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The CasMini tool is less than half the size of other CRISPR systems and could overcome current barriers in the gene-editing technology.
The common analogy used to explain CRISPR gene editing technology is a DNA scissors. By relying on adapted CRISPR-associated (Cas) proteins, scientists cut strands of DNA to modify its function. This could be used to address biological problems ranging from curing Huntington’s to creating an immunity to HIV. These approaches haven’t been without issue, however.
Stanley Qi, an assistant professor of bioengineering at Stanford University, wanted to challenge the limited concept of the technology and address its current barriers. In a new paper published in Molecular Cell, Qi and his team set out a new CRISPR system called CasMini, which is half the size of other versions and has the potential to bypass previous issues.
“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,” said Qi.
“CRISPR can be as simple as a cutter, or more advanced as a regulator, an editor, a labeller or imager. Many applications are emerging from this exciting field.”
CRISPR, but smaller
Size is a central factor in the CRISPR field. Previous Cas proteins have been limited by their bulk, resulting in an inability to be delivered into human cells and tissues. These version, such as Cas9 and Cas 12a (the Cas number refers to the protein that they’re based on), range from 1,000 amino acids up to 1,500.
‘After iterations of bioengineering, we saw some engineered proteins start to turn on, like magic. It made us really appreciate the power of synthetic biology and bioengineering’
–DR XIAOSHU XU
CasMini uses Cas14 (also known as Cas12f), an archaea-based protein with somewhere between 400 and 700 amino acids. But the first hurdle for Qi and his team was that Cas proteins coming from these single-celled organisms are not suited to working in human bodies.
“We thought, ‘OK, millions of years of evolution have not been able to turn this CRISPR system into something that functions in the human body. Can we change that in just one or two years?’” said Qi.
Xiaoshu Xu, lead author of the paper and a postdoctoral scholar at Qi’s lab, saw no activity when Cas14 was used in human cells. The team hypothesised that the issue lay in the complexity of human DNA. They guessed that Cas14 couldn’t find its target because of how inaccessible the human genome was.
Hitting its mark
By using a computationally predicted structure of Cas14, Xu chose around 40 mutations in the Cas14 protein that could help deal with the complexity. She created a system of testing these variants where a working modification would turn a human cell green by activating an embedded green fluorescent protein in the genome.
“At first, this system did not work at all for a year,” said Xu. “But after iterations of bioengineering, we saw some engineered proteins start to turn on, like magic. It made us really appreciate the power of synthetic biology and bioengineering.”
While the first results were unexceptional, they were enough to push Xu to continue the avenue of research. After more and more trials, she was able to fine-tune her system’s performance.
“We started with seeing only two cells showing a green signal, and now after engineering, almost every cell is green under the microscope,” Xu explained.
Applying their science
Satisfied with the lab’s progress, Qi set his eyes on the next step. “At some moment, I had to stop [Xu]. I said ‘That’s good for now. You’ve made a pretty good system. We should think about how this molecule can be used for applications.’”
By engineering RNA to guide Cas14 to its target, the lab further developed the system’s efficacy is human cells. The researchers trialled their system to delete and edit genes in lab-based human cells, including those related to HIV, tumour response and anaemia. The lab reported that the system worked on nearly every gene that was tested.
With this progress, the scientists have set their eyes on collaborations with other groups to pursue potential applications in gene therapy. They are also investigating how to work with RNA technologies, where molecular size is often an issue.
“This ability to engineer these systems has been desired in the field since the early days of CRISPR, and I feel like we did our part to move toward that reality,” concluded Qi. “And this engineering approach can be so broadly helpful. That’s what excites me – opening the door on new possibilities.”
