Small CRISPR Is Effective and Easier to Deliver to Cells

Bioengineers at Stanford University have built a smaller CRISPR molecule for DNA editing. Its small size should make it easier to deliver into human cells, tissues, and the body for gene therapy.

The new "CasMINI" molecule (protein) developed by the Stanford scientists has only 529 amino acids. That's much less than the 1000 to 1500 amino acids in the Cas9 and Cas12a CRISPR proteins commonly used in CRISPR gene editing.

A paper is published in Molecular Cell. The scientists report that CasMINI could delete, activate, and edit genetic code. The smaller size of CasMINI means it should be easier to deliver into human cells and the human body.

The bioengineers started with the small CRISPR protein Cas12f. It contains only about 400 to 700 amino acids. But it's not suitable for editing the DNA of mammalian and human cells.

"We thought, 'Okay, 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?'" says Stanley Qi in a press release issued by Stanford University. "To my knowledge, we have, for the first time, turned a nonworking CRISPR into a working one."

The bioengineers selected about 40 modifications of the molecule that could make it suitable for editing human cells. And they implemented the modifications. They also engineered the RNA that guides Cas12f to its target DNA. Modifications to both components were crucial to making the CasMINI system work in human cells.

"At first, this system did not work at all for a year," says researcher Xiaoshu 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."

The scientists tested CasMINI's ability to delete and edit genes in lab-based human cells. They included genes related to HIV infection, anti-tumor immune response, and anemia. It worked on almost every gene they tested, with robust responses in several.

The Stanford bioengineers “used protein engineering to transform a Cas12f protein that didn’t appear to work in mammalian cells into one that did,” summarizes SingularityHub. They also noted that two other recent research papers indicate that Cas12f molecules could be packaged inside viruses along with their guide RNA. And they could be delivered to human cells to make effective edits.

"This engineering approach can be so broadly helpful," concludes Qi. "That's what excites me - opening the door on new possibilities." Qi is persuaded that we should start thinking about how this new CasMini molecule can be applied for practical benefits.