Achieving single nucleotide sensitivity in direct hybridization genome imaging

Yanbo Wang, W. Taylor Cottle, Haobo Wang, Momcilo Gavrilov, Roger S. Zou, Minh Tam Pham, Srinivasan Yegnasubramanian, Scott Bailey, Taekjip Ha

Research output: Contribution to journalArticlepeer-review

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, 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.

Original languageEnglish (US)
Article number7776
JournalNature communications
Volume13
Issue number1
DOIs
StatePublished - Dec 2022

ASJC Scopus subject areas

  • General Physics and Astronomy
  • General Chemistry
  • General Biochemistry, Genetics and Molecular Biology

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