Miniature CRISPR-Cas System Developed

CasMINI, a newly developed compact and efficient CRISPR-Cas system, could help facilitate cell-engineering and gene-therapy applications because it is easier to deliver into cells, according to a study published today in Molecular Cell.

“This is a critical step forward for CRISPR genome-engineering applications,” says senior study author Stanley Qi of Stanford University. “The work presents the smallest CRISPR to date, according to our knowledge, as a genome-editing technology. If people sometimes think of Cas9 as molecular scissors, here we created a Swiss knife containing multiple functions. It is not a big one, but a miniature one that is highly portable for easy use.”

The development of CRISPR-Cas systems for human cells has revolutionized genome engineering. These systems offer opportunities for the development of gene therapies for a variety of genetic diseases, but their large sizes often restrict delivery into cells. As a result, there is a need to engineer highly efficient, compact Cas systems to facilitate the next generation of genome-engineering applications.

One potential solution is Cas12f, also known as Cas14. Ranging between 400 and 700 amino acids, the protein is less than half the size of currently used CRISPR systems such as Cas9 or Cas12a. But until now, it was not clear whether this compact protein could be used in mammalian cells. “Recent years have identified thousands of CRISPRs, which are known as bacteria’s immunity defense system,” Qi explains. “More than 99.9% of discovered CRISPRs, however, cannot work in human cells, limiting their use as genome-editing technologies.”

In the new study, Qi and his team applied RNA and protein engineering to the Cas12f system to generate an efficient miniature Cas system for mammalian genome engineering. Derived from archaea, the natural Cas12f protein and its single-guide RNA showed no detectable activity in mammalian cells. By optimizing the single-guide RNA design and performing multiple rounds of iterative protein engineering and screening, the researchers generated a class of Cas12f variants named CasMINI.

The engineered Cas12f protein variants combined with engineered single-guide RNAs exhibited efficient gene-regulation and gene-editing activity. The researchers demonstrated that CasMINI can drive high levels of gene activation comparable to those associated with Cas12a and allows for robust base editing and gene editing. Moreover, it is highly specific and does not produce detectable off-target effects, they say.