CRISPR-based interference in Prokaryotes from exploration to exploitation

Speaker: John van der Oost
Department: Microbiology, Wageningen University
Subject: CRISPR-Cas system
Location: TU Delft

Date: 12 November 2015
Author: Gabriele Kockelkoren


Professor John van der Oost works in the laboratories of Microbiology of Wageningen UR and is extremely interested in the CRISPR system. Originally having a background in microbiology, John van der Oost is very fascinated by bacteria. The CRISPR system, subject of this BN talk, is owned by 85% of the archaea and by 40% of the bacteria.

Normally viruses infect prokaryotes, then the viral replication cycle occurs and new viral phages are formed which can infect new prokaryotes. This cycle repeats itself over and over again. However, the potential hosts have evolutionary developed skills to overcome this. These established mechanisms are: inhibition of absorption, inhibition of DNA injection, degradation of DNA and abortive infection systems. In 1987 researchers in a Japanese lab described a remarkable repetitive structure in the DNA of bacteria. These repeat were interspaced by variable spaces, these spaces are called spacers. However, at that time its function was not known. This unknown structure was later called CRISPR, which is an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats. So a new defence mechanism was found.

It was found that the repetitive stretches are co-localised with the repetitive genes and that many CRISPR spacers are homologous to viruses or plasmids. Next to the CRISPR loci, the CRISPR associated (Cas) genes are located. Therefore, the whole pathway is often referred to as the CRISPR-Cas9 pathway. By experimental evidence it provides an adaptable and inheritable immunity.

The process of the CRISPR-Cas9 system can be divided into three main stages. The first stage is the spacer acquisition state. When an infection occurs and this infection does not end successfully for the virus, then the foreign DNA of the virus, will be cut into pieces. Parts of this DNA will be incorporated as spacers in the DNA of the host as a spacer. This way the CRISPR system has time to acquire an additional unit in its CRISPR. During the second stage the Cas genes are transcribed and pre-crRNA (CRISPR pre-RNA) is formed. Afterwards the pre-crRNA is changed into mature crRNA. The third and final stage is the target interference. This stage involves activation and targeting of Cas-proteins and crRNA for neutralisation of viral DNA.

It is essential for this process to be very specific. There has to be a clear discrimination between the sequences of the host and those of the virus. As the sequences contain both the DNA of the bacterium itself and of the virus, looking only at the sequence is not enough. Hence the discrimination is made due to the protospacer adjacent motif, called PAM. This PAM is only found the DNA of the host. If the motif is present, only then the process starts attacking the other system. This forms a very subtle, but important difference. There is also the Seed sequence which serves as a second control point. The Seed sequence may not have any mismatches. If any mismatches are found, then the crRNA will not bind.



Figure 1: General overview of CRISPR-Cas-system. Source:

There is a huge diversity in the CRISPR-Cas systems. This diversity is mainly related to the CRISPR-associated proteins and genes. The types of CRISPR systems can be divided into three main categories and several subtypes. The type I and III are very similar and belong to Class1. Type II and the more recently discovered type V belong to Class2. The Class1 system relies on the Cascade system, where different Cas genes work together forming a complex named CRISPR-associated complex for antiviral defence (Cascade). Researching Class1 in the E. Coli system, it is observed that when all 8 Cas proteins are expressed, the results are not successful. However, by expressing 6 genes with designed CRISPR with spacers which correspond to a certain phage lambda sequence, immunity is produced. While looking at the Cas proteins, Cas1 and Cas2 are most likely involved in the acquisition of spacers. As Cas1 and 2 make up a complex and bind to dsDNA, they recognize the PAM sequence.  Cas 3 appears to be a helicase-nuclease hybrid, which is recruited by a conformational change of the Cascade due to an increasing density of the nucleic acid. Together with Cas6, Cascade is responsible for the pre-crRNA processing. As aforementioned, Class1 contains the type I and type III complex. The type III targets RNA rather than DNA.  In Class 2 no Cascade is present, there is only a single protein which is Cas9 for Type II. Type II is remarkable for two aspects: First the use of tracrRNA, which stands for trans-activating CRISPR RNA and secondly, the nuclease which is used to cut the DNA is RNaseIII. Most recently a new type has been discovered, type V, also called Cpf1. This system also has one single unit like Cas9. The difference is that there is no tracrRNA and the PAM is at the 5’-side instead of the 3’-side. Furthermore, type V uses a non-Cas RNase and the dsDNA break that it makes is staggered. In all the other types this break is cut.

The applications of the CRISPR-Cas system are great. Not only is it an engineering tool for creating viral immunity for bacteria, but also viruses can be made that target “bad” bacteria. Ultimately, CRISPR-Cas can be used as a powerful genome engineering tool which can lead to great consequences for human evolution.



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