Systems Analysis of Highly Multiplexed CRISPR-Base Editing in Streptomycetes

Christopher M. Whitford, Tetiana Gren, Emilia Palazzotto, Sang Yup Lee, Yaojun Tong*, Tilmann Weber*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review


CRISPR tools, especially Cas9n-sgRNA guided cytidine deaminase base editors such as CRISPR-BEST, have dramatically simplified genetic manipulation of streptomycetes. One major advantage of CRISPR base editing technology is the possibility to multiplex experiments in genomically instable species. Here, we demonstrate scaled up Csy4 based multiplexed genome editing using CRISPR-mcBEST in Streptomyces coelicolor. We evaluated the system by simultaneously targeting 9, 18, and finally all 28 predicted specialized metabolite biosynthetic gene clusters in a single experiment. We present important insights into the performance of Csy4 based multiplexed genome editing at different scales. Using multiomics analysis, we investigated the systems wide effects of such extensive editing experiments and revealed great potentials and important bottlenecks of CRISPR-mcBEST. The presented analysis provides crucial data and insights toward the development of multiplexed base editing as a novel paradigm for high throughput engineering of Streptomyces chassis and beyond.

Original languageEnglish
JournalACS Synthetic Biology
Pages (from-to)2353−2366
Number of pages14
Publication statusPublished - 2023


  • base editor
  • cytidine deaminase
  • multiplexed genome engineering
  • Streptomyces


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