Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases.

Genome editing technologies based on DNA-dependent polymerases (DDPs) could offer several benefits compared with other types of editors to install diverse edits. Here, we develop click editing, a genome writing platform that couples the advantageous properties of DDPs with RNA-programmable nickases to permit the installation of a range of edits, including substitutions, insertions and deletions. Click editors (CEs) leverage the 'click'-like bioconjugation ability of HUH endonucleases with single-stranded DNA substrates to covalently tether 'click DNA' (clkDNA) templates encoding user-specifiable edits at targeted genomic loci. Through iterative optimization of the modular components of CEs and their clkDNAs, we demonstrate the ability to install precise genome edits with minimal indels in diverse immortalized human cell types and primary fibroblasts with precise editing efficiencies of up to ~30%. Editing efficiency can be improved by rapidly screening clkDNA oligonucleotides with various modifications, including repair-evading substitutions. Click editing is a precise and versatile genome editing approach for diverse biological applications.