PE3b

PE3b is a refinement of prime editing used in genome editing to improve efficiency and accuracy. It builds on the PE3 approach by adding a second guide RNA that nicks the opposite DNA strand, but with design features intended to limit indel formation. The goal is to bias repair toward the edited sequence.

Mechanism: Like PE2, PE3b uses a Cas9 nickase fused to reverse transcriptase and a pegRNA to introduce a desired edit. The second nicking guide in PE3b is designed so that nicking occurs preferentially only after the edit has been incorporated, typically by requiring the edited sequence to create or preserve a PAM or a binding site. This conditional nicking reduces nicking events in unedited cells and aims to minimize unwanted insertions or deletions while preserving editing efficiency.

Design considerations: Successful PE3b implementation depends on careful design of the pegRNA and the second nicking guide. The edit must create a sequence context that allows selective nicking after editing. Because of locus-specific factors, the relative benefits of PE3b can vary, and extensive optimization may be needed for different targets.

Performance and use: In demonstrated cases, PE3b showed improved editing efficiency with lower indel rates compared with PE3 and PE2 in certain mammalian cells. However, outcomes are context-dependent, and PE3b is one option among several prime editing strategies for precise sequence changes, small insertions, or deletions.

Limitations: Off-target effects, variable efficiency by locus and cell type, and increased design complexity remain considerations for PE3b applications.

See also: prime editing, PE3, Cas9 nickase, pegRNA.