Speaker: Prof. dr. John van der Oost
Subject: CRISPR-based interference in prokaryotes
Author: Carolien Bastiaanssen
Prof. John van der Oost from the Laboratory of Microbiology of Wageningen University gave a BN seminar about CRISPR. This is a defence system which about 85% of archaea and 40% of bacteria have. The topics that were covered in this seminar were the discovery and mechanism of CRISPR, the different classes and their characteristics and the applications of the system.
Bacteria and archaea have developed various ways of dealing with viral threats: inhibition of adsorption, inhibition of DNA injection, degradation of DNA and abortive infection. In the 1980s a Japanese research group discovered a peculiar structure in the DNA of bacteria. At that time they did not know its function, but it turned out to be yet another defence mechanism of archaea and bacteria. The name of this mechanism, CRISPR, is an acronym that stands for Clustered Regularly Interspaced Short Palindromic Repeats. Between these repeats there are sequences, called spacers, which are homologous to viral DNA. Situated next to the CRISPR loci are the CRISPR associated (Cas) genes. The whole pathway is often referred to as CRISPR-Cas and it provides a form of adaptive and heritable immunity.
The process can be divided into three stages. The first stage is the acquisition of spacers. If a virus injects DNA into a bacteria, this DNA is recognized as foreign and it will be cut into pieces. Part of it will be incorporated in the bacteria’s own genome as a spacer. In the next stage the Cas genes are transcribed and pre-crRNA (CRISPR RNA) is produced. This is processed into mature crRNA. The final stage is the actual target interference where the Cas proteins and the crRNA locate foreign DNA and disable it. A very important aspect is that the system has to discriminate between non-self and self DNA. Because the foreign sequences are incorporated into the DNA of the bacterium itself, only looking at the sequence is not enough. CRISPR therefore relies on a protospacer adjacent motif (PAM). This short sequence is present in the viral DNA but not in the spacer. A second control point is the seed sequence, if there are any mismatches in this sequence the crRNA will not bind.
The figure above gives an overview of the CRISPR-Cas system. Source: Van der Oost, J. et all, Unravellling the structural and mechanistic basis of CRISPR-Cas systems, Nature Reviews Microbiology (2014)
There is a huge diversity of CRISPR-Cas systems, primarily in the Cas genes and proteins. There are three main types and each of them has several subtypes. Type I and Type III are very similar and are also referred to as Class I. Type II and a recently discovered Type V are referred to as Class 2. In Class I the different Cas genes produce a multisubunit complex, named CRISPR-associated complex for antiviral defence (Cascade). Together with Cas6, Cascade is responsible for pre-crRNA processing. When a target is near, Cascade undergoes a conformational change, probably to recruit Cas3. Together they target the DNA. The spacer acquisition is done by Cas1 and Cas2. In Class 2 there is no Cascade but only a single protein, for Type II this is Cas9. The unique features of Type II are that there is tracrRNA, which stands for trans-activating CRISPR RNA and that the nuclease used to cut the DNA is RNaseIII. The most recently discovered Type V (or Cpf1) also has a single subunit like Cas9. However it has no tracrRNA and here the PAM is at the 5’ side instead of at the 3’ side. It uses a non-Cas RNase and the double-strand DNA break that it makes is staggered unlike the cut in the other types. All types target DNA except for Type III which targets RNA.
Apart from protecting bacteria from viral attacks CRISPR can be used for various other purposes. Prof. Van der Oost and his co-workers engineered spacers from the lambda phage and introduced them into E. Coli. In this way they made E. coli immune for the lambda phage. Another application of CRISPR could be an interesting alternative for antibiotics. Using CRISPR to engineer good viruses to target harmful bacteria. And last but not least CRISPR has great possibilities as a genome editing tool.