Kinetics of dCas9 target search in Escherichia coli

• Published in: Science (American Association for the Advancement of Science) Vol. 357; no. 6358; pp. 1420 - 1424
• Main Authors: Jones, Daniel Lawson, Leroy, Prune, Unoson, Cecilia, Fange, David, Ćurić, Vladimir, Lawson, Michael J., Elf, Johan
• Format: Journal Article
• Language: English
• Published: United States American Association for the Advancement of Science 29.09.2017; The American Association for the Advancement of Science
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; CRISPR; CRISPR-Associated Protein 9; Deoxyribonucleic acid; DNA; E coli; Endonucleases - genetics; Endonucleases - metabolism; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Fluorescence; Fluorescence microscopy; Gene Editing; Genome editing; Genomes; Kinetics; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Nucleotide sequence; Oligonucleotides; Protein Binding; Reaction kinetics; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Ribonucleic acid; RNA; Searching; Streptococcus pyogenes - enzymology; Streptococcus pyogenes - genetics; Time Factors
• ISSN: 0036-8075; 1095-9203; 1095-9203
• DOI: 10.1126/science.aah7084

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.