CAMBRIDGE, Mass.—The promise of genome editing—being able to adjust the activity of different genes precisely and efficiently—could have a variety of applications in health management, but as with all emerging technologies, there are hurdles to overcome. Fortunately, a multi-institute group of scientists may have solved one of the primary issues impeding the advancement of this approach.
Researchers from the Massachusetts Institute of Technology (MIT), the Broad Institute of MIT and Harvard and the National Center for Biotechnology Information (NCBI) of the National Institutes of Health conducted a collaborative study that led to the discovery of a highly efficient Cas9 nuclease that can address one of the leading roadblocks facing in-vivo genome editing. The results were published in a Nature paper titled “In-vivo genome editing using Staphylococcus aureus Cas9,” which appeared in the journal April 1.
The CRISPR-Cas9 system was originally discovered in bacteria. This approach enables the cutting of DNA as a defense mechanism against viral infection, and while a multitude of microbial species have this system, the Cas9 enzyme from Streptococcus pyogenes (SpCas9) was the first to be engineered for changing the DNA of higher organisms. CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are used in genome editing, and the CRISPR-Cas9 system makes it possible to mutate or change the expression of genes in living cells. The Cas9 nucleases in particular recognize DNA targets in complex with RNA guides, and it is now possible to use this engineered system to target specific nucleic acid sequences and cut the DNA at those points, which in turn modifies the genes' activity to enable further study of their function.
One of the hurdles researchers face with this technology is delivering the necessary parts of the CRISPR-Cas9 system into cells. While adeno-associated virus (AAV) is one of the primary candidates, it has a small capacity in terms of what it can “carry” into cells, and as such, SpCas9 and the necessary RNA accompaniments tax its limits. The new Cas9 nuclease from Staphylococcus aureus (SaCas9) detailed in the Nature paper, however, is 25 percent smaller than SpCas9, which speaks well for its potential.
Feng Zhang, a core member of the Broad Institute and investigator at the McGovern Institute for Brain Research at MIT, led the Broad/MIT group in working with researchers from MIT, led by MIT Institute Prof. Phillip Sharp, with Eugene Koonin leading the NCBI team. The collaborators were seeking Cas9 enzymes that could match SpCas9's efficiency with a smaller size, and identified SaCas9 by using comparative genomics to analyze Cas9s from more than 600 different bacteria types.
Though they found several that looked promising, SaCas9 was the only candidate whose DNA cutting efficiency matched that of SpCas9 in mammalian cells. Using the BLESS method, which was developed by Nicola Crosetto of the Karolinska Institute and Ivan Dikic at the Goethe University Medical School, they were able to determine the presence of unintended “off-targets,” and found that SaCas9's DNA targeting accuracy was comparable to that of SpCas9.
“This study highlights the power of using comparative genome analysis to expand the CRISPR-Cas9 toolbox,” said Zhang. “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.”
To test SaCas9's in-vivo gene editing capabilities, the researchers used AAV/SaCas9 to target PCSK9, or proprotein convertase subtilisin/kexin type 9, “a therapeutically relevant gene involved in cholesterol homeostasis. Inhibitors of the human convertase PCSK9 have emerged as a promising new class of cardioprotective drugs after human genetic studies revealed that loss of PCSK9 is associated with a reduced risk of cardiovascular disease and lower levels of low-density lipoprotein (LDL) cholesterol,” as noted in the paper. The researchers administered AAV/SaCas9 to a mouse model, and found that “The AAV-SaCas9 system is able to mediate efficient and rapid editing of PCSK9 in the mouse liver, resulting in reductions of serum PCSK9 and total cholesterol levels.” A week after administration, PCSK9 levels were almost completing depleted, with total cholesterol decreased 40 percent. Additionally, no obvious signs of inflammation or immune response were seen in the mice.
“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, who is co-first author of the study, along with Le Cong and Winston Yan.
Moving forward, Zhang says they hope to compare and contrast SpCas9 and SaCas9 to identify ways of further optimizing the system.