20 September 2019 Vol 365, Issue 6459
CRISPR-mediated live imaging of genome editing and transcription
By Haifeng Wang, Muneaki Nakamura, Timothy R. Abbott, Dehua Zhao, Kaiwen Luo, Cordelia Yu, Cindy M. Nguyen, Albert Lo, Timothy P. Daley, Marie La Russa, Yanxia Liu, Lei S. Qi
Science20 Sep 2019 : 1301-1305 Restricted Access
A CRISPR-mediated imaging approach, LiveFISH, enables real-time tracking of DNA and RNA in living cells.
Tracking nucleic acids in living cells
Fluorescence in situ hybridization (FISH) is a powerful molecular technique for detecting nucleic acids in cells. However, it requires cell fixation and denaturation. Wang et al. found that CRISPR-Cas9 protects guide RNAs from degradation in cells only when bound to target DNA. Taking advantage of this target-dependent stability switch, they developed a labeling technique, named CRISPR LiveFISH, to detect DNA and RNA using fluorophore-conjugated guide RNAs with Cas9 and Cas13, respectively. CRISPR LiveFISH improves the signal-to-noise ratio, is compatible with living cells, and allows tracking real-time dynamics of genome editing, chromosome translocation, and transcription.
We report a robust, versatile approach called CRISPR live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligonucleotides for genome tracking in a broad range of cell types, including primary cells. An intrinsic stability switch of CRISPR guide RNAs enables LiveFISH to accurately detect chromosomal disorders such as Patau syndrome in prenatal amniotic fluid cells and track multiple loci in human T lymphocytes. In addition, LiveFISH tracks the real-time movement of DNA double-strand breaks induced by CRISPR-Cas9–mediated editing and consequent chromosome translocations. Finally, by combining Cas9 and Cas13 systems, LiveFISH allows for simultaneous visualization of genomic DNA and RNA transcripts in living cells. The LiveFISH approach enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.