A team of scientists from the University of Massachusetts (UMass) Medical School has created a new CRISPR/Cas9 technology called CRISPRainbow, which allows researchers to tag and track up to seven different genomic locations in live cells.
"Most people are using CRISPR for editing genomes," said Hanhui Ma, UMass research specialist and co-author of the study. "We are using it to label DNA and track the movement of DNA in live cells."
The ability to pinpoint locations of genomic elements in live cells is crucial for our understanding of chromosome dynamics due to the fact that the genes that guide our biology and health act in a three-dimensional space. Where our DNA is positioned in the nucleus plays a huge role in numerous biological processes from embryonic development to cancer.
Despite this importance, current technology can, at most, follow three genomic locations at a time in live cells. In order to track more, more cells must be covered in formaldehyde, leading to their death and preventing scientists from being able to observe chromosome structure changes over time.
In order to overcome this challenge, Ma and his team created a Cas9 mutation that makes the nucleus inactive so that it only binds to DNA and does not cut the genome. A guide RNA, which can be programmed by researchers, then brings this deactivated CRISPR/Cas9 element to specific genomic locations.
Ma engineered the guide RNA with one of three primary fluorescent proteins - red, green or blue - in order to allow the team to track the modified complex. Using a microscope, these proteins can be tracked and observed in real time. Furthermore, by attaching a second fluorescent protein to the guide RNA, the team was able to create three additional colors: cyan, magenta and yellow. White is created 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," said Thoru Pederson, professor of biochemistry and molecular pharmacology at UMass and co-author of the study. "For example, red and green can be yellow. Using the three primary colors and this approach called computational coloring we can generate an additional three colors."
The team's unique new technology can locate as many as seven different DNA sites at the same time, each one tagged in a distinctive color, allowing scientists to track dynamic, topological genome movements that are important for our biological functioning.
"With this technology, we can visualize different chromosome loci at different points in time," said Li-Chun Tu, an assistant professor of biochemistry and pharmacology at UMass and co-author of the study. "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."
The findings were published in the April 18 issue of the journal Nature.
