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

(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...

(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!

(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

(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!

(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

(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

(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

(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

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

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)

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)

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)

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)

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)

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)