More detailed information about the machinery behind the popular CRISPR-Cas9 genome editing tool has been revealed in a study by scientists at the University of Massachusetts Medical School published in the Journal of Cell Biology.

The important new details about the inner workings of CRISPR-Cas9 in live cells might have implications for the development of therapeutics that use this powerful gene editing tool.

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CRISPR-Cas9 is a powerful gene editing system and is a component of the bacterial immune system that protects genes from viral invasion.

CRIPR-Cas9 complex, which is more efficient and precise than previous technologies, is being adapted in the lab as scientists find ways to program and deliver it quickly to selectively edit specific genetic sequences for study.

To cut a piece of double-stranded DNA, CRISPR-Cas9 makes use of a guide RNA made of some 20 nucleotides to target specific regions of a genome at which the Cas9 complex then makes the cut. This allows scientists to remove or insert genetic sequences into the genome.

But since the underlying dynamics of how the CRISPR/Cas9 system works inside live cells aren't well understood, some delivery systems and techniques have been more successful than others.

"We don't know a lot about the details of how the CRISPR-Cas9 complex gets around the genome of a live cell and finds its target," said Thoru Pederson, PhD, Vitold Arnett Professor of Cell Biology and professor of biochemistry and molecular pharmacology.

"What we've learned in this study about how this machinery works is important and useful for gene editors looking to develop tools for the lab and potentially the clinic."

To observe the actions of the CRISPR-Cas9 system at work in a live cell, Dr. Pederson and colleagues developed a technique for labeling the guide RNA and Cas9 elements with different florescent molecules so they could be tracked simultaneously.

What they found is the guide RNA, when not bound to Cas9, is extremely short-lived.

When the Cas9 and guide RNA do assemble, the complex is much more stable. About half show a lifetime in the cell nucleus of some 15minutes with the remainder being considerably more stable.

"Cas9 stabilizes the guide RNA," said Hanhui Ma, PhD, research specialist in the Pederson laboratory and co-first author of the Journal of Cell Biology article.

"If you're delivering the guide RNA and Cas9 separately into the cell for them to then assemble it won't be as efficient because some of the guide RNA will degrade. If you deliver them both into the cell, already assembled, you'll see more activity."

The other observation made was that the duration of target residence by the Cas9-guide RNA complex determines whether the DNA will be cut. When the guide RNA sequence perfectly matched the target DNA sequence, the Cas9-guide RNA complex remained bound for as long as two hours before leaving, with cleavage completed.

With mismatched guide RNA-target DNA sequences, the complex lingered for as little as a few minutes and cleavage was impaired. The team behind this observation included David Grunwald, PhD, assistant professor of the RNA Therapeutics Institute and Li-Chun Tu, PhD, postdoctoral fellow in the Grunwald lab.

Knowing this, scientists can potentially predict mathematically where an off- target cut may happen based on how long the CRISPR complex it sits on the genome, said Dr. Ma.

"We still don't know the rules of CRISPR," said Dr. Ma.

"Everybody wants to know what will happen when it's delivered into a live cell. This study helps write a piece of that operating manual."

TagsCRISPR/Cas9, genome editing tool, University of Massachusetts Medical School, DNA, RNA, Thoru Pederson, live cells, Cells