CRISPR-Cas9 is a promising gene editing tool that makes it far easier for scientists to make edits to the genome. Rather than using more established gene editing techniques that require the total replacement of a defective gene, CRISPR-Cas9 can be used to make edits to faulty genes. Genetic mutations are eliminated, which makes the genes fully functional.
The Cas9 component of the system is an enzyme that binds to a specific section of DNA where the faulty gene is located and cuts the DNA to remove the faulty DNA. Cas9 is highly specific, but it is not 100% accurate. The Cas9 enzyme can also remove sections of DNA in areas of the genome that do not contain the defective gene, and that is problematic. These off-target effects could, for instance, result in the formation of cancers.
CRISPR-Cas9 has already been used in trials on humans, the first of which are now underway in the United States and have so far produced promising results. However, for CRISPR-Cas9 to be used to safely treat a much wider range of diseases, the specificity of the Cas9 enzyme, or similar enzymes, needs to be improved.
Currently, research has focused on two types of Cas9 enzymes, obtained from two species of bacteria: Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9). Both of these Cas9s can perform edits to the genome with high specificity, but both also have off-target effects.
SpCas9 is favored as it allows accurate genome-wide targeting, but it is much larger than SaCas9. SaCas9 is smaller and easier to deliver into cells, but it falls short on genome-wide targeting accuracy.
Modified SpCas9s have been engineered to reduce off-target effects and improve the precision of gene editing but delivering these into cells is difficult. They are too large to be carried into cells by the adeno-associated viral (AAV) vectors necessary for in vivo gene editing. Consequently, they would need to be split and reassembled in cells, which is a major challenge.
Researchers at City University of Hong Kong and the Karolinska Institutet in Sweden may have the solution. They have created a modified SaCas9 called SaCas9-HF which is small enough to be carried into cells via AAV vectors, and it has high genome-wide targeting accuracy in human cells, without reducing on-target efficiency.
The researchers, led by Dr. Zheng Zongli, Assistant Professor of Department of Biomedical Sciences at CityU and the Ming Wai Lau Centre for Reparative Medicine of Karolinska Institutet in Hong Kong, tested the accuracy of the new SaCas9-HF at 24 locations on the genome and compared the results to the wild type SaCas9. Many of those locations have very similar genetic sequences, so are often associated with off-target effects.
Compared to the wild type, the SaCas9-HF made far fewer errors. There were 90% fewer off-target effects with SaCas9-HF making this engineered Cas9 suitable for gene editing in locations that require highly precise edits to be made.
The study is detailed in the paper – Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity – which was recently published in the journal Proceedings of the National Academy of Sciences (PNAS). DOI: 10.1073/pnas.1906843116