NEW YORK – Using the Cas14 enzyme, researchers at Stanford University have engineered a miniature Cas system that was less than half the size of currently used CRISPR systems such as Cas9 or Cas12a, but which had levels of gene activation comparable to Cas12a.
In a paper published on Friday in Molecular Cell, the researchers described their efforts to develop what they called the CasMINI system. They began with the Cas14 enzyme, which was first discovered in November 2018 by a team of researchers at the University of California, Berkeley, led by Jennifer Doudna. At the time, Cas14 was most notable for its exceptionally small size of only 400 to 700 amino acids in length. Also, Cas14 didn't seem to need a specific protospacer adjacent motif in order to bind or cleave a target DNA sequence. However, the Stanford researchers noted, it was not clear until now whether Cas14 could be used in mammalian cells.
After conducting several rounds of RNA and protein engineering to introduce beneficial mutations, they developed the CasMINI system at approximately 529 amino acids in length. In subsequent experiments, they demonstrated that it could drive up to thousands-fold increases in gene activation, while the natural Cas14 system failed to function in mammalian cells.
Importantly, when they compared CasMINI to Lachnospiraceae bacterium-derived Cas12a, a large Cas effector of about 1,228 amino acids in length, they found that the gene activation efficiency of CasMINI outperformed Cas12a by twofold.
The researchers also showed that CasMINI was highly specific and allowed for robust base editing and gene editing.
"This is a critical step forward for CRISPR genome-engineering applications," senior study author and Stanford researcher Stanley Qi said in a statement. "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."
Qi also emphasized that this study represented the power of rational RNA engineering and protein engineering, which he and his colleagues were able to use to turn a nuclease that didn't function in mammalian cells into one that was highly efficient in those same cells.
"There were previous efforts from others to improve the performance of working CRISPRs," Qi said. "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 study authors noted that these results suggested there were other Cas12 systems that could be optimized for better efficiency using protein and guide RNA engineering. The large size of many CRISPR-Cas proteins has proven particularly challenging for efficient cell engineering and in vivo delivery, particularly for therapeutic applications. But CasMINI's small size — 62 percent and 57 percent smaller than the commonly used SpCas9 and LbCas12a, respectively — makes it suitable for a wide range of therapeutic applications, they added.
"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 said in his statement. "It is on our wish list that it will become a therapy to treat genetic diseases, to cure cancer, and to reverse organ degeneration."