Structural basis for pegRNA-guided reverse transcription by a prime editor.

Area of Science:

• Molecular Biology
• Genetics
• Structural Biology

Background:

• Prime editing enables precise genome modifications using a Cas9 nickase and reverse transcriptase guided by a prime editing guide RNA (pegRNA).
• The precise molecular mechanism of prime editing remains unclear due to a lack of structural data.

Purpose of the Study:

• To elucidate the structural basis of prime editing mechanism using cryo-electron microscopy.
• To understand the role of Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) in pegRNA-guided reverse transcription.
• To engineer improved prime editing systems based on structural insights.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine the structures of the prime editor complex with target DNA in various states.
• Functional analyses were performed to assess the activity and specificity of the prime editor.
• Rational engineering of pegRNA and prime editor variants was conducted.

Main Results:

• Structures revealed that M-MLV RT can extend reverse transcription beyond the intended site, leading to undesired edits.
• M-MLV RT maintains a stable position relative to SpCas9 during reverse transcription.
• The prime editing guide RNA-synthesized DNA heteroduplex accumulates on the SpCas9 surface.
• Engineered pegRNA and prime editor variants were developed.

Conclusions:

• The study provides unprecedented structural insights into the stepwise mechanism of prime editing.
• Understanding the structural basis of off-target edits facilitates the development of more precise genome editing tools.
• The findings pave the way for an enhanced prime editing toolbox for versatile genome engineering.