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 and Taekjip Ha ()
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Yanbo Wang: Johns Hopkins University School of Medicine
W. Taylor Cottle: Johns Hopkins University School of Medicine
Haobo Wang: Johns Hopkins University School of Medicine
Momcilo Gavrilov: Johns Hopkins University School of Medicine
Roger S. Zou: Johns Hopkins University
Minh-Tam Pham: Johns Hopkins University School of Medicine
Srinivasan Yegnasubramanian: Johns Hopkins University School of Medicine
Scott Bailey: Johns Hopkins University School of Medicine
Taekjip Ha: Johns Hopkins University School of Medicine

Nature Communications, 2022, vol. 13, issue 1, 1-12

Abstract: 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.

Date: 2022
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DOI: 10.1038/s41467-022-35476-y

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