Fine-tuning the regulation of Cas9 expression levels for efficient CRISPR-Cas9 mediated recombination in Streptomyces

Abstract

CRISPR-Cas9 has proven as a very powerful gene editing tool for Actinomyces, allowing scarless and precise genome editing in selected strains of these biotechnologically relevant microorganisms. However, its general application in actinomycetes has been limited due to its inefficacy when applying the system in an untested strain. Here, we provide evidence of how Cas9 levels are toxic for the model actinomycetes Streptomyces coelicolor M145 and Streptomyces lividans TK24, which show delayed or absence of growth. We overcame this toxicity by lowering Cas9 levels and have generated a set of plasmids in which Cas9 expression is either controlled by theophylline-inducible or constitutive promoters. We validated the targeting of these CRISPR-Cas9 system using the glycerol uptake operon and the actinorhodin biosynthesis gene cluster. Our results highlight the importance of adjusting Cas9 expression levels specifically in strains to gain optimum and efficient gene editing in Actinomyces.

Characterization and Engineering ofStreptomyces griseofuscusDSM 40191 as a Potential Host for Heterologous Expression of Biosynthetic Gene Clusters

“…Carbon sources uniquely utilized by S. griseofuscus include 2-aminoethanol, alpha-keto-valeric acid, D-malic acid. On the contrary, the number of nitrogen sources utilized were higher in S. griseofuscus (60) as compared to S. coelicolor (52) and S. venezuelae (49). These could be primarily attributed to the categories amino acid metabolism and other non-defined classes of metabolism.…”

“…S. griseofuscus is accessible to all these methods, implemented for single or multiplexed knockouts, which generate minimal off-target effects. Even though CRISPR-based systems were shown to be applicable in Actinobacteria, their use is still largely limited to model strains and typically requires further adjustments (60). It is, therefore, highly beneficial that S. griseofuscus does not require any of such additional efforts.…”

An Update on Molecular Tools for Genetic Engineering of Actinomycetes—The Source of Important Antibiotics and Other Valuable Compounds

“…The biggest advantage of the CRISPR/Cas technology is the fact that basically any site of interest can be targeted without laborious requirements. However, disadvantages like off-target effects [ 222 ] and Cas9 toxicity [ 223 , 224 , 225 ] must be considered.…”

“…One of the biggest drawbacks while working with CRISPR/Cas9 genome editing tools is the toxicity of Cas9 expression for the cells [ 223 , 224 , 225 ]. The level of Cas9 expression, therefore, must be tightly controlled.…”

“…This allows for controlling the Cas9 expression during CRISR-Cas9-mediated recombination experiments. The expression is either translationally regulated using theophylline or under the control of a relatively weak constitutive promoter [ 225 ].…”