CaExTun: Mitigating Cas9-Related Toxicity in Streptomyces through Species-Specific Expression Tuning with Randomized Constitutive Promoters.

ABSTRACT

The CRISPR/Cas9 system provides an efficient tool for engineering genomes. However, its application to Streptomyces genome engineering has been hampered by excessive toxicity associated with overexpression of Cas9 protein. As the level of Cas9 toxicity varies significantly between Streptomyces species, species-specific optimization of Cas9 expression is a strategy to mitigate its toxicity while maintaining sufficient double-strand break (DSB) activity for genome engineering. Using a pool of randomized constitutive promoters and a blue pigment indigoidine biosynthetic gene (IndC) as a reporter, we developed the CaExTun (Cas9 Expression Tuning) platform, which enables rapid screening of a large pool of promoter-Cas9 constructs to quickly recover the one with high DSB activity and no apparent toxicity. We demonstrate the utility of CaExTun using four model Streptomyces species. We also show that CaExTun can be applied to the CRISPRi system by allowing the construction of a library of promoter-dCas9 constructs that confer a wide range of gene repression levels. As demonstrated here, CaExTun is a versatile tool for the rapid optimization of the CRISPR/Cas9 system in a species-specific manner and thus will facilitate CRISPR/Cas9-based genome engineering efforts in Streptomyces.