Bacillus subtilis gene expression regulation and control tool based on I-F3 type CRISPR (clustered regularly interspaced short palindromic repeats) system

Abstract

The invention discloses a bacillus subtilis gene expression regulation and control tool based on an I-F3 type CRISPR (clustered regularly interspaced short palindromic repeats) system, and belongs to the field of gene engineering. The CRISPRi inhibition capability of a VchQCas system derived from Vibrio cholera in bacillus subtilis WS9C is verified through expression optimization, and a VcaQCas system with high CRISPRi regulation and control efficiency and PAM broad-spectrum property is identified and obtained from QCascade systems of eight types of I-F3 on the basis of bioinformatics methods such as gene mining and CRISPR element prediction. And then, the targeting preference of the system in the bacillus subtilis WS9C and the PAM suitability are analyzed in detail. The bacillus subtilis provided by the invention provides a new gene expression regulation strategy as a foreign protein expression host, and also provides reference for site selection of gene regulation.

Description

Technical Field

[0001] This invention relates to a gene expression regulation tool for Bacillus subtilis based on the I-F3 CRISPR system, belonging to the field of biotechnology. Background Technology

[0002] Bacillus subtilis ( Bacillus subtilis Bacillus subtilis is a Gram-positive strain widely distributed in the natural environment. Due to its non-pathogenicity and lack of endotoxin production, it has been designated a GRAS (Generally Recognized as Safe) microorganism by the U.S. Food and Drug Administration (FDA) and is widely used in the food and pharmaceutical industries. Bacillus subtilis is an important producer of α-amylase and neutral protease. Because it lacks an outer membrane structure, it can efficiently secrete proteins into the culture medium, significantly reducing downstream purification costs in industrial applications. Furthermore, Bacillus subtilis has a clear genetic background, mature gene editing methods, and no obvious codon bias.

[0003] Achieving precise regulation of gene expression has become a key issue in further improving biosynthetic capacity. Gene expression regulation tools based on the CRISPR-Cas system offer advantages such as programmability and ease of construction. CRISPR-Cas proteins are divided into Class I and Class II. Class I proteins utilize multi-protein effector complexes to function, while Class II proteins rely on single effector proteins. Existing CRISPR-Cas regulation tools in Bacillus subtilis are mainly constructed based on Class II CRISPR-Cas systems, primarily including dCas9 and dCas12a. In recent years, the continuous development of different types of CRISPR-Cas systems has provided a wealth of enabling tools for gene editing and expression regulation in microbial cell factories. Types I-F3 CRISPR-Cas systems have been used to mediate CAST-directed transposition and achieve large-fragment genome integration at multiple sites. This transposition system includes two components: the TniQ-Cascade (QCascade) and the Tns operon. The QCascade includes TniQ, Cas6 responsible for cleavage to generate mature crRNA, Cas7 responsible for assembling the complex backbone, Cas8 responsible for PAM site recognition, and the crRNA array. Meanwhile, this complex naturally lacks DNA strand cleavage activity, and the Cas8 subunit has low PAM dependence. Therefore, the I-F3 type QCascade system holds promise for developing CRISPR gene expression regulation tools with low PAM dependence. Summary of the Invention

[0004] To address the limitation in the application of Bacillus subtilis gene expression regulation tools in existing technologies, this invention provides a Bacillus subtilis gene expression regulation tool based on the I-F3 CRISPR system.

[0005] The first technical solution provided by this invention is a gene expression regulation tool for Bacillus subtilis. The regulation tool includes a QCascade system and crRNA. The elements of the QCascade system include TniQ, Cas8, Cas7, and Cas6. The QCascade system is... Vch QCas、 Pse QCas or Vca QCas system.

[0006] In one embodiment, the... Vch QCas、 Pse QCas or Vca The QCas system originates from... Vibrio cholerae HE-45 Pseudoalteromonas sp. S983 and Vibrio campbellii FXH286.

[0007] In one embodiment, the Vch In the QCas system, the amino acid sequence of TniQ is shown in SEQ ID NO.1, the amino acid sequence of Cas8 is shown in SEQ ID NO.2, the amino acid sequence of Cas7 is shown in SEQ ID NO.3, and the amino acid sequence of Cas6 is shown in SEQ ID NO.4.

