An enhanced Eco1 retron editor enables precision genome engineering in human cells from a single-copy integrated lentivirus

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 as major contributors to poor retron editor efficiency. 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 liberate the sgRNA and ncRNA. These modifications yielded a ncRNA with 5'- and 3'-end protection and an sgRNA with minimal 5' extension. This strategy increased steady-state ncRNA levels and rescued Cas9 activity leading to enhanced efficiency of the Eco1 retron editor in human cells. The enhanced Eco1 retron editor enabled the insertion of missense mutations in human cells from a single integrated lentivirus, thereby ensuring genotype-phenotype linkage over multiple cell divisions. This work reveals a previously unappreciated role for ncRNA stability in retron editor efficiency in human cells. Here we present an enhanced Eco1 retron editor that enables efficient introduction of missense mutations in human cells from a single heritable genome copy.