Christopher Whitford

A cloning strategy as depicted in the OpenCloning website

#OpenCloning is a an Open Source alternative to SnapGene/Benchling that supports automation and integration with other software

✅ Free
🔓 Open Source
🧬 More cloning methods than SnapGene
🤖 Can be automated with python
👨‍🔬 Built by a researcher — for researchers!

👉 Check it out at opencloning.org

Check out this new amazing preprint by David, @tuesparholt.bsky.social , @thombooth.bsky.social, and @tilmweber.bsky.social!

Check out the webserver (lnkd.in/eK4MnutJ) if you prefer a GUI, or GitHub (lnkd.in/euj6HcSM) for the code!

We are already using StreptoCAD to significantly speed up experimental workflows, and are already actively working on adding new capabilities to the pipeline! Stay tuned for more!

All design parameters are automatically tracked, making it very easy to reproduce & compare experimental outcomes. All workflows further come with html protocols, so it's super easy to just get started in the lab.

StreptoCAD: An Open-Source Software Toolbox Automating Genome Engineering Workflows in Streptomycetes Streptomycetes hold immense potential for discovering novel bioactive molecules for applications in medicine or sustainable agriculture. However, high-throughput exploration is hampered by the current Streptomyces genetic engineering methods that involve the manual design of complex experimental molecular biological engineering strategies for each targeted gene. Here, we introduce StreptoCAD, an open-source software toolbox that automates and streamlines the design of genome engineering strategies in Streptomyces, supporting various CRISPR-based and gene overexpression methods. Once initiated, StreptoCAD designs all necessary DNA primers and CRISPR guide sequences, simulates plasmid assemblies (cloning) and the resulting modification of the genomic target(s), and further summarizes the information needed for laboratory implementation and documentation. StreptoCAD currently offers six design workflows, including the construction of overexpression libraries, base-editing, including multiplexed CRISPR-BEST plasmid generation, and genome engineering using CRISPR-Cas9, CRISPR-Cas3, and CRISPRi systems. In addition to automating the design process, StreptoCAD further secures compliance with the FAIR principles, ensuring reproducibility and ease of data management via standardized output files. To experimentally demonstrate the design process and output of StreptoCAD, we designed and constructed a series of gene overexpression strains, and performed CRISPRi knockdowns in Streptomyces Gö40/10, underscoring the tool’s efficiency and user-friendliness.. This tool simplifies complex genetic engineering tasks and promotes collaboration through standardized workflows and design parameters. StreptoCAD is set to transform genome engineering in Streptomyces, making sophisticated genetic manipulations accessible for all and accelerating natural product discovery.

New paper alert!!

Led by Lucas, we automated all the design steps for genome engineering experiments in streptomycetes. If you are using our Streptomyces CRISPR toolbox, you can now design hundreds of experiments in a matter of minutes.

@tilmweber.bsky.social @kblin.bsky.social
....

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.

CASCADE-Cas3 enables highly efficient genome engineering in Streptomyces species Abstract. Type I clustered regularly interspaced short palindromic repeat (CRISPR) systems are widespread in bacteria and archaea. Compared to more widely

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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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

getphylo: rapid and automatic generation of multi-locus phylogenetic trees - BMC Bioinformatics Background The increasing amount of genomic data calls for tools that can create genome-scale phylogenies quickly and efficiently. Existing tools rely on large reference databases or require lengthy d...

📢 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!

Pangenome mining of the Streptomyces genus redefines species’ biosynthetic potential - Genome Biology Background Streptomyces is a highly diverse genus known for the production of secondary or specialized metabolites with a wide range of applications in the medical and agricultural industries. Several...

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....

StreptoCAD: An open-source software toolbox automating genome engineering workflows in streptomycetes Streptomycetes hold immense potential for discovering novel bioactive molecules for applications in medicine or sustainable agriculture. However, high-throughput exploration is hampered by the current...

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