthermore, the activating factor is selected from preferred ω-activating factors, and the nucleotide sequence of the gene encoding the ω-activating factor is shown in SEQ ID NO. 6. The ω-activating factor is the ω subunit of wild-type *Pseudomonas putida* KT2440 itself. ω is highly conserved and, as a component of the RNA polymerase structure, binds to RNA polymerase after the initiation complex is formed, thereby promoting subsequent transcription and regulating the transcription efficiency and stability of the gene.
[0012] Furthermore, the nucleotide sequence of the Ptrc promoter is shown in SEQ ID NO.7.
[0013] Furthermore, the nucleotide sequence of the xylR-pxylA promoter is shown in SEQ ID NO.8.
[0014] Furthermore, the nucleotide sequence of the ph5 promoter is shown in SEQ ID NO.9.
[0015] Furthermore, the regulatory vector is selected from the pPROBE-GT vector.
[0016] The method for preparing the gene editing-transcriptional regulation system suitable for Pseudomonas is as follows: First, sgRNA is designed for the gene to be regulated or the gene to be edited, and the DNA corresponding to the sgRNA is synthesized. The DNA is then ligated to the regulatory vector by enzyme digestion and ligation. Then, the coding genes of Cas9 protein, N4, sc, activator, Ptrc promoter, xylR-pxylA promoter, and ph5 promoter are also ligated to the regulatory vector, thus constructing the gene editing-transcriptional regulation system suitable for Pseudomonas.
[0017] The application of the gene editing-transcriptional regulation system applicable to Pseudomonas in gene regulation or gene editing of Pseudomonas; the gene editing includes gene knockout, gene insertion and / or gene replacement.
[0018] Furthermore, the target gene for gene regulation is the ph2 promoter, and the nucleotide sequence of the DNA corresponding to the sgRNA designed for the ph2 promoter is shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, or SEQ ID NO.14.
[0019] Furthermore, the target gene for gene regulation is the ph3 promoter, and the nucleotide sequence of the DNA corresponding to the sgRNA designed for the ph3 promoter is shown in SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, or SEQ ID NO.19.
[0020] Furthermore, the target gene for the gene editing is pyrF.
[0021] Furthermore, the target gene for gene editing is the promoter corresponding to the natural genes PP_0181, PP_0679, PP_2322, PP_4793, PP_5540, PP_1840, PP_2268, PP_3611, PP_4876, PP_5181, PP_1206, PP_3950, PP_4055, PP_4217, or PP_4488 of Pseudomonas KT2440.
[0022] The CRISPR-AIE system of this invention, when applied, expresses relevant elements—sgRNA, Cas9 protein-N4, and sc-activator—in Pseudomonas bacteria to be regulated or edited in plasmid form. The sgRNA (designed according to the gene to be regulated or edited) can bind to the target site of the genomic DNA and guide Cas9 protein-N4 to that site. N4 specifically binds to sc, and the activator fused with sc also reaches the target site. The system designed in this invention can recruit multiple activators, and a large number of activators can further recruit more RNA polymerases, thereby activating the transcription of the target gene. When sgRNA binds to the RBS (ribosome binding site) region, it can occupy the binding site of the original related activator, thereby achieving the inhibitory effect on the specific gene. When used for gene editing, another plasmid with homologous arms is required.
[0023] Specifically, when used for gene regulation, the application method is as follows:
[0024] (1) Construction of the regulatory vector: sgRNA is designed and synthesized according to the gene to be regulated, and is ligated to the regulatory vector backbone by enzyme digestion and ligation; DNA fragments corresponding to other elements are also ligated to the regulatory vector backbone.
[0025] (2) Transplantation: The regulatory vector constructed above was transferred into competent cells of Pseudomonas aeruginosa by electroporation and cultured until the stationary phase.
[0026] Specifically, when used for gene editing, the application method is as follows:
[0027] (1) Construction of the regulatory vector: Design and synthesize sgRNA based on the gene to be edited, and link it to the regulatory vector backbone by enzyme digestion and ligation; link the DNA fragments corresponding to other elements to the regulatory vector backbone; at the same time, construct a homologous fragment vector containing the upstream homologous arm of the gene to be edited, the target fragment, and the downstream homologous arm.
