Making sense of the genome’s three-dimensional structure is like trying to untangle a set of Christmas tree lights…in a darkened room…and none of the bulbs is alight. If just a few bulbs were to glow, however, it might be easier to distinguish between tight coils and loose strands. And if bulbs of multiple colors were available, the whole task might become considerably less knotty.

Similarly, in investigations of chromosome dynamics, fluorescent labels are useful. To date, however, researchers interested in tracking the genome’s movements in living cells have been limited to just three colors. But these researchers may soon have access to a broader palette.

A new technology, aptly named CRISPRainbow, has been developed by scientists at the University of Massachusetts Medical School (UMMS). CRISPRainbow can allow researchers to tag and track up to seven different genomic locations in live cells.

The technology was described April 18 in Nature Biotechnology, in an article entitled, “Multiplexed Labeling of Genomic Loci with dCas9 and Engineered sgRNAs Using CRISPRainbow.” The article describes how CRISPR technology, well known for its gene editing and gene regulation applications, may also be used to apply fluorescent labels to genomic loci.

“Here we describe CRISPRainbow, a system for labeling DNA in living cells based on nuclease-dead (d) Cas9 combined with engineered single guide RNA (sgRNA) scaffolds that bind sets of fluorescent proteins,” wrote the article’s authors. “We demonstrate simultaneous imaging of up to six chromosomal loci in individual live cells and document large differences in the dynamic properties of different chromosomal loci.”

Although it is possible to use multiple colors with existing technologies, going beyond the three-color limit imposes a dire requirement. Cells must be fixed by bathing them in formaldehyde, thus killing them and making it impossible to observe how the chromosome’s structure changes over time or in response to stimuli.

To overcome this technological hurdle, the UMMS scientists used a CRISPR/Cas9 system to tag specific locations along the genome. They created a Cas9 mutation that makes the nuclease inactive so it only binds to the DNA and doesn't cut the genome. Once deactivated, the CRISPR/Cas9 element is ferried to a specific location on the genome by a guide RNA that can be programmed by the researchers.

To see and track the CRISPR/Cas9 complex once it is bound to the genome, the scientists engineered the guide RNA to include one of three primary fluorescent proteins: red, green, or blue. These proteins can then be observed and tracked in real time under a microscope. By attaching a second fluorescent protein to the guide RNA, the scientists were able to combine these three primary colors to generate three additional labels: cyan, magenta, and yellow. A seventh label, white, is achieved by combining all three primary colors.

“Computers cooperating with spectral filters in the microscope read out combinations of colors and display them as a color that you request,” explained Thoru Pederson, Ph.D., the study’s senior author and a professor of cell biology and professor of biochemistry and molecular pharmacology. “For example, red and green can be yellow. Using the three primary colors and this approach, which is called computational coloring, we can generate an additional three colors.”

CRISPRainbow can simultaneously locate as many as seven different DNA sites, each in a distinctive color. Deploying it in living cells adds to the power of the technology, as it can track dynamic, topological movements of the genome that may have important biological consequences.

“With this technology, we can visualize different chromosome loci at different points in time,” said study co-author Li-Chun Tu, Ph.D., postdoctoral associate in the lab of David Grunwald, Ph.D., assistant professor of biochemistry and pharmacology at UMMS. “And we can monitor them to see how far and fast these loci move. With this, we can see how these structural changes affect the genes being expressed and their relation to health and disease.”

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