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CRISPR or RNAi? Which gene switch is better?

I knew almost nothing about the much vaunted tool for editing genes called CRISPR when a journal article on the topic showed up in my inbox last week. CRISPR is supposed to be precise and amazingly easy to use. But every journal article about CRISPR I've seen has been, for me, totally impenetrable.

The paper, titled “Systematic comparison of CRISPR and RNAi screens for essential genes,” was coming out in Nature Biotechnology soon. I was afraid, but I had to bite the bullet. So I made appointments with first author and doctoral student David Morgens and senior author and Mike Bassik, PhD, a professor of genetics. Then I stopped at Wikipedia to look up phrases like “gene knockdown” and tried not to worry.

Patiently, Bassik and Morgens ran me through the basics, then added details that brought their paper to life.

As they explained to me, their screens were designed to find out what thousands of genes do by turning them off one at a time and looking to see which ones affect cell growth. Such screens can be used for, among other things, finding targets for pharmaceutical drugs.

RNA interference, which has been in use for a while, is a system in which an RNA molecule is used to interfere with the function of a gene — a “knockdown” of gene function. It's like throwing light switches to see which ones turn off which lights. But, as Bassik explained to me, RNAi technology had problems such as false positives and false negatives. He said it was a “messy reagent,” sometimes knocking down a gene by 90 percent and sometimes by only 50 percent. It’s hard to know what to expect. Think of a mischievous child playing with the dimmer switch.

Lately, researchers have been using the much newer technology CRISPR to precisely inactivate genes. CRISPR is a combination of an enzyme that can cut DNA and a “guide RNA” that takes the enzyme exactly where you want to make the cut. So CRISPR can turn off genes at the DNA level, creating a “knockout.”

The assumption has been that CRISPR is much more accurate. But was it? Nobody had actually compared the two technologies head to head, said Morgens.

When the researchers started looking at the results of this study, they weren't what the researchers expected. “We actually got quite different answers from the two technologies, and that’s kind of scary because we like to think that there’s some ‘correct’ answer," Morgens said.

When I asked him for an example, he said the CRISPR screen identified molecules involved in transcription — the mediator complex and RNA polymerase — as being very important for the growth of the cell. Meanwhile, the RNAi screen implied they weren’t important in cell growth. “RNAi failed to find these really important complexes,” said Morgens.

And, on the flip side, CRISPR missed stuff, too.

Morgens said that either both technologies are producing false results, or the biology is much more complicated that previously thought. Either way, the technology used to ask a question seems to determine, to some extent, the answers researchers are getting.

“That’s actually quite cool,” said Morgens. But, yes, also scary.

Ideally, said Morgens and Bassik, researchers should use both screens together to get the best possible information. And Morgens has made it easier to do that by creating a statistical tool that combines the results from RNAi and CRISPR into one answer. His plan is to now use both technologies to look for therapeutic targets.

Their story left me with the feeling that there’s lots more to be learned about gene regulation, and not only by the likes of me.

Previously: CRISPR critters and CRISPR conundrums and Policing the editor: Stanford scientists devise way to monitor CRISPR effectiveness
Photo by saaby

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