owbugs, armadillos, hedgehogs … and DNA? The same strategy that some animals use to avoid being attacked — roll into a ball and keep your vulnerable bits beyond predators’ reach — turns out to let genes avoid being sliced up by the genome-editing molecules of CRISPR, scientists reported on Monday. When a segment of DNA wraps itself around a protein into what’s called a nucleosome, CRISPR-Cas9 can no more cut it than a hungry hawk can bite a rolled-up hedgehog.
If CRISPR “can’t see DNA when it’s wrapped around a nucleosome,” said biochemist Dana Carroll of the University of Utah, who led the study, “it could be an issue.”
Nucleosomes exist because fitting the 6-foot-long human genome into a cell nucleus is akin to packing 30 miles of yarn into a basketball: It needs to be rolled up. When a cell doesn’t need a particular gene at a particular moment, that gene rolls around a protein, becoming inaccessible to the cell’s gene-activating machinery. Most human DNA gets packaged into a nucleosome at some point, with nucleosomes sprinkled across the human genome at every 200 or so of its 3 billion A, T, C, and G “letters.”
The nucleosome issue is only the latest in an expanding list of challenges, including genomic havoc and concerns about cancer, to using CRISPR to treat diseases. As with those others, it’s too early to know whether rolled-up DNA will be an impediment to the use of CRISPR to repair disease genes in people.
This is not the first study to find that nucleosomes affect whether CRISPR can cut its target. But other research showed that effect in test tubes, or found that CRISPR-Cas9 is much better at cutting regions of the genome with few nucleosomes than regions chock-a-block with them, but without proving that nucleosomes are why. The earlier research also used surrogate measures of DNA cleavage rather than directly measuring it.
Those previous experiments “didn’t try to change the accessibility of some region directly” and see if that affected CRISPR’s ability to cut, said James Haber of Brandeis University, who reviewed the paper, in Proceedings of the National Academy of Sciences. “These scientists did precisely that.”
Carroll’s team used various guide RNAs, the molecules that lead CRISPR-Cas9 to its target, and aimed first at a nucleosome-rich region in the genome of yeast. They got, as expected, a piddling amount of editing. “Then we took exactly the same guide RNA and measured how well it cut where the nucleosome was largely gone,” said Carroll, who has been developing genome-editing technologies for 20 years. (He used a biochemical trick to evict the nucleosome.)
Voila: lots of editing. That showed causality. They also measured cleavage directly, rather than with a surrogate readout. “Cas9 cleavage is strongly inhibited when the DNA target is within a nucleosome,” the scientists wrote.
Most algorithms that predict whether CRISPR-Cas9 will work on a given gene are based on the gene’s DNA sequence. It would be a good idea, Carroll said, to take into account whether DNA is wrapped into a hard-to-reach nucleosome.
Although the research was done in yeast, the findings likely pertain to human genomes, too: Yeast and humans are both eukaryotes, meaning their genes reside inside a nucleus.
The previous generation genome editor, called zinc fingers, had no problem reaching DNA that’s wrapped around nucleosomes, Carroll’s team reported. That hadn’t been directly shown before either, and Carroll called that finding “surprising” and “remarkable.” CRISPR is an easier way to edit genomes, but in the great race to develop therapies based on gene editing, one company, Sangamo Therapeutics, is betting on zinc fingers rather than CRISPR. Sangamo did not reply to a request for comment. Carroll receives license royalties from Sangamo based on his zinc finger discoveries and is on the scientific advisory board of Recombinentics, which is using CRISPR to edit the genomes of livestock to produce, for example, dehorned cattle.
Is DNA that’s wrapped into a nucleosome forever off-limits to CRISPR, or just temporarily? DNA wraps and unwraps itself as a cell moves through its growth cycle. If CRISPR has enough time to work, the natural ebb and flow of nucleosomes might make every target accessible eventually, like a hedgehog tiring of being rolled up while a hawk awaits.
The trouble is, the longer CRISPR hangs around a genome, the more unintentional havoc it could wreak. Also, while DNA in dividing cells forms and un-forms nucleosomes, in cells that don’t divide (such as neurons) “some regions may be always inaccessible,” said Brandeis’ Haber.
In many of the therapeutic uses of CRISPR that are being developed or contemplated, “not only will the target cells likely not be dividing,” Carroll and his colleagues wrote, but the fact that only some CRISPR molecules even make it into the cells at all “make[s] it imperative” that editing work when they do enter.