Cracking the CRISPR Code
By Mick Kulikowski | PDF
Imagine you’re a virus — but not just any virus. You’re a bacteriophage, or a virus that specifically attacks bacteria.
You operate by invading a bacterium and hijacking its cellular machinery to overwhelm and devour it while producing more copies of yourself. You grow more powerful by moving on to an adjacent bacterium and devouring it too. Soon, you’ll conquer the world!
Or you might, if not for one problem: Many bacteria have an adaptive defense system that can recognize foreign DNA and use a special molecular scalpel to cleave it from their cells and destroy it.
Moreover, this adaptive defense mechanism performs double duty as a sort of genetic tape recording that remembers previous phage attacks and how to respond to them, providing immunization against future phage attacks of a similar type.
You, virus, have been foiled by a tool known as CRISPR, a microbial adaptive immune system that has been co-opted into a cut-and-replace gene-editing system currently taking the scientific world by storm. CRISPR has potential applications for crop production, agricultural biotechnology and a myriad of other important processes and industries. It promises to be a Swiss army knife for genome editing in life forms ranging from bacteria to humans.
While researchers across the globe are quickly jumping on the bullet train that is the CRISPR bandwagon, a pioneering NC State professor has spent more than a decade helping move the bandwagon forward, authoring dozens of refereed journal articles and chalking up around 20 patents along the way.
Beyond Cheese and Yogurt
Food scientist Rodolphe Barrangou clearly remembers his CRISPR “eureka” moments.
Shortly after earning his Ph.D. in functional engineering under Todd Klaenhammer in NC State’s food science department in 2004, Barrangou worked to produce better bacterial starter cultures for cheese and yogurt at Danisco, a food ingredients company in Madison, Wisconsin.
Barrangou and his colleagues sequenced genomes — the complete set of genes — for dairy bacteria such as Streptococcus thermophilus. That particular bacterium breaks down lactose, a sugar in milk, and turns it into lactic acid, an important ingredient in cheese and yogurt as well as in pickled vegetables and fermented products.
But Barrangou’s team had difficulty putting the dairy bacterium genome puzzle together. Think of assembling a multimillion-piece jigsaw puzzle without having a picture template to know exactly what it should look like. The reason? CRISPRs.
In the alphabet soup of cellular DNA sequences, which are represented by the letters A, T, G and C that correspond to the chemical building blocks of DNA — adenine, thiamine, guanine and cytosine — a CRISPR looks like a relatively short, repetitive DNA sequence, after which comes a sequence of two or three dozen letters known as “spacer” DNA, followed by another palindromic sequence just like the first.
“They weren’t called CRISPRs a decade ago. We called them SPIDRs, for ‘spacers interspersed direct repeats,’” Barrangou says. “They kept breaking down the assembly process as we sequenced these bacterial genomes. We knew they were part of the DNA repeat family, but we didn’t know what they did or why they mattered so much.”
At the same time, Barrangou and his Danisco colleagues also were sequencing genomes of important phages involved in cheese and yogurt manufacturing. While comparing and contrasting these phage DNA sequences with the bacterial DNA sequences, patterns emerged.
“Some of the spacer DNA sequences we found in bacteria were close matches to monkey pox, frog pox and ringworm virus sequences,” Barrangou says. “One of the key moments was when we realized that those were actually real viral sequences. In hindsight we know now that these elements were a record of viruses that the bacterium had vaccinated itself against.
“The CRISPR content of those bacteria, the way they clustered together by similarity, is correlated with their profiles of resistance to the phage,” Barrangou adds. “That was a key element showing that there was a link between the function of phage resistance and the genetic makeup of CRISPR content.”
At the same time in summer 2005, three independent research groups published papers suggesting that CRISPRs and their spacer DNA were indeed important. The research found that bacterial spacer DNA matched foreign genetic elements, which provided clues that it could be involved in bacterial immunity.
Armed with the knowledge from these papers and their own lab work, Barrangou and his Danisco colleagues published a seminal paper in the journal Science in 2007, showing that the S. thermophilus bacterium could be engineered to either resist or succumb to attack from phages by altering the spacer DNA that matched the phage DNA.
“That paper showed CRISPR is indeed an adaptive immune system with the functional ability to acquire genetic snapshots of phage attacks,” Barrangou says.
Cutting with CRISPR
If you’ve ever used a word processing program, you’re probably familiar with highlighting a block of text and using the Control-X (or Command-X) keystroke combination to delete that text. CRISPR uses the same principle to defend bacteria against the DNA of attacking viruses.
CRISPR stands for “clustered regularly interspaced short palindromic repeats” that appear in non-random patterns. CRISPR systems exist in prokaryotes — single-celled bacteria and archaea — and within the last two years have been put to work by scientists in eukaryotes, or plants and animals.
There are several different types of CRISPR systems. Barrangou is concerned mostly with CRISPR-Cas systems that use Cas9 proteins as scalpels to cleave away unwanted foreign DNA.
Simply put, the CRISPR system recognizes invasive foreign DNA and creates CRISPR RNAs that look for and mimic the DNA letter sequences of the invaders. When the CRISPR RNAs find a matching sequence, they guide the scalpels — in this case, Cas9 proteins — to cut the exact sequence of invading DNA from the cell.
