CRISPR (Clustered Regularly Interspaced Palindromic Repeats) gene editing technology allows permanent modification of genes within organisms. It is considered a breakthrough in biotechnology ever since its discovery. Researchers from the University of Copenhagen (Denmark), led by Spanish researcher Guillermo Montoya, now went one step further. They discovered how Cpf1, a new type of molecular scissors, unzip and cleave DNA.
In a study published in the journal Nature, the scientific team has described how this new system for genome editing works for the first time. The Cpf1 protein enables the cleavage of double stranded DNA, allowing the initiation of the genome modification process. It is a member of the CRISPR-Cas family and acts like a GPS in order to identify its destination within the genome. Because the molecular scissors are highly precise in identifying the target DNA sequence, they enable safer modifications and editing instructions written in genome.
Researchs from Novo Nordisk Foundation Center for Protein Research used an X-ray Crystallography, one of the main biophysical techniques, to illuminate molecular structures and observe structures at atomic resolution. The structure reveals the machinery involved in DNA unwinding to form a CRISPR RNA (crRNA)–DNA hybrid and a displaced DNA strand.
In Montoya’s opinion, “the main advantage of Cpf1 lies in its high specificity and the cleaving mode of the DNA, since it is possible to create staggered ends with the new molecular scissors, instead of blunt-ended breaks as is the case with Cas9, which facilitates the insertion of a DNA sequence.”
Applications for CRISPR/Cas9 system for cutting and pasting genome sequences are growing fast. The technology is already being used to modify animal and plant genomes and is starting to be adopted for human therapy. Wide applicability has been proven by researches: eliminating the HIV virus in living mice, bringing the woolly mammoth back to life, CRISPR pill that could replace antibiotics, first therapeutic use for Retinitis Pigmentosa, better understanding of our brain and many more. The CRISPR/Cpf1 system could take genomic editing to the new level.
“The high precision of this protein for recognizing 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,” Montoya adds.
Genome editing is cheaper. Cpf1 requires only crRNA, reducing the size of the engineered gRNA molecule required by half compared to Cas9. Another advantage is that Cpf1 cuts a comfortable distance away from its protospacer adjacent motif – the PAM site. Cutting does not disrupt the PAM site, allowing for multiple rounds of DNA cleavage and increased chance for the desired genomic editing to occur. Also, Cas9 relies on G-rich PAM sites, while Cpf1 recognizes T-rich PAM sequences. This significantly expands the breadth of possible genomic targets.
Cpf1 properties make this system especially “suitable for its use in the treatment of genetic diseases and tumors.“ The new technology “can also be used to modify microorganisms, with the aim of synthesizing the metabolites required in the production of drugs and biofuels,” adds Montoya.
Learn more about molecular scissors in video below:
By Andreja Gregoric, MSc