But Cas9 isn’t perfect. Sometimes, it will cut a target that mostly matches its guide RNA, but not entirely. It’s as if it sees “MUSTARD” and thinks: “Eh, close enough, let’s slice and dice.” These off-target cuts are a nuisance to CRISPR users, who have flocked to the technique precisely because it’s meant to be precise.
They also raise serious questions about proposed (speculative) uses for the technique, such as editing the genomes of embryos to prevent inherited disorders. If you’re going to try that, you’d better make sure that you’re not inadvertently activating a cancer gene or disrupting an essential one. Indeed, when a Chinese team recently (and controversially) used CRISPR to edit a disease gene in (inviable) human embryos, they found surprising and worrying levels of off-target cuts.
That’s why the many position statements about the ethics of CRISPR have universally highlighted the risks of off-target cuts, and the need to identify, understand, and avoid them. And it’s why several groups of scientists have been trying to develop ways of making CRISPR more specific, so that they make fewer off-target cuts.
The simplest involve lowering the levels of active Cas9. But this reduces the frequency of both off-target cuts, and on-target ones—you get a more specific editor, but also a less efficient one.
You could also change the guide. Keith Joung from the Massachusetts General Hospital has shown that using shorter guide RNAs leads to fewer off-target cuts. When guides are long, the Cas9 enzyme will tolerate several mismatches before it fails; with shorter ones, any single mismatch threatens to derail the editor.
CRISPR pioneers Feng Zhang and George Church are trying to change the Cas9 editor itself. First, they created half-hearted versions. The usual enzyme checks a DNA sequence against its guide RNA and then cuts both strands, but the mutant versions cut just one strand. So you need two of them, checking their own guide RNAs, to fully sever a stretch of DNA. It’s unlikely that both enzymes will get things wrong, so together, they become more specific.
Today, Zhang has unveiled yet another strategy for engineering Cas9. First, his team searched for mutations that will make Cas9 more discerning in its attacks, so that it only cuts DNA that perfectly matches its guide RNA template. They found five, which make the enzyme more specific but no less efficient. They then tested these mutations in combinations, until they settled on one particularly judicious version of Cas9, which they called esCas9 (“enhanced specificity Cas9”). This upgraded Cas9 cuts its targets just as well as Cas9 Classic, but never veers off target—at least, not that the team could detect. It’s a precision weapon that inflicts no collateral damage.
These methods are complementary, Zhang told me at the International Summit on Human Gene Editing. They could all be fused together—for example, by building a version of esCas9 that cuts just one strand and relies on shorter guides. “Maybe some kind of superposition will lead to the ultimate system,” he says. “And maybe you’ll have a whole toolbox of tools and you’ll pick the right one for the application in mind.”