한빛사 논문
Yanbo Wang1, Wayne Taylor Cottle1, Haobo Wang2, Xinyu Ashlee Feng3, John Mallon2, Momcilo Gavrilov1, Scott Bailey1,2, Taekjip Ha1,4,5,6,7,*
1Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
2Bloomberg School of Public Health, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
3Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA
4Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA
5Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA
6Howard Hughes Medical Institute, Baltimore, MD 21205, USA
*Corresponding author
Abstract
Cas9 in complex with a programmable guide RNA targets specific double-stranded DNA for cleavage. By harnessing Cas9 as a programmable loader of superhelicase to genomic DNA, we report a physiological-temperature DNA fluorescence in situ hybridization (FISH) method termed genome oligopaint via local denaturation (GOLD) FISH. Instead of global denaturation as in conventional DNA FISH, loading a superhelicase at a Cas9-generated nick allows for local DNA denaturation, reducing nonspecific binding of probes and avoiding harsh treatments such as heat denaturation. GOLD FISH relies on Cas9 cleaving target DNA sequences and avoids the high nuclear background associated with other genome labeling methods that rely on Cas9 binding. The excellent signal brightness and specificity enable us to image nonrepetitive genomic DNA loci and analyze the conformational differences between active and inactive X chromosomes. Finally, GOLD FISH could be used for rapid identification of HER2 gene amplification in patient tissue.
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