bioRxiv : the preprint server for biologyMay 10, 2023

How DNA packaging and sequence details affect prime editing efficiency

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Mar 25, 2026

Abstract

Editing efficiency in prime editing may be influenced by specific sequence features, including the presence of G/C nucleotides.

Regional constitutive heterochromatin and local nucleosome occlusion can hinder editing efficiency.
High editing efficiency is associated with position-specific G/C nucleotides in the primer binding site and reverse transcription template regions of pegRNA.
The presence of G/C nucleotides is most critical immediately 5' to the protospacer adjacent motif (PAM) site.
Different last templated nucleotides affect editing efficiency based on the lengths of the primer binding site and reverse transcription template.
AGG is identified as the preferred PAM, with a guanine nucleotide four bases downstream enhancing editing.
A neural network analysis indicates multi-nucleotide preferences, showing dependencies among several bases across pegRNA.

Simplified

Prime editor (PE) is a highly versatile CRISPR-Cas9 genome editing technique. The current constructs, however, have variable efficiency and may require laborious experimental optimization. This study presents statistical models for learning the salient epigenomic and sequence features of target sites modulating the editing efficiency and provides guidelines for designing optimal PEs. We found that both regional constitutive heterochromatin and local nucleosome occlusion of target sites impede editing, while position-specific G/C nucleotides in the primer binding site (PBS) and reverse transcription (RT) template regions of PE guide-RNA (pegRNA) yield high editing efficiency, especially for short PBS designs. The presence of G/C nucleotides was most critical immediately 5' to the protospacer adjacent motif (PAM) site for all designs. The effects of different last templated nucleotides were quantified and seen to depend on both PBS and RT template lengths. Our models found AGG to be the preferred PAM and detected a guanine nucleotide four bases downstream of PAM to facilitate editing, suggesting a hitherto-unrecognized interaction with Cas9. A neural network interpretation method based on nonextensive statistical mechanics further revealed multi-nucleotide preferences, indicating dependency among several bases across pegRNA. Our work clarifies previous conflicting observations and uncovers context-dependent features important for optimizing PE designs.

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How DNA packaging and sequence details affect prime editing efficiency

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