[0008] In one embodiment, the Pse In the QCas system, the amino acid sequence of TniQ is shown in SEQ ID NO.5, the amino acid sequence of Cas8 is shown in SEQ ID NO.6, the amino acid sequence of Cas7 is shown in SEQ ID NO.7, and the amino acid sequence of Cas6 is shown in SEQ ID NO.8.

[0009] In one embodiment, the Vca In the QCas system, the amino acid sequence of TniQ is shown in SEQ ID NO.9, the amino acid sequence of Cas8 is shown in SEQ ID NO.10, the amino acid sequence of Cas7 is shown in SEQ ID NO.11, and the amino acid sequence of Cas6 is shown in SEQ ID NO.12.

[0010] In one implementation, the QCascade system is integrated into the genome of Bacillus subtilis. mprThe crRNA is expressed using the pAD123 plasmid as the expression vector.

[0011] In one embodiment, the TniQ and Cas7 are connected via a constitutive promoter P as shown in SEQ ID NO.14. sunA Regulation; Cas8 and Cas6 are regulated via the constitutive promoter P as shown in SEQ ID NO.15. groES Regulation; crRNA is regulated by the constitutive promoter P, as shown in SEQ ID NO.16. veg Regulate expression.

[0012] In one embodiment, the CRISPR array sequence matched by the QCascade system is identified by CRISPRCasFinder software and has the CRISPR array sequence shown in SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.19.

[0013] The second technical solution provided by this invention is a method for regulating gene expression in Bacillus subtilis. This method involves expressing crRNA via plasmid in Bacillus subtilis that integrates the QCascade system to regulate the expression of a target gene. The QCascade system comprises TniQ, Cas8, Cas7, and Cas6. Vch QCas、 Pse QCas or Vca QCas system.

[0014] In one embodiment, the target gene includes, but is not limited to, green fluorescent protein.

[0015] In one embodiment, the Bacillus subtilis includes, but is not limited to, Bacillus subtilis WS9C, which is disclosed in patent CN117965411A.

[0016] In one embodiment, the... Vch QCas、 Pse QCas or Vca The QCas system originates from... Vibrio cholerae HE-45 Pseudoalteromonas sp. S983 and Vibrio campbellii FXH286.

[0017] In one embodiment, the VchIn the QCas system, the amino acid sequence of TniQ is shown in SEQ ID NO.1, the amino acid sequence of Cas8 is shown in SEQ ID NO.2, the amino acid sequence of Cas7 is shown in SEQ ID NO.3, and the amino acid sequence of Cas6 is shown in SEQ ID NO.4.

[0018] In one embodiment, the Pse In the QCas system, the amino acid sequence of TniQ is shown in SEQ ID NO.5, the amino acid sequence of Cas8 is shown in SEQ ID NO.6, the amino acid sequence of Cas7 is shown in SEQ ID NO.7, and the amino acid sequence of Cas6 is shown in SEQ ID NO.8.

[0019] In one embodiment, the Vca In the QCas system, the amino acid sequence of TniQ is shown in SEQ ID NO.9, the amino acid sequence of Cas8 is shown in SEQ ID NO.10, the amino acid sequence of Cas7 is shown in SEQ ID NO.11, and the amino acid sequence of Cas6 is shown in SEQ ID NO.12.

[0020] In one implementation, the target gene is integrated into the genome. nprE The site is defined by a constitutive promoter P, as shown in the nucleotide sequence SEQ ID NO.13. amyQ’ Regulation.

[0021] Furthermore, the target gene is a fluorescent protein gene. gfpmut3 fluorescent protein gene gfpmut3 It has a sequence as shown in SEQ ID NO.26.

[0022] In one implementation, gene expression is regulated through the genome. mpr The QCascade system is integrated at the site, and crRNA is expressed via the pAD123 plasmid.

