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A compact and efficient CRISPR-Cas system, named CasMINI, could be broadly useful for cell-engineering and gene-therapy applications because it is easier to deliver into cells. The findings appear in a study publishing September 3 in the journal Molecular Cell.

This is a critical step forward for CRISPR genome-engineering applications. The work presents the smallest CRISPR to date, cheap cartia 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."

Stanley Qi, Senior Study Author, Stanford University

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 and thus impede clinical applications. For example, adeno-associated virus (AAV), a vector widely applied for in vivo delivery, has limited packaging capacity of the payload (less than 4.7 kb), and many Cas fusion proteins are beyond this limit. 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.

"Here we turn a non-working CRISPR in mammalian cells, via rational RNA engineering and protein engineering, into a highly efficient working one," Qi says. "There were previous efforts from others to improve the performance of working CRISPRs. But our work is the first to make a non-working one working. This highlights the power of bioengineering to achieve something evolution has not yet done."

The size of the engineered CasMINI molecule is only 529 amino acids. This small size makes it suitable for a wide range of therapeutic applications. For example, the CasMINI fusion proteins are well suited for AAV packaging. In addition, CasMINI mRNA can be easily packaged into lipid nanoparticles or other RNA-delivery modalities, potentially enhancing its entry into cells. Its small size and non-human pathogen source might make it less likely to produce immune responses than large protein payloads would be.

More work is needed to further optimize the efficiency of CasMINI for base editing and gene editing and to test the performance of the system in vivo with different delivery modalities. The researchers plan to test the system for in vivo gene-therapy applications.

"The availability of a miniature CasMINI enables new applications, ranging from in vitro applications such as engineering better tumor-killing lymphocytes or reprogramming stem cells to in vivo gene therapy to treat genetic diseases in the eye, muscle, or liver," Qi says. "It is on our wish list that it will become a therapy to treat genetic diseases, to cure cancer, and to reverse organ degeneration."