Zhang Lab Produces Engineered CRISPR Complex with Greater Precision

By Aaron Krol

December 3, 2015 | CRISPR-Cas9 gene editing has quickly become a basic tool of molecular biology, but don’t let that fool you into thinking we know exactly how it works. Armed with the DNA-slicing Cas9 molecule and a single guide RNA (sgRNA) sequence, biologists can and enthusiastically do make small, precise cuts almost anywhere in the genome of any species, altering the genetic code at will. But the steps to the intricate molecular tango of Cas9 and DNA ― and the reasons their footwork sometimes slips ― are still being worked out.

In fact, while Jennifer Doudna and Emmanuelle Charpentier first demonstrated how to program Cas9 for gene editing in 2012, it wasn’t until early last year that they were able to propose a 3D structure for the molecule, based on x-ray crystallography images. Knowing the shape of Cas9, and which substructures it uses to link hands with DNA, is crucial to learning how it makes its cuts. (Interestingly, at least one group, working at Montana State University, had gotten images of a CRISPR-associated molecule as early as 2010.)

As a new report published this week in Science shows, Cas9 will only become more powerful as we puzzle out exactly how it interacts with DNA. The paper, a product of Feng Zhang’s lab at the Broad Institute of MIT and Harvard, presents a tweaked version of Cas9 that slightly loosens its grip on DNA. It’s a feat of “rational engineering,” using knowledge of a molecule’s form and function to deliberately change its behavior ― as opposed to the random plug-and-play search for better molecules that has sometimes characterized CRISPR-Cas9 research.

Zhang and his colleagues, including lead author Ian Slaymaker, offer their new Cas9 as a solution to the problem of off-target cuts, where an sgRNA sequence mistakenly sets up the Cas9 guillotine over the wrong area of the genome. These misdirected cuts crop up at low levels in almost every CRISPR-Cas9 experiment. In the lab they’re basically harmless, and most fall across meaningless strings of DNA that the genome can easily do without. But if we were to start using CRISPR as a therapy, to correct rare diseases embedded in people’s DNA, the one-in-a-hundred chance of snipping out something vital becomes an all too real threat.

Zhang has a big stake in wiping out off-target errors. Not only is his lab a powerhouse of CRISPR research (he’s sometimes mentioned as a likely contender for a Nobel Prize in connection with the technology), he is also a scientific founder of Editas Medicine, one of three companies hoping to get Cas9-based cures into the clinic. So it’s not surprising to see his group scouring the structure of Cas9 for ways to make it more discerning about the DNA it chops up.

Holding On With Both Hands

The Science paper started with an observation about how Cas9 contends with damaged DNA.

The authors noticed that, when the target DNA itself is weakened by a mismatch between its two strands, even poorly designed Cas9 complexes are able to cut it effectively. It seemed that a gap between DNA strands provided an opening for a Cas9 molecule that wouldn’t otherwise be up to the job.

They reasoned that Cas9 needs to separate both DNA strands before it can slice them ― and that a Cas9 molecule that strains to separate those strands would be less promiscuous with its scissors.

Cas9 appears to attach to DNA at two junctions on opposite strands. First, Cas9 uses an sgRNA molecule to bind to a specified sequence of DNA; that’s the reason its edits can be aimed so precisely. Close but imperfect sgRNA matches are what cause off-target cuts: a mismatch will weaken Cas9’s grip on the DNA molecule, but not always enough to stop it from pulling the two strands apart, especially if it’s found a part of the genome with many similarities to its real target.

Second, once Cas9 has latched on to its DNA target, it pulls at the opposite strand by clutching it in a groove between two structures called the RuvC and HNH domains. This second grip doesn’t care about the DNA sequence at all. Instead, the groove has a strong positive charge, which creates a powerful bond with the negatively charged DNA.

Images of the Cas9 molecule, with RuvC and HNH domains colored, show how it interacts with sgRNA and DNA to pull DNA strands apart before cutting them. Image credit: Broad Institute

That was the inspiration for the Zhang lab’s engineering of a new Cas9 molecule. The team found 32 sites inside the RuvC-HNH groove where they could replace positively-charged components with alanine, a neutrally-charged amino acid. One by one, they made these switches to create 32 different mutant Cas9 molecules, each of which should theoretically have had a weaker clutch than the natural version. The lab reasoned that this would make a perfect sgRNA match even more important. In an off-target match, already weakened by a poor bond between DNA and sgRNA, the two DNA strands might be too hard to separate for Cas9 to make its cut.

The hunch was borne out when the Zhang lab used its mutant Cas9 molecules to make cuts that they knew would normally cause several off-target glitches, and checked whether their new molecules were making the same mistakes. After a few rounds of experiments, they started mixing the most promising mutations together, searching for a Cas9 molecule that performed well in multiple gene regions known to be problematic for natural Cas9.

Finally, the authors settled on a modified Cas9 molecule they named “eSpCas9,” which appears to cause especially few off-target cuts, without dulling its blade for its real DNA targets. This new molecule actually has three different alanine substitutions, a product of both rational engineering and simple trial-and-error.

Inching Toward the Clinic

The Broad Institute will be releasing eSpCas9 for outside scientists to use, something it has also done with past CRISPR innovations like libraries of useful sgRNA sequences. Future studies, however, covering more tricky regions across the genome, will probably turn up even more precise Cas9 variants; we may even find that different Cas9 mutants are better suited for different tasks.

Zhang himself has been very interested in the search for new and better Cas9 molecules for particular jobs. Just this April, his lab published research on the incredible variety of CRISPR complexes found in nature, where hundreds of bacterial species use their own molecular armaments to slice up the DNA of invading viruses. While most work with Cas9 has used a molecule found in Streptococcus pyogenes, Zhang and Editas have high hopes for Staphylococcus aureus Cas9 ― a version of the molecule so compact that it can be encoded in a virus, the better to deliver to patients’ cells for Cas9-based therapies.

Indeed, while the experiments described in the Science paper all work with S. pyogenes Cas9, the authors also note that they’ve performed similar fine-tuning of Cas9 molecules from other bacteria, S. aureus among them. So while scientists around the world may find eSpCas9 very useful in the lab, it’s a fair guess that, at Editas, the research staff will be following other avenues from this research.

Meanwhile, Zhang and several of his co-authors are currently in Washington, D.C., at a summit to discuss the future of gene editing in light of CRISPR-Cas9’s fast-evolving possibilities. For now, most experts would agree that risks from off-target errors make gene editing of humans a dangerous proposition. But these problems are swiftly clearing up ― leaving the ethical and political questions around engineering the human genome more salient than ever before.