[0023] In one embodiment, the TniQ and Cas7 are connected via a constitutive promoter P as shown in SEQ ID NO.14. sunA Regulation; Cas8 and Cas6 are regulated via the constitutive promoter P as shown in SEQ ID NO.15. groES Regulation; crRNA is regulated by the constitutive promoter P, as shown in SEQ ID NO.16. veg Regulate expression.

[0024] In one embodiment, the CRISPR array sequence matched by the QCascade system is identified by CRISPRCasFinder software and has the CRISPR array sequence shown in SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.19.

[0025] In one implementation, the fluorescent protein gene is targeted. gfpmut3 The n32 sequences are shown in SEQ ID NO.20~SEQ ID NO.25 respectively.

[0026] In one implementation, the crRNA used to verify PAM dependence has a sequence as shown in SEQ ID NO.27.

[0027] The third technical solution provided by this invention is the application of the regulatory tool described in the first technical solution or the method described in the second technical solution in the gene expression regulation of Bacillus subtilis.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention constructs a gene expression regulation tool based on the I-F3 type CRISPR system, which can effectively suppress the expression level of genomic genes.

[0029] (2) The gene expression regulation tool provided by the present invention can effectively guide the modification of Bacillus subtilis cell factories. Attached Figure Description

[0030] Figure 1 Optimization of single-expression cassette promoters for the Vibrio cholerae-derived regulatory system; A represents the fluorescence inhibition rate of single-expression cassettes regulated by maltose-inducible promoters; B represents the fluorescence inhibition rate of single-expression cassettes regulated by xylose-inducible promoters; C represents the fluorescence inhibition rate of single-expression cassettes regulated by IPTG-inducible promoters; D represents the fluorescence inhibition rate of single-expression cassettes regulated by various constitutive promoters.

[0031] Figure 2 The effect of expression module optimization on the fluorescence inhibition ability of the Vibrio cholerae-derived regulatory system; A is a schematic diagram of expression module construction; B is a comparison of the system inhibition ability in single expression cassette and dual expression cassette modes; C is the effect of different culture media on the inhibition ability of the dual expression cassette system.

[0032] Figure 3 The effect of promoter optimization on the inhibitory capacity of the Vibrio cholerae-derived regulatory system.

[0033] Figure 4The effect of copy number optimization of single elements in the Vibrio cholerae-derived regulatory system on fluorescence inhibition ability is shown in Figure A; Figure B is a schematic diagram of the construction of single element copy number optimization; Figure B shows the effect of overexpression of different single elements on the system's inhibition ability.

[0034] Figure 5 Analysis of 90 Cas8 phylogenetic trees output from gene mining.

[0035] Figure 6 Construction of low PAM-dependent CRISPR systems and identification of CRISPRi activity; A is a schematic diagram of the construction of CRISPR systems from different sources; B is an evaluation of the CRISPRi inhibition ability of CRISPR systems from various sources.

[0036] Figure 7 Target preference analysis of the source regulatory system of Vibrio cholerae, Pseudomonas pseudoalteromonas and Vibrio kanehirae.

[0037] Figure 8 Validation of PAM dependence in the Vibrio kanehirae source regulatory system; A is a schematic diagram of PAM library construction; B is... Vca The inhibitory activity of the QCas system under all PAM conditions; C is Vca The QCas system can efficiently identify the PAM base distribution. Detailed Implementation

[0038] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0039] Test method: Fluorescence measurement: GFP fluorescence intensity was measured using the TECAN SPARK fluorescence enzyme-linked immunosorbent assay (ELISA). First, the cultured bacterial solution was centrifuged at 4000 rpm for 15 min, the supernatant was discarded, and the solution was washed with PBS buffer. This centrifugation and washing process was repeated twice. Then, 200 μL of the bacterial suspension in PBS was added to a 96-well ELISA plate. Fluorescence intensity was detected using a TECAN SPARK fluorescence ELISA reader. GFP / split GFP fluorescence intensity was excited using a 488 nm semiconductor laser, and the emission channel was filtered using a 530 / 30 nm filter. After vortexing, the fluorescence value was detected using an automatically optimized gain value. The absorbance at 600 nm was also measured simultaneously. The relative fluorescence value was calculated using the following formula, where the background fluorescence GFP... BG It is obtained by detecting the fluorescence value and background absorbance OD of strains that do not contain fluorescent proteins. BG This is the absorbance detected in the blank culture medium: .

