Abstract
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.
🔬 Techniques
🧬 Organisms
✨ Fluorophores
🧪 Sample Preparation
🔬 Cell Lines
💾 Data Repositories
🏛️ Research Organizations (ROR)
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📊 Figures
Fig. 1
Comparison of Cas9 and transcription factor search
Cas9 (in yellow) must unwind dsDNA at every PAM (red dots) to test for sgRNA complementarity to the protospacer (detail, lower right). In contrast, a transcription factor (in blue) scans the dsDNA by ...
Fig. 2
Association
(A) Upper: Schematic illustration of the single molecule assay, where dCas9-YPet is expressed at low copy number in a strain containing pSMART plasmids each with 36 lacO1-binding sites. In the absence...
Fig. 3
Dissociation and repression
(A) Fluorescence images of cells with dCas9-YPet at three different time points (t in min) after removing IPTG. Scale bar: 2u00b5m. (B) Two examples from a set of dissociation curves acquired at diffe...
Fig. 4
Non-specific Binding
Cells containing dCas9-YPet but no specific chromosomal target are imaged in the presence (strain:PL42H9) or absence (strain:PL42H8) of sgRNA-a, using exposure times ranging from 2ms to 1s while keepi...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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