Since the discovery of the CRISPR-Cas9 system in 2012, the potential of editing genes for improved agricultural output and treatment of human diseases has been immense. However, Cas proteins, which function to target and cut genes, are often large and difficult to enter targeted cells via viral vectors, such as adeno-associated virus (AAV).
Scientists at the University of California, Berkeley recently identified a hypercompact Cas protein, CasΦ (Cas-phi), which measures approximately 70-80-kilodalton, about half the size of Cas9 and Cas12, in a paper published in Science. This marks the discovery of the most compact working Cas variant as of today.
CasΦ’s unusual small size is attributed to its limited spacer sequences. In other Cas proteins, spacer sequences functions to store genetic information of viral DNA sequences for potential future targeting. As it is more compact, this means that the gene editing protein can more easily enter a cell through small delivery vehicles, including the most commonly used AAV. It also leaves more space inside the viral vector for additional cargo, such as fusing different proteins to the Cas protein, DNA repair templates or other factors that function to regulate the gene editing mechanism. This allows for an enhanced precision in the gene editing tool.
Traditionally, the Cas proteins originate from bacteria as a natural immune defence mechanism against bacteriophages. The new variety, CasΦ, evolved in bacteriophages, a group of viruses that infect bacteria. Previously, a study published in Nature in February 2020 surprisingly revealed that some huge phages also have CRISPR-Cas systems by genetically sequencing bacteriophages from diverse ecosystems. The study marks the first time a new type of CRISPR-Cas system is found in viral genomes, offering a genetically unique and exclusive variant.
In the above image, a megaphage, a member of a bacteriophage family Biggiephage, introduce viral genes into the host cell, including genes for CasΦ. The megaphage utilise the CasΦ protein to trick the bacteria that has been infected to eliminate rival viruses (competing phage), instead of itself, an intelligent survival mechanism. The CRISPR-Cas systems of phages also have the capacity to silence host transcription and translation activity to redirect biosynthesis to favour phage survival.
Compared to Cas9, it is just as selective and efficient in targeting specific regions of DNA in bacteria, animal, and plant cells. CasΦ is capable of producing mature crRNA (CRISPR RNA), which functions to guide the Cas protein to target and recognise the specific DNA sequence to be edited. CasΦ also has the ability to cut double-stranded DNA using only a single active site. Other Cas enzymes usually use one or two active sites to cut DNA and rely on additional factors or a separate active site for crRNA processing. Because of its smaller size, it can also target a wider range of genetic sequences compared to other Cas proteins. As CasΦ originates from a bacteriophage, potential allergic reactions from traditional Cas proteins can be mitigated as some patients may have antibodies that can reject the Cas9 gene-editing procedure.
This means future application can take advantage of CasΦ’s improved properties to deliver promising applications for stem cell engineering, gene therapy, engineering tissue and animal disease models for research, and the development of disease-resistant transgenic plants. The next step is to optimise CasΦ for gene editing to eliminate any off-target effects. Researchers can also genetically amend this protein to create new functionalities for genetic manipulation – the sky is the limit.
Image credit: Freepix