Optimizing genome editing efficiency in Streptomyces fradiae via a CRISPR/Cas9n-mediated editing system

Abstract

Streptomyces fradiae is an important bioresource to produce various antibacterial natural products, however, the time-consuming and labor-intensive genome editing toolkits hindered the construction and application of engineered strains, and this study aimed to establish an efficient CRISPR/Cas9n genome editing system in S. fradiae. Initially, the CRISPR/Cas9-mediated editing tool was employed to replace those awkward genome editing tools that relied on homologous recombination, while the off-target Cas9 exhibited high toxicity to S. fradiae Sf01. Therefore, the nickase mutation D10A, high-fidelity mutations including N497A, R661A, Q695A, and Q926A, and thiostrepton-induced promotor P_tipA were incorporated into the Cas9 expression cassette, which reduced its toxicity. The deletion of single gene neoI and long fragment sequence (13.3 kb) were achieved with efficiencies of 77.8% and 44%, respectively. Additionally, the established tool was applied to facilitate the rapid deletion of nagB, replacement of P_frr with P_ermE*, and integration of exogenous vgbS, with respective efficiencies of 77.8%, 100%, and 67.8%, and all of the above modification strategies benefited neomycin synthesis in S. fradiae. Taken together, this research established an efficient CRISPR/Cas9n-mediated genome editing toolkit in S. fradiae, paving the way for developing high-performance neomycin-producing strains and facilitating the genetic modification of Streptomyces.IMPORTANCEThis study describes the development and application of a genome editing system mediated by CRISPR/Cas9n in Streptomyces fradiae for the first time, which overcomes the challenges associated with genome editing caused by high GC content (74.5%) coupling with complex genome structure, and reduces the negative impact of "off-target effect." Our work not only provides a facile editing tool for constructing S. fradiae strains of high-yield neomycin but also offers the technical guidance for the design of a CRISPR/Cas9n mediated genome editing tool in those creatures with high GC content genomes.

Keywords: CRISPR/Cas9D10A; Streptomyces fradiae; metabolic engineering; neomycin; single strand breaks repair.

Fig 1

Expression cassette maps of Cas9 and sgRNA.

Fig 2

Construction and ability assessment of Cas9 mediated vectors. (A) pTHF-Cas9 plasmid map. (B) Conjugation efficiency of vectors in S. fradiae Sf01. (C) Conjugation efficiency of vectors in S. coelicolor M145. (D) The effect of D10A mutation introduction on conjugation efficiency (statistical analyses by t-test; * P < 0.05, ** P < 0.01, *** P < 0.001; N.S., no significant difference).

Fig 3

Evaluation of pEHF-Cas9n and pTHF-Cas9n for the construction of Sf01ΔneoI. (A) Editing processing of Sf01ΔneoI using pHF-Cas9n. (B) PCR validation of Sf01ΔneoI through pEHF-Cas9n. Line M, DL 5000 DNA marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); line W, PCR product of Sf01 using ΔneoI-YF/ΔneoI-YR, 948 bp; line 1–9, PCR product of Sf01ΔneoI using ΔneoI-YF/ΔneoI-YR, 597 bp for expected size. (C) Effect of thiostrepton concentration on the editing capability of pTHF-Cas9n.

Fig 4

Deletion of gene nagB and its effect on neomycin production. (A) Assembly procession of pTC9n-ΔnagB. (B) PCR validation of Sf01ΔnagB. Line W, PCR product of Sf01 using ΔnagB-YF/ΔnagB-YR, 994 bp; line M, DL 5000 DNA marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); line 1–9, PCR product of Sf01ΔnagB using ΔnagB-YF/ΔnagB-YR, 447 bp for expected size. (C) Neomycin titer assessment (statistical analyses by t-test; * P < 0.05).

Fig 5

Replacement of frr promotor and its effect. (A) Assembly procession of pTC9n-ΔP_frr::P_ermE*. (B) PCR validation of Sf01ΔP_frr::P_ermE*. Line W, PCR product of Sf01 using P_frr-YF/P_frr-YR, 286 bp; line M, DL 5000 DNA marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); line 1–7, PCR product of Sf01ΔP_frr::P_ermE* using P_frr-YF/P_frr-YR, 619 bp for expected size. (C) Transcription levels of gene frr. (D) Neomycin titer assessment (statistical analyses by t-test; * P < 0.05, ** P < 0.01, *** P < 0.001).

Fig 6

Integration of vgbS expression cassette and its effect on neomycin production. (A) Assembly procession of pTC9n-ΔnagB::P_ermE*-SP_phoD-vgbS. (B) PCR validation of SF2. Line W, PCR product of Sf01 using ΔnagB-YF/ΔnagB-YR, 994 bp; line M, DL 5000 DNA marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); line 1–7, PCR product of SF2 using ΔnagB-YF/ΔnagB-YR, 1,442 bp for expected size. (C) Neomycin titer assessment (statistical analyses by t-test; * P < 0.05; N.S., no significant difference).