A method for enhancing the gene editing efficiency of actinomycetes and its application

A gene editing and efficiency technology, applied in the field of genetic engineering and genetic modification, can solve the problems of genetic modification failure, off-target effect and toxic activity hindering the application, and the reduction of transformants, so as to improve editing efficiency, inhibit cytotoxicity and off-target probability Effect

Active Publication Date: 2021-02-19
ZHEJIANG UNIV
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Problems solved by technology

[0004] However, off-target effects and toxic activities of the CRISPR / Cas9 system would greatly hinder its application
A visible consequence of Cas9 toxicity in microbial manipulations is a drastic reduction of transformants, e.g. in excellent genetic engineering hosts Escherichia coli, yeast and Streptomyces, most often leading to genetic modification failure

Method used

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  • A method for enhancing the gene editing efficiency of actinomycetes and its application
  • A method for enhancing the gene editing efficiency of actinomycetes and its application
  • A method for enhancing the gene editing efficiency of actinomycetes and its application

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Embodiment 1

[0033] The bacterial species selected in Example 1 is Streptomyces coelicolor A3 (2) (Bentley SD et al.Nature, 2002) (preservation number: CGMCC 4.7168), its whole genome sequence (sequence number: GenBank: NC_003888.3 ). Taking the knockout of actII-ORF4 gene in Streptomyces coelicolor A3(2) by this method as an example, the present invention will be described in detail, wherein Streptomyces coelicolor A3(2) is used as a model bacterium. Specific steps are as follows:

[0034] (1) According to literature reports on the regulation of related Streptomyces promoters (He Huang et al.Acta BiochimBiophys Sin, 2015), the tipAp promoter and ermEp* promoter induced by thiostrepton were screened out to obtain the Constitutive promoter of strain Streptomyces coelicolor A3(2) and strong promoter of atpD gene expression. (See Table 1 and attached figure 1 )

[0035] (2) According to the plasmid library of Streptomyces coelicolor A3(2) and the size and position of the determined elemen...

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Abstract

The present invention provides a method for enhancing the gene editing efficiency of actinomycetes and its application. Taking the deletion of actII-ORF4 in Streptomyces coelicolor as an example, the inducible promoter tipAp, ribosome switch and split Cas9 are used to control the activity of Cas9. Compared with the traditional constitutive Cas9 expression, the conversion efficiency is increased by about 260 times in the conversion process; the editing process adds inducers and increases the ATP concentration, so that the editing efficiency reaches 80%. The method of the present invention establishes a CRISPR / Cas9 system that can be induced by small chemical molecules and reversibly regulated by blue light, so as to ensure that the transformation efficiency of the plasmid and the normal growth and metabolism of the target host are not affected to the greatest extent, and realize the double-stranded The precise and high-efficiency gene editing of fragmentation, the invention is applied to the genetic engineering and genetic modification of other models including Streptomyces or industrial actinomycetes.

Description

technical field [0001] The invention belongs to the field of genetic engineering and genetic modification, and specifically relates to a method and application for enhancing gene editing efficiency by controlling Cas9 activity and ATP concentration in actinomycetes. Background technique [0002] CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) system is a unique immune system in prokaryotes, accompanied by RNA-mediated specific sequences, it recognizes and degrades foreign DNA, causing functional loss of foreign DNA its inactivation. In recent years, CRISPR / Cas9 system, as a gene-directed editing technology for specific sites, has the advantages of simple and convenient operation, low investment, high efficiency, good specificity and universality. The most powerful genome editing toolkit for molecular mechanism dissection and gene expression control, showing great potential in cellular reprogramming for mammalian protein engineering...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N15/76C12R1/465
Inventor 毛旭明李永泉刘一帆王凯罗帅
Owner ZHEJIANG UNIV
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