1. Structural basis for pegRNA-guided reverse transcription by a prime editor.
- Author
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Shuto Y, Nakagawa R, Zhu S, Hoki M, Omura SN, Hirano H, Itoh Y, Zhang F, and Nureki O
- Subjects
- Humans, Cryoelectron Microscopy, DNA chemistry, DNA metabolism, DNA genetics, DNA ultrastructure, Models, Molecular, Ribonuclease H deficiency, Ribonuclease H genetics, Viral Proteins chemistry, Viral Proteins metabolism, Viral Proteins ultrastructure, Viral Proteins genetics, HEK293 Cells, CRISPR-Associated Protein 9 chemistry, CRISPR-Associated Protein 9 metabolism, CRISPR-Associated Protein 9 genetics, CRISPR-Associated Protein 9 ultrastructure, Gene Editing, Moloney murine leukemia virus enzymology, Moloney murine leukemia virus genetics, Reverse Transcription, RNA, Guide, CRISPR-Cas Systems chemistry, RNA, Guide, CRISPR-Cas Systems genetics, RNA, Guide, CRISPR-Cas Systems metabolism, RNA, Guide, CRISPR-Cas Systems ultrastructure, RNA-Directed DNA Polymerase chemistry, RNA-Directed DNA Polymerase metabolism, RNA-Directed DNA Polymerase ultrastructure, Streptococcus pyogenes enzymology, Streptococcus pyogenes genetics
- 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
1 . 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., (© 2024. The Author(s).)- Published
- 2024
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