[0040] The culture medium used in the examples: LB medium: yeast extract (OXOID) 5.0 g / L, tryptone (OXOID) 10.0 g / L, NaCl (Sinopharm Chemical Reagent Co., Ltd.) 10.0 g / L.

[0041] YN culture medium: yeast extract (OXOID) 7.0 g / L, nutrient broth (Qingdao Haibo Biotechnology Co., Ltd.) 18.0 g / L.

[0042] The primers involved in this embodiment are:

[0043]

[0044] Continued table

[0045] Continued table

[0046] The underlined letters represent the homologous arm regions used in one-step cloning ligation, and the wavy letter X represents the PAM mutation site. Example 1 Bacillus subtilis Vch QCas expression system construction Will Vibrio cholerae DNA targeting module in HE-45-derived Tn6677 Vch QCas integration into the WS9C genome (disclosed in patent application CN117965411A) mpr The recombinant strain WS9C6677 was obtained by identifying the target site. All elements in the expression cassette are regulated by a single promoter, and the crRNA plasmid expressed in the integrative strain targets and inhibits green fluorescent protein. gfpmut3 The fluorescence changes were measured. The specific method is as follows: the coding genes in the Tn6677 system were amplified in vitro by PCR, and then these coding genes were processed using overlap PCR. tniQ (SEQ ID NO.28)- cas8 (SEQ ID NO.29)- cas7 (SEQ ID NO.30)- cas6 The fragments were ligated in the order of (SEQ ID NO.31), and then constructed into the integration vector pET24a containing different promoters using a one-step cloning method. After correct sequencing, the fragments were integrated into the genome with spectinomycin as the resistance gene. mpr Site selection. The integrated strain was first cultured in 10 ml of LB broth at 37°C and 200 rpm for 10 h. Then, 500 μL of the bacterial culture was transferred to a new 10 mL LB broth, and 250 μL of 400 g / L xylose solution was added. The culture was then incubated at 37°C and 200 rpm for 4 h to prepare competent cells. Single-stranded DNA containing crRNA and array sequences was synthesized separately using primers. 15 bp homologous arms were added to both ends. PCR annealing was used to pair the two complementary single strands. The vector fragment carrying the same homologous arm ends was amplified by PCR. Subsequently, the crRNA was ligated into the pAD123 plasmid vector using a one-step cloning method. After transformation into JM109, sequencing yielded a positive plasmid. The target... gfp The recombinant crRNA plasmid of the template strand was transformed into WS9C6677 competent cells integrating the Tn6677 CRISPR cassette, and positive clones were obtained by plating on LB agar plates containing chloramphenicol resistance. Clones were obtained by overnight culture at 37°C on LB agar plates containing chloramphenicol (5 µg / mL). Seed culture was obtained by culturing at 37°C and 200 rpm for 12 h, followed by inoculation with 10 mL of LB medium at a 5% inoculum. After culturing at 37°C and 750 rpm for 2 h, an inducer was added, and the temperature was lowered to 33°C for 24 h. Fluorescence values ​​were then measured.

[0047] Among them, three constitutive promoters (P) were selected. citz (SEQ ID NO.32), P groES (SEQ ID NO.33), P gsiB (SEQ ID NO.34) and 3 inducible promoters (P xylA (SEQ ID NO.35), P grac100 (SEQ ID NO.36), P glv (SEQ ID NO.37) regulates Tn6677 CRISPR cassette ( Figure 1 A). Strains WS9Vch1, WS9Vch2, WS9Vch3, WS9Vch4, WS9Vch5, and WS9Vch6 were obtained respectively.

[0048] Different promoter regulation Vch QCas system control capabilities such as Figure 1 As shown, the inhibition intensity of CRISPRi mediated by the three constitutive promoters and the three inducible promoters was all less than 20%, and the strain was severely affected by the inducer, indicating that inducible promoters are not suitable for use.