[0028] (2) Transplantation: The regulatory vector and homologous fragment vector constructed above were simultaneously transformed into competent cells of Pseudomonas aeruginosa by electroporation and cultured until the stationary phase.
[0029] The CRISPR-AIE system of this invention can achieve three functions: activation, inhibition, and gene editing. Experiments show that the activation multiplier can reach up to 17 times, the inhibition multiplier can reach up to 100%, and the gene editing efficiency can reach 99%.
[0030] The CRISPR-AIE system of this invention is presented in plasmid form. During use, only the sgRNA needs to be changed and electroporated into the target cell to achieve different levels of activation and inhibition of the target gene, enabling fine-grained regulation. The phenotypic changes following the target gene alteration can indicate the construction of cell factories. Compared with conventional gene editing methods, it is simple to operate, has low time and economic costs.
[0031] To reduce off-target effects, this invention also performed codon optimization on the Cas9 sequence from S. pyogenes SF370. The optimization algorithm was performed on the jcat website (http: / / www.jcat.de / ), resulting in the Cas9n(D10A) sequence. The CAI-Value (codon fitness index) increased from 0.109 to 0.992 (a CAI-Value closer to 1 indicates better optimization). Then, H840A was mutated on Cas9n(D10A) to obtain dcas9; based on dcas9, mutations were performed at sites A262T, R324L, S409I, E480K, E543D, M694I, and E1219V to obtain Xdcas9.
[0032] The CRISPR-AIE system of the present invention achieves the following beneficial effects:
[0033] First, existing technologies typically require the replacement of multiple systems to achieve different regulatory / gene editing functions, which is cumbersome. This invention only requires changing the sgRNA at the plasmid level to achieve alternating activation and inhibition functions.
[0034] Second, existing transcriptional regulation technologies lack gene editing capabilities; this invention adds gene editing capabilities, allowing editing of different genes simply by changing the sgRNA at the plasmid level.
[0035] Third, existing CRISPR-Cas9 technologies exhibit strong off-target effects, which reduce the efficiency of activation, inhibition, and gene editing, leading to unstable regulatory effects. This invention uses mutants of Cas9 proteins such as Cas9n, dcas9, and Xdcas9, which reduces lethality, significantly lowers off-target effects, and makes the regulatory or editing effects more stable.
[0036] Fourth, the CRISPR-AIE system of the present invention can achieve both activation and inhibition functions (enabling general regulation of natural promoters in model organism KT2440) and gene editing functions in the model organism KT2440, exhibiting diverse functions and good and stable effects. Attached Figure Description
[0037] Figure 1 Schematic diagram of the CRISPR-AIE system.
[0038] Figure 2 : A schematic diagram illustrating the working principle of the CRISPR-AIE system.
[0039] Figure 3 : Schematic diagram of colony PCR detection results, where M is the DNA marker.
[0040] Figure 4 : Schematic diagram of gene editing sequencing results.
[0041] Figure 5 : Schematic diagram of the CRISPR-AIE system's regulation effect on the promoter corresponding to PP_1840. Detailed Implementation
[0042] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.
[0043] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.
[0044] The gene editing-transcriptional regulation system for Pseudomonas of the present invention, named the CRISPR-AIE system, has the following structure: Figure 1 As shown, it includes a regulatory vector containing the following elements: the DNA sequence corresponding to sgRNA, the gene encoding Cas9 protein, the gene encoding N4, the gene encoding sc, the gene encoding the activator, the Ptrc promoter, the xylR-pxylA promoter, and the ph5 promoter.
