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Kinetics of dCas9 target search in Escherichia coli
Daniel Lawson Jones1, Prune Leroy1, Cecilia Unoson1
1Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
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.
Insights
It takes a single Cas9 protein six hours to find its target DNA sequence in E. coli. High concentrations of Cas9 and guide RNA are necessary for rapid DNA targeting.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- The Cas9 protein, guided by RNA, can target specific DNA sequences.
- This process requires DNA unwinding to verify base pairing.
- Understanding the speed of this search is crucial for gene editing applications.
Purpose of the Study:
- To determine the time it takes for Cas9 protein to locate a specific chromosomal DNA sequence within a living cell.
- To investigate the intracellular search mechanisms of catalytically inactive Cas9 (dCas9).
Main Methods:
- Utilized single-molecule fluorescence microscopy.
- Employed bulk restriction-protection assays.
- Studied dCas9 in living Escherichia coli.
Main Results:
- A single fluorescently labeled dCas9 protein requires 6 hours to find its target DNA sequence.
- Each potential target DNA sequence is bound for less than 30 milliseconds.
- Once bound, dCas9 remains associated with the DNA until replication.
- High concentrations of both Cas9 and guide RNA are essential for fast targeting.
Conclusions:
- The intracellular search for specific DNA sequences by Cas9 is a slow process.
- Efficient and rapid DNA targeting by Cas9 necessitates high intracellular concentrations of both the protein and its guide RNA.
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