Mol Cell 2021 04 2;81(7):1566-1577.e8. Epub 2021 Mar 2.
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA; Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Howard Hughes Medical Institute, Baltimore, MD 21205, USA. Electronic address:
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. Read More