The most common CRISPR-Cas9 system used by biomedical scientists to edit genes is derived from Streptococcus pyogenes; however, a team of scientists have developed a much smaller CRISPR-Cas9 system – the smallest CRISPR-Cas9 to date.
While the Streptococcus pyogenes CRISPR-Cas9 is being used, there is a problem with its size. The protein contains 1,368 amino acids which is too large for it to be introduced intact via an adeno-associated virus (AAV). In order to get around this problem, it is possible to split the proteins and introduce them separately via two separate AAVs; however, doing so requires much larger numbers of viruses to be introduced and the process is less efficient than introducing an intact SpCas9.
Scientists have searched for other Cas9 systems that are smaller, with a 1,053- amino acid version obtained from Staphylococcus aureus. The Staphylococcus aureus can be introduced intact via AAV although it is still too large for other essential proteins to be included. A much smaller Cas9 is therefore required and scientists at the South Korea’s Center for Genome Engineering at the Institute for Basic Science (IBS) have found one such candidate.
The new gene editing tool has been derived from bacteria best known for causing food poisoning – Campylobacter jejuni. The researchers worked with the Seoul National University and ToolGen to develop the Campylobacter jejuni (CjCas9) system, which was used in muscle cells and the eyes of mice to modify a gene that causes blindness.
The researchers say CjCas9, at just 984 amino acids, can be packed into an AAV with the associated gRNAs necessary for gene editing and a fluorescent reporter protein. The team says the CjCas9 system is both small and highly efficient.
The CjCas9 is also highly specific, only cleaving genes at a limited number of sites in both the mouse and human genome. The scientists were able to introduce highly targeted mutations at high frequencies in both mouse muscle cells and retinal pigment epithelium (RPE) cells.
The researchers say the CjCas9 can be used to target the Vegfa or Hif1a gene in RPE cells and could potentially be used for the treatment of age-related macular degeneration – the leading cause of blindness in adults.
The teams paper, In Vivo Genome Editing with a Small Cas9 Orthologue Derived from Campylobacter jejuni, has recently been published in the journal Nature.