%AWang, Yanbo%ACottle, W.%AWang, Haobo%AGavrilov, Momcilo%AZou, Roger%APham, Minh-Tam%AYegnasubramanian, Srinivasan%ABailey, Scott%AHa, Taekjip%BJournal Name: Nature Communications; Journal Volume: 13; Journal Issue: 1; Related Information: CHORUS Timestamp: 2022-12-15 13:12:00 %D2022%INature Publishing Group; None %JJournal Name: Nature Communications; Journal Volume: 13; Journal Issue: 1; Related Information: CHORUS Timestamp: 2022-12-15 13:12:00 %K %MOSTI ID: 10385807 %PMedium: X %TAchieving single nucleotide sensitivity in direct hybridization genome imaging %XAbstract

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, single guide genome oligopaint via local denaturation fluorescence in situ hybridization (sgGOLDFISH), which leverages the high cleavage specificity of eSpCas9(1.1) variant combined with a rationally designed guide RNA to load a superhelicase and reveal probe binding sites through local denaturation. The guide RNA carries an intentionally introduced mismatch so that while wild-type target DNA sequence can be efficiently cleaved, a mutant sequence with an additional mismatch (e.g., caused by a point mutation) cannot be cleaved. Because sgGOLDFISH relies on genomic DNA being cleaved by Cas9 to reveal probe binding sites, the probes will only label the wild-type sequence but not the mutant sequence. Therefore, sgGOLDFISH has the sensitivity to differentiate the wild-type and mutant sequences differing by only a single base pair. Using sgGOLDFISH, we identify base-editor-modified and unmodified progeroid fibroblasts from a heterogeneous population, validate the identification through progerin immunofluorescence, and demonstrate accurate sub-nuclear localization of point mutations.

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