joanna wysocka

Joanna Wysocka and her lab team found that DNA twitches during transcription to bring distant regions in contact and enhance gene expression. (Credit: Misha Gravenor)

DNA flails about during transcription like a strand of spaghetti being sucked through pursed lips, Standford researchers have found. Like the resulting of out-of-control flying globs of sauce, the surprising discovery flies in the face of conventional wisdom, which posits that static loops of DNA are required to bring together distant regions that enhance and promote gene expression. 

A new DNA labelling technique, which can precisely tag any individual stretch of DNA with fluorescent molecules to track their three-dimensional locations and movements, revealed this genetic dance. The technique, which the researchers have termed CARGO, for chimeric array of gRNA oligo, is a variation of the CRISPR/Cas9 gene-editing tool, and it promises to revolutionise the study of genome dynamics. 

 

‘Entirely Unexpected and Surprising’ 

“We’ve found that, as the polymerase ploughs across the DNA, it provides a source of molecular agitation that increases mobility within a local chromosome domain and can repeatedly bring distant regions of the genome together,” said Joanna Wysocka, PhD, professor of developmental biology and of chemical and systems biology. “This was entirely unexpected and surprising, and directly counters the prevailing beliefs about transcription. It’s just one example of what we and others can now learn by using CARGO to label specific DNA regions.” 

The paper describing the research is published in Science. Wysocka is the senior author, and graduate student Bo Gu is the lead author. 

Allele Specific Gene Editing with CRISPR

CRISPR is most commonly used to seek out and replace specific DNA sequences in the genome with other DNA sequences. To do so, an enzyme called Cas9 uses a short RNA sequence to guide the DNA sequences to the correct spot in the genome. 

A variation of the technique that was developed by other researchers instead uses guide RNAs and the CRISPR system with a catalytically inactive form of Cas9 to recognise and label specific stretches of DNA with fluorescent molecules. But that works best on highly repetitive regions where a single guide RNA can marshal the critical mass of fluorescent tags necessary to generate enough light to be seen through a microscope. 

Wysocka, Gu and co-author, Tomasz Swigut, PhD, devised a way to introduce an array of many different guide RNAs into a cell to precisely recognise nonrepetitive, unique stretches of DNA and label them with multiple fluorescent tags so they can be easily visualised under a microscope. 

 

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‘CARGO Solves the Delivery Problem’ 

“All the most interesting stuff in the genome present as single copies,” Wysocka said. “People have been trying unsuccessfully to label single regions, or loci, for some time. But CARGO solves the delivery problem. Now we can label any region, or locus, that we want by using many different guide RNAs to blanket the DNA so we can see it clearly.” Wysocka and Gu emphasise that the CARGO technique will be useful to researchers pursuing many different questions about the genome or gene expression. 

How it Actually Looks When CRISPR Snips a Strand of DNA

Already it’s opened their eyes about the process of transcription, which is often simulated when distant enhancer regions are brought into close proximity with other DNA regions called promoters. 

“We found that any locus we looked at moved about four times faster in its active state, when nearby genes are being transcribed into RNA,” Wysocka said. “We propose that this enhanced movement, or diffusion is likely to bring distant regions of the DNA together and further promote transcription.” 

Gu, Swigut and Wysocka have filed a U.S. provisional patent application relating to the CARGO methodology. 

 


Materials provided by Krisa Conger, Standford Medicine News Center. Note: Content may be edited for style and length.