Light, CRISPR and DNA Repair
Johns Hopkins University
As there is only one or two copies of chromosomes in most cells, genome dynamics and processing are intrinsically single-molecule events. We recently developed a generalizable strategy, termed very fast (vf)CRISPR, that uses caged guide (cg)RNAs for synchronous, light-activated cleavage of genomic DNA on the seconds time scale. Imaging-guided vfCRISPR further controls CRISPR activation at the subnuclear level, down to the single-allele resolution, enabling new types of mechanistic studies of DNA repair and genome editing processes.
We also developed GOLD FISH (Genome Oligopaint via Local Denaturation for Fluorescence in-situ Hybridization), a technology to image selected genomic loci with ultralow background. GOLD FISH loads a superhelicase at a Cas9-generated nick so that the unwinding of downstream DNA creates binding sites for FISH probes without global DNA denaturation or harsh treatments. GOLD FISH achieves at least tenfold reduction in background while better preserving chromosomal ultrastructure’s in cultured cells and human patient tissues.
In this talk, I will discuss our efforts to further improve vfCRISPR and GOLD FISH