How fast can a cell locate a specific chromosomal DNA sequence specified by a single stranded oligonucleotide? This question is relevant not only because the whole CRISPER/Cas9 system depends on this process happening reasonably fast; it also defined how efficiently a cell can locate and fix a double stranded break in its DNA and is closely related to how the Argonaut protein family can locate RNA and exert their functions in embryonic development and cell differentiation, just to give a few examples.
We have previously shown that a DNA binding protein typically finds a specific target sequence within a couple of minutes. Given that there are usually many proteins involved in the search process, this time average makes biological sense.
In the case of CRISP/Cas9 we expect the search time to be significantly longer. The reason for this is that the protein-nucleic acid complex does not simply rely on interactions in the DNA groves to find the target site, but actually needs to unwind the double helix and interrogate the DNA sequence to discriminate right from wrong. The search problem is somewhat reduced by the fact the potential targets are defined by the presence of a PAM sequence, but still presents a daunting task.
We developed two new methods to measure how long it takes for Cas9 to find its target sequence. The first method showed that it takes a staggering six hours for Cas9 to search through a bacterial genome of four million base pairs. The results could also be verified using the second, independent technology. The time also match how many milliseconds Cas9 spends at each position, which is measured by following single labeled Cas9 molecules in real time. Compared to the search time of a transcription factor 6 hours is an eternity, but it is the prize that the CRISP/Cas9 system has to pay in order to be reprogrammable to target any sequence.
Weather used by bacteria as a defence against invading phages or by scientists as a method to repress a specific gene, the system requires both Cas9 and its guide RNA to be present in high concentrations in order to work efficiently.
This study was published in Science: Daniel Lawson Jones, Prune Leroy, Cecilia Unoson, David Fange, Vladimir Curic, Michael J. Lawson, Johan Elf (2017) Kinetics of dCas9 target search in Escherichia coli. Science 357 (6358) pp1420-1424.
Following the publication of the search kinetics of Cas9 in living cells we have continued to investigate the sequence dependance of target recognition, but these results will no be published within the scope of this project report.
