CRISPR-associated Cas9 (white), Staphylococcus ausreus, based on Protein Database ID (5AXW). Credit: Thomas Splettstoesser (Wikipedia, CC BY-SA 4.0)
CRISPR gene editing can be described as a molecular scissors that cuts out certain sections of DNA. This is the common analogy. Stanley Qi, Stanford University assistant professor of bioengineering, likes this analogy. However, he believes it's time for CRISPR to be reimagined as a Swiss Army Knife.
CRISPR can be as simple or as complex as a cutter. It can also be more sophisticated as an editor, regulator, labeler, imager, and editor. This exciting field is generating many applications," Qi said. He is also an assistant professor of chemical-system biology at Stanford School of Medicine, and a Stanford ChEM–H institute scholar.
However, the many CRISPR systems currently in clinical use for gene therapy of eye, liver, and brain diseases are limited in scope. They all have the same problem: they are too big and difficult to deliver into living cells, tissues, or organisms.
Qi and his colleagues announce what they consider a significant step forward in CRISPR. They have published a paper Sept. 3rd in Molecular Cell. It is a mini-CRISPR system that is efficient, multi-purpose and versatile. While the most common CRISPR systems, with names like Cas9a and Cas12a denoting different versions of CRISPR proteins (Cas), are made up of approximately 1000 to 1500 amino acid. Their "CasMINI", however, has 529.
CasMINI can delete, activate, and edit genetic codes just like its beefier counterparts. This was confirmed by the researchers in experiments. Because it is smaller, it is easier to inject into human cells. This makes it an ideal tool for treating many ailments, such as eye disease and organ degeneration.
Persistent effort
The researchers chose to begin with Cas12f, also known as Cas14, a CRISPR protein that contains approximately 400 to 700 amino acid. Cas12f, like all CRISPR proteins is derived from Archaeasingle-celled animals. This means that it is not well-suited for mammalian cells. Only a handful of CRISPR proteins have been shown to be effective in mammalian cells. CAS12f isn't one of these. Bioengineers such as Qi find it a challenging challenge.
"We thought that millions of years of evolution had not been able turn this CRISPR system to function in the human body. Is it possible to change this in a matter of years? Qi said. Qi said, "To my knowledge we have turned a nonworking CRISPR to a working one for the first-ever time."
Indeed, Xiaoshu Xu (a postdoctoral scholar in Qi lab) and the lead author of this paper saw no activity from natural Cas12f cells. Xu and Qi argued that Cas12f is unable to locate its target cells because human genome DNA is more complex and less accessible than microbial. She carefully selected 40 mutations within the Cas12f protein structure to be able to bypass this limitation. The pipeline will allow her to test many variants of the protein at once. The theory is that a working variant could turn a human cell (or any other cell) green by activating green fluorescent proteins (GFP).
Xu stated that the system didn't work for a year at first. "But, after many iterations in bioengineering, some engineered proteins started to turn on like magic." This made us appreciate the power and potential of bioengineering and synthetic biology."
Although the first successes were modest, they encouraged Xu to keep going because it meant that the system was working. She was able to improve the performance of the protein over many more iterations. Xu stated that she started by only seeing two cells with a green signal. Now, after engineering, nearly every cell is green under a microscope.
Qi recalled, "At some point, I had to stop him." I said, "That's fine for now. This is a very good system. It's important to think about the applications of this molecule.
Researchers also created the RNA that guides Cas protein to its target DNA. CasMINI worked in human cells thanks to modifications to these components. CasMINI was tested for its ability to delete or edit genes in human cells grown in a laboratory. This included genes that are related to HIV, anti-tumor immunity response, and anemia. They tested almost all the genes it worked with strong responses in many.
Open the door
Researchers have already started to form collaborations with scientists in order to develop gene therapies. They are also keen to learn how they can contribute to advancements in RNA technology, such as the COVID-19 vaccines. Size is also a limiting factor.
Qi stated, "This ability to engineer such systems has been sought in the field ever since the early days CRISPR. I feel like we did our part towards that reality." This engineering approach can be so helpful. This is what excites me.
Learn more about the New CRISPR/Cas system that cuts virus RNA
More information: Engineered Miniature-CRISPR-Cas System to Mammalian Genome Regulation & Editing, Molecular Cell (2021). https://www.cell.com/molecular-cell/fulltext/S1097-2765(21)00648-1 Journal information: Molecular Cell Engineered Miniature CRISPR-Cas System for Mammalian Genome Regulation and Editing,(2021). DOI: 10.1016/j.molcel.2021.08.008
