Manipulate single nucleic acid molecules in live cells

Perturbation is a powerful method to investigate biological process. By synchronously manipulating biomolecules, we can study their functions and follow cellular response. We develop fast inducible methods to manipulate DNA and RNA in live cells. First, we have established a very fast light inducible CRISPR/Cas9 system (vfCRISPR) to create DNA double strand break (DSB) on demand. We utilize chemically synthesized guide RNA with caged nucleotide placed strategically at positions that allows Cas9 binding to target DNA without cutting it. After light exposure, the target DNA is cut within seconds. The synchronous induction of DSB allows us to study how cells respond to it. For example, actively transcribed genes at and around the DSB are repressed. We used MS2 system to monitor transcription of a single gene and vfCRISPR to create single DSB. We observed rapid transcription repression after DSB induction. We investigated the molecular pathways that mediate rapid transcription repression and its spatial propagation. Second, we established a rapid inducible decay of RNA (RIDR) technology by recruiting an RNA decay factor to the target mRNA. We applied RIDR to endogenous β-actin RNA and observed rapid appearance and gradual dissolution of RNA granules, which we identified as processing bodies (p-bodies). We measured the RNA recruitment and decay kinetics in p-bodies by fitting a mathematical model of compartmentalized RNA counts in single cells as a function of time. This allows us to determine the decay kinetics of specific RNAs in p-bodies for the first time. Together, the synchronous manipulation of single molecules is a powerful tool to study the spatial and temporal dynamics of biological processes in cells.