9GS9

Tn7016 PseCAST QCascade

これはPDB形式変換不可エントリーです。

9GS9 の概要

• エントリーDOI: 10.2210/pdb9gs9/pdb
• EMDBエントリー: 51543
• 分子名称: crRNA, T-DNA, NT-DNA, ... (7 entities in total)
• 機能のキーワード: crispr, transposon, genome, rna
• 由来する生物種: Pseudoalteromonas; 詳細
• タンパク質・核酸の鎖数: 13
• 化学式量合計: 486966.95

構造登録者

Lampe, G.D.,Liang, A.R.,Zhang, D.J.,Fernandez, I.S.,Sternberg, S.H. (登録日: 2024-09-13, 公開日: 2024-10-16, 最終更新日: 2024-11-20)

主引用文献

Lampe, G.D.,Liang, A.R.,Zhang, D.J.,Fernandez, I.S.,Sternberg, S.H.
Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells.
Biorxiv, 2024

PubMed Abstract: 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 Tn transposon (CAST) for DSB-free, RNA-guided DNA integration in human cells, taking advantage of its programmability and large payload capacity. CAST 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 CAST 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.

PubMed: 39345383
DOI: 10.1101/2024.09.19.613948

実験手法

ELECTRON MICROSCOPY (2.6 Å)