Viewing genes inside living cells!

Viewing genes inside living cells!
 

DNA is often depicted as tidy strands stretched out in straight lines. But in reality, our DNA is clumped up inside the nuclei of our cells like cooked spaghetti. "We have two meters of DNA folded into a nucleus that is so tiny that 10,000 of them will fit onto the tip of a needle," author explained. "We know that DNA is not linear but forms these loops, these large, three-dimensional loops. We want to basically image those kind of interactions and get an idea of how the genome is organized in three-dimensional space, because that's functionally important."

Researchers developed a way to track genes inside living cells. They can set them aglow and watch them move in three dimensions, allowing him to map their positions much like star charts record the shifting heavens above. And just as the moon influences the tides, the position of genes influences the effects they have; thus, 3D maps of gene locations could lead scientists to a vastly more sophisticated appreciation of how our genes work and interact -- and how they affect our health.

"This has been a dream for a long time," author said. "We are able to image basically any region in the genome that we want, in real time, in living cells. It works beautifully. ... With the traditional method, which is the gold standard, basically you will never be able to get this kind of data, because you have to kill the cells to get the imaging. But here we are doing it in live cells and in real time."

The new approach uses the CRISPR gene editing system that has proved a sensation in the science world. The researchers flag specific genomic regions with fluorescent proteins and then use CRISPR to do chromosome imaging.

They designed single-guide RNAs (sgRNAs) integrated with up to 16 MS2 binding motifs to enable robust fluorescent signal amplification. These engineered sgRNAs enable multicolour labelling of low-repeat-containing regions using a single sgRNA and of non-repetitive regions with as few as four unique sgRNAs.

Authors were able to achieve tracking of native chromatin loci throughout the cell cycle and determine differential positioning of transcriptionally active and inactive regions in the nucleus. 

The new method overcomes longstanding limitations of gene imaging. "We were told we would never be able to do this," author said. "There are some approaches that let you look at three-dimensional organization. But you do that experiment on hundreds of millions of cells, and you have to kill them to do it. Here, we can look at the single-cell level, and the cell is still alive, and we can take movies of what's happening inside."

https://www.nature.com/articles/ncomms14725

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