A new, compact CRISPR-Cas system can now precisely and efficiently shuttle gene-editing tools into mammalian cells, overcoming present challenges in genome engineering.
Most gadgets today are designed to be compact yet efficient, small yet powerful, simple yet elegant. But that is not wholly limited to electronic devices. In a new study published in Molecular Cell, researchers from Standford University have developed a compact and efficient CRISPR-Cas system called CasMINI that shows immense potential for broad applications in cell engineering and gene-therapy.
“This is a critical step forward for CRISPR genome-engineering applications,” said 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.”
Since it was first adapted for human cells, the CRISPR-Cas system has revolutionised genome engineering, opening doors to treat various genetic diseases and making medicine more precise and personalised than ever. However, due to their large sizes, the CRISPR platform has yet to become a fully functional and reliable therapeutic agent, thus impeding clinical applications. For instance, many Cas fusion proteins exceed the packaging capacity (4.7 kb) of the adeno-associated virus, a widely used vector for in vivo delivery. As such, it is crucial to develop highly efficient, compact Cas systems if we are to facilitate next-generation genome engineering.
One promising solution is Cas12f (also known as Cas14). With a size ranging between 400 to 700 amino acids, the protein is less than half the size of CRISPR systems like Cas9 or Cas12a that are currently used in most research and clinical trials. However, until recently, scientists have yet to determine whether the compact Cas12f could be used in mammalian cells. According to Qi, scientists “have identified thousands of CRISPRs, which are known as bacteria’s immunity defence system […] More than 99.9 per cent of discovered CRISPRs, however, cannot work in human cells, limiting their use as genome-editing technologies.”
In the current study, Qi’s team set out to apply RNA and protein engineering to the Cas12f system to create an efficient, miniature Cas system that can be used for mammalian genome engineering. In its natural state, the archaea-derived Cas12f protein and its single-guide RNA demonstrate no detectable activity in mammalian cells. To solve this, the scientists optimised the single-guide RNA design and performed multiple rounds of iterative protein engineering and screening, ultimately generating a new class of Cas12f variants called CasMINI.
The newly engineered Cas12f protein variants combined with engineered single-guide RNAs reportedly exhibited efficient gene-regulation and gene-editing activity. Through a series of experiments, the researchers also demonstrated that CasMINI can drive high levels of gene activation that are comparable to those associated with Cas12a. Furthermore, because the CasMINI is highly specific, precise, and produces no detectable off-target effects, this latest development can allow for robust base editing and gene editing.
“Here we turn a non-working CRISPR in mammalian cells, via rational RNA engineering and protein engineering, into a highly efficient working one,” Qi explained. “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.”
Because the CasMINI molecule is only 529 amino acids long and originates from a non-human pathogen source, it is less likely to trigger immune responses than large protein payloads. This makes it highly suitable for therapeutic applications. Besides that, it is also expected to solve a number of present challenges faced in genome engineering today. For one, the CasMINI fusion proteins are small enough to be packaged in the adeno-associated virus, therefore resolving the incompatibility between the CRISPR platform and adeno-associated virus. The CasMINI mRNA can also be easily packaged into lipid nanoparticles or other RNA-delivery modalities, potentially enhancing its ability to penetrate cells.
More work is needed to further optimise the efficiency of CasMINI for base editing and gene editing as well as to test its performance in vivo with different methods of delivery. Nonetheless, this innovative machinery shows promise to overcome the limits of current CRISPR gene editing and revolutionise biomedical research. At present, the researchers are planning 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 tumour-killing lymphocytes or reprogramming stem cells to in vivo gene therapy to treat genetic diseases in the eye, muscle, or liver,” said Qi. “It is on our wish list that it will become a therapy to treat genetic diseases, to cure cancer, and to reverse organ degeneration.”
Source: Xu et al. (2021). Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Molecular Cell.