CRISPR study reveals unexpected roles of non-coding RNAs

December 16, 2016 by Nicholas Weiler
DNA
Credit: NIH

UC San Francisco researchers have taken a major step toward understanding the function of the tens of thousands of human genes that do not code for proteins, a phenomenon considered one of the key remaining mysteries of the human genome. New findings , which focused on the roles of these genes in human cancer cells, suggest a possible new strategy for targeting the disease.

In recent years, researchers have recognized that non-coding regions of the genome—long dismissed as "junk DNA"—are actually key players in cell biology, development, and disease. However, the vast majority of these regions have not yet been extensively studied.

Now UCSF scientists have developed an approach to studying the function of genes that produce RNA transcripts but no protein – called long non-coding RNAs (lncRNAs)—at an unprecedented scale. In a proof of principle experiment, the researchers set out to look for common lncRNAs required for the growth of many different types of cancer cells. To their surprise, they found that each cancer cell line they tested relied upon a different set of IncRNAs for growth and survival, suggesting that these molecules might be promising targets for precision cancer therapies.

The new study—published online by the journal Science on Thursday, Dec. 15, 2016—was conducted jointly by the UCSF labs of Daniel Lim, MD, PhD, an associate professor of neurological surgery and member of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCSF and Jonathan Weissman, PhD, a Howard Hughes Medical Investigator and professor of cellular and molecular pharmacology at UCSF.

Researchers from the two labs teamed up to create a genome-scale platform to study lncRNA biology using CRISPR-based interference (CRISPRi), a technique developed in the Weissman lab in collaboration with Stanley Qi, PhD, now of Stanford University, that enables researchers to precisely tune the activity of specific genes up or down.

The UCSF team assembled a large library of CRISPR guide RNAs against 16,401 different lncRNA genes—a large number of those that are currently known—then used CRISPRi to selectively inactivate each gene in an array of seven human cell lines, including six cancer cell types and a line of induced (iPSCs) supplied by co-author Bruce Conklin, MD, a professor of medicine at UCSF and the academically affiliated Gladstone Institute of Cardiovascular disease.

In collaboration with the lab of Howard Chang, MD, PhD, of Stanford University, the team found that although about 5,000 different lncRNA genes were actively expressed in each cell line, overall only 499 lncRNAs significantly impacted cellular growth when they were inactivated. Moreover, although the researchers had initially thought that there would be a set of lncRNAs that were essential to the survival of all cells, no such core set of essential lncRNAs were found. In fact, they found that the opposite was true: 89 percent of the "essential" lncRNAs were crucial for only one cell type, and had no effect on the others.

"This was a big surprise," said Lim, whose lab has been studying the role of lncRNAs in brain development. "Typically, protein-coding genes that are essential for one type of cell are also crucial for a broad range of cell types. But with lncRNAs we see an unexpected but exquisite specificity."

The researchers also examined how silencing particular lncRNAs impacted gene expression in the seven . They found that when an lncRNA was "essential" to a particular cell type silencing it strongly modulated the activity of up to hundreds of other genes. In other cells, silencing the same lncRNA had no effect on the expression of any other genes.

"I think of it like the power-supply under your desk," Lim said. "If you've got your computer and a lot of peripherals plugged into it, tripping that switch can cause a very bad day. But if nothing's plugged in, you can flip the switch as much as you like and nothing happens."

Platform opens up study of non-coding RNA's role in evolution, development and disease

The seed of the new approach to studying lncRNAs came from conversations between the paper's two lead authors, John Liu and Max Horlbeck, who are MD-PhD students in the Lim and Weissman labs, respectively, and who also happened to be housemates at the outset of the project.

"The Lim lab had been spending a lot of time testing different lncRNAs one at a time to see if they were relevant to brain tumors," said Liu. "But that was incredibly slow. We had so few examples of lncRNA function, and it was very difficult to predict which lncRNAs would be important."

For their part, the Weissman lab had also grown interested in lncRNAs: "We had been working for several years to develop CRISPRi as a way of screening protein-coding ," Horlbeck said. "But we realized that because the technique works by tuning down how much RNA a specific gene makes, it's a perfect tool to investigate the role of non-coding RNAs, which are hard to study with other methods at this scale."

The researchers say they are eager to broaden the study of lncRNA biology by sharing their techniques, which open up many fundamental questions about the biological function of lncRNAs in development and evolution, as well as their potential utility for precision medicine approaches to cancer treatment.

"One critical question is whether different lncRNAs are essential for the growth of cancerous cells in particular, but not healthy ," Weissman said. "If so, they could potentially be targeted to impede tumors without being toxic to the rest of the body, and that would be very attractive for the pharmaceutical industry."

Explore further: First comprehensive profile of non-protein-coding RNAs in human cancers

More information: S. J. Liu et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells, Science (2016). DOI: 10.1126/science.aah7111

Related Stories

First comprehensive profile of non-protein-coding RNAs in human cancers

October 12, 2015
Growing insights about a significant, yet poorly understood, part of the genome - the "dark matter of DNA"—have fundamentally changed the way scientists approach the study of diseases. The human genome contains about 20,000 ...

Long non-coding RNA molecules necessary to regulate differentiation of embryonic stem cells into cardiac cells

January 25, 2013
When the human genome was sequenced, biologists were surprised to find that very little of the genome—less than 3 percent—corresponds to protein-coding genes. What, they wondered, was all the rest of that DNA doing?

Function of mysterious RNAs may often lie in their genes

April 7, 2016
A new genetic clue discovered by a team co-led by a researcher at the Perelman School of Medicine at the University of Pennsylvania is shedding light on the functions of the mysterious "long non-coding RNAs" (lncRNAs). These ...

Long non-coding RNA prevents the death of maturing red blood cells

December 7, 2011
A long non-coding RNA (lncRNA) regulates programmed cell death during one of the final stages of red blood cell differentiation, according to Whitehead Institute researchers. This is the first time a lncRNA has been found ...

Recommended for you

An architect gene is involved in the assimilation of breast milk

October 17, 2017
A family of "architect" genes called Hox coordinates the formation of organs and limbs during embryonic life. Geneticists from the University of Geneva (UNIGE) and the Swiss Federal Institute of Technology in Lausanne (EPFL), ...

Study identifies genes responsible for diversity of human skin colors

October 12, 2017
Human populations feature a broad palette of skin tones. But until now, few genes have been shown to contribute to normal variation in skin color, and these had primarily been discovered through studies of European populations.

Genes critical for hearing identified

October 12, 2017
Fifty-two previously unidentified genes that are critical for hearing have been found by testing over 3,000 mouse genes. The newly discovered genes will provide insights into the causes of hearing loss in humans, say scientists ...

Team completes atlas of human DNA differences that influence gene expression

October 11, 2017
Researchers funded by the National Institutes of Health (NIH) have completed a detailed atlas documenting the stretches of human DNA that influence gene expression - a key way in which a person's genome gives rise to an observable ...

Genetic advance for male birth control

October 10, 2017
When it comes to birth control, many males turn to two options: condoms or vasectomies. While the two choices are effective, both methods merely focus on blocking the transportation of sperm.

Researchers uncover new congenital heart disease genes

October 9, 2017
Approximately one in every 100 babies is born with congenital heart disease (CHD), and CHD remains the leading cause of mortality from birth defects. Although advancements in surgery and care have improved rates of survival ...

0 comments

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.