CRISPR/Cas9-Based Editing of Streptomyces for Discovery, Characterization, and Production of Natural Products

Abstract

Microbial natural products (NPs) especially of the Streptomyces genus have been regarded as an unparalleled resource for pharmaceutical drugs discovery. Moreover, recent progress in sequencing technologies and computational resources further reinforces to identify numerous NP biosynthetic gene clusters (BGCs) from the genomes of Streptomyces. However, the majority of these BGCs are silent or poorly expressed in native strains and remain to be activated and investigated, which relies heavily on efficient genome editing approaches. Accordingly, numerous strategies are developed, especially, the most recently developed, namely, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) system reveals remarkable higher accuracy and efficiency for genome editing in various model organisms including the Streptomyces. In this mini review, we highlight the application of CRISPR/Cas9-based approaches in Streptomyces, focus on the editing of BGCs either in vivo or in vitro, as well as target cloning of large-sized BGCs and heterologous expression in a genetically manipulatable host, for discovery, characterization, reengineering, and production of potential pharmaceutical drugs.

Characterization of Cas proteins for CRISPR-Cas editing in streptomycetes

“…Here, we present alternative CRISPR-Cas systems for genome editing in streptomycetes and showcase their potential to circumvent some of the limitations associated with the widely used and well characterized S. pyogenes CRISPR-Cas system (Zhang, et al, 2018, Tao, et al, 2018. While Cpf1 systems have recently examined for streptomycetes (Li, et al, 2018), this would be the first application of sth1Cas9 and saCas9 systems in streptomycetes.…”

“…The rapid adoption of this technology is due to its capability for precise editing and ease of use -a short RNA transcript is sufficient to guide the nuclease to its desired target, as well as the wide range of organisms that it can be applied in. These advantages have led us and other labs to explore CRISPR-Cas editing in streptomycetes (Li, et al, 2018, Zhang, et al, 2018, Tao, et al, 2018, which are prolific producers of bioactive secondary metabolites. Previously, we demonstrated that CRISPR-Cas increases the speed and throughput of genome editing compared to the traditional two step selection-counterselection protocol, allowing rapid genetic access to natural products encoded in the genomes of hitherto genetically intractable streptomycetes (Zhang, et al, 2018).…”

“…To date, Streptomyces strains have been mostly engineered using the well-characterized Streptococcus pyogenes Cas9 (spCas9, Tao, et al, 2018). Yet as we have observed in our labs and others (Moreb, et al, 2017, Cui, et al, 2018, a major bottleneck is that many bacteria strains still cannot be edited by S. pyogenes Cas9.…”

Recent advances in natural products exploitation in Streptomyces via synthetic biology

“…Luckily, with the advent of CRISPR systems, tools for site‐specific genome editing in Streptomyces were rapidly developed (Figure ). Those CRISPR‐based gene manipulation tools complement traditional approaches and facilitate discovery of new compounds with significant bioactivities . Ryan Cobb et al.…”

“…Luckily, with the advent of CRISPR systems, tools for site-specific genome editing in Streptomyces were rapidly developed ( Figure 2). Those CRISPR-based gene manipulation tools complement traditional approaches and facilitate discovery of new compounds with significant bioactivities [19,23,24]. Ryan Cobb et al [25] introduced the CRISPR/Cas9 system into Streptomyces and achieved fast multiplex genome editing via selected gene knock-in and knock-out.…”