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CN-122012461-A - Bacillus subtilis gene expression regulation and control tool based on I-F3 CRISPR system

CN122012461ACN 122012461 ACN122012461 ACN 122012461ACN-122012461-A

Abstract

The invention discloses a bacillus subtilis gene expression regulation and control tool based on an I-F3 CRISPR system, and belongs to the field of genetic engineering. The CRISPRi inhibition capability of a VchQCas system derived from Vibrio cholerae in bacillus subtilis WS9C is verified through expression optimization, and a VcaQCas system with high CRISPRi regulation efficiency and PAM broad spectrum is identified from QCascade systems of 8I-F3 types based on bioinformatics methods such as gene mining, CRISPR element prediction and the like. Subsequently, the targeting preference and PAM suitability of the system in bacillus subtilis WS9C were analyzed in detail. The bacillus subtilis provides a novel gene expression regulation strategy as an exogenous protein expression host, and provides a reference for site selection of gene regulation.

Inventors

  • ZHANG KANG
  • WU JING
  • ZHU XIONGYANG
  • CHEN SHUMIN
  • CHEN YINGYING

Assignees

  • 江南大学

Dates

Publication Date
20260512
Application Date
20260128

Claims (10)

  1. 1. A bacillus subtilis gene expression regulation tool is characterized by comprising a QCascade system and crRNA, wherein elements of the QCascade system comprise TniQ, cas8, cas7 and Cas6, the QCascade system is VchQCas, pseQCas or VcaQCas system, the VchQCas system comprises a TniQ amino acid sequence shown in SEQ ID NO.1, a Cas8 amino acid sequence shown in SEQ ID NO.2, a Cas7 amino acid sequence shown in SEQ ID NO.3 and a Cas6 amino acid sequence shown in SEQ ID NO.4, the PseQCas system comprises a TniQ amino acid sequence shown in SEQ ID NO.5, a Cas8 amino acid sequence shown in SEQ ID NO.6, a Cas7 amino acid sequence shown in SEQ ID NO.7, a Cas6 amino acid sequence shown in SEQ ID NO.8, a VcaQCas system comprises a TniQ amino acid sequence shown in SEQ ID NO.9, a Cas8 amino acid sequence shown in SEQ ID NO.10, a Cas7 amino acid sequence shown in SEQ ID NO.3 and a Cas 12 amino acid sequence shown in SEQ ID NO. 6.
  2. 2. The gene expression control tool according to claim 1, wherein QCascade is integrated at the mpr locus of the bacillus subtilis genome, and the crRNA uses the pAD123 plasmid as an expression vector.
  3. 3. The gene expression control tool of claim 1, wherein TniQ and Cas7 are regulated by constitutive promoter P sunA as shown in SEQ ID No.14, cas8 and Cas6 are regulated by constitutive promoter P groES as shown in SEQ ID No.15, and crRNA is regulated by constitutive promoter P veg as shown in SEQ ID No. 16.
  4. 4. The gene expression control tool of claim 1, wherein the QCascade system matches CRISPR ARRAY sequence having the sequence shown as SEQ ID No.17, SEQ ID No.18, SEQ ID No. 19.
  5. 5. A bacillus subtilis gene expression regulation method is characterized in that crRNA is expressed through plasmids in bacillus subtilis integrated with a QCascade system to regulate expression of a target gene, elements of the QCascade system comprise TniQ, cas8, cas7 and Cas6, the QCascade system is VchQCas, pseQCas or VcaQCas, the amino acid sequence of TniQ in the VchQCas system is shown as SEQ ID NO.1, the amino acid sequence of Cas8 is shown as SEQ ID NO.2, the amino acid sequence of Cas7 is shown as SEQ ID NO.3, the amino acid sequence of Cas6 is shown as SEQ ID NO.4, the amino acid sequence of TniQ in the PseQCas system is shown as SEQ ID NO.5, the amino acid sequence of Cas8 is shown as SEQ ID NO.6, the amino acid sequence of Cas7 is shown as SEQ ID NO.7, the amino acid sequence of Cas6 is shown as SEQ ID NO.8, the amino acid sequence of TniQ in the VchQCas system is shown as SEQ ID NO.9, the amino acid sequence of Cas8 is shown as SEQ ID NO.8, and the amino acid sequence of Cas8 is shown as SEQ ID NO. 12.
  6. 6. The method according to claim 5, wherein the gene of interest is integrated at the nprE locus of the genome and is regulated by a constitutive promoter P amyQ' as shown in SEQ ID NO. 13.
  7. 7. The method of claim 5, wherein the gene of interest is fluorescent protein gene gfpmut, fluorescent protein gene gfpmut having the sequence shown in SEQ ID NO. 26.
  8. 8. The method of claim 5, wherein the bacillus subtilis comprises bacillus subtilis WS9C.
  9. 9. The method of claim 5, wherein the n32 sequence of the targeted fluorescent protein gene gfpmut is shown as SEQ ID NO. 20-SEQ ID NO.25, respectively.
  10. 10. Use of the regulation tool of any one of claims 1 to 4 or the method of any one of claims 5 to 9 in regulation of gene expression of bacillus subtilis.

