Biotech

From infant poop to trance music, here’s a play-by-play of a CRISPR experiment

Scientists in Jennifer Doudna’s lab pull back the veil on their gene-editing process.
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Francis Scialabba

· 4 min read

Ten years ago, scientists developed CRISPR—and since then, the gene-editing tool has been hailed as one of the biggest biological breakthroughs of our time.

For scientists, CRISPR is a key way to study cause and effect between strands of DNA and organisms. Its lab applications have ranged from modifying plants to editing cells for cancer treatment, and CRISPR-based startups have collectively raised billions of dollars.

Much has been made of the technology’s revolutionary potential, but today we’re lifting the veil on what CRISPR looks like in a hands-on setting, via a lab at the University of California, Berkeley. In the lab, which is part of the university’s Innovative Genomics Institute—founded by CRISPR co-inventor Jennifer Doudna—scientists are using a new variant of CRISPR called DART to make changes to the human-gut microbiome.

We spoke with two of the institute’s investigators, Benjamin Rubin and Brady Cress, for a look at how they use the tech day to day.

Cut-and-paste DNA

So, what’s the advantage of using CRISPR for gut microbiomes anyway? The hope, Rubin said, is that one day, it will allow for more precision than the current approach: prescribing antibiotics for bacterial infections.

Although the initial problem may be fixed by that path, he added, antibiotics can also decimate the microbial community and cause collateral damage, like microbiome imbalances.

“The future we’re hoping to drive towards is not this one where we’re using the sledgehammer of antibiotics—we’d be using the scalpel of CRISPR-Cas9 editing to just fix the problem while leaving the rest of the microbial community intact,” Rubin said.

In particular, the team is using DART—short for “DNA-editing all-in-one RNA-guided CRISPR–Cas transposase”—for a two-part task: 1) identify a specific section of DNA to edit and 2) hold and then insert an entirely new piece of DNA in the chosen space.

“You can think of [DART] as a cut-and-paste,” Cress said. “So it’s really, really handy for pasting in really big pieces of DNA, which is what we need to do to be able to manipulate communities well.”

A few days in the lab

There’s lots of custom software involved in the team’s DNA-editing machinery, but depending on the experiment they’re running, Rubin and Cress start by customizing the targeted DNA section (think: the address written on a package) and the edit they plan to make (think: the contents of the package). That requires adding two pieces of custom DNA to their preexisting gene-editing tool, and the team typically orders this dried DNA from a DNA-synthesis company, like Twist Bioscience or Integrated DNA Technologies.

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Within a few days or a week, both pieces of DNA arrive in the mail, and Rubin and Cress essentially use enzymes to paste the new pieces together with the gene-editing tool in one test tube.

After that, it’s time for the experiment to really begin—and the process typically starts in the early evening. That’s because, the night before the lab work starts, Rubin and Cress need to prepare one of the experiment’s primary materials: infant poop.

The feces are donated by a hospital, and the UC Berkeley team stores them in extremely cold freezers (-80 degrees Celsius). The night before an experiment, Rubin or Cress stick their gloved hands into an oxygen-free chamber that mimics the human gut. They move the feces into flasks to let bacteria grow overnight, and they also ready a safe variant of E. coli—which they use to ferry the gene-editing tool into the microbe community they’re targeting, Cress said.

The next morning, the team returns to the lab to mix the E. coli with the infant feces and let the mixture sit in a petri dish for ~12 hours.

That night, Rubin and Cress return again to check on whether the bacteria mixed together, since the gene-editing tool would have already been delivered to a certain number of the cells. While testing how many were successfully edited, Rubin or Cress turn on some music (EDM or trance for Rubin, usually rock or metal for Cress), and spend a couple of hours using a spatula-like tool to carefully scrape the cells from the petri dish into a test tube.

The next morning, they check to see how many of the cells were edited and what’s changed in the bacterial community.

“It’s a brand-new field [with] only a few entries,” Rubin said. “Both of us believe very strongly that in five years, probably, people will be using this to better understand microbial communities, and probably in 10 years…people will be using this to modify microbial communities to have positive impact, whether on human guts or on plant microbial communities.”

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