A method for enhancing genome editing efficiency of Streptomyces and its application

Abstract

The invention provides a method for enhancing the genome editing efficiency of Streptomyces and its application. By adding elements that control Cas9 activity, the triple control of Cas9 protein activity at the transcription, translation, and protein levels can be achieved, the cytotoxicity of Cas9 protein to Streptomyces can be inhibited, and the transformation efficiency of plasmids in Streptomyces can be improved. Achieve efficient gene editing. The present invention establishes a gene editing system that is both induced by small chemical molecules and regulated by inhibitory proteins, which can inhibit the toxicity of Cas9 in vivo, and realize the efficient introduction of genetic elements without affecting the transformation efficiency of edited plasmids and the growth and metabolism of host cells. and subsequent gene editing. Small-molecule chemical inducers are easy to add, and can realize efficient and precise genome editing based on double-strand breaks and directional repair. The present invention can be applied to Streptomyces genetic engineering and genetic modification including model or industrial Streptomyces.

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 genome editing method for inducible Streptomyces. Methods and applications for enhancing gene transformation efficiency and editing efficiency. Background technique [0002] The CRISPR(clustered regularly interspaced short palindromic repeats) / Cas(CRISPR-associated) system is a prokaryotic-specific immune system found in prokaryotes. The main function of this system is that the RNA-mediated (single-guide RNA, sgRNA) accompanied by a specific targeting sequence recognizes and degrades the foreign invaded DNA, causing the loss of function of the foreign invaded DNA and its inactivation. Due to such characteristics, the CRISPR / Cas9 system has been developed as a gene-directed editing technology targeting specific sites, with recognized advantages such as high efficie...

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

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