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Volume 7, Issue 11

Genome editing reveals that pSCL4 is required for chromosome linearity in Open Access

Abstract

is an industrially important actinomycete whose genetic manipulation is limited by low transformation and conjugation efficiencies, low levels of recombination of introduced DNA, and difficulty in obtaining consistent sporulation. We describe the construction and application of versatile vectors for Cas9-mediated genome editing of this strain. To design spacer sequences with confidence, we derived a highly accurate genome assembly for an isolate of the type strain (ATCC 27064). This yielded a chromosome assembly (6.75 Mb) plus assemblies for pSCL4 (1795 kb) and pSCL2 (149 kb). The strain also carries pSCL1 (12 kb), but its small size resulted in only partial sequence coverage. The previously described pSCL3 (444 kb) is not present in this isolate. Using our Cas9 vectors, we cured pSCL4 with high efficiency by targeting the plasmid’s gene. Five of the resulting pSCL4-cured isolates were characterized and all showed impaired sporulation. Shotgun genome sequencing of each of these derivatives revealed large deletions at the ends of the chromosomes in all of them, and for two clones sufficient sequence data was obtained to show that the chromosome had circularized. Taken together, these data indicate that pSCL4 is essential for the structural stability of the linear chromosome.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): actinomycetes genomics , Bionano optical mapping , CRISPR-Cas9 , next-generation sequencing , PacBio and plasmid curing
Funding
This study was supported by the:
  • Biotechnology and Biological Sciences Research Council (Award BB/N023536/1)
    • Principle Award Recipient: JasonMicklefield
  • Biotechnology and Biological Sciences Research Council (Award BB/N02351X/1)
    • Principle Award Recipient: BarrieWilkinson
  • Biotechnology and Biological Sciences Research Council (Award BBS/E/J/000PR9790)
    • Principle Award Recipient: NotApplicable
© 2021 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2021-11-08
2024-04-24
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Genome editing reveals that pSCL4 is required for chromosome linearity in Streptomyces clavuligerus
M Gen 7, 000669 (2021); https://doi.org/10.1099/mgen.0.000669
/content/journal/mgen/10.1099/mgen.0.000669
/content/journal/mgen/10.1099/mgen.0.000669
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