When it comes to expressing genes, having a functioning DNA sequence that codes for protein is only part of the battle. A suite of regulatory and transcriptional elements are responsible for making sure that the DNA gets expressed appropriately as well as for controlling its frequency. If all of these components aren’t working in concert and the gene is creating too much, too little, or defective proteins, disease can set in. A team of bioengineers have managed to manipulate some of this genetic machinery into a tool that will allow them to alter a gene’s expression, potentially avoiding the progression of disease. Lei Stanley Qi from Stanford University is one of the senior authors of the paper, published in the journal Cell.
This new reprogramming tool was inspired by the clustered regularly interspaced short palindromic repeats (CRISPRs) that many prokaryotes use to eliminate any viral or damaged DNA that it might pick up. RNA finds the desired location on the DNA, and an enzyme called cas9 cuts the strand of DNA. While this effectively disables viral DNA and protects prokaryotes, scientists have found a way to make it work to their advantage in genomes of living eukaryotic cells as well, just by altering the sequence targeted by the CRISPRs.
The system can be designed to remove damaged or disease-causing mutations from the DNA sequence and insert another variation of the gene, altering the protein it makes. CRISPRs can also target regulatory regions, can increase or decrease the amount of protein the gene makes, or turn it off completely.
"It's like driving a car. You control the wheel to control direction, and the engine to control the speed, and how you balance the two determines how the car moves," Qi said in a press release. "We can do the same thing in the cell by up- or down-regulating genes, and produce different outcomes.”
Manipulating genes with CRISPR has really only been around since 2013, and Qi’s team expanded the technique by allowing two genes to be altered in the same shot. This increases the variety of possible outcomes produced by the gene, and could be useful in targeting certain diseases that affect multiple genes.
By altering the expression of two genes in the metabolic pathways of yeast, it produced four different products. The speed and direction in which mammalian cells moved were directed by tweaking a pair of genes regulating the cell’s motility. While these two experiments don’t have any obvious implications for disease treatments, they demonstrate proof of concept of the team’s dual gene targeting technique.
"Our technique allows us to directly control multiple specific genes and pathways in the genome without expressing new transgenes or uncontrolled behaviors, such as producing too much of a protein, or doing so in the wrong cells," Qi continued. "We could eventually synthesize tens of thousands of RNA molecules to control the genome over a whole organism.”
In order for this technique to work in a living organism, the researchers will need to develop a new way of introducing the engineered CRISPRs, which are currently injected directly into cells. A long-term goal is for these gene-editing tools to be administered into the bloodstream, and to make its way into the cells to perform the necessary edits. This is no small task, as a number of challenges exist. The CRISPRS need to travel through the bloodstream without alerting the immune system, pass through the selectively permeable cell membrane, pass through the nuclear envelope, and then target the gene in the correct location.
Qi explained that his lab will be working in conjunction with colleagues from other disciplines, increasing the odds of overcoming those obstacles. "I'm optimistic because everything about this system comes naturally from cells, and should be compatible with any organism.”
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