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Savage Lab @savagecatsonly.bsky.social · Aug 5

Congrats to our savage lab undergrads on their research and presentations in our inaugural undergrad research symposium 🎉🥳

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Reposted by Savage Lab

Doudna Lab @doudna-lab.bsky.social · Jul 29

‼️ New pre-print from co-leads @owentuck.bsky.social and Jason Hu! Check out this fascinating example of how coevolution enables defense system innovation.

Owen Tuck @owentuck.bsky.social · Jul 29

Excited to finally share this work!
We noticed a pair of genes - a nuclease and a protease - shuffles between antiviral systems. We show how proteolysis activates the nuclease, triggering defense in known and unknown immune contexts.
tinyurl.com/2uwwy4ty

Recurrent acquisition of nuclease-protease pairs in antiviral immunity

Antiviral immune systems diversify by integrating new genes into existing pathways, creating new mechanisms of viral resistance. We identified genes encoding a predicted nuclease paired with a trypsin...

tinyurl.com

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Savage Lab @savagecatsonly.bsky.social · Jun 1

We love generating data! But what should we do with it? We collaborated w/ the Listgarten lab on ProteinGuide - a new method for using experimental data to guide protein generative models. Congrats to @junhaobearxiong.bsky.social, @hnisonoff.bsky.social, @marialukarska.bsky.social, Ishan, & Luke! ⬇️

Junhao (Bear) Xiong @junhaobearxiong.bsky.social · May 31

Guide your favorite protein generative model with experimental data? Meet ProteinGuide - a method to condition pre-trained models on properties without retraining. We validated it both in silico by guiding ProteinMPNN and ESM3 on 3 tasks and in vitro by engineering base editors.

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Reposted by Savage Lab

brittneywthornton.bsky.social @brittneywthornton.bsky.social · Mar 4

Excited to share our new preprint on mutational scanning of the TnpB RNP! We identify several highly activating, single-position mutations in TnpB’s protein and reRNA. These datasets enabled us to engineer enhanced TnpB protein variants (>50x plant editing levels compared to WT).

Savage Lab @savagecatsonly.bsky.social · Feb 26

What's better than 1 deep mutational scanning (DMS) library? 2! In a new pre-print @brittneywthornton.bsky.social and @rfw.bsky.social et al. map the mutational landscape of ISDra2 TnpB protein and reRNA and leverage these datasets to engineer highly active variants (1/9)

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Reposted by Savage Lab

Ben Adler @benadler.bsky.social · Feb 26

It is finally out!

Muntathar Al-Shimary, @doudna-lab.bsky.social , @cresslab.bsky.social and I present a gene knockdown tool that works in diverse bacteriophages: CRISPRi through antisense RNA Targeting (CRISPRi-ART)!

Check it out @naturemicrobiol.bsky.social
www.nature.com/articles/s41...

CRISPRi-ART enables functional genomics of diverse bacteriophages using RNA-binding dCas13d - Nature Microbiology

Leveraging RNA-targeting dCas13d enables selective interference with phage protein translation and facilitates measurement of phage gene fitness at a transcriptome-wide scale.

www.nature.com

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Savage Lab @savagecatsonly.bsky.social · Feb 26

Congrats to the whole team!! (Whole team not pictured)

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Savage Lab @savagecatsonly.bsky.social · Feb 26

(9/9) In addition to laying the groundwork for engineering, the data could provide valuable insights into TnpB evolution and function in transposable elements. If you want to read more check out the pre-print here: www.biorxiv.org/content/10.1...

Latent activity in TnpB revealed by mutational scanning

TnpB is an evolutionarily diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Due to their small size and putative evolutionary relationship to Cas12s, TnpB holds signi...

www.biorxiv.org

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Savage Lab @savagecatsonly.bsky.social · Feb 26

(8/9) They then chose 33 enriched muts across 19 AA positions & made & assayed a combinatorial protein library. 6 enriched variants were followed up on in HEK293T cells & N. benthamiana editing. All variants increased editing, with a max of 50-fold increase over WT in benthi!

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Savage Lab @savagecatsonly.bsky.social · Feb 26

(7/9) Enrichment varied by domain - many muts in RuvC and WED were enriched, most ZnF muts were depleted, muts in the unstructured C-terminal tail were ~neutral. Additionally, positive AAs were enriched w/i the vicinity of nucleic acids, particularly near the heteroduplex

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Savage Lab @savagecatsonly.bsky.social · Feb 26

(6/9) For the protein library, the authors made all single AA mutations and all stop codons. Surprisingly, ~20% of all mutations were enriched over WT!

