Engineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite production

Abstract

The CRISPR-Cas9 system has frequently been used for genome editing in Streptomyces; however, cytotoxicity, caused by off-target cleavage, limits its application. In this study, we implement innovative modification to Cas9, strategically addressing challenges encountered during gene manipulation using Cas9 within strains possessing high GC content genome. The Cas9-BD, a modified Cas9 with the addition of polyaspartate to its N- and C-termini, is developed with decreased off-target binding and cytotoxicity compared with wild-type Cas9. Cas9-BD and similarly modified dCas9-BD have been successfully employed for simultaneous biosynthetic gene cluster (BGC) refactoring, multiple BGC deletions, or multiplexed gene expression modulations in Streptomyces. We also demonstrate improved secondary metabolite production using multiplexed genome editing with multiple single guide RNA libraries in several Streptomyces strains. Cas9-BD is also used to capture large BGCs using a developed in vivo cloning method. The modified CRISPR-Cas9 system is successfully applied to many Streptomyces sp., providing versatile and efficient genome editing tools for strain engineering of actinomycetes with high GC content genome.

Fig. 1. Modified Cas9s developed in this study and their in vitro DNA cleavage efficiencies.

a Cas9 modification scheme and proposed differential binding effects on the on- and off-target DNAs. b Representation of the wild-type and various modified Cas9s. c The 1.3 kb double stranded DNAs showing the specified target sequences used for in vitro cleavage reactions. d Cleavage efficiencies of various modified Cas9s on target DNAs. The efficiency of reactions was measured after 30 min of reaction and visualized using agarose gel electrophoresis. All reactions were performed in triplicates (n = 3). The mean was plotted with error bar representing standard deviation. P values were determined by unpaired two-tailed Student’s t-test based on cleavage efficiency of each protein with on-target linear DNA (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Source data are provided as a Source Data file.

Fig. 2. Cytotoxicity of wild-type Cas9 and Cas9-BD.

S. coelicolor M1146 colonies on agar plates after conjugation with various types of plasmids to compare toxicity between Cas9 and Cas9-BD.

Fig. 3. Improved metabolite production in Streptomyces sp. with multiplexed genome editing by Cas9-BD.

a The design of the promoter for the exchange of genes in the oviedomycin BGC in S. antibioticus NRRL 3238. The target genes, ovmOI, ovmOII, and ovmF, are colored red, purple, and green, respectively. The inserted promoters, ermE*p, kasO*p, and sp44p, are also represented. b Results of flask cultivation of the wild-type and engineered S. antibioticus NRRL 3238. Engineered strains with activated oviedomycin BGCs exhibited a dark brown color. c Exchanged promoters of each strain are listed. Blank fields in the table indicate that the promoter was not exchanged. d The design of the promoter for the exchange of genes in rapamycin BGC in S. rapamycinicus NRRL 5491. The genes of polyketide synthase, rapA, rapB, and rapC, are colored green, and the BGC activator gene and rapH are colored red. The inserted promoters, ermE*p, kasO*p, and sp44p, are also presented. e Results of flask cultivation with the wild-type and engineered S. rapamycinicus NRRL 5491 are shown. f Exchanged promoters of each strain are listed. Blank fields in the table indicate that the promoter was not exchanged. All cultivations were performed in triplicates (n = 3). The mean was plotted with error bar representing standard deviation. P values were determined by unpaired two-tailed Student’s t-test (ns, not significant; *, P < 0.05; **, P < 0.01, ***, P < 0.001). Source data are provided as a Source Data file.

Fig. 4. BGCs deletions in S. coelicolor M1146.

a Representation of the plasmids constructed for BGC deletions in S. coelicolor using Cas9-BD. Insertion of sgRNA to create a cleavage of BGC, and two homologous regions for recombination of ~1 kb were inserted after cleavage into a plasmid containing the gene Cas9-BD. To perform multiple deletion, sets of sgRNA and homologous regions were inserted in the plasmids. b–d Scheme of BGC deletion of desferrioxamine B, SapB, and SCO-2138 and confirmation via nested PCR. e Result of double deletion of desferrioxamine B and SapB. f Triple deletion, including desferrioxamine B, SapB, and SCO-2138 BGC. All conjugations were performed in a single replicate (n = 1). Source data are provided as a Source Data file. Ctl, product of PCR amplification using the genomic DNA of S. coelicolor M1146 as the template. Mar, DNA ladder marker; Des. B, desferrioxamine B.

Fig. 5. Capturing of FK506 BGC and production of FK506 in S. coelicolor M1146.

a The scheme for capturing, cloning, and refactoring large BGC to produce FK506 in S. coelicolor M1146. b HPLC plots of different strains harvested 8 d after cultivation. Metabolite peaks shown in both S. tsukubaensis NRRL 18488 and S. coelicolor-FKKN strains are pointed by dotted gray lines. FK506 peak is highlighted by a star. c FK506 production during flask cultivation of different strains was presented. All cultivations were performed in triplicates (n = 3). The mean was plotted with error bar representing standard deviation. P values were determined by unpaired two-tailed Student’s t-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Source data are provided as a Source Data file.

Fig. 6. Measurement of toxicity and inhibition efficiency of dCas9 and dCas9-BD in S. coelicolor M1146.

a Cell growth measured by phenylamine staining and (b) Expression of the gusA gene measured by GUS assay at 48 h after cultivation. The results of the strain without dCas9 and sgRNA are represented in black columns. Strains expressing wild-type dCas9 and dCas9-BD are represented in red and blue, respectively. Expressed sgRNA listed as C describes the absence of both dCas9 and sgRNA; 0 represents dCas9 without sgRNA; 1 describes the sgRNA targeted promoter region of the gusA gene; 2 describes the sgRNA targeted 5’ region of a gusA gene; 3 describes the sgRNA targeting the middle of a gusA gene; and 3 g describes expressing all three sgRNAs (1-3). All cultivations were performed in triplicates (n = 3). The mean was plotted with an error bar representing the standard deviation. P values were determined by unpaired two-tailed Student’s t-test with results obtained from strain lacking both dCas9 and sgRNA (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Source data are provided as a Source Data file.

Fig. 7. Secondary metabolite production in S. coelicolor A3(2) via utilization of dCas9-BD with multiple sgRNAs.

a The central metabolic pathway involved in the secondary metabolite production of S. coelicolor A3(2). The malonyl-CoA and acetyl-CoA highlighted in blue are precursors of target metabolites, actinorhodin, undecylprodigiosin, and coelimycin P2. The genes highlighted in red, pyc, gltA, adhE, fabD, and fabH, present the inhibition targets to achieve precursor accumulation. b The secondary metabolites actinorhodin, prodigiosin, and coelimycin P2, were produced by the 23 strains engineered using dCas9-BD. The five strains showing a dramatic improvement in secondary metabolite production are marked with blue arrows. c ACT, actinorhodin; d RED, undecylprodigiosin; e CPK P2, coelimycin P2 productions of the five strains in a flask cultivation; WT, S. coelicolor A3(2); WT*, S. coelicolor A3(2) harboring pdCIRSPomyces-2BD without sgRNA; S. coelicolor A3(2)*, S. coelicolor A3(2) harboring pdCRISPomyces-2BD without sgRNA. f The harbored sgRNAs in five high-producing strains. Flask cultivations were performed in triplicates (n = 3). The mean was plotted with an error bar representing the standard deviation. Source data are provided as a Source Data file.