Systems Analysis of Highly Multiplexed CRISPR-Base Editing in Streptomycetes

Area of Science:

• Microbiology
• Molecular Biology
• Synthetic Biology

Background:

• CRISPR base editing tools, like CRISPR-BEST, have streamlined genetic manipulation in streptomycetes.
• Multiplexed genome editing is advantageous, especially in genomically unstable species.

Purpose of the Study:

• To demonstrate scaled-up multiplexed genome editing using Csy4-based CRISPR-mcBEST in Streptomyces coelicolor.
• To evaluate the performance of multiplexed editing at various scales, targeting up to all 28 specialized metabolite biosynthetic gene clusters.

Main Methods:

• Utilized Csy4-based CRISPR-mcBEST for multiplexed genome editing in Streptomyces coelicolor.
• Performed simultaneous targeting of 9, 18, and 28 gene clusters.
• Conducted multiomics analysis to assess system-wide effects.

Main Results:

• Successfully demonstrated scaled-up multiplexed genome editing targeting all 28 gene clusters in a single experiment.
• Provided insights into the performance and scalability of Csy4-based multiplexed genome editing.
• Multiomics analysis revealed both the potential and limitations of CRISPR-mcBEST.

Conclusions:

• CRISPR-mcBEST is a powerful tool for multiplexed genome editing in Streptomyces.
• This technology has significant potential for high-throughput engineering of Streptomyces and other microbial chassis.
• Further development is needed to overcome identified bottlenecks for broader application.