AddTag, a two-step approach with supporting software package that facilitates CRISPR/Cas-mediated precision genome editing

Abstract

AbstractCRISPR/Cas-induced genome editing is a powerful tool for genetic engineering, however, targeting constraints limit which loci are editable with this method. Since the length of a DNA sequence impacts the likelihood it overlaps a unique target site, precision editing of small genomic features with CRISPR/Cas remains an obstacle. We introduce a two-step genome editing strategy that virtually eliminates CRISPR/Cas targeting constraints and facilitates precision genome editing of elements as short as a single base-pair at virtually any locus in any organism that supports CRISPR/Cas-induced genome editing. Our two-step approach first replaces the locus of interest with an “AddTag” sequence, which is subsequently replaced with any engineered sequence, and thus circumvents the need for direct overlap with a unique CRISPR/Cas target site. In this study, we demonstrate the feasibility of our approach by editing transcription factor binding sites within Candida albicans