[0049] The CRISPR cassette was expressed in two separate expression cassettes, first by PCR amplification. tniQ , cas7 , cas8 , cas6 In addition to promoter sequences and other elements, corresponding 15 bp homologous arm sequences were added to the ends. These fragments were then ligated in vitro in the following order using Overlap-PCR: P gsiB -tniQ - cas7- P groES -cas8 - cas6 The fragment was constructed into an integration vector using a one-step cloning method, with an endogenous Bacillus subtilis RBS sequence (AAGGAGTGTCAAGA) inserted between TniQ and Cas7, and between Cas8 and Cas6 for tandem expression. The vector was then transformed into E. coli JM109, plated on LB agar plates containing kanamycin resistance, and cultured overnight to obtain positive clones. The crRNA was expressed via the constitutive promoter P... veg Regulation of expression. The inhibitory effects of single and dual expression cassette regulation were compared using the above construction methods, and the results are as follows: Figure 2 As shown in B. The dual expression cassette was significantly better than the single expression cassette, with a 41% fluorescence inhibition rate observed. The effect of fermentation conditions on CRISPRi activity was investigated using LB and YN media. The results of changing culture conditions are shown in Figure B. Figure 2 As shown in C, the LB medium showed better regulatory effect than YN.

[0050] Example 2 Bacillus subtilis Vch QCas expression optimization Regulation Vch The two expression box promoters of the QCas system were optimized, and the second promoter was fixed as the strongly constitutive promoter P. groES Referring to the method in Example 1, the first promoter is changed to P. sunA (SEQ ID NO.38), P amyQ’ (SEQ ID NO.39) and P gsiB (SEQ ID NO.34). The above construction method will... Vch The QCas system is integrated into the genome and transferred to targeted... gfpmut3 The crRNA plasmid of the gene was used, and the fluorescence inhibition level was measured after fermentation. The fluorescence inhibition results are as follows: Figure 3 As shown, P sunA With P groES The regulation effect was optimal, reaching a maximum fluorescence inhibition rate of 60.80%. The regulation effect was related to the promoter strength; stronger promoters resulted in stronger inhibition.

[0051] For research Vch The optimal expression level of a single element in the QCas system was attempted to be increased. Vch The copy number of QCas as a whole and of individual element genes within it. First, the genome was analyzed according to the method described in Example 1. lacA The site integrates a complete Vch The QCas unit eliminates resistance through the Cre / lox method, specifically by... SPC The resistance gene was flanked by lox71 / lox66 sequences, and the integrative plasmid was transformed into competent cells of *Bacillus subtilis* WS9C (disclosed in patent application CN117965411A) for integration. Positive integrons were obtained using 100 µg / mL spectinomycin as the selection pressure. Subsequently, the plasmid was transformed into a Cre enzyme expression plasmid and induced to express on 0.1 mM IPTG plates. The plasmids were then photocopied onto antibiotic-free and spectinomycin-resistant plates, and strains that successfully eliminated resistance were selected. Then, the genome was analyzed... mpr Site integration of different single elements TniQ, Cas7, Cas8, Cas6 or Vch A single copy of QCas. Overexpression of a single element is achieved by a strongly constitutive promoter P. sunA Regulation was performed. Referring to Example 1, a crRNA expression plasmid was introduced, and changes in fluorescence values ​​were studied. Figure 4 As shown, when increasing Vch When the QCas copy number was increased, the fluorescence inhibition rate increased slightly to 70.89%. However, when the copy number of other single elements increased, the fluorescence inhibition ability decreased. Therefore, the original dual expression cassette system was kept unchanged.