[0045] The CRISPR-AIE system of this invention, when applied, expresses relevant elements—sgRNA, Cas9 protein-N4, and sc-activator—in Pseudomonas bacteria to be regulated or edited in plasmid form. The sgRNA (designed according to the gene to be regulated or edited) can bind to the target site on the genomic DNA and guide Cas9 protein-N4 to that site. N4 specifically binds to sc, and the activator fused with sc also reaches the target site. The system designed in this invention can recruit multiple activators, and a large number of activators can further recruit more RNA polymerases, thereby activating the transcription of the target gene, such as... Figure 2 As shown; when sgRNA binds to the RBS (ribosome binding site) region, it can occupy the binding site of the original related activator, thereby achieving the inhibitory effect on the specific gene; when used for gene editing, another plasmid with homologous arms needs to be provided.
[0046] Example 1: Activating the transcription of a target gene using the CRISPR-AIE system
[0047] The steps are as follows:
[0048] (1) Construction of regulatory carriers
[0049] The regulatory vector was pPROBE-GT, and the target gene was the ph2 promoter (nucleotide sequence shown in SEQ ID NO. 10). Four sgRNAs were designed, and the DNA sequences corresponding to these four sgRNAs were numbered A-1, A-2, A-3, and A-4, respectively. The nucleotide sequence of A-1 is shown in SEQ ID NO. 11, the nucleotide sequence of A-2 is shown in SEQ ID NO. 12, the nucleotide sequence of A-3 is shown in SEQ ID NO. 13, and the nucleotide sequence of A-4 is shown in SEQ ID NO. 14.
[0050] The DNA corresponding to these four sgRNAs was artificially synthesized and ligated to the regulatory vector backbone by enzyme digestion and ligation. The coding genes for dCas9 protein, N4, sc, ω activator, Ptrc promoter, xylR-pxylA promoter, and ph5 promoter were also ligated to the regulatory vector backbone to obtain four different regulatory vectors with different sgRNAs. At the same time, a regulatory vector without sgRNA was constructed as a control.
[0051] (2) Transfer in
[0052] The constructed regulatory vector was transferred into competent cells of the ph2 reporter strain via electroporation, inoculated onto Gm resistant plates, and incubated upside down in a 30°C incubator for 16 hours.
[0053] The ph2 reporter strain was obtained by replacing the DNA fragment at positions 4922274-4922368 of the genomic locus of the model strain KT2440 with the ph2-mcherry gene.
[0054] (3) Detection of regulation effect
[0055] Single colonies grown on Gm-resistant plates were inoculated into 5 ml of LB liquid medium containing Gm and cultured at 30°C and 200 rpm until the stationary phase (approximately 20 h). The resulting bacterial suspensions were used to assess the regulatory effect. Based on the different sgRNAs, the bacterial suspensions were numbered A-1, A-2, A-3, A-4, and the control. Fluorescence intensity was measured in samples using a microplate reader (excitation wavelength 550 nm, emission wavelength 600 nm), with three biological replicates for each measurement. The results are shown in Table 1.
[0056] Table 1
[0057]
[0058]
[0059] Comparing the average values of the data from each group, it can be seen that different sgRNAs can achieve different levels of activation. Among them, compared with the ph2 reporter strain without sgRNA, A-1 was activated 3.3 times, A-2 was activated 10 times, A-3 was activated 17 times, and A-4 was activated 3.9 times.
[0060] Then, the present invention designed and synthesized several sgRNAs, and regulated transcription using the above method, ultimately achieving precise regulation at 11 levels, including 2-fold inhibition, 1.6-fold activation, 2-fold activation, 2.7-fold activation, 3.3-fold activation, 3.9-fold activation, 4.4-fold activation, 5.8-fold activation, 8.2-fold activation, 10-fold activation, and 17-fold activation.
[0061] Example 2: Using the CRISPR-AIE system to inhibit the transcription of a target gene
[0062] The steps are as follows:
[0063] (1) Construction of regulatory carriers
[0064] The regulatory vector was pPROBE-GT, and the target gene was the ph3 promoter (nucleotide sequence shown in SEQ ID NO.15). Four sgRNAs were designed, and the DNA sequences corresponding to these four sgRNAs were numbered R-1, R-2, R-3, and R-4, respectively. The nucleotide sequence of R-1 is shown in SEQ ID NO.16, the nucleotide sequence of R-2 is shown in SEQ ID NO.17, the nucleotide sequence of R-3 is shown in SEQ ID NO.18, and the nucleotide sequence of R-4 is shown in SEQ ID NO.19.
