Researchers develop a hypercompact CRISPR

The common analogy for CRISPR gene editing is that it works like molecular scissors, cutting selected sections of DNA. Stanley Qi, assistant professor of bioengineering at Stanford University, likes this analogy, but he thinks it’s time to reimagine CRISPR as a Swiss army knife.

“CRISPR can be as simple as a cutter, or more advanced than a regulator, editor, labeler or imager. Many applications are emerging in this exciting field, ”said Qi, who is also an assistant professor of chemical and systems biology at the Stanford School of Medicine and a researcher at the Stanford ChEM-H institute.

The many different CRISPR systems used or clinically tested for gene therapy of diseases of the eye, liver and brain, however, remain limited in scope because they all suffer from the same flaw: they are too big and, therefore, too difficult. to be introduced into cells, tissues or living organisms.

In an article published on September 3 in Molecular cell, Qi and its collaborators announce what they consider to be a big step forward for CRISPR: an efficient, versatile tool, mini CRISPR system. While commonly used CRISPR systems – with names like Cas9 and Cas12a denoting various versions of CRISPR-associated proteins (Cas) – consist of around 1000 to 1500 amino acids, their “CasMINI” has 529.

The researchers confirmed in experiments that CasMINI could suppress, activate and modify the genetic code, just like its beefier counterparts. Its small size means that it should be easier to deliver into human cells and the human body, making it a potential tool for treating a variety of ailments, including eye diseases, organ degeneration, and genetic diseases in general.

Persistent efforts

To make the system as small as possible, the researchers decided to start with the CRISPR Cas12f protein (also known as Cas14), as it only contains around 400-700 amino acids. However, like other CRISPR proteins, Cas12f is naturally derived from archaea – single-celled organisms – which means it is not well suited to mammalian cells, let alone human cells or bodies. Only a few CRISPR proteins are known to function in mammalian cells without modification. Unfortunately, CAS12f is not one of them. This makes it an attractive challenge for bioengineers like Qi.

“We thought, ‘Okay, millions of years of evolution haven’t been able to turn this CRISPR system into something that works in the human body. “Can we change that in just one or two years?” Qi said. “To my knowledge, we have, for the first time, transformed a non-functional CRISPR into a functional CRISPR. “

Indeed, Xiaoshu Xu, postdoctoral researcher at the Qi laboratory and lead author of the article, did not see any activity of natural Cas12f in human cells. Xu and Qi hypothesized that the problem was that DNA from the human genome is more complicated and less accessible than microbial DNA, making it difficult for Cas12f to find its target in cells. By examining the computational predicted structure of the Cas12f system, she carefully selected about 40 mutations in the protein that could potentially work around this limitation and established a pipeline to test many protein variants at once. A functional variant would, in theory, make a human cell green by activating green fluorescent protein (GFP) in its genome.

“At first, this system didn’t work at all for a year,” Xu said. “But after iterations of bioengineering, we saw some modified proteins start to activate, like magic. It really made us appreciate the power of synthetic biology and bioengineering.”

The first positive results were modest, but they excited Xu and encouraged her to move on because it meant the system was working. Over many more iterations, she was able to further improve the performance of the protein. “We started out by seeing only two cells showing a green signal, and now after engineering, almost all cells are green under the microscope,” Xu said.

“At one point, I had to stop it,” Qi recalls. “I said, ‘It’s okay for now. You have made a very good system. We should think about how this molecule can be used for applications.

In addition to protein engineering, the researchers also engineered the RNA that guides the Cas protein to its target DNA. The modifications made to both components were crucial for the CasMINI system to function in human cells. They tested the ability of CasMINI to suppress and modify genes in human cells in the laboratory, including genes linked to HIV infection, anti-tumor immune response and anemia. It worked on almost all of the genes they tested, with robust responses in several.

To open the door

Researchers have already started to set up collaborations with other scientists to pursue gene therapies. They are also interested in how they might contribute to advances in RNA technologies – like what has been used to develop COVID-19 mRNA vaccines – where size can also be a limiting factor.

“This ability to design these systems has been desired in the field since the early days of CRISPR, and I feel like we have done our part to move towards this reality,” Qi said. “And this engineering approach can be very helpful. That’s what excites me – opening the door to new possibilities.

Additional co-authors of the paper at Stanford are graduate students Augustine Chemparathy and Hannah Kempton, and postdoctoral fellows Leiping Zeng, Stephen Shang, and Muneaki Nakamura. Qi is also a member of Stanford Bio-X. the Maternal and Child Health Research Institute (MCHRI), the Stanford Cancer Institute and the Wu Tsai Institute of Neuroscience. This research was funded by the Li Ka Shing Foundation.

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