[0052] Example 3: Mining of I-F3 CRISPR Systems from Different Sources The construction in Example 2 Vch While the QCas system can regulate CRISPRi, its inhibitory ability is relatively weak. Therefore, we selected the Cas8 protein sequence responsible for PAM recognition in the CRISPR transposition system of *E. coli*, which possesses both high transposition efficiency and broad PAM spectrum, for PSI-BLAST search. The Cas8 proteins are derived from... Vibrio cholerae , Pseudoalteromonas sp. S983 and Psychromonassp. RZ5. A PSI-BLAST search was performed on the non-redundant protein sequences (nr) database using the following parameters: -evalue 0.005 -num_alignments 9999999 -num_iterations 15. From the 90 output results, eight Cas8 proteins containing the Cas8 protein were selected for synthesis using the I-F3 CRISPR system. System components were placed in two expression cassettes and integrated into the genome of Bacillus subtilis WS9C. mpr Sites, TniQ and Cas7 via constitutive promoter P sunA Regulation; Cas8 and Cas6 via the constitutive promoter P groES Regulation. crRNA is expressed via plasmid pAD123 and is regulated by the constitutive promoter P. veg Regulation of expression. The crRNA array sequence corresponding to each system was obtained by searching the corresponding genome using CRISPRCasFinder software, as shown in Table 1. In the output results, only the alignment results with the highest similarity for each species were counted to avoid excessive redundancy in the collected genes. After data filtering and deduplication, a total of 90 output results were collected, and phylogenetic analysis was performed on these 90 Cas8 protein sequences. Figure 5 The three Cas8 sequences used in PSI-BLAST are marked in red. In some of these 90 results, the QCas system of some of the Cas8 sequences has been shown in other studies to be present in type strains. E . coli The results show clear transposable activity, and these identified activities are marked in blue. After removing incompletely annotated results, eight QCas systems from different sources were selected. Pse QCas and Psy Genes were synthesized using the QCas system and constructed into the aforementioned dual expression cassette framework for CRISPRi activity characterization.

[0053] Table 1. Statistical information on Cas proteins of types I-F3.

[0054] The underlined sequence represents the stem-loop structure in an array. Green fluorescent protein gfpmut3 Gene integration into the genome aprE Site and constitutive promoter P amyQ’ Regulation as a reporter gene. P amyQ - gfp Expression box and P spoVG - mcherryThe expression cassette was constructed in the early stage and subsequently amplified from the Bacillus subtilis WS9C genome and the pHY300PLK vector, respectively. nprE Homologous arm sequences and tetracycline resistance genes TcR Build " using overlap pcr nprE - P spoVG - mcherry- P amyQ - gfp - TcR-nprE (as shown in SEQ ID NO.40) and integrated into the genome. nprE The site was selected, and tetracycline was used as a resistance marker. Resistance was eliminated after successful integration using the aforementioned method. When assessing the inhibitory capacity of each system, the crRNA targeted the same site, i.e. gfpmut3 At a position 38 nt downstream of the start codon, 5´-NCC was selected as the fluorescence suppression capability of the PAM recognition sequence detection system. The targeted sequence... gfp The crRNA plasmid of the template strand was transformed into WS9C competent cells integrating different I-F3 type CRISPR system elements. Clones were obtained by overnight culture at 37°C on LB plates containing chloramphenicol (5 µg / mL). Seed culture was obtained by culturing at 37°C and 200 rpm for 12 h, followed by inoculation with 10 mL of LB medium at a 5% inoculum. The cells were then cultured at 37°C and 750 rpm for 2 h, followed by cooling to 33°C and culturing for 24 h, during which fluorescence values ​​were measured. Results are as follows: Figure 6 As shown, in all QCascade systems, only Vch Qcas、 Pse QCas、 Vca QCas and Rhe QCas detected a significant downregulation of fluorescence intensity. The inhibition rates reached 60.80%, 64.56%, 92.15%, and 57.45%, respectively. Vca The QCas system exhibited the highest gene expression repression intensity. Pse QCas and Rhe QCas mediated a change compared to the control group. Vch The fluorescence downregulation level is similar to that of QCas, but its mediated fluorescence intensity inhibition rate is lower. Vca The QCas system shows significant differences. Furthermore, observation of colony morphology revealed that expression... Rhe When QCas strains grew on LB plates, the colonies became noticeably stickier, suggesting... Rhe The expression of the QCas system has a significant impact on the physiology of the strain. Therefore, it was selected... Vch QCas、 Pse QCas and Vca The three QCas systems were further characterized.

