Method for enhancing streptomyces genome editing efficiency and application of method

Abstract

The invention provides a method for enhancing streptomyces genome editing efficiency and application of the method. By adding an element for controlling Cas9 activity, triple control of Cas9 protein activity on transcription, translation and protein levels is realized, cytotoxicity of Cas9 protein to streptomycete is inhibited, transformation efficiency of plasmids in streptomycete is improved, and efficient gene editing can be realized under induction conditions. According to the invention, a gene editing system which is induced by chemical small molecules and regulated and controlled by inhibitory proteins is established, in-vivo toxicity of Cas9 is inhibited, and efficient introduction of genetic elements and efficient editing of subsequent genes are realized on the premise of not influencing transformation efficiency of editing plasmids and growth and metabolism of host cells. The small molecule chemical inducer is convenient to add, and efficient and accurate genome editing based on double-strand breakage and directional repair can be achieved. The method of the inventioon can be applied to gene engineering and genetic modification transformation of streptomycetes including mode or industrial streptomycetes.

Description

technical field [0001] The invention belongs to the field of genetic engineering and genetic modification, and relates to a method for enhancing the genome editing efficiency of Streptomyces, which is an efficient inducible Streptomyces genome editing method. Methods and applications for enhancing gene transformation efficiency and editing efficiency. Background technique [0002] CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) system is a prokaryote-specific immune system found in prokaryotes. The main function of this system is RNA-mediated (single-guide RNA, sgRNA) accompanied by a specific targeting sequence, which recognizes and degrades the foreign invading DNA, causing the function loss of the foreign invading DNA and leading to its inactivation. Due to such characteristics, the CRISPR / Cas9 system was developed as a gene-directed editing technology for specific sites, which has recognized advantages such as high efficiency, ...

AI Technical Summary

Problems solved by technology

However, the genetic operating systems of most Streptomyces, especially Streptomyces for industrial production, are immature, and some industrial Streptomyces cannot even use the CRISPR / Cas9 system for gene editing

The reason is that in addition to the low efficiency of genetic manipulation of industrial Streptomyces itself, the cytotoxicity of CRISPR / Cas9 system to Streptomyces is also an important reason

At present, there are few regulated Streptomyces CRISPR / Cas9 systems developed, or the developed regulatory system is single, such as only at the level of transcription or translation, and the regulatory effect is not obvious; using light-induced regulation of Cas9 activity is difficult to control and operate, and it is difficult for Gene editing efficiency is difficult to effectively control

Images