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Method capable of enhancing actinomycete gene editing efficiency and application thereof

A technology of gene editing and actinomycetes, which is applied in the field of genetic engineering and genetic modification, can solve problems such as off-target effects and toxic activities hindering application, genetic modification failure, and reduction of transformants, so as to inhibit cytotoxicity and off-target probability, and improve The effect of editing efficiency

Active Publication Date: 2019-05-28
ZHEJIANG UNIV
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AI Technical Summary

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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  • Method capable of enhancing actinomycete gene editing efficiency and application thereof
  • Method capable of enhancing actinomycete gene editing efficiency and application thereof
  • Method capable of enhancing actinomycete gene editing efficiency and application thereof

Examples

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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 invention provides a method capable of enhancing actinomycete gene editing efficiency and application thereof. According to the method, actII-ORF4 in streptomyces coelicolor is deleted, and an inducible promoter, a ribosome switch and split C as9 are utilized to control Cas9 activity; compared with traditional constitutive Cas9 expression, the converting efficiency of a converting process is improved by about 260 times; in an editing process, an inducer is added, the ATP concentration is improved, and the editing efficiency can reach 80%. By means of the method disclosed by the invention,a CRISPR / Cas9 system which not only can be induced by chemical micromolecules, but also can be reversibly regulated by blue light is established; thus, accurate high-efficiency gene editing based on breakage of double strands can be achieved under the situation that the plasmid transformation efficiency and the maximum limit of normal growth of a target host are not affected. The method disclosedby the invention can be applied to other modes including streptomycete or gene engineering and genetic modification transformation of 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 Applications(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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