Researchers see rapidly hunting CRISPR system in action

Researchers around the world are using CRISPR systems for genome editing, with CRISPR-Cas9 as the best known example. However, there is still a great deal of uncertainty about how these types of systems work in the cell. Researchers at Delft University of Technology and Wageningen University & Research have now followed one such system, the so-called CRISPR Cascade complex, during the hunt for hostile DNA for the first time. The bacterial defence mechanism is astonishingly fast and efficient: it checks no less than 100 different pieces of DNA every second.

In the past decade, CRISPR-Cas has revolutionized gene editing. The adaptation of DNA in a living cell used to take a lot of effort. However, with this relatively new tool researchers can very easily switch off genes, or cut open a strand of genetic material with almost surgical precision in order to insert a new piece of DNA.    

Biological factory
CRISPR-Cas is often seen as a single system, but in reality there are many such systems. They are defense mechanisms that protect bacteria from attacks by tiny virus particles called bacteriophages. These moonlander-shaped particles bind to the surface of a bacterium and pump their DNA into the cell. They then use the bacterium as a biological factory in order to make thousands of copies of themselves. Once that is done, they let the bacterium explode and the search for a new victim begins.

CRISPR-Cas systems are the response of bacteria to this threat. They consist of two parts: a system that hunts down enemy DNA, and a system that cuts the DNA apart. In some cases, nature has linked these systems together to form an all-in-one solution, which has led to Cas9.

Molecular tools
Researchers have converted this ingenious biological mechanism into a molecular tool that allows them to rewrite DNA in living cells. Strangely enough, however, there are still many outstanding fundamental questions about how CRISPR systems function. Only recently, for example, it became apparent that rewriting DNA with CRISPR-Cas9 can lead to unwanted mutations. Before we can use the technique to start eliminating genetic diseases from our DNA, we need to know exactly how the system works.

Researchers at Delft University of Technology have now seen a CRISPR system at work in a living bacterium for the first time. They looked at the so-called Cascade complex. "We have known this system since 2008. It was the first CRISPR system we discovered," says group leader Stan Brouns. "Nevertheless, we did not yet know how the Cascade complex moves through the cell. It was also unclear to us how quickly the system works and how many complexes are needed to properly protect a bacterium.

Considerable investment
What was clear already was that CRISPR systems are looking for a needle in a haystack. "A bacterial cell contains an awful lot of DNA, about 5 million base pairs, the letters of the genetic code. By far the largest part of that is our own DNA," says TU Delft researcher Jochem Vink, who led the research. "The Cascade complex is looking for a tiny piece, about thirty base pairs".

How does the system do that? One possibility would be for the bacteria to create a whole host of Cascade complexes. Many hands make light work. "However, creating and maintaining these systems is a big investment for a bacterium," says Vink. "So we didn't expect them to need a lot of these complexes in order to be well protected.” This premonition proved to be correct: the researchers' calculations show that a hundred Cascade complexes give a bacterium a good chance of survival.

Hard workers
But how can just one hundred Cascade complexes check such a huge mass of DNA? The answer is obvious: they are hard workers. The Delft researchers were able to take a picture of the Cascade complexes one hundred times a second using a quick laser pulse. Thus, they could see whether the complexes were moving or standing still. "if they didn't move for a moment, they had logically attached themselves to a piece of DNA in order to check it," explains Brouns.

The speed at which Cascade complexes check strains of DNA is almost impossible to comprehend. It turns out that such a complex checks no less than a hundred pieces of DNA per second. This is as fast as the laser pulse, so the researchers had to develop a new calculation method to fill the holes in their observations. "At first, we were surprised at how fast they were working," says Vink. "But on the other hand, it's not that strange. There is so much DNA to check and so little time for a phage to take over the cell, that the complexes need to act quickly."

What is the optimal amount of CRISPR systems for a bacterium? "That depends," says Brouns. "With twenty complexes, the chance of survival is fifty-fifty. But since bacterial colonies usually consist of billions of copies, they may also be able to survive with fewer." The environment in which a bacterium lives plays an important role. In places where many phages live, they will invest more in good protection than in places where the threat is less acute. "Bacteria undoubtedly strike a balance," says Vink. "They are very ingenious creatures."

***

More information:

Jochem N.A. Vink, Koen J.A. Martens, Marnix Vlot, Rebecca E. McKenzie, Cristóbal Almendros, Boris Estrada Bonilla, Daan J.W. Brocken, Johannes Hohlbein, Stan J.J. Brouns, 'Direct Visualization of Native CRISPR Target Search in Live Bacteria Reveals Cascade DNA Surveillance Mechanism', Molecular Cell.

DOI: https://doi.org/10.1016/j.molcel.2019.10.021

Contact:

Jochem Vink
J.N.A.Vink@tudelft.nl
06 – 43 10 48 29

Read more: Tinkering under the bonnet of life - about the upcoming revolution called CRISPR-Cas9 and the work of the Stan Brouns Lab.