Inner Workings of Revolutionary Gene Editing Technology Revealed

The CRISPR-Cas9 gene editing system has broken onto the scene pushing full steam ahead in the science world. Now, new research reveals important details on the inner workings of the machinery in live cells, which may help fuel the development of therapeutics that use this tool.

This powerful gene editing system is more precise and efficient compared with other technologies. Currently, the CRISPR-Cas9 complex is being adapted in the lab, and scientists are exploring different ways to program and deliver it quickly to selectively edit specific genetic sequences for the study, according to a study published in The Journal of Cell Biology.

CRISPR uses a guide of RNA made up of about 20 nucleotides to target specific regions of a genome at which the Cas9 complex can cut a piece of double-stranded DNA. The underlying dynamics of how the CRISPR-Cas9 system works inside live cells was unclear, and certain delivery systems and techniques have been more successful than others.

For the study, the researchers set out to uncover these underlying dynamics.

“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 researcher Thoru Pederson, PhD. “What we’ve3 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.”

The researchers developed a new technique for labeling the guide RNA and Cas9 elements with different florescent molecules that could be tracked simultaneously.

The results showed that when the guide RNA is not bound to Cas9, it is very short lived, but when they are bound, the complex becomes more stable. Researchers reported that about half showed an approximate 15-minute lifetime in the cell nucleus, while the rest were considerable more stable.

“Cas9 stabilizes the guide RNA,” said co-first study author Hanhui Ma, PhD. “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.”

An additional finding was that the duration of target residence by the Cas9 guide RNA complex is responsible for determining whether the DNA will be cut. Researchers found that the Cas9-guide RNA complex was able to remain bound for up to 2 hours before leaving, with cleavage completed, when the guide RNA sequence matched the target DNA sequence.

However, when the RNA and DNA sequences were mismatched, the complex stayed for only a few minutes and the cleavage was impaired.

“We still don’t know the rules of CRISPR,” Ma said. “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.”

The authors noted that the study’s findings can potentially help scientists to mathematically predict where an off-target cut may happen, based on how long the CRISPR complex sits on the genome.