Speaker: Prof. Dr. Jennifer Doudna
Department: UC Berkely
Subject: CRISPR Systems: Nature’s Toolbox for Genome Protection
Location: Wageningen University
Author: Mirte Golverdingen
On Friday September 30th, I travelled to Wageningen to join the Lecture of prof. dr. Jennifer Doudna. She is one of the pioneers on the very promising CRISPR field. She started her lecture by explaining what CRISPR is and how it was discovered. CRISPR, Clustered regularly interspaced short palindromic repeats, are hallmarks of acquired immunity in bacteria.
The discovery of CRISPR started with curiosity on how bacteria defend themselves from viruses. 10 years ago, it emerged that many bacteria have an array of repetitive arrays in their genome, CRISPRs. In 2005 it was shown that the arrays contained virus RNA’s, this showed that CRISPR indeed could be a viral immune system. Over the next several years they did research to the way bacteria adapted to the viral DNA. It turned out that bacteria use a mechanism that adapts the viral DNA, then, crRNA is transcribed from this DNA and these CRISPR-RNAs can target complexes of virus DNA.
The Cas9 gene is the only gene that is necessary to fight of viruses by using CRISPR. From CRISPR to CRISPR systems there is much variation in how the DNA guides the DNA cutting work. These variations are classified into two classes, Class 1 and Class 2 systems.
Doudna did research to Class 2, consisting of CRISPR systems that contain one single enzyme responsible for detecting and destroying DNA. Cas 9 is a dual RNA guided DNA endonuclease. It is programmed by the crRNA, tracrRNA duplex and holds on to an unwound double stranded DNA helix. (See Figure 1) TracrRNA is important for creating crRNA but it also works in the complex with the crRNA. It trims away short molecules of RNA that are still functional. You can simplify this into a single guide instead of a dual crRNA-tracrRNA chimera. This Cas9 protein still worked.
Figure 1: Cas9 can be programmed using a single engineered RNA molecule combining tracrRNA and crRNA features. (A) (Top) In type II CRISPR/Cas systems, Cas9 is guided by a two-RNA structure formed by activating tracrRNA and targeting crRNA to cleave site-specifically–targeted dsDNA (see fig. S1). (Bottom) A chimeric RNA generated by fusing the 3′ end of crRNA to the 5′ end of tracrRNA. Jinek & Chylinski et al. Science 337, 816 (2012).
Cas9 and its guide RNA act like a molecular scalpel to cut DNA. It searches on all the DNA in the cell to look if it can find DNA that match. When a match occurs the DNA is opened and cut with a double stranded break.
For the biotechnology, this is very interesting. Genome editing begins with dsDNA cleavage and results in non-homogenous end joining or Homologous recombination with a donor DNA between the two ends. Normally it is very hard to fix the double stranded break. However, the Cas9 is simple and cheap, it uses the DNA as donor so you can easily build in new genes. The CRISPR-Cas9 genome editing technology is useful because of the power of base pairing. It is a very convenient way to recognize DNA and to reprogram the specificity of the systems. All CRIPSR systems are based on this simple idea. Secondly, there are many applications of this technique in animal and plants. Moreover, it is a programmable DNA manipulation program with all the possibilities occurring from this idea.
The DNA target recognition is driven by pam motifs. Cas9 works by unwinding the DNA, however it does not hydrolize ATP. Cas9 first binds to PAM motives in the theta DNA curtains of the phage. PAM contact triggers the DNA unwinding, Cas9 is therefore a catalysator. In this way, the DNA can be unwound. The conformational change when Cas9 binds to the DNA is partly responsible for this DNA unwinding.
Cas9 searching mechanism is based on finding PAM motives, that are only seen in phage DNA. When there is recognition, unwinding finds place. The DNA will be cut and the virus is killed. Cas9 therefore does not slide along the DNA strand, instead it binds and searches very quickly and then leaves to another spot on the DNA.
I was very excited about this talk, it showed how the CRIPSR-Cas9 system worked. And it also showed something from research work done behind this revolutionary discovery. I hope there will be very renewing applications of this technique. The decade in this field will be even more exciting compared to the first decade.