9gs9: Difference between revisions
New page: '''Unreleased structure''' The entry 9gs9 is ON HOLD Authors: Lampe, G.D., Liang, A.R., Zhang, D.J., Fernandez, I.S., Sternberg, S.H. Description: Tn7016 PseCAST QCascade [[Category: U... |
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==Tn7016 PseCAST QCascade== | |||
<StructureSection load='9gs9' size='340' side='right'caption='[[9gs9]], [[Resolution|resolution]] 2.60Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[9gs9]] is a 13 chain structure with sequence from [https://en.wikipedia.org/wiki/DNA_molecule DNA molecule], [https://en.wikipedia.org/wiki/Pseudoalteromonas Pseudoalteromonas], [https://en.wikipedia.org/wiki/Pseudoalteromonas_agarivorans_S816 Pseudoalteromonas agarivorans S816] and [https://en.wikipedia.org/wiki/RNA_satellites RNA satellites]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=9GS9 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=9GS9 FirstGlance]. <br> | |||
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">Electron Microscopy, [[Resolution|Resolution]] 2.6Å</td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=9gs9 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=9gs9 OCA], [https://pdbe.org/9gs9 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=9gs9 RCSB], [https://www.ebi.ac.uk/pdbsum/9gs9 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=9gs9 ProSAT]</span></td></tr> | |||
</table> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in a heterogeneous mixture of undesirable editing outcomes. We recently leveraged a type I-F CRISPR-associated transposase (CAST) from the Pseudoalteromonas Tn7016 transposon (PseCAST) for DSB-free, RNA-guided DNA integration in human cells, taking advantage of its programmability and large payload capacity. PseCAST is the only characterized CAST system that has achieved human genomic DNA insertions, but multiple lines of evidence suggest that DNA binding may be a critical bottleneck that limits high-efficiency activity. Here we report structural determinants of target DNA recognition by the PseCAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), which revealed novel subtype-specific interactions and RNA-DNA heteroduplex features. By combining our structural data with target DNA library screens and rationally engineered protein mutations, we uncovered CAST variants that exhibit increased integration efficiency and modified PAM stringency. Structure predictions of key interfaces in the transpososome holoenzyme also revealed opportunities for the design of hybrid CASTs, which we leveraged to build chimeric systems that combine high-activity DNA binding and DNA integration modules. Collectively, our work provides unique structural insights into type I-F CAST systems while showcasing multiple diverse strategies to investigate and engineer new RNA-guided transposase architectures for human genome editing applications. | |||
Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells.,Lampe GD, Liang AR, Zhang DJ, Fernandez IS, Sternberg SH bioRxiv [Preprint]. 2024 Sep 19:2024.09.19.613948. doi: , 10.1101/2024.09.19.613948. PMID:39345383<ref>PMID:39345383</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
[[Category: | <div class="pdbe-citations 9gs9" style="background-color:#fffaf0;"></div> | ||
[[Category: | == References == | ||
[[Category: | <references/> | ||
[[Category: Fernandez | __TOC__ | ||
[[Category: Lampe | </StructureSection> | ||
[[Category: DNA molecule]] | |||
[[Category: Large Structures]] | |||
[[Category: Pseudoalteromonas]] | |||
[[Category: Pseudoalteromonas agarivorans S816]] | |||
[[Category: RNA satellites]] | |||
[[Category: Fernandez IS]] | |||
[[Category: Lampe GD]] | |||
[[Category: Liang AR]] | |||
[[Category: Sternberg SH]] | |||
[[Category: Zhang DJ]] | |||