[0065] The DNA corresponding to these four sgRNAs was artificially synthesized and ligated to the regulatory vector backbone by enzyme digestion and ligation. The coding genes for dCas9 protein, N4, sc, ω activator, Ptrc promoter, xylR-pxylA promoter, and ph5 promoter were also ligated to the regulatory vector backbone to obtain four different regulatory vectors with sgRNAs. At the same time, a regulatory vector without sgRNA was constructed as a control.
[0066] (2) Transfer in
[0067] The constructed regulatory vector was transferred into competent cells of the ph3 reporter strain via electroporation, inoculated onto Gm resistant plates, and incubated upside down in a 30°C incubator for 12 hours.
[0068] The ph3 reporter strain was obtained by replacing the DNA fragment at positions 4922274-4922368 of the genomic locus of the model strain KT2440 with the ph3-GFP gene.
[0069] (3) Detection of regulation effect
[0070] Single colonies grown on Gm-resistant plates were inoculated into 5 ml of LB liquid medium containing Gm and cultured at 30°C and 200 rpm until the stationary phase (approximately 20 h). The resulting bacterial suspensions were used to assess the regulatory effect. Based on the different sgRNAs, the bacterial suspensions were numbered R-1, R-2, R-3, R-4, and the control. Fluorescence intensity was measured in samples using a microplate reader (excitation wavelength 485 nm, emission wavelength 528 nm), with three biological replicates for each measurement. The results are shown in Table 2, with all data presented after removing background values (1810).
[0071] Table 2
[0072] serial number fluorescence intensity Comparison 22932，23280.5，21976.5 Bacterial solution R-1 20132.5，19575，18819 Bacterial solution R-2 19065，19516，19867.5 Bacterial solution R-3 193，194.5，223.5 Bacterial solution R-4 -478，-185，-167.5
[0073] Comparing the average values of the above groups of data, it can be seen that different sgRNAs can achieve different levels of inhibition, with R-4 achieving complete inhibition compared to the original strain.
[0074] Example 3: Editing the target gene using the CRISPR-AIE system
[0075] The steps are as follows:
[0076] (1) Construction of regulatory carriers
[0077] The editing vector was pPROBE-GT, the gene to be edited was pyrF, and the homologous fragment vector containing the upstream homologous arm, target fragment, and downstream homologous arm of the gene to be edited was pBBR1. sgRNA was designed (the DNA sequence corresponding to sgRNA is shown in SEQ ID NO.20).
[0078] The DNA corresponding to the artificially synthesized sgRNA was ligated to the editing vector backbone via enzyme digestion and ligation; the coding genes for Cas9n protein, N4, sc, ω activator, Ptrc promoter, xylR-pxylA promoter, and ph5 promoter were also ligated to the editing vector backbone.
[0079] The pPROBE-GT vector contains the Gm resistance gene, and the pBBR1 vector contains the Kan resistance gene. The use of two resistance genes is to ensure that the relevant elements and fragments can be stably expressed in the model organism and gene editing can be performed. The reason for not using a single vector is that the editing vector itself is nearly 20,000 bp. If homologous sequences are added, the vector will be too large, prone to collapse, and unable to exist stably in the cell.
[0080] (2) Transfer in
[0081] The constructed regulatory vector and the homologous fragment vector containing the upstream homologous arm of the gene to be edited, the target fragment, and the downstream homologous arm were transformed into competent KT2440 cells by electroporation and cultured upside down in a 30°C incubator for 16 hours on Gm+Kan dual-resistance plates.
[0082] (3) Editing effect detection
[0083] Single colonies grown on Gm+Kan double-antibiotic plates were inoculated into LB+Ura (uracil) liquid medium containing Gm+Kan and cultured at 30°C and 200 rpm until the late stationary phase (approximately 20 h). 100 μl of bacterial culture was spread onto 5-FOA plates and incubated upside down at 30°C for 14 h. Single colonies were randomly selected for colony PCR detection and sent for analysis. The colony PCR primers were pyrF D1 (nucleotide sequence shown in SEQ ID NO. 21) and pyrF D2 (nucleotide sequence shown in SEQ ID NO. 22).
