Beyond Cas9: 4 Ways to Edit DNA

Crispr scientists are, essentially, muggers in lab coats. When they need a new pair of DNA-slicing scissors, called a nuclease, they just steal one from a germ. But repurposing microbial machinery isn’t so simple: Some nucleases are too big; some are too blunt; some don’t work well inside human cells. So, as Crispr wends its way out of the petri dish and into our genes, the search is on for slimmer, sharper tools. With trillions of muggable microbes, there are plenty to choose from. Here are just a few, from the stalwarts to the up-and-comers.


It started with Streptococcus pyogenes. Seven years ago, the bug that causes strep throat, toxic shock syndrome, and flesh-­eating disease supplied researchers with their first gene-snipping nuclease, Cas9. Though still widely used, Cas9 isn’t perfect. For one thing, it’s bulky. To get a nuclease to its target gene, you sometimes have to smuggle it across the cell border inside a virus. The larger the nuclease is, the less editing you can squeeze into a single trip. Another problem: S. pyogenes has afflicted humans for so long that many of us may carry an immunity to Cas9, making it an iffy editing tool.


The US Department of Energy’s Joint Genome Institute maintains a database of microbial DNA from various unsavory places, including a Superfund site in California and a shuttered uranium mill in Colorado. A couple of years ago, after poring through 155 million gene sequences, scientists found three previously undiscovered nucleases, including one they called CasX. Now renamed Cas12e, it has a couple of things going for it: It’s tiny, which makes it easy to deliver into a cell, and it doesn’t appear to be related to Cas9, which makes it less likely to trigger the same immune response in humans.


If you give a mouse fermented shrimp compost, you get Bacillus hisashii, a heat-­loving gut bacterium with a nuclease called Cas12b. (Other microbes produce the same enzyme, but their versions don’t work well at human body temperature.) Earlier this year, in the journal Nature, researchers reported they’d created a streamlined version of Cas12b that’s especially well suited to editing human immune cells. Best of all, their lab-grown mutant is about 20 percent smaller than that first Cas9 and way less prone to off-target snipping.


This handy little nuclease is smaller and more accurate than Cas9, and it has shown promise in speeding up the production of biofuels and bioplastics. Last year, researchers announced that Cas12a can also screen for HPV in STD swabs. First they programmed it to seek out two cancer-causing strains of the virus. Then, in a test tube, they combined it with a kind of fluorescent alarm system. When the nuclease came into contact with HPV, it would hack apart both the virus and the alarm compound, causing the test tube to light up. Voilà, a positive result.

This article appears in the April issue. Subscribe now.