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How new 'molecular scissors' may give crisper CRISPR

10 July 2017

By Rikita Patel

Appeared in BioNews 908

Researchers have revealed the structure and action of a new type of 'molecular scissors' known as Cpf1, which may improve CRISPR genome editing.

Using X-ray crystallography, the team were able to view the three-dimensional structure of Cpf1, a bacterial protein from the CRISPR-Cas family of molecules. Like CRISPR/Cas9, the CRISPR-Cpf1 tool has potentially widespread applications in biotechnology.

They were also able to visualise exactly how Cpf1 unzips and cuts double-stranded DNA, saying that Cpf1 'acts like a GPS' across the genome to recognise and cleave DNA (genetic) sequences with high precision. 

The new CRISPR-Cpf1 technique has therapeutic potential for treating cancers and genetic diseases, and could also 'be used to modify microorganisms, with the aim of synthesising the metabolites required in the production of drugs and biofuels', according to lead researcher, Professor Guillermo Montoya, from the Novo Nordisk Foundation Center for Protein Research (NNF-CPR) at the University of Copenhagen, Denmark.

The researchers, who published their study in Nature, first created Cpf1 gene constructs and inserted these into Escherichia coli bacteria to trigger Cpf1 gene expression which they observed. 'We radiated the crystals of the Cpf1 protein using X-rays to be able to observe its structure at atomic resolution, enabling us to see all its components. X-ray diffraction is one of the main biophysical techniques used to elucidate biomolecular structures,' said Professor Montoya.

While cutting DNA using Cas9 results in blunt ended strands, the study showed that Cpf1 cleaves DNA in a staggered fashion leaving 'sticky ends', which makes insertion of a DNA sequence easier. Furthermore, in comparison to Cas9, the structural component for DNA recognition and binding for CRISPR-Cpf1 (called the Protospacer Adjacent Motif or PAM) is located far from the DNA cleavage site. The resulting physical separation may prevent damage of PAM site and offer new possibilities for DNA cleavage.

Professor Montoya said: 'The high precision of this protein recognising the DNA sequence on which it is going to act functions like a GPS, directing the Cpf1 system within the intricate map of the genome to identify its destination. In comparison with other proteins used for this purpose, it is also very versatile and easy to be reprogrammed.'

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