Abstract A major challenge in human genomics is to decipher the context specific relationship of sequence to function. However, existing tools for locus specific hypermutation and evolution in the native genome context are limited. Here we present a novel programmable platform for long-range, locus-specific hypermutation called helicase-assisted continuous editing (HACE). HACE leverages CRISPR-Cas9 to target a processive helicase-deaminase fusion that incurs mutations across large (&gt;1000 bp) genomic intervals. We applied HACE to identify mutations in MEK1 that confer kinase inhibitor resistance, to dissect the impact of individual variants in SF3B1-dependent mis-splicing, and to evaluate noncoding variants in a stimulation-dependent immune enhancer of CD69. HACE provides a powerful tool for investigating coding and noncoding variants, uncovering combinatorial sequence-to-function relationships, and evolving new biological functions. One Sentence Summary We developed a tool for continuous, long-range, targeted diversification of endogenous mammalian genomes and used it to explore the function of genetic variants in both coding and non-coding regions.

Helicase-assisted continuous editing for programmable mutagenesis of endogenous genomes