9gs9 | pdb_00009gs9

Tn7016 PseCAST QCascade

spinqualitylabelsall models 9gs9, resolution 2.60Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 9gs9 is a 13 chain structure with sequence from DNA molecule, Pseudoalteromonas, Pseudoalteromonas agarivorans S816 and RNA satellites. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: Electron Microscopy, Resolution 2.6Å Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT 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 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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