Kinetics of dCas9 target search in Escherichia coli.
Daniel Lawson JonesPrune LeroyCecilia UnosonDavid FangeVladimir ĆurićMichael J LawsonJohan ElfPublished in: Science (New York, N.Y.) (2017)
How fast can a cell locate a specific chromosomal DNA sequence specified by a single-stranded oligonucleotide? To address this question, we investigate the intracellular search processes of the Cas9 protein, which can be programmed by a guide RNA to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays. We find that it takes a single fluorescently labeled dCas9 6 hours to find the correct target sequence, which implies that each potential target is bound for less than 30 milliseconds. Once bound, dCas9 remains associated until replication. To achieve fast targeting, both Cas9 and its guide RNA have to be present at high concentrations.
Keyphrases
- single molecule
- crispr cas
- genome editing
- escherichia coli
- nucleic acid
- living cells
- atomic force microscopy
- amino acid
- cancer therapy
- binding protein
- single cell
- high throughput
- biofilm formation
- cell therapy
- staphylococcus aureus
- stem cells
- computed tomography
- mesenchymal stem cells
- dna methylation
- cystic fibrosis
- climate change
- reactive oxygen species
- dna binding
- optical coherence tomography
- positron emission tomography