8/8 Finally, a big thank you to all co-authors - Peter, David, Renata, Tetiana, and @tilmweber.bsky.social ! This has been a fun collaboration!

7/8 We further further sequenced hosts after BAC integration and verified multicopy integrations, and showed that the genomes remain stable, even in non-selective medium.

If your are engineering streptomycetes, please give it a try and let us know how it goes! All plasmids are on Addgene!

6/8We then demonstrated how the high efficiencies of CASCADE-Cas3 can be used for streamlined host construction by substitution of large genomic regions with PhiC31 integration sites - allowing multicopy expression of BGCs. Reintroduction of the act BGC resulted in significantly higher production.

5/8 In all three strains, CASCADE-Cas3 installed the desired deletions with very high efficiencies (up to 100%)! This did not require modifications to the plasmids or modified methods, showcasing that CASCADE-Cas3 can be used "out of the box" across different species.

4/8 Furthermore, efficiencies appeared to be less dependant on which spacer was used, indicating less background/off-target activity. What about other streptomycetes? We picked S. albidoflavus, S. venezuelae, as well as NBC1270, an isolate from our collection, for further experiments.

3/8 So we were wondering - is CASCADE-Cas3 much better suited for genome engineering of streptomycetes? Targeting the act BGC of S. coelicolor, we got some very strong initial data supporting this hypothesis. We obtained very precise deletions with robustly high efficiencies.

2/8 The minimal type I-C CASCADE-Cas3 is fascinating. A complex consisting of Cas5, Cas7, and Cas8 binds the target site and recruits the processive nuclease Cas3, which generates recombinogenic overhangs! Sounds great for homologous recombination!

1/8 We found that type I CRISPR systems (like CASCADE-Cas3) are much more widespread in streptomycetes than type II CRISPR systems (e.g. Cas9). CASCADE-Cas3 also has a TA rich PAM, which translates to way fewer potential off-target sites.

Interested in engineering streptomycetes? Struggling with existing genome engineering tools? Then check out our newest paper on CASCADE-Cas3 based genome engineering in streptomycetes!

Some highlights below:

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research.state-of-denmark.com?utm_campaign...

It’s been a tough few weeks. My 10yo daughter was diagnosed with a very rare, aggressive cancer called interdigitating dendritic cell sarcoma (IDCS). I’m reaching out to identify clinicians/patients who have encountered pediatric IDCS or other (non-LCH) dendritic or histiocytic sarcomas cases.

If you’re at a European institution and are looking to poach American scientists, this would be a very good week to do it

📢 Check out this new tool from @thombooth.bsky.social, @wemakemolecules.bsky.social and @tilmweber.bsky.social!

Build phylogenies of BGCs to primates! 🙊

All you need are Gbk files of your chosen sequences. It's super fast and in my regular rotation of bioinformatic tools

#phylo #secmet #micro

Great starter pack! Would love to be added!

In July, we tripled the number of high-quality Streptomyces genomes in the NCBI. Today, we have published an updated pangenomic analysis of their biosynthetic potential! Very greatful to Omkar for including me in this project!!

🧪🦠 #microbiology

genomebiology.biomedcentral.com/articles/10....

Are you working with #streptomycetes? Then you may be interested in trying our new #StreptoCAD platform: StreptoCAD is an open-source software
toolbox automating genome engineering
workflows in streptomycetes. www.biorxiv.org/content/10.1...

StreptoCAD: An open-source software toolbox automating genome engineering workflows in streptomycetes https://www.biorxiv.org/content/10.1101/2024.12.19.629370v1