One-step high-efficiency CRISPR/Cas9-mediated genome editing in <italic>Streptomyces</italic>

Abstract

The RNA-guided DNA editing technology CRISPRs (clustered regularly interspaced short palindromic repeats)/Cas9 had been used to introduce double-stranded breaks into genomes and to direct subsequent site-specific insertions/deletions or the replacement of genetic material in bacteria, such as Escherichia coli, Streptococcus pneumonia, and Lactobacillus reuteri. In this study, we established a high-efficiency CRISPR/Cas9 genome editing plasmid pKCcas9dO for use in Streptomyces genetic manipulation, which comprises a target-specific guide RNA, a codon-optimized cas9, and two homology-directed repair templates. By delivering pKCcas9dO series editing plasmids into the model strain Streptomyces coelicolor M145, through one-step intergeneric transfer, we achieved the genome editing at different levels with high efficiencies of 60%-100%, including single gene deletion, such as actII-orf4, redD, and glnR, and single large-size gene cluster deletion, such as the antibiotic biosynthetic clusters of actinorhodin (ACT) (21.3 kb), undecylprodigiosin (RED) (31.6 kb), and Ca 2+ -dependent antibiotic (82.8 kb). Furthermore, we also realized simultaneous deletions of actII-orf4 and redD, and of the ACT and RED biosynthetic gene clusters with high efficiencies of 54% and 45%, respectively. Finally, we applied this system to introduce nucleotide point mutations into the rpsL gene, which conferred the mutants with resistance to streptomycin. Notably, using this system, the time required for one round of genome modification is reduced by one-third or one-half of those for conventional methods. These results clearly indicate that the established CRISPR/Cas9 genome editing system substantially improves the genome editing efficiency compared with the currently existing methods in Streptomyces, and it has promise for application to genome modification in other Actinomyces species.

Direct Involvement of the Master Nitrogen Metabolism Regulator GlnR in Antibiotic Biosynthesis in Streptomyces

“…The ⌬glnR mutant was generated previously using the CRISPR/Cas9-mediated genome editing system (15). In comparison with M145, the ⌬glnR mutant exhibited impaired bacterial growth on both culture conditions, particularly during the early growth stage (supplemental Figs.…”

“…The first step is to delete the upstream region of actII-ORF4 (294 bp with respect to the translation start codon), and the second step involved in situ complementation of the deleted region using the mutated actII-ORF4 upstream region. Both steps were performed using the CRISPR/Cas9-mediated method (15). For deletion of the upstream region of actII-ORF4, a cassette for the transcription of a specific single guide RNA (sgRNA; in this case, actIIpdelsgRNA) was amplified from the plasmid pCB003 using the primer pair actIIpdel-gRNA-fw/rv.…”

“…The mutant with base mutations in the GlnR-binding site upstream of redZ was generated in one step using CRISPR/ Cas9-mediated genome editing technology (15). The primer pair redZpmu-gRNA-fw/rv was used for amplification of the specific sgRNA (redZpmu-sgRNA) transcription cassette.…”

Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

“…Using the CRISPR/Cas9 system for selection of E. coli genome editing, up to 65% of cells were found to possess the desired chromosomal mutation specified by the synthetic oligonucleotide (24), rendering CRISPR/Cas9 recombineering the most powerful methodology for engineering bacterial genomes to date (26). In addition to E. coli and Streptococcus (24,27), this technology has recently been adapted for use with lactic acid bacteria (28), Streptomyces (29), and Clostridium (30).…”

Bio-based production of fuels and industrial chemicals by repurposing antibiotic-producing type I modular polyketide synthases: opportunities and challenges

“…Finally, CRISPR-Cas9 systems have been developed for genome editing of Streptomyces strains. [35][36][37] Efficient generation of gene deletions were demonstrated in S. coelicolor, S. lividans, S. albus, Streptomyces viridochromogenes and Streptomyces pristinaespiralis. This promising tool should also be tested for the efficacy of introducing single or multiple insertions in various Streptomyces hosts.…”