Nav: Home

New tools allow rapid ID of CRISPR-Cas system PAMs

March 31, 2016

CRISPR-Cas systems are widely heralded as a new generation of genetic tools. But development of these tools requires researchers to identify the protospacer-adjacent motifs (PAMs) that unlock each system's functionality. A new set of techniques expedites PAM identification -- and early testing finds that many CRISPR-Cas systems actually have multiple PAMs of varying strength.

CRISPR-Cas systems protect bacteria from invaders such as viruses. They do this by creating small strands of RNA that match DNA sequences specific to a given invader. When those CRISPR RNAs find a match, they unleash proteins that chop up the invader's DNA, preventing it from replicating. However, the first step in the process isn't comparing the RNA to target DNA. The first step involves PAM recognition and binding.

PAMs are short genetic sequences adjacent to the target DNA in viruses or other invaders. When the protein in a CRISPR-Cas system identifies a PAM, that identification tells the protein to bind to that DNA and begin comparing the adjacent DNA sequence to the CRISPR RNA. If the DNA and RNA match, then the protein cleaves the target DNA.

"For researchers to make use of a CRISPR-Cas system for gene editing, gene regulation, or other techniques, you first need to identify the relevant PAM sequences that trigger that specific CRISPR-protein combination," says Chase Beisel, an assistant professor of chemical and biomolecular engineering at NC State University and a senior author of a paper describing the work.

"For example, the CRISPR-Cas9 tool derived from Streptococcus pyogenes has a different PAM than the CRISPR-Cas9 tool derived from Staphylococcus aureus," Beisel says. "There are thousands of potential CRISPR tools out there; to make use of them we need an efficient way to identify their PAMs. And we think we've developed tools to do that."

PAM identification is tricky because it is difficult to predict which genetic sequences function as PAMs for a given CRISPR-Cas system. And the genetic sequences that function as PAMs vary widely, even between closely related CRISPR-Cas systems. For example, the PAM that triggers the Cas9 protein from S. pyogenes consists of only three nucleotides, but the PAM that triggers the Cas9 protein from S. aureus contains six nucleotides -- none of which overlap with those from S. pyogenes.

"To address this challenge, we developed a tool called PAM-SCANR," says Ryan Leenay, a graduate student in Beisel's lab and lead author on the paper. "PAM-SCANR allows us to identify PAM sequences for any given CRISPR-protein combination."

Here's how PAM-SCANR works. First, researchers start with a CRISPR-Cas system they want to find the PAM for. The relevant CRISPR-protein pair is then used as the reactive agent in a high-throughput screen that exposes the CRISPR-protein pair to many different gene sequences simultaneously. The gene sequences are part of a genetic construct engineered to light up -- they literally fluoresce -- when the CRISPR-protein pair binds to them. And that can only happen if a functional PAM is present.

"One thing that makes this tool unique is that it could be used to screen and identify PAMs across a wide range of CRISPR-Cas systems," Leenay says.

The researchers tested PAM-SCANR in five CRISPR-Cas systems across three of the four CRISPR types that are known to rely on PAMs to function. Different CRISPR types use different proteins and rely on different mechanisms of action.

The researchers also developed a tool called a PAM wheel that helps researchers visualize the output of PAM-SCANR screens. It also allows researchers to see if some PAMs are better than others and by how much.

This is important because, in testing PAM-SCANR, the researchers found that there can be multiple PAMs for a given CRISPR-Cas system -- which was a surprise -- and that some PAMs trigger a much stronger response than others.

"When we first discovered PAMs nearly a decade ago, we initially thought that only one PAM worked," says Rodolphe Barrangou, an associate professor in NC State's Department of Food, Bioprocessing and Nutrition Sciences and a senior author on the manuscript. "However, our tools revealed there can be multiple PAMs for a single CRISPR-Cas system, and some PAMs clearly performed better than others as part of CRISPR recognizing its target DNA."

The researchers are already using PAM-SCANR to identify PAMs for CRISPR-Cas systems that may be the next generation of CRISPR tools. They're also working to determine how different PAMs for a specific CRISPR-Cas system may affect that system's effectiveness in any given application.

"For example, we want to know if the variability among PAMs has implications for genome editing and our ability to predict off-target sites -- places a CRISPR-Cas system might attack other than the target DNA," Beisel says.
-end-
The paper, "Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems," will be published online at 12 noon (EST) on March 31 in the journal Molecular Cell. Co-senior author of the paper is Rodolphe Barrangou, an associate professor of food science at NC State. Lead author of the paper is Ryan Leenay, a Ph.D. student at NC State. The paper was co-authored by Kenneth Maksimchuk, a former postdoctoral researcher at NC State; Rebecca Slotkowski and Roma Agrawal, undergraduates at NC State; and Ahmed Gomaa and Alexandra Briner, Ph.D. students at NC State. The work was supported by the National Science Foundation under grants CBET-1403135 and MCB-1452902; and by the National Institutes of Health under grant 5T32GM008776-15.

North Carolina State University

Related Dna Articles:

A new spin on DNA
For decades, researchers have chased ways to study biological machines.
From face to DNA: New method aims to improve match between DNA sample and face database
Predicting what someone's face looks like based on a DNA sample remains a hard nut to crack for science.
Self-healing DNA nanostructures
DNA assembled into nanostructures such as tubes and origami-inspired shapes could someday find applications ranging from DNA computers to nanomedicine.
DNA design that anyone can do
Researchers at MIT and Arizona State University have designed a computer program that allows users to translate any free-form drawing into a two-dimensional, nanoscale structure made of DNA.
DNA find
A Queensland University of Technology-led collaboration with University of Adelaide reveals that Australia's pint-sized banded hare-wallaby is the closest living relative of the giant short-faced kangaroos which roamed the continent for millions of years, but died out about 40,000 years ago.
More Dna News and Dna Current Events

Best Science Podcasts 2019

We have hand picked the best science podcasts for 2019. Sit back and enjoy new science podcasts updated daily from your favorite science news services and scientists.
Now Playing: TED Radio Hour

Erasing The Stigma
Many of us either cope with mental illness or know someone who does. But we still have a hard time talking about it. This hour, TED speakers explore ways to push past — and even erase — the stigma. Guests include musician and comedian Jordan Raskopoulos, neuroscientist and psychiatrist Thomas Insel, psychiatrist Dixon Chibanda, anxiety and depression researcher Olivia Remes, and entrepreneur Sangu Delle.
Now Playing: Science for the People

#537 Science Journalism, Hold the Hype
Everyone's seen a piece of science getting over-exaggerated in the media. Most people would be quick to blame journalists and big media for getting in wrong. In many cases, you'd be right. But there's other sources of hype in science journalism. and one of them can be found in the humble, and little-known press release. We're talking with Chris Chambers about doing science about science journalism, and where the hype creeps in. Related links: The association between exaggeration in health related science news and academic press releases: retrospective observational study Claims of causality in health news: a randomised trial This...