[0084] Three biological parallel sets were set up, and 32 single colonies were picked from each of the three parallel plates for colony PCR detection (if the editing was successful, a 1545bp electrophoresis band should appear). The results are as follows: Figure 3 As shown, only one monoclonal clone in these three parallel groups showed no target band. The PCR products showing the target band were sent for sequencing, and the results showed that all were successfully edited (editing here refers to knocking out the target gene), with an editing efficiency of 31 / 32 + 32 / 32 + 32 / 32 = 99%. The gene editing sequencing results of a randomly selected PCR product with the target band are shown below. Figure 4 As shown.
[0085] Example 4: Regulating the natural promoter of Pseudomonas using the CRISPR-AIE system
[0086] The steps are as follows:
[0087] (1) Construction of regulatory carriers
[0088] The regulatory vector was pPROBE-GT, and the target genes were the promoters corresponding to the natural genes PP_0181, PP_0679, PP_2322, PP_4793, PP_5540, PP_1840, PP_2268, PP_3611, PP_4876, PP_5181, PP_1206, PP_3950, PP_4055, PP_4217, and PP_4488 of Pseudomonas KT2440 (in this example, 15 promoters were randomly selected from the natural promoters of KT2440 for general regulatory experiments). Four sgRNAs were designed for each promoter for regulatory experiments.
[0089] Taking 1840 as an example, 10 sgRNAs were designed. The DNA sequences corresponding to these 10 sgRNAs are numbered 1840-1, 1840-2, 1840-3, 1840-4, 1840-5, 1840-6, 1840-7, 1840-8, 1840-9, and 1840-10, respectively. The nucleotide sequence of 0181-1 is shown in SEQ ID NO.23, the nucleotide sequence of 0181-2 is shown in SEQ ID NO.24, the nucleotide sequence of 0181-3 is shown in SEQ ID NO.25, the nucleotide sequence of 0181-4 is shown in SEQ ID NO.26, the nucleotide sequence of 0181-5 is shown in SEQ ID NO.27, the nucleotide sequence of 0181-6 is shown in SEQ ID NO.28, the nucleotide sequence of 0181-7 is shown in SEQ ID NO.29, and the nucleotide sequence of 0181-8 is shown in SEQ ID NO.29. As shown in NO.30, the nucleotide sequence of 0181-9 is shown in SEQ ID NO.31, and the nucleotide sequence of 0181-10 is shown in SEQ ID NO.32.
[0090] The DNA corresponding to these 10 sgRNAs was artificially synthesized and ligated to the regulatory vector backbone by enzyme digestion and ligation. The coding genes for XdCas9 protein, N4, sc, ω activator, Ptrc promoter, xylR-pxylA promoter and ph5 promoter were also ligated to the regulatory vector backbone to obtain 10 different regulatory vectors with sgRNAs. At the same time, a regulatory vector without sgRNA was constructed as a control.
[0091] (2) Transfer in
[0092] The constructed regulatory vector was transferred into competent KT2440 cells via electroporation, seeded onto Gm resistant plates, and incubated upside down in a 30°C incubator for 16 hours.
[0093] (3) Detection of regulation effect
[0094] Single colonies grown on Gm-resistant plates were inoculated into 5 ml of LB liquid medium containing Gm and cultured at 30°C and 200 rpm until the stationary phase (approximately 20 h). The resulting bacterial culture was used to assess the regulatory effect. Fluorescence intensity was measured in samples using a microplate reader (excitation wavelength 550 nm, emission wavelength 600 nm), with three biological replicates for each measurement. The regulatory effect is as follows: Figure 5 As shown.