[0055] Example 4 Vch QCas、 Pse QCas and Vca Target chain preference verification in QCas system The three systems obtained in Example 3 that showed significant CRISPRi inhibitory activity in Bacillus subtilis WS9C: Vch QCas、 Pse QCas and Vca QCas was used for targeting preference analysis. The inhibitory activity of the systemic CRISPRi was validated by designing crRNAs targeting different sites. A total of six crRNAs (SEQ ID NO.20~SEQ ID NO.25) targeting different sites were designed, including three crRNAs targeting the template strand (A1: 650 nt downstream of the start codon; A2: 267 nt downstream of the start codon; A3: 38 nt downstream of the start codon) and three crRNAs targeting the non-template strand (S1: 5 nt upstream of the start codon; S2: 347 nt downstream of the start codon; S3: 651 nt downstream of the start codon). All crRNAs were designed using a uniform PAM sequence (5´-NCC), and the targeting sites on both the template and non-template strands were kept as consistent as possible for subsequent comparisons. Results are as follows: Figure 7 As shown, regarding chain bias, the inhibition efficiency of all three systems was significantly higher when targeting the template chain than when targeting non-template chains. When targeting the template chain, Vch QCas、 Pse QCas and Vca The average inhibition rates of QCas were 56.12%, 69.76%, and 82.25%, respectively. Therefore, the one with stronger inhibitory ability was selected. Vca The QCas system was further investigated for its dependence on PAM.

[0056] Example 5 Vca QCas system PAM dependency verification A design such as Figure 8 A shows a small PAM library used to verify all 64 PAM sequences for... Vca The effect of the QCas system on the ability to repress CRISPRi gene expression. After integrating... Vca A dual plasmid expression system was constructed in the QCas strain, expressing green fluorescent protein. gfpmut3pHTGFP and pADG0 plasmid expressing crRNA were used, where pHTGFP is a derivative vector of pHT01. Using Fkan and Rkan primers, the chloramphenicol resistance gene in pHTGFP was replaced with the kanamycin resistance gene via one-step cloning, and the IPTG inducible promoter P was modified. grac Replace with constitutive promoter P aprE The specific steps are as follows: First, amplify the genome separately... gfpmut3 Genes and promoter P aprE The sequences were then ligated using overlap-PCR, and the promoter P was removed. grac The pHT01 vector sequence and its P aprE - gfpmut3 The sequence was cloned and ligated in one step. Subsequently, the kanamycin resistance gene was amplified by PCR using pUB110 plasmid as a template, and the above-mentioned P... aprE - gfpmut3 The vector sequence with the chloramphenicol gene removed was amplified using a plasmid as a template. The kanamycin resistance gene fragment was ligated using a one-step cloning method and transformed into JM109 to obtain positive clones. crRNA was immobilized and targeted to... gfpmut3 The first 32 bases of the template strand were mutated to all 64 possible base combinations by altering three bases at positions -3 to -1 upstream of the start codon. Strains containing the same PAM plasmid (pHTGFP) and non-targeting crRNA plasmids were used as controls to avoid errors in the inhibition rate calculation caused by changes in the upstream bases of the start codon affecting fluorescent protein expression.

[0057] For all 64 PAM cases Vca The inhibition ability of the QCas system against CRISPRi was measured, and the results are as follows: Figure 8 As shown in B. Among all PAM combinations, Vca The QCas system can identify 35 different PAM sequences and mediate effective CRISPRi inhibition regulation (inhibition rate >50%). Besides the typical 5'-NCC PAM sequence of I-F3 type Cas proteins, several other usable PAMs were detected. Information on PAM sequences capable of mediating effective regulation was collected, and base distribution probability statistics were performed. PAMs with inhibition rates >50% were enriched and a Weblogo conservation analysis plot was plotted, with the vertical axis representing the probability of base occurrence. The results are shown below. Figure 8 As shown in C. It was found that the base distribution at the -3 site is not conserved, while at the -2 and -1 sites, the presence of G / C makes the PAM sequence easier to identify.

