Targeted genome editing with a DNA-dependent DNA polymerase and exogenous DNA-containing templates

Reverse transcriptases, used in prime editing systems, exhibit lower fidelity, processivity and dNTP affinity than many DNA-dependent DNA polymerases. We report that a DNA-dependent DNA polymerase (phi29), untethered from Cas9, enables editing from a synthetic, end-stabilized DNA-containing template at up to 60% efficiency in human cells. Compared to prime editing, DNA polymerase editing avoids autoinhibitory intramolecular base pairing of the template, facilitates template synthesis and supports larger insertions (>100 nucleotides).