The potential of molecular biology to shape the outcome of diseases is most evident when observing the elegance and versatility of CRISPR. Now, a collaborative study from researchers at the Broad Institute, MIT, and the National Center for Biotechnology Information (NCBI) has identified an ortholog of the Cas9 nuclease that will allow the genome editing tool to be packaged for in vivo use—a major hurdle that needed to be addressed for CRISPR to be used therapeutically.    

The findings from this study were released online recently in Nature through an article entitled “In vivo genome editing using Staphylococcus aureus Cas9.”

The CRISPR-Cas9 system was originally identified in the bacteria Streptococcus pyogenes as a defense mechanism against viral DNA. The S. pyogenes Cas9 (SpCas9) enzyme was then engineered to be able to alter DNA from an array of higher organisms. In the time since its discovery however, the CRISPR-Cas9 pathway has been found in a number of microbial species.   

For this genome editing tool to be effective in live animals and humans the components must be packaged into a vector for delivery to the cells of interest. Many scientists have their eye on the Adeno-associated virus (AAV) as a delivery candidate, since it is not known to cause any disease within humans and it has recently received regulatory approval in Europe.

Unfortunately, the small cargo capacity of AAV makes it extremely difficult to package the large SPCas9 enzyme along with the other necessary CRISPR components—sort of like stuffing a watermelon into a sausage casing. However, the discovery from the current study outlines how this conundrum can be circumvented.

“Sifting through the 600 or so available Cas9 sequences, we identified a group of small variants in which the enzymatic domains were intact whereas the non-enzymatic portion was substantially truncated,” explained Eugene Koonin, Ph.D., senior investigator with NCBI and co-author on the study. “Luckily, one of these smaller Cas9 proteins turned out to be suitable for the development of the methodology described in this paper. We are now actively exploring the diversity of Cas9 proteins and their relatives in the hope to find new varieties that could potentially lead to even more powerful tools.”

The investigators discovered a Cas9 nuclease form Staphylococcus aureus (SaCas9) that functioned with similar efficiency in vitro as SpCas9, but was 25% smaller. Their next step was to test the packaged AAV/SaCas9 vector to determine if it could be utilized as a therapeutic tool.

The team chose PCKS9 as their test gene target, since its loss has been associated with decreased levels of LDL cholesterol and a reduced risk of cardiovascular diseases. Using a mouse model that mimics high cholesterol the researchers observed a nearly complete depletion of serum PCKS9 and a 40% reduction in total cholesterol one week after administering AAV/Cas9.   

“While we have chosen a therapeutically relevant target, PCSK9, in this proof-of-principle study, the greater goal here is the development of a versatile and efficient system that expands our ability to edit genomes in vivo,” said Fei Ann Ran, Ph.D., postdoctoral fellow at the Broad and co-first author of the study.

The scientists were very excited about the results from the mouse studies and say that their next step is to compare the two Cas9 enzymes in great detail with the hope of identifying areas where AAV/SaCas9 system can be further optimized.

“This study highlights the power of using comparative genome analysis to expand the CRISPR-Cas9 toolbox,” stated Feng Zhang, Ph.D., professor of biomedical engineering at MIT and senior author of the current study. “Our long-term goal is to develop CRISPR as a therapeutic platform. This new Cas9 provides a scaffold to expand our Cas9 repertoire, and help us create better models of disease, identify mechanisms, and develop new treatments.”

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