Imagine a Swiss Army knife. Within its compact case, it holds a variety of cutters and openers for everyday use. Stanley Qi, an assistant professor of Bioengineering at Stanford, analogizes this knife to the new CRISPR system: the CasMINI.
On September 3, Qi and his colleagues introduced the mini CRISPR system, a new addition to the CRISPR-associated (Cas) proteins that would efficiently edit and activate genetic code. The CasMINI is made of 529 amino acids, compared to the 1000 to 1500 amino acids necessary for the commonly used CRISPR systems like the Cas9 and Cas12a. This new system would allow for easier access to human cells throughout the body, benefiting various organs as genetic diseases can now be treated. But what exactly is a CRISPR?
CRISPR was previously only known as the Clustered Regularly Interspaced Short Palindromic Repeats of genetic information found in bacterial species. It is now named to be a tool for genome editing founded and co-invented by Dr. Emmanuelle Charpentier, a French professor and researcher in microbiology, genetics, and biochemistry. In theory, the commonly used CRISPR-Cas9 system edits genes by disrupting a DNA sequence at a specific location designated by a guide RNA bound to the enzyme. The system also removes and inserts fragments of DNA or simply corrects the sequence as a form of manipulation for treatment. Although the research still remains at its early stages of development, these functionalities of the CRISPR-Cas9 system has been applied to the research in the treatment of diseases such as cancer and blood disorders.
Going back to the CRISPR-CasMINI, the efforts to decrease the size of the system first began with another CRISPR system, the Cas12f, as it only consists of significantly less amino acids compared to other commonly used systems. Qi encountered a problem, however, as Cas12f was not one of the few CRISPR proteins that could function in mammalian cells even without any modifications. Subsequently, Xiaoshu Xu, a postdoctoral scholar in Qi lab and lead author of the publication announcing the Cas-MINI, was unable to find any activity in the natural CRISPR-Cas12f systems in human cells. After hypothesizing that finding the target in these cells would be too difficult for the Cas12f proteins, Xu carefully mutated about 40 iterations of the protein in hopes of avoiding this limit. These iterations were set so that the green fluorescent protein (GFP) would be activated in the genome and turn a human cell green as a sign of success. Despite the weak first results, the system soon began to outperform its previous iteration until “almost every cell [was] green under the microscope”, as Xu comments. The guide RNA responsible for setting a target for this system had also been bioengineered, and as a result, the CasMINI protein was complete and ready to be announced as the new CRISPR system.