The CRISPR-Cas system has lots of advantages over other gene-editing systems, Barrangou says. It’s quick, efficient, precise, relatively inexpensive and, as the scientific community has shown over the past two years, transferable to many types of living things.
Barrangou ticks off the list of possible applications: genome editing, antibacterial and antimicrobial production, food safety, food production, plant breeding and perhaps even animal breeding.
“Some of these things sound like science fiction, but there is real science behind it,” Barrangou says.
There are still a few gaps to overcome before CRISPR-Cas becomes a ubiquitous tool, however. Cas9 proteins are rather large and cumbersome to work with, Barrangou says. In addition, all guides — the CRISPR RNAs that guide the Cas9 scalpels to their target — are not created equally. Some do a better job than others of homing in on an invasive target.
One way of bridging these gaps, Barrangou said, is to collaborate with others, across campus and across the globe. One of his collaborators, NC State chemical and biomolecular engineer Chase Beisel, brings specializations in bacteria, systems biology and regulatory RNAs to bear on the issue.
A Better Bacterium
Beisel first learned about CRISPR systems as a postdoctoral researcher in 2009 at the National Institutes of Health in Bethesda, Maryland. He was working in a microbiology lab that focused on regulatory RNAs in bacteria — how they function and what they do.
“What got me excited was how these CRISPR RNAs can direct proteins,” he says. “CRISPR is a naturally occurring system for which you can design an RNA and direct it to a specific DNA sequence. As an engineer, you think, ‘How can we use this?’”
Beisel began his CRISPR research when he started his career at NC State in fall 2011.
“I was not only launching a new lab here at the university but also entering an entirely new field from my projects at NIH, so it was challenging,” Beisel says.
He met Barrangou in October 2012, when they ran into each other at an NC State biotechnology symposium on CRISPR led by Barrangou, who was the guest speaker.
“I knew of Rodolphe as a name on papers I had read in the CRISPR literature,” Beisel says. “At that point, my lab had some initial results of using CRISPR systems as antimicrobials. In a conversation with Rodolphe at the symposium, we realized we had a number of overlapping interests and began thinking of ways we could collaborate.”
Barrangou joined the NC State faculty a year later, in 2013.
‘A Ridiculous Record of Collaboration’
That collaborative spirit has yielded a number of important results for the two NC State professors in the past few years. Beisel calls Barrangou’s capacity to work with various groups “a ridiculous record of collaboration.”
“I’m an engineer by training, so I understand engineers — not all of them, obviously,” Barrangou says with a smile. “But when you bring different and complementary skills together, you get more than the sum of your ingredients.”
Beisel’s academic pedigree is in chemical engineering but with a biological focus. “As an engineer, you say, ‘We have a problem, how do we solve it?’ A biologist says, ‘Nature created this solution. We just need to figure out what it is and how it works.’”
Their work, together and apart, has made great advances in CRISPR-related science.
In 2014, Barrangou and Beisel co-authored a paper in mBio that showed CRISPR can be used as a “smart bomb” in bacteria. They also co-authored a 2014 paper in Molecular Cell that delved into the mechanisms of how the CRISPR RNAs guide the Cas9 scalpels to the targeted foreign DNA.
Working separately from Barrangou, Beisel and NC State colleagues this summer published findings in the journal Angewandte Chemie on a new way to get CRISPR-Cas9 into a cell by using so-called “nanoclews,” or tiny single-bound strands of DNA.
And Barrangou, working apart from Beisel, published with NC State colleagues a paper in Proceedings of the National Academy of Sciences on using CRISPR to identify key bacterial genomic regions and whether these regions are expendable or required for bacterial survival.
Business attention — and the attendant venture capital — flows to CRISPR research, technologies and applications. The academic literature crackles with CRISPR-related findings.
As new studies have shown, CRISPR systems can be utilized to not only delete DNA sequences from a cell, but also to add sequences. Since the CRISPR system is essentially an immune system, it can also be turned against itself to cause cell death. You read that right: A bacteria’s defensive machinery can be directed against itself to make it commit cellular suicide. Think of the antibacterial potential of unleashing that power.
“Resistant bacteria can be targeted by CRISPR systems,” Barrangou says. “The literature has already shown staph being knocked out in mice. Basically it can work on any sequence in any cell in any way.”
When asked about CRISPR’s future, Barrangou says the past is prologue.
“I think it’s back to the basics — back to bacteria,” he says. “That means using CRISPR machinery to kill cells. There’s lots of promise here and less engineering needed because CRISPR comes from bacteria. In the short term, I would look for applications in antimicrobials and antibiotics.”
The biggest technical challenge now, Barrangou says, is how to efficiently deliver the CRISPR payload into specific cells.
“Chase and I think using phages is the way to go,” he says. “You can inject phage DNA containing CRISPR like a syringe; the CRISPR will selectively target the exact DNA sequence to kill whatever it is you want to kill. And it won’t target the good bacteria, unlike antibiotics, which kill everything.
“I think this system is astonishingly cool on so many levels — its practicality, its technological promise, its power. We’re only limited by our imagination.”