CRISPR-Cas9 Based Engineering of Actinomycetal Genomes

Abstract

Bacteria of the order Actinomycetales are one of the most important sources of pharmacologically active and industrially relevant secondary metabolites. Unfortunately, many of them are still recalcitrant to genetic manipulation, which is a bottleneck for systematic metabolic engineering. To facilitate the genetic manipulation of actinomycetes, we developed a highly efficient CRISPR-Cas9 system to delete gene(s) or gene cluster(s), implement precise gene replacements, and reversibly control gene expression in actinomycetes. We demonstrate our system by targeting two genes, actIORF1 (SCO5087) and actVB (SCO5092), from the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor A3(2). Our CRISPR-Cas9 system successfully inactivated the targeted genes. When no templates for homology-directed repair (HDR) were present, the site-specific DNA double-strand breaks (DSBs) introduced by Cas9 were repaired through the error-prone nonhomologous end joining (NHEJ) pathway, resulting in a library of deletions with variable sizes around the targeted sequence. If templates for HDR were provided at the same time, precise deletions of the targeted gene were observed with near 100% frequency. Moreover, we developed a system to efficiently and reversibly control expression of target genes, deemed CRISPRi, based on a catalytically dead variant of Cas9 (dCas9). The CRISPR-Cas9 based system described here comprises a powerful and broadly applicable set of tools to manipulate actinomycetal genomes.

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…Due to the lack of technology to freely engineer this species, discovery of novel drugs or enhancing the production of already produced natural products has been hampered from further development. However, along with the advances in the genetic tools for Streptomyces species engineering such as the recently enabled CRISPR for actinomycetes, [243] the prospect with which these native producers hold is bright. [5,244] Then, E. coli itself also has a large room to be developed further as a prominent heterologous host for polyketide biosynthesis.…”

Toward Systems Metabolic Engineering of Streptomycetes for Secondary Metabolites Production

“…However, efficiency and specificity are greatly improved if a homology template for HR is provided. [45][46][47] This has been demonstrated in independent studies, in which Cobb et al [45] and Huang et al [46] reported 60%100% and 70%100% efficiencies, respectively, whereas Tong et al [47] reported near 100% efficiency for their studies on the actinorhodin gene cluster in S. coelicolor.…”

“…The CRISPR-Cas9 technique can be applied for deletion of genes and gene clusters [45][46][47] in a multiplex manner, [45] reversible gene expression control, [47] and induction. [48] The optimized system relies on two components: the single guide RNA (sgRNA), which is a synthetic RNA consisting of a CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) complex, and is required for guiding Cas9 to modify the targeted genome sequence; and the endonuclease Cas9 or catalytically inactive dCas9, which upon interaction with the sgRNA, scans the genome for protospacer adjacent motif (PAM) sequences and the corresponding 20 bp recognition site.…”

“…Here, the dCas9 presents an easy-to-clone tool for reversible control of gene expression, and thus is useful for investigating regulation without the risk of off-target effects or subjecting the cells to the stress associated with gene replacements or mutagenesis procedures. [47] CRISPR-Cas9-mediated mutagenesis can be used in the absence of templates for HR. In this case, the repair mechanism is dependent on the nonhomologous end joining (NHEJ) of the DNA in Streptomyces species.…”

Genome-Editing Technologies: Principles and Applications

“…However, many Actinomycetales species are recalcitrant to genetic manipulation, a fact that has severely hampered their use for metabolic engineering. CRISPRCas9 has been used to inactivate multiple genes in actinomycetes (Tong et al 2015), indicating its ability to enable the creation of designer bacterial strains with enhanced metabolite production capabilities. CRISPR has also facilitated multiplexed metabolic pathway engineering in yeast at high efficiencies (Jakociunas et al 2015a,b), as well as random mutagenesis of yeast chromosomal DNA for phenotypic screening of desired mutants (Ryan et al 2014).…”