An enhanced Eco1 retron editor enables precision genome engineering in human cells without double-strand breaks.

ABSTRACT: Retrons are a retroelement class found in diverse prokaryotes that can be adapted to augment CRISPR-Cas9 genome engineering technology to efficiently rewrite short stretches of genetic information in bacteria and yeast. However, efficiency in human cells has been limited by unknown factors. We identified non-coding RNA (ncRNA) instability and impaired Cas9 activity due to 5' sgRNA extension as key contributors to low retron editor efficiency in human cells. We re-engineered the Eco1 ncRNA to incorporate an exoribonuclease-resistant RNA (xrRNA) pseudoknot from the Zika virus 3' UTR and devised an RNA processing strategy using Csy4 ribonuclease to minimize 5' sgRNA extension. This strategy increased steady-state ncRNA levels and rescued sgRNA activity, leading to increased templated repair.

SUBMITTER: Cattle MA 

PROVIDER: S-EPMC11326160 | biostudies-literature | 2025 May

REPOSITORIES: biostudies-literature