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Combining Single Molecule and Ensemble approaches To Investigate Cas9 Target Search

Periodic Reporting for period 1 - CasMETICS (Combining Single Molecule and Ensemble approaches To Investigate Cas9 Target Search)

Reporting period: 2016-04-01 to 2018-03-31

The primary objectives of the action was to measure in vivo the search kinetics of Cas9 using multiple complementary approaches. Here, search kinetics is taken to encompass both the length of time taken for an individual dCas9 molecule to find its binding site, and the sequence of intermediate steps involved in finding the binding site.

The search time was measured using both a single-molecule fluorescence microscopy based approach and a bulk assay based on detecting Cas9 protection against cleavage by restriction enzymes. The use of orthogonal but complementary approaches is an important step to ensure the accuracy and reliability of measurements. With these search time measurement techniques in place we have the possibility to investigate the dependence of search time on various factors such as guide rna sequence and whether transcription of the target region in the genome facilitates Cas9 binding by opening up the DNA (as in Type III CRISPR systems).

Cas9 is rapidly becoming the genome editing tool of choice for most biology research lab and is predicted to have many important medical applications. In order to execute Cas9 mediated gene editing in a safe and effective manner it is crucial to have comprehensive information about the underlying molecular mechanisms.
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.
In less than a decade, CRISPR-Cas9 has revolutionized biological research. Cas9 makes it possible to accurately correct or change, in principle any DNA sequence, and the hope is that the protein will also make it possible to cure and prevent genetic diseases.

The Molecule Cas9 can be programmed with a piece of artificial genetic code that allows it to find the corresponding sequence in the genome. Most proteins searching for DNA code can only recognize a specific sequence by probing the outside of the DNA helix. Cas9 can search for an arbitrary code, but to determine if it has found the right sequence, the molecule must open the DNA helix and compare the sequence with the programmed code. The search process is random and consumes no energy, making it very slow.
Within the scope of this project, we have now learned how Cas9 looks for the right DNA sequence.

Our results show that the price Cas9 pays for its flexibility is time. To find the target faster, more Cas9 molecules that look for the same sequence are needed. The very high concentrations of Cas9 needed to find the right sequence within a reasonable period of time can cause serious problems for the cells you try to alternate, but the results from the study give us important clues to how the system can be improved.

By sacrificing some of the flexibility of Cas9, it is possible to construct a gene snipper that is still sufficiently versatile to edit different genes but at the same time fast enough to be medically useful. The key lies in the so-called PAM sequences that determine where and how often Cas9 opens the DNA helix. A gene snipper that does not need to open the DNA helix as often is not only faster but would also reduce the risk of side effects.
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