+Search query
-Structure paper
Title | Structural basis for pegRNA-guided reverse transcription by a prime editor. |
---|---|
Journal, issue, pages | Nature, Year 2024 |
Publish date | May 29, 2024 |
Authors | Yutaro Shuto / Ryoya Nakagawa / Shiyou Zhu / Mizuki Hoki / Satoshi N Omura / Hisato Hirano / Yuzuru Itoh / Feng Zhang / Osamu Nureki / |
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 ...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. |
External links | Nature / PubMed:38811740 |
Methods | EM (single particle) |
Resolution | 2.9 - 3.2 Å |
Structure data | EMDB-37858, PDB-8wus: EMDB-37859, PDB-8wut: EMDB-37860, PDB-8wuu: EMDB-37861, PDB-8wuv: EMDB-39253, PDB-8ygj: |
Source |
|
Keywords | RNA BINDING PROTEIN/RNA/DNA / CRISPR-Cas / RNA BINDING PROTEIN-RNA-DNA complex / RNA BINDING PROTEIN / RNA BINDING PROTEIN/DNA/RNA / RNA BINDING PROTEIN-DNA-RNA COMPLEX |