Novel Method May Treat Age-Related Macular Degeneration in the Future

Scientists have engineered the smallest CRISPR-Cas9 to-date, which is expected to become a useful therapeutic tool in the future.

The CRISPR-Cas9 technique has gained popularity for its ability to edit genes by creating cuts on the target gene in precise locations indicated by the guide RNA. Cas9 is used by several bacteria as an immunity weapon and is needed to cut viral DNA, which could damage the bacteria.

Cas9 is derived from the bacterium Streptococcus pyogenes. Unfortunately, the protein is made of 1368 aminoacids, and is too big to be delivered and packaged in AAV.

“AAV is an efficient and safe vector to express a gene of interest in vivo and has been used widely in gene therapy,” said author KIM Jin-Soo.

If it were to be split into 2 parts, it would require twice the number of viruses to be delivered. Furthermore, the split Cas9 is less active than the intact SpCas9. Although Staphylococcus aureus Cas9 is also used in gene editing and is slightly smaller with 1053 aminoacids, it does not leave enough space inside the AAV for other proteins.

In a study published in Nature Communications, investigators found that CjCas9 is both efficient and small, with only 984 aminoacids. Furthermore, CjCas9 can be packaged in AAV together, with more than 1 guide RNA, as well as with a fluorescent reporter protein.

To use a bacterial protein for gene editing, the investigators designed a short DNA sequencing immediately following the DNA sequence targeted by the Cas9 Protospacer Adjacent Motif (PAM).

For Cas9 to bind and cleave to the targeted DNA sequence, each different Cas9 requires a specific PAM sequence, according to the authors. Additionally, the length of the guide RNA needed to be modified.

The investigators packaged the new CRISPR-Cas9 complex with 2 guide RNAs and a fluorescent reporter protein into AAV, to mutate genes in the muscles and eyes of mice. The primary focus was on 2 genes involved in the age-related macular degeneration (AMD), which is one of the lead causes of blindness in adults. The genes were Vascular endothelial growth factor A (VEGF A) and HIF-1a.

HIF-1a has not been considered as a drug target like VEGF A has, according to the authors.

In the study, the investigators demonstrated that CjCas9 delivered to the retina via AAV can inactivate Hif1a and VEGF A in mice efficaciously. Furthermore, it reduced the area of choroidal neovascularization.

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

Because Hif1a gene target sequences are the same in both mice and humans, the findings suggest that the approach could be used in the future to treat AMD in humans.