SAN FRANCISCO, Sept. 3 (Xinhua) -- A research team led by University of California (UC) scientists has used a modified version of the gene-editing technique CRISPR to find enhancers, or regulatory sequences that turn on and off a gene that affects development of the immune cells known as T cells.
Each one of our cells has the same 22,000 or so genes in its genome, but each uses different combinations of those same genes, turning them on and off, or allowing them expressed and repressed, as their role and situation demand, which determines what kind of cell - kidney, brain, skin, heart - each will become.
The new study, published online in Nature by the team of researchers mostly from UC San Francisco and UC Berkeley, used a tool called CRISPR activation (CRISPRa), to search for the enhancers.
The most common application of CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, an enzyme called Cas9 snips DNA at particular sequences specified by the sequence of a "guide RNA." With the technology, scientists can excise or edit any gene, and observe how these changes affect cells or whole organisms.
However, sequences that code directly for proteins make up only 2 percent of our genome. Enhancers and other regulatory DNA elements spread throughout the other 98 percent. Scientists can look for potential enhancer sequences based on how they interact with proteins that bind to DNA, but figuring out which enhancers work with which genes is much more challenging.
Thinking of the genome as a model home with 22,000 lightbulbs (the genes) and hundreds of thousands of switches (the enhancers), the challenges have been finding all of the switches and figuring out which lightbulbs they control and when. Previously, CRISPR has been used to cut out wires looking for those that would cause a bulb to go dark, giving a good idea of what that section of the circuit was doing. But cutting out a light switch when it's off doesn't tell you anything about what it controls.
A better approach would be a universal "on" switch that could target any part of the genome and, if that part included an enhancer, could activate that enhancer. CRISPRa, developed in 2013 by Jonathan Weissman, professor of cellular and molecular pharmacology at UC San Francisco, uses a "blunted" version of the DNA-cutting Cas9 protein, strapped to a chain of activating proteins. Instead of cutting DNA, it can activate any enhancers in the area.
By targeting the CRISPRa complex to thousands of different potential enhancer sites, the research team behind the new study reasoned, they would be able to determine which had the ability to turn on a particular gene.
The gene studied by the team produces a protein called IL2RA, which is critical to the function T cells. Depending on conditions in the body, T cells have the ability to either trigger inflammation or suppress it. The IL2RA gene produces a protein that tells T cells that it's time to put on their anti-inflammatory hats. If the enhancers that should turn on the gene have errors, the cells fail to suppress inflammation, potentially leading to autoimmune disorders like Crohn's disease.
To track down locations of the enhancers that control IL2RA, the team produced over 20,000 different guide RNAs and put them into T cells with a modified Cas9 protein, essentially performing 20,000 experiments in parallel to find all the sequences that turn on the gene. Targeting some of the sequences with CRISPRa increased IL2RA production, yielding a short list of locations that might be important for regulating the fate of T cells.
The research team hopes to expand the method, perhaps by finding ways to search for enhancers of many different genes at once, according to a news release from UC San Francisco. And they expect the method to be a widely applicable tool for untying genetic interactions in all kinds of cells.