CRISPR gene editing offers new possibilities for treatment of conditions such as cancer and congenital diseases through gene therapy. CRISPR proteins Cas9 and Cas12a can perform the functions of gene editing and activation in mammalian cells, but their large size can limit their ability to enter the cells of living organisms. This is why researchers at Stanford University began to investigate a smaller CRISPR protein, Cas12f, and ultimately managed to engineer a “mini” CRISPR system with editing and regulation abilities.
Cas12f, also known as Cas14, contains only about 400 to 700 amino acids compared to the 1000 to 1500 that comprise Cas9 and Cas12a, but does not naturally function in mammalian cells. The researchers used computational predictions of a Cas12f system structure to select about 40 mutations that could potentially allow the system to function in human cells. After testing these mutations, and over several iterations of bioengineering, the resulting Cas12f variant was able to function well in human cells.
“At first, this system did not work at all for a year. But after iterations of bioengineering, we saw some engineered proteins start to turn on, like magic. It made us really appreciate the power of synthetic biology and bioengineering,” said first author Xiaoshu Xu.
The variant was paired with guide RNA, also engineered by the team, which together formed a system dubbed CasMINI. The researchers report that the CasMINI system is highly specific and efficient, allows for robust base and gene editing, and is comparable to Cas12a in gene activation activity. The system’s deletion and editing abilities were tested in human cells, including on genes related to HIV infection, anti-tumor immune response and anemia, and found to work on almost every gene tested, with robust responses in several. The team published their research in Molecular Cell.
“This ability to engineer these systems has been desired in the field since the early days of CRISPR, and I feel like we did our part to move toward that reality,” said corresponding author Stanley Qi. “And this engineering approach can be so broadly helpful. That’s what excites me – opening the door on new possibilities.”
The researchers have begun assembling collaborations with other scientists to pursue potential gene therapies utilizing the compact CRISPR system. They are also interested in how their research could contribute to RNA technologies, such as the development of mRNA vaccines, where molecule size can also present limitations.