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Plasmid for crispri system, method for its construction and application in targeted gene silencing of Yersinia pestis

A gene and purpose technology, applied in the application of gene-directed silencing, the plasmid field of CRISPRi system, can solve the problems of complex operation and low efficiency, and achieve the effect of reducing transcription level and protein level

Active Publication Date: 2022-07-05
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the research on Yersinia pestis, the traditional gene knockout technology is based on the principle of homologous recombination. In recent years, reports have confirmed the feasibility of the application of the CRISPR / Cas system in Yersinia pestis ( Yan, M.Y., Yan, H.Q., Ren, G.X., Zhao, J.P., Guo, X.P., & Sun, Y.C. (2017). CRISPR-Cas12a-Assisted Recombineering in Bacteria. Appl Environ Microbiol, 83(17).), these gene editing techniques It can realize gene knockout in Yersinia pestis and modify and edit its genome. Although it can be used to study the gene function of Yersinia pestis, it has the disadvantages of complex operation, low efficiency, and irreversibility

Method used

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  • Plasmid for crispri system, method for its construction and application in targeted gene silencing of Yersinia pestis
  • Plasmid for crispri system, method for its construction and application in targeted gene silencing of Yersinia pestis
  • Plasmid for crispri system, method for its construction and application in targeted gene silencing of Yersinia pestis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: sgRNA expression plasmid and pdCas9-tetO plasmid

[0045] 1.1. Construction of plasmid pgRNA-phoP: The pgRNA vector (empty vector, its schematic structure is shown in 1A, obtained from Addgene#44251, website: https: / / www.addgene.org / ) as the template, using the following table 1 The primers phop-1o-F (SEQ ID NO: 21) and phop-1o-R (SEQ ID NO: 22) shown in Table 2 were applied to the pgRNA vector according to the system shown in Table 2 and the PCR program shown in Table 3 below. Carry out PCR amplification to obtain PCR amplification fragments, and then use restriction nucleases Spe I and HindIII to digest the pgRNA vector and its PCR amplification fragments respectively according to the restriction nucleases Spe I and HindIII according to the following table 4. The ligation system shown in Table 5 below connects the two restriction fragments to obtain an sgRNA expression plasmid (control plasmid) for the target gene phoP of Yersinia pestis, named plasmid pg...

Embodiment 2

[0086] Example 2: Use of Inducing Agents

[0087] 2.1. The inventors selected 9 genes (ail, cobB, cspB, hdeD, hmsH, ibpB, pla, slyA, yscB, each of which are related to the Yersinia pestis phenotype on the chromosome or plasmid in Yersinia pestis). The function of the gene is shown in Table 10 above), and the sgRNA expression plasmid constructed in Example 1 (see Table 10) and the dCas9-tetO plasmid are transformed into bacteria by electric shock to construct the corresponding target gene silencing strain, and use 1 μg / ml of aTc was used as the inducer, and the constructed silent strains were divided into silent strains under induced conditions (the inducer was added at the time of transformation) and silent strains under non-induced conditions. Meanwhile, wild control strains were set up. Each strain was grown to mid-log phase, or OD 620 is around 1.0.

[0088] Using the nucleotide sequences shown in Table 12 above, the qRT-PCR (real-time fluorescence quantitative PCR) exp...

Embodiment 3

[0092] Example 3: Protein Level Expression

[0093] The inventors selected 4 target genes hmsH, phoP, pla, slyA of Yersinia pestis (the respective gene functions are shown in Table 10 above), using the dual plasmid system corresponding to the target gene constructed in Example 1, transformed into the plague by electric shock The corresponding target gene silencing strain was constructed, and the induction culture was performed under non-inducing conditions and inducing conditions with 1 μg / ml aTc as the inducer (the inducer was added at the time of transformation), and the wild strain of Yersinia pestis was established at the same time. As a control, each strain tested was grown to mid-log phase, or OD 620 is around 1.0. Western blotting was used to verify the protein level changes of the target gene in the wild strain and the corresponding silenced strain, and the GroEL protein was used as the internal reference to keep the same amount of samples. The result is as Figur...

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Abstract

The invention discloses a plasmid for CRISPRi system, its construction method and its application in directional silencing of a gene of Yersinia pestis, belonging to the technical field of gene editing, wherein the plasmid comprises a sgRNA expression plasmid and a pdCas9-tetO plasmid. The double-plasmid CRISPRi system constructed by the invention can be transformed into Yersinia pestis, and the target gene can be silenced in the presence of the inducer anhydrotetracycline hydrochloride. The CRISPRi system of the invention can greatly reduce the transcription level and protein level of the target gene, and make Yersinia pestis show the relevant phenotype of the target gene deletion, and can be used as a novel tool for high-throughput gene function research in Yersinia pestis.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the field of gene editing technology, and particularly relates to a plasmid used in a CRISPRi system, a construction method thereof, and its application in directional silencing of a target gene of Yersinia pestis. Background technique [0002] Yersinia pestis (also referred to as Yersinia pestis hereinafter) is a causative bacterium of the natural foci of severe infectious disease-plague. As far back as 2,000 years ago, there were records of plague epidemics, which triggered three worldwide pandemics and brought huge disasters to mankind. [0003] The plague is a legal Class A infectious disease in my country, and Yersinia pestis is also one of the Class A biological warfare agents with strong infectivity and high pathogenicity. With the improvement of the level of epidemic prevention, the incidence of plague has been controlled, but in terms of treatment, there is no other effecti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/66C12N9/22
Inventor 王桐杜宗敏杨瑞馥宋亚军
Owner ACADEMY OF MILITARY MEDICAL SCI
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