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.

Keywords: AID system; CRISPR dCas9; Streptomyces; UGI; actinorhodin.

FIGURE 1

Plasmid construction. (A) Step to prepare ermE promoter and gRNA region. (B) The construction of vector backbones integrating the fused dCasPmCDA1‐UGI. (C) Complete vector.

FIGURE 2

Overview of genome editing using CRISPR‐dCas9‐PmCDA‐UGI in S. coelicolor.

FIGURE 3

Essential genes for actinorhodin production. (A) Actinorhodin gene cluster. (B) Mutated target gene actVA‐ORF4. The positions of PAM sequence (GGC) and target 10 are given. (C) The position of SCO5079 protein (actVA‐ORF4) in actinorhodin biosynthesis.

FIGURE 4

Genome editing using dCas9‐PmCDA‐UGI‐AAV system. (A) Phenotypical analysis of five mutants selected and grew on ISP2 medium agar, the mutant with targeted actVA‐ORF4. Wild‐type S. coelicolor, pGM1190, and Empty vector (dcas9‐PmCDA‐UGI‐AAV without target) were used as control experiments. (B) Sequence alignment between Wild type and target 10 (actVA‐ORF4) edited by dCas9‐PmCDA‐UGI‐AAV. The PAM sequence is shown in the red box, and the site targeted by sgRNA is indicated by a brown underline. Uncolored bases are seen in single mutation results. (C) A typical sequencing chromatogram showing the mutation from C to T in the target location. S. coelicolor wild‐type strain was used as a control.

FIGURE 5

The actinorhodin color differences from the cultures of WT (Wild type), Empty (plasmid CRISPR system with no expected target), Target 10 (plasmid CRISPR system with 20 nucleotides from gen actVA‐ORF4 as targets) At a pH of 2, actinorhodin undergoes a color change, shifting from its initial blue to a distinct red are depicted in the photograph (A) and observed at various absorbances (B).

FIGURE 6

The outcome of assessing mutations in the open reading frame (ORF) genome plays a crucial role in producing actinorhodin. (A) The mutated gene critical for actinorhodin production was analyzed and compared to the reference sequence from the S. coelicolor wild type. The study recorded the total count of single nucleotide variants (SNVs) in this analysis. (B) The number of mutations based on the mutation types, including SNVs, deletions, and insertions, were observed in the critical genes. (C) The single nucleotide variations (SNPs) are observed in multiple ORFs, these mutations do not result in the creation of a stop codon.