[0095] Depend on Figure 5As can be seen, the CRISPR-AIE system of the present invention can achieve good regulation (activation and inhibition) of the KT2440 natural promoter PP_1840. The regulation effects are as follows: 1840-1 achieves 8.58-fold inhibition, 1840-2 achieves 1.65-fold activation, 1840-3 achieves 1.25-fold activation, 1840-4 achieves 5.50-fold inhibition, 1840-5 achieves 1.89-fold activation, 1840-6 achieves 1.57-fold inhibition, 1840-7 achieves 1.18-fold inhibition, 1840-8 achieves 2.65-fold inhibition, 1840-9 achieves 9.83-fold activation, and 1840-10 achieves 4.79-fold activation.
[0096] Meanwhile, the CRISPR-AIE system of the present invention also exhibits good control effects on promoters such as PP_0679, PP_2322, PP_4793, PP_5540, PP_0181, PP_2268, PP_3611, PP_4876, PP_5181, PP_1206, PP_3950, PP_4055, PP_4217, and PP_4488.
[0097] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.
Claims
1. A gene editing-transcriptional regulation system suitable for Pseudomonas, characterized in that: This includes regulatory vectors, which contain the following elements: DNA corresponding to sgRNA, gene encoding Cas9 protein, gene encoding N4, gene encoding sc, gene encoding activator, Ptrc promoter, xylR-pxylA promoter and ph5 promoter. When the gene editing-transcriptional regulation system applicable to Pseudomonas is applied, relevant elements—sgRNA, cas9 protein-N4, and sc-activator—are expressed in plasmid form in the Pseudomonas to be regulated or edited. Specifically, the sgRNA, designed according to the gene to be regulated or edited, binds to the target site of the genomic DNA and guides cas9 protein-N4 to the target site. N4 specifically binds to sc, thereby causing the activator fused with sc to also reach the target site. The Cas9 protein is Cas9n, dcas9, or Xdcas9. The nucleotide sequence of the gene encoding Cas9n is shown in SEQ ID NO.1, the nucleotide sequence of the gene encoding dcas9 is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding Xdcas9 is shown in SEQ ID NO.
3. The activator is selected from ω-activators, and the nucleotide sequence of the gene encoding the ω-activator is shown in SEQ ID NO.6; The nucleotide sequence of the gene encoding N4 is shown in SEQ ID NO.4; The nucleotide sequence of the gene encoding sc is shown in SEQ ID NO.
5.
2. The gene editing-transcriptional regulation system for Pseudomonas according to claim 1, characterized in that: The nucleotide sequence of the Ptrc promoter is shown in SEQ ID NO.7; The nucleotide sequence of the xylR-pxylA promoter is shown in SEQ ID NO. 8; The nucleotide sequence of the ph5 promoter is shown in SEQ ID NO.
9.
3. The gene editing-transcriptional regulation system for Pseudomonas according to claim 1, characterized in that: The regulatory vector is selected from the pPROBE-GT vector.
4. The method for preparing a gene editing-transcriptional regulation system suitable for Pseudomonas aeruginosa according to any one of claims 1 to 3, characterized in that: First, sgRNAs were designed for the genes to be regulated or edited, and the corresponding DNA was synthesized. These sgRNAs were then ligated into the regulatory vector via enzyme digestion and ligation. Next, the coding genes for Cas9 protein, N4, sc, activator, Ptrc promoter, xylR-pxylA promoter, and ph5 promoter were also ligated into the regulatory vector, thus constructing a gene editing-transcriptional regulation system suitable for Pseudomonas.
5. The application of the gene editing-transcriptional regulation system for Pseudomonas according to any one of claims 1 to 3 in gene regulation or gene editing of Pseudomonas.
6. The application according to claim 5, characterized in that: The target gene for gene regulation is the ph2 promoter, and the nucleotide sequence of the DNA corresponding to the sgRNA designed for the ph2 promoter is shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, or SEQ ID NO.
14. Alternatively: The target gene for gene regulation is the ph3 promoter, and the nucleotide sequence of the DNA corresponding to the sgRNA designed for the ph3 promoter is shown in SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, or SEQ ID NO.
19.
7. The application according to claim 5, characterized in that: The target gene for the gene editing is pyrF.