[0058] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A gene expression regulation tool for Bacillus subtilis, characterized in that, The regulatory tool includes the QCascade system and crRNA. The components of the QCascade system include TniQ, Cas8, Cas7, and Cas6. The QCascade system is... Vch QCas、 Pse QCas or Vca QCas system; The Vch In the QCas system, the amino acid sequence of TniQ is shown in SEQ ID NO.1, the amino acid sequence of Cas8 is shown in SEQ ID NO.2, the amino acid sequence of Cas7 is shown in SEQ ID NO.3, and the amino acid sequence of Cas6 is shown in SEQ ID NO.

4. The Pse In the QCas system, the amino acid sequence of TniQ is shown in SEQ ID NO. 5, the amino acid sequence of Cas8 is shown in SEQ ID NO. 6, the amino acid sequence of Cas7 is shown in SEQ ID NO. 7, and the amino acid sequence of Cas6 is shown in SEQ ID NO.

8. Vca In the QCas system, the amino acid sequence of TniQ is shown in SEQ ID NO.9, the amino acid sequence of Cas8 is shown in SEQ ID NO.10, the amino acid sequence of Cas7 is shown in SEQ ID NO.11, and the amino acid sequence of Cas6 is shown in SEQ ID NO.

12.

2. The gene expression regulation tool according to claim 1, characterized in that, The QCascade system is integrated into the genome of Bacillus subtilis. mpr The crRNA is expressed using the pAD123 plasmid as the expression vector.

3. The gene expression regulation tool according to claim 1, characterized in that, The TniQ and Cas7 are connected via a constitutive promoter P as shown in SEQ ID NO.

14. sunA Regulation; Cas8 and Cas6 are regulated via the constitutive promoter P as shown in SEQ ID NO.

15. groES Regulation; crRNA is regulated by the constitutive promoter P, as shown in SEQ ID NO.

16. veg Regulate expression.

4. The gene expression regulation tool according to claim 1, characterized in that, The CRISPR array sequence matched by the QCascade system has sequences as shown in SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.

19.

5. A method for regulating gene expression in Bacillus subtilis, characterized in that, The method involves expressing crRNA via plasmid in Bacillus subtilis that integrates the QCascade system to regulate the expression of a target gene. The QCascade system comprises TniQ, Cas8, Cas7, and Cas6. Vch QCas、 Pse QCas or Vca QCas system; The Vch In the QCas system, the amino acid sequence of TniQ is shown in SEQ ID NO.1, the amino acid sequence of Cas8 is shown in SEQ ID NO.2, the amino acid sequence of Cas7 is shown in SEQ ID NO.3, and the amino acid sequence of Cas6 is shown in SEQ ID NO.

4. The Pse In the QCas system, the amino acid sequence of TniQ is shown in SEQ ID NO.5, the amino acid sequence of Cas8 is shown in SEQ ID NO.6, the amino acid sequence of Cas7 is shown in SEQ ID NO.7, and the amino acid sequence of Cas6 is shown in SEQ ID NO.

8. Vca In the QCas system, the amino acid sequence of TniQ is shown in SEQ ID NO.9, the amino acid sequence of Cas8 is shown in SEQ ID NO.10, the amino acid sequence of Cas7 is shown in SEQ ID NO.11, and the amino acid sequence of Cas6 is shown in SEQ ID NO.

12.

6. The method according to claim 5, characterized in that, The target gene is integrated into the genome. nprE The site is defined by a constitutive promoter P, as shown in the nucleotide sequence SEQ ID NO.

13. amyQ’ Regulation.

7. The method according to claim 5, characterized in that, The target gene is a fluorescent protein gene. gfpmut3 fluorescent protein gene gfpmut3 It has a sequence as shown in SEQ ID NO.

26.

8. The method according to claim 5, characterized in that, The Bacillus subtilis mentioned includes Bacillus subtilis WS9C.

9. The method according to claim 5, characterized in that, Targeting fluorescent protein genes gfpmut3 The n32 sequences are shown in SEQ ID NO.20 to SEQ ID NO.25, respectively.

10. The application of the regulatory tool according to any one of claims 1 to 4 or the method according to any one of claims 5 to 9 in the regulation of gene expression in Bacillus subtilis.

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