CRISPR-based interference: from Cas9 to Cpf1

BioNanoscience seminar, 12.11.15

Title: CRISPR-based interference in prokaryotes: from exploration to exploitation

Speaker: John van der Oost (WUR)

Author: Edgar Schönfeld

CRISPR/CAS9 is a new genome editing tool that has revolutionized biology. It is better than previously used methods in terms of cost, time and precision. The tool is based on a bacterial defense mechanism against viruses that was discovered in the 80’s. Then, researchers found clusters of identical repeating DNA sequences that were separated by variable regions in bacterial genomes. Much later, this pattern was named “clustered regularly interspaced short palindromic repeats”, or CRISPR. Despite from regarding it as a funny structure, nobody saw the significance of these repeats until it was discovered that some of the variable regions were homologous to viral genes. Soon scientists realized that it was part of an adaptive immune system in bacterial cells, which works in the following way: When viral DNA enters the cell, Cas-proteins (CRISPR-associated proteins) fragment the invading sequence and select a certain region, which is inserted as a spacer in the CRISPR locus. The variable regions (called spacers) in the CRISPR locus constitute a library of previously invading DNA sequences. The CRISPR locus is preceded by a leader sequence and an operon of genes encoding the Cas-proteins, which have individual functions in the viral defence mechanism. After the CRISPR locus is transcribed into pre-RNA it is cut into individual spacer crRNAs (CRISPR-RNA) by a Cas-protein. Thereby the repeating RNA regions mark the borders between different spacer crRNA-sequences. Each such crRNA associates with other specific Cas-proteins. Finally, these CRISPR ribonucleoprotein-complexes (crRNPs) scan the DNA of invading viruses. Thereby the RNA-component acts as a guide that recognizes the invading DNA sequences it descended from, through its homology. Eventually, the Cas protein degrades the targeted sequence.

From: “Revenge of the phages: defeating bacterial defences” Julie E. Samson, Alfonson H. Magadán, Mourad Sabri & Sylvain Moineau, Nature Reviews Microbiology 11, 675-687 (2013)

There are two classes of CRISPR mechanisms. Class I genes contain an operon representing different Cas-proteins called “cascade”, whereas class II systems contain only Cas9. In such a system Cas9 does the trimming of the pre-RNA and also associates with the resulting guide RNAs. In both processes, Cas9 requires the interaction of tracrRNA, which is transcribed together with the Cas-genes and the CRISPR locus. In contrast to a crRNA-crRNP complex involving Cas3 and the cascade, the crRNA only connects to a single unit (Cas9) in a class II system. Cas9 induces a double strand break in the targeted DNA, which is repaired by means of the error-prone homologous end-joining. This CRISPR-Cas9 system is employed by researchers to knock out genes. In order to insert a new gene, a template DNA sequence must be provided as well, which will be automatically inserted via homologous end joining.
Interestingly, there is another Class II effector protein that offers several advantages compared to Cas9, and John van der Oost was involved in its recent discovery. This protein is called Cpf1. Its most distinct feature is its independence from tracrRNA. This simplifies the design of the CRISPR genome editing system, because less RNA need to be synthesized. In addition, Cpf1 creates sticky ends when cutting DNA, as opposed to the blunt ends resulting from Cas9-mediated cleavage. That facilitates the insertion of new genetic sequences, and also allows to choose the orientation of insertion.
The CRISPR-Cas9 system induced a patent fight between two American universities, which might come to an end if the CRISPR-Cpf1 system proves to be superior to CRISPR-Cas9.


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