Achieving single nucleotide sensitivity in direct hybridization genome imaging

Abstract

Direct visualization of point mutations in situ can be informative for studying genetic diseases and nuclear biology. We describe a direct hybridization genome imaging method with single-nucleotide sensitivity, sgGOLDFISH, which leverages the high cleavage specificity of enhanced Cas9 combined with a single extended guide RNA to load a superhelicase and reveal probe binding sites through local denaturation. Using sgGOLDFISH, we identified base-editor-modified and unmodified progeroid fibroblasts from a heterogeneous population, validated the identification through progerin immunofluorescence, and demonstrated accurate sub-nuclear localization of point mutations.