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Stanford researchers have engineered a new miniature CRISPR system that should be easier to deliver into human cells.
Bioengineers have repurposed a “non-working” CRISPR system to make a smaller version of the genome engineering tool. Its diminutive size should make it easier to deliver into human cells, tissues, and the body for gene therapy.
The common analogy for CRISPR gene editing is that it works like molecular scissors, cutting out select sections of
“>amino acids, their “CasMINI” has 529.
The researchers confirmed in experiments that CasMINI could delete, activate and edit genetic code just like its beefier counterparts. Its smaller size means it should be easier to deliver into human cells and the human body, making it a potential tool for treating diverse ailments, including eye disease, organ degeneration and genetic diseases generally.
To make the system as small as possible, the researchers decided to start with the CRISPR protein Cas12f (also known as Cas14), because it contains only about 400 to 700 amino acids. However, like other CRISPR proteins, Cas12f naturally originates 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 work in mammalian cells without modification. Unfortunately, CAS12f is not one of them. This makes it an enticing challenge for bioengineers like Qi.
“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?’” said Qi. “To my knowledge, we have, for the first time, turned a nonworking CRISPR into a working one.”
Indeed, Xiaoshu Xu, a postdoctoral scholar in the Qi lab and lead author of the paper, saw no activity of the natural Cas12f in human cells. Xu and Qi hypothesized that the issue was that human genome DNA is more complicated and less accessible than microbial DNA, making it hard for Cas12f to find its target in cells. By looking at the computationally predicted structure of the Cas12f system, she carefully chose about 40 mutations in the protein that could potentially bypass this limitation and established a pipeline for testing many protein variants at a time. A working variant would, in theory, turn a human cell green by activating green fluorescent protein (GFP) in its genome.
“At first, this system did not work at all for a year,” Xu said. “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 first successful results were modest, but they excited Xu and encouraged her to push forward because it meant the system worked. Over many additional iterations, she was able to further improve the protein’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 said.
“At some moment, I had to stop her,” recalled Qi. “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.’”
In addition to protein engineering, the researchers also engineered the (function(d, s, id){
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