Minimalist CRISPR ‘Gene Scissors’ Make Way For Undruggable Targets

By using a smaller version of Cas9, researchers from South Korea have made it possible to edit genes in the eyes of live mice.

AsianScientist (Mar. 1, 2017) – Scientists have made the CRISPR-Cas9 gene editing system smaller, delivered it to muscle cells and the eyes of mice and used it to modify a gene that causes blindness. These findings have been published in Nature Communications.

CRISPR-Cas9 is now a buzzword among molecular biologists. It is an innovative, cheap and precise technique to edit genes. The Cas9 enzyme acts as a pair of ‘gene scissors,’ creating cuts on the target gene in precise locations indicated by guide RNA. In order for CRISPR-Cas9 complex to reach the target DNA, it has to be delivered via plasmids or viruses.

“Adeno-associated viruses (AAV) are efficient and safe vectors widely used to express a gene of interest in vivo and in gene therapy,” explained Professor Kim Jin-Soo, director of the Institute for Basic Science (IBS) Center for Genome Engineering and corresponding author of the study.

In nature, Cas9 is used by several bacteria as an immune defense mechanism, cutting viral DNA which could damage the bacteria. The most common version of the CRISPR-Cas9 technique uses Cas9 derived from the bacterium Streptococcus pyogenes. However, this protein is made of 1,368 amino acids and it is too large to be delivered and packaged in AAV.

Even if scientists split it up into two parts—each packaged in a different virus—other issues arise: Double the amount of viruses need to be delivered and the split Cas9 is less active than the intact SpCas9. Staphylococcus aureus Cas9 is also used for gene editing. It is slightly smaller (1,053 aminoacids), so that it can just fit inside the AAV, but does not leave enough space for other proteins.

In this study, the team found that Campylobacter jejuni Cas9 (CjCas9) is both efficient and small. It has 984 amino acids and it can be packed in AAV together with more than one guide RNAs as well as with a fluorescent reporter protein.

Using AAV, the IBS scientists delivered a modified version of CjCas9, two guide RNAs and a fluorescent reporter protein to the muscles and eyes of mice. They concentrated on two genes involved in the age-related macular degeneration (AMD), one of the leading causes of blindness in adults. One gene is a common therapeutic target for ADM, called vascular endothelial growth factor A (VEGF A); the other is a transcription factor that activates the transcription of VEGF A and it is known as HIF-1a.

Unlike VEGF A, HIF-1a has not been considered as a drug target. So-called ‘undruggable’ genes, such as transcription factors in general, cannot be targeted directly by antibodies and other biological or chemical drugs. In this study, the research team proved that CjCas9 delivered to the retina via AAV can inactivate HIF-1a and VEGF A in mice efficiently and reduced the area of choroidal neovascularization.

Intraocular injections of AAV-packaged CRISPR-CjCas9 could be beneficial to treat various retinal diseases and systemic diseases, the authors concluded.

“CjCas9 is highly specific and does not cause off-target mutations in the genome,” said Kim.

Hif1a gene target sequences are the same in both mice and humans, thereby the method presented in this study could be used in the future for the treatment of ADM in human patients. By paving the way to the application of CjCas9 against ‘undruggable’ genes or non-coding sequences, this technology can broaden the range of therapeutic targets, making the entire human genome potentially druggable.

The article can be found at: Kim et al. (2017) In Vivo Genome Editing with a Small Cas9 Orthologue Derived from Campylobacter Jejuni.


Source: Institute for Basic Science.
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