8YGJ

SpCas9-MMLV RT-pegRNA-target DNA complex (elongation 28-nt)

8YGJ の概要

• エントリーDOI: 10.2210/pdb8ygj/pdb
• EMDBエントリー: 39253
• 分子名称: RNA (137-MER), DNA (51-MER), DNA (5'-D(P*TP*GP*AP*TP*GP*GP*CP*AP*GP*AP*GP*TP*AP*CP*TP*AP*G)-3'), ... (6 entities in total)
• 機能のキーワード: crispr-cas, rna binding protein-dna-rna complex, rna binding protein/dna/rna
• 由来する生物種: Streptococcus pyogenes; 詳細
• タンパク質・核酸の鎖数: 6
• 化学式量合計: 298370.82

構造登録者

Shuto, Y.,Nakagawa, R.,Hoki, M.,Omura, S.N.,Hirano, H.,Itoh, Y.,Nureki, O. (登録日: 2024-02-26, 公開日: 2024-06-05, 最終更新日: 2024-09-11)

主引用文献

Shuto, Y.,Nakagawa, R.,Zhu, S.,Hoki, M.,Omura, S.N.,Hirano, H.,Itoh, Y.,Zhang, F.,Nureki, O.
Structural basis for pegRNA-guided reverse transcription by a prime editor.
Nature, 631:224-231, 2024

PubMed Abstract: The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing guide RNA (pegRNA) to facilitate a wide variety of precise genome edits in living cells. However, owing to a lack of structural information, the molecular mechanism of pegRNA-guided reverse transcription by the prime editor remains poorly understood. Here we present cryo-electron microscopy structures of the SpCas9-M-MLV RTΔRNaseH-pegRNA-target DNA complex in multiple states. The termination structure, along with our functional analysis, reveals that M-MLV RT extends reverse transcription beyond the expected site, resulting in scaffold-derived incorporations that cause undesired edits at the target loci. Furthermore, structural comparisons among the pre-initiation, initiation and elongation states show that M-MLV RT remains in a consistent position relative to SpCas9 during reverse transcription, whereas the pegRNA-synthesized DNA heteroduplex builds up along the surface of SpCas9. On the basis of our structural insights, we rationally engineered pegRNA variants and prime-editor variants in which M-MLV RT is fused within SpCas9. Collectively, our findings provide structural insights into the stepwise mechanism of prime editing, and will pave the way for the development of a versatile prime editing toolbox.

PubMed: 38811740
DOI: 10.1038/s41586-024-07497-8

実験手法

ELECTRON MICROSCOPY (3.2 Å)