CRISPRoff Silences Genes without DNA Damage

A new gene editing technology allows researchers to control gene expression with high specificity while leaving the sequence of the DNA unchanged. CRISPRoff, which was designed by Whitehead Institute scientists, is stable enough to be inherited through hundreds of cell divisions, and is also fully reversible.

"The big story here is we now have a simple tool that can silence the vast majority of genes," says Jonathan Weissman, senior author of a paper published in Cell today. "We can do this for multiple genes at the same time without any DNA damage, with great deal of homogeneity, and in a way that can be reversed. It's a great tool for controlling gene expression."

Weissman and collaborators had previously created two other epigenetic editors—CRISPRi and CRISPRa—but both of these came with a caveat. In order for them to work in cells, the cells had to be continually expressing artificial proteins to maintain the changes.

"With this new CRISPRoff technology, you can [express a protein briefly] to write a program that's remembered and carried out indefinitely by the cell," says co-author Luke Gilbert. "It changes the game so now you're basically writing a change that is passed down through cell divisions—in some ways we can learn to create a version 2.0 of CRISPR-Cas9 that is safer and just as effective, and can do all these other things as well."

To build an epigenetic editor that could mimic natural DNA methylation, the researchers created a tiny protein machine that, guided by small RNAs, can tack methyl groups onto specific spots on the strand. These methylated genes are then "silenced," hence the name CRISPRoff. Because the method does not alter the sequence of the DNA strand, the researchers can reverse the silencing effect using enzymes that remove methyl groups, a method they called CRISPRon.

As they tested CRISPRoff in different conditions, the researchers discovered a few interesting features of the new system. For one thing, they could target the method to the vast majority of genes in the human genome—and it worked not just for the genes themselves, but also for other regions of DNA that control gene expression but do not code for proteins.

Also, surprisingly to the researchers, CRISPRoff was even able to silence genes that did not have CpG islands, which had previously been thought necessary to any DNA methylation mechanism. "What was thought before this work was that the 30 percent of genes that do not have a CpG island were not controlled by DNA methylation," Gilbert says. "But our work clearly shows that you don't require a CpG island to turn genes off by methylation."