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Savage Lab @savagecatsonly.bsky.social · Feb 26

(5/9) The hinge forms a sharp bend in the reRNA in stem 2, which is hypothesized to act as a regulatory switch for TnpB. Multiple groups have shown that stem 2 truncations increase TnpB editing, and our data suggests that hinge mutations may activate TnpB by a similar mechanism

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Savage Lab @savagecatsonly.bsky.social · Feb 26

(4/9) Starting with the reRNA– the library contained all single substitutions, 1-2 nt deletions, and larger secondary structure truncations. Many mutations were enriched over WT. Particularly, some (especially 1-2 nt dels) in the “hinge” region were highly enriched

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Savage Lab @savagecatsonly.bsky.social · Feb 26

(3/9) In the native genomic context, TnpB’s RNA scaffold (reRNA) is encoded in the protein’s mRNA. To map the mutational landscapes of the RNP components separately, they assayed two DMS libraries for the protein and RNA in an in vivo selection for endonuclease activity in yeast

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Savage Lab @savagecatsonly.bsky.social · Feb 26

(2/9) TnpB is an RNA-guided endonuclease found in prokaryotic transposons. Given its compact size, complex scaffold RNA, and putative ancestral relationship to Cas12s, they thought the TnpB ribonucleoprotein would be an ideal model for DMS

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Savage Lab @savagecatsonly.bsky.social · Feb 26

What's better than 1 deep mutational scanning (DMS) library? 2! In a new pre-print @brittneywthornton.bsky.social and @rfw.bsky.social et al. map the mutational landscape of ISDra2 TnpB protein and reRNA and leverage these datasets to engineer highly active variants (1/9)

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Reposted by Savage Lab

Innovative Genomics Institute @innovativegenomics.bsky.social · Feb 7

New research from IGI Investigator Dave Savage @savagecatsonly.bsky.social and first author Noam Prywes @prywes.bsky.social (pictured) details the landscape of possible Rubisco variants, suggesting ways to make the world's most abundant enzyme better at its job. 🌱🧪🧬

Read more: ow.ly/t2JO50ULh10

Photo of Noam Prywes standing in from of bamboo plants

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Savage Lab @savagecatsonly.bsky.social · Jan 24

Check out the summary and "behind the paper" here doi.org/10.1038/d415...

Mass testing of mutant enzymes involved in photosynthesis

Insights from a high-throughput screening assay of rubisco variants could be used to engineer improved versions of this key enzyme.

doi.org

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Savage Lab @savagecatsonly.bsky.social · Jan 24

Small changes in sequence can have big consequences in function, and not only negatively! We hope that this data is useful to other enzyme engineers and that this result expands our view of what is possible with even small changes. (7/7)

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Savage Lab @savagecatsonly.bsky.social · Jan 24

Several single AA mutations increased the CO2 affinity! Given sequence divergence and different oligomeric states of Form I & II, we were surprised to find single muts led to CO2 affinity outside of the range of Form II & at the edge of the distribution of plants and algae (6/7)

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Savage Lab @savagecatsonly.bsky.social · Jan 24

Rubisco is, after all, an enzyme, so we wanted to biochemically characterize each mutation. By repeating the assay over CO2 titration, key biochemical parameters (e.g. Vmax, Kc) are estimated for each mutant! (5/7)

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Savage Lab @savagecatsonly.bsky.social · Jan 24

This assay neatly identified known structural, functional, and evolutionary effects, confirming the robustness of the screen. For example, key residues in Loop 6 and the active site, are mutation-intolerant (4/7)

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Savage Lab @savagecatsonly.bsky.social · Jan 24

To address this, @prywes.bsky.social et. al developed an assay to estimate the fitness of 99% of single mutations in a high-throughput E. coli screen. E. coli growth rate is linked to enzyme behavior! (3/7)

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Savage Lab @savagecatsonly.bsky.social · Jan 24

As the entry-point for nearly all CO2 to the biosphere, rubisco is arguably one of the most important enzymes. Yet, Rubisco catalysis is slow. While there are 100s of biochemical measurements, our understanding of catalysis is limited by low-throughput in vitro assays (2/7)

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Savage Lab @savagecatsonly.bsky.social · Jan 24

What limits rubisco function? Is it the chemical mechanism? Evolution? In this paper, @prywes.bsky.social et. al explore this question by assaying >99% of single amino acid mutants in Form II rubisco (1/7) doi.org/10.1038/s415...

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Reposted by Savage Lab

Innovative Genomics Institute @innovativegenomics.bsky.social · Jan 22

New research from IGI Investigator Dave Savage and first author Noam Prywes details the landscape of possible Rubisco variants, suggesting ways to make the world's most abundant enzyme better at its job. 🌱

Read more: ow.ly/t2JO50ULh10

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