Description

Bacillus subtilis gene expression regulation and control tool based on I-F3 CRISPR system Technical Field The invention relates to a bacillus subtilis gene expression regulation tool based on an I-F3 CRISPR system, and belongs to the technical field of biology. Background Bacillus subtilis (Bacillus subtilis) is a gram-positive model strain widely distributed in natural environment, and is authenticated by the U.S. food and drug administration (Food and Drug Adminisration, FDA) as a GRAS (Generally Recognized as Safe) microorganism and widely applied to industries such as food, medicine and the like due to characteristics such as non-pathogenicity and non-endotoxin production. Bacillus subtilis is an important production strain of alpha-amylase and neutral protease, and because of its lack of outer membrane structure, proteins can be secreted into the culture medium with high efficiency, thereby greatly reducing downstream purification costs in industrial applications. In addition, the bacillus subtilis has clear genetic background, the gene editing method is mature, and no obvious codon preference exists. How to realize accurate regulation of gene expression has become a key problem whether the biosynthesis capacity can be further improved at present. The gene expression regulation and control tool based on the CRISPR-Cas system has the advantages of being programmable, easy to construct and the like. CRISPR-Cas proteins are classified into class I and class II proteins, with class I proteins functioning using a polyprotein effector complex and class II proteins functioning dependent on a single effector protein. Existing CRISPR-Cas regulatory tools in bacillus subtilis are mainly constructed based on class II CRISPR-Cas systems, mainly including dCas9 and dCas12a. In recent years, the continued development of different types of CRISPR-Cas systems provides a rich enabling tool for gene editing and expression regulation in microbial cell factories. I-F3 type CRISPR-Cas systems have been used to mediate CAST directed transposition and enable multi-site large fragment genomic integration. The transposition system comprises two components of TniQ-Cascade (QCascade) and a Tns operon. Wherein QCascade includes TniQ, cas6 responsible for cleavage to generate mature crrnas, cas7 responsible for assembly of complex backbones, cas8 responsible for PAM site recognition, and CRRNA ARRAY. Meanwhile, the complex naturally lacks activity to cleave DNA strands, and Cas8 subunit has low PAM dependence. Thus, the I-F3 type QCascade system is expected to be used for developing low PAM dependent CRISPR gene expression regulation tools. Disclosure of Invention In order to solve the problem that the application of a bacillus subtilis gene expression regulation tool in the prior art is limited, the invention provides the bacillus subtilis gene expression regulation tool based on an I-F3 CRISPR system. The first technical scheme provided by the invention is a bacillus subtilis gene expression regulation tool, the regulation tool comprises a QCascade system and crRNA, elements of the QCascade system comprise TniQ, cas8, cas7 and Cas6, and the QCascade system is VchQCas, pseQCas or VcaQCas system. In one embodiment, the VchQCas, pseQCas or VcaQCas systems are derived from Vibrio choleraeHE-45, pseudoalteromonas sp.s983 and Vibrio campbelliiFXH286, respectively. In one embodiment, in the VchQCas 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. In one embodiment, in the PseQCas 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. In one embodiment, in the VcaQCas 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. In one embodiment, QCascade is integrated at the mpr locus of the bacillus subtilis genome, and the crRNA uses the pAD123 plasmid as an expression vector. In one embodiment, tniQ and Cas7 are regulated by constitutive promoter P sunA as shown in SEQ ID No.14, cas8 and Cas6 are regulated by constitutive promoter P groES as shown in SEQ ID No.15, and crRNA is expressed by constitutive promoter P veg as shown in SEQ ID No. 16. In one embodiment, the CRISPR ARRAY sequence to which the QCascade system is matched is identified by CRISPRCASFINDER software and has the CRISPR ARRAY sequence as shown in SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO. 19. The second technical scheme provided by the invention is a bacillus subtilis gene expression regulation method, which is characte