Editing Streptomyces genome using target AID system fused with UGI-degradation tag

Abstract

The utilization of Streptomyces as a microbial chassis for developing innovative drugs and medicinal compounds showcases its capability to produce bioactive natural substances. Recent focus on the clustered regularly interspaced short palindromic repeat (CRISPR) technology highlights its potential in genome editing. However, applying CRISPR technology in certain microbial strains, particularly Streptomyces, encounters specific challenges. These challenges include achieving efficient gene expression and maintaining genetic stability, which are critical for successful genome editing. To overcome these obstacles, an innovative approach has been developed that combines several key elements: activation-induced cytidine deaminase (AID), nuclease-deficient cas9 variants (dCas9), and Petromyzon marinus cytidine deaminase 1 (PmCDA1). In this study, this novel strategy was employed to engineer a Streptomyces coelicolor strain. The target gene was actVA-ORF4 (SCO5079), which is involved in actinorhodin production. The engineering process involved introducing a specific construct [pGM1190-dcas9-pmCDA-UGI-AAV-actVA-ORF4 (SCO5079)] to create a CrA10 mutant strain. The resulting CrA10 mutant strain did not produce actinorhodin. This outcome highlights the potential of this combined approach in the genetic manipulation of Streptomyces. The failure of the CrA10 mutant to produce actinorhodin conclusively demonstrates the success of gene editing at the targeted site, affirming the effectiveness of this method for precise genetic modifications in Streptomyces.