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

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 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. This work reveals a previously unappreciated role for ncRNA stability in retron editor efficiency in human cells and presents an enhanced Eco1 retron editor capable of precise genome editing in human cells from a single integrated lentivirus and, in the context of the nCas9 H840A nickase, without creating double-strand breaks.