Corporate Banner
Satellite Banner
Stem Cells, Cellular Therapy & Biobanking
Scientific Community
Become a Member | Sign in
Home>News>This Article

Powerful Gene-Editing Tool Appears to Cause Off-Target Mutations in Human Cells

Published: Wednesday, June 26, 2013
Last Updated: Wednesday, June 26, 2013
Bookmark and Share
Results indicate need to improve precision of CRISPR-Cas RNA-guided nucleases.

In the past year a group of synthetic proteins called CRISPR-Cas RNA-guided nucleases (RGNs) have generated great excitement in the scientific community as gene-editing tools.

Exploiting a method that some bacteria use to combat viruses and other pathogens, CRISPR-Cas RGNs can cut through DNA strands at specific sites, allowing the insertion of new genetic material.  However, a team of Massachusetts General Hospital (MGH) researchers has found a significant limitation to the use of CRISPR-Cas RGNs, production of unwanted DNA mutations at sites other than the desired target.

"We found that expression of CRISPR-Cas RGNs in human cells can have off-target effects that, surprisingly, can occur at sites with significant sequence differences from the targeted DNA site," says J. Keith Joung, MD, PhD, associate chief for Research in the Massachusetts General Hospital (MGH) Department of Pathology and co-senior author of the report receiving online publication in Nature Biotechnology.  "RGNs continue to have tremendous advantages over other genome editing technologies, but these findings have now focused our work on improving their precision."

Consisting of a DNA-cutting enzyme called Cas 9, coupled with a short, 20-nucleotide segment of RNA that matches the target DNA segment, CRISPR-Cas RGNs mimic the primitive immune systems of certain bacteria.  When these microbes are infected by viruses or other organisms, they copy a segment of the invader's genetic code and incorporate it into their DNA, passing it on to future bacterial generations.  If the same pathogen is encountered in the future, the bacterial enzyme called Cas9, guided by an RNA sequence the matches the copied DNA segment, inactivates the pathogen by cutting its DNA at the target site.

About a year ago, scientists reported the first use of programmed CRISPR-Cas RGNs to target and cut specific DNA sites.  Since then several research teams, including Joung's, have succesfully used CRISPR-Cas RGNs to make genomic changes in fruit flies, zebrafish, mice and in human cells – including induced pluripotent stem cells which have many of the characteristics of embryonic stem cells.  The technology's reliance on such a short RNA segment makes CRISPR-Cas RGNs much easier to use than other gene-editing tools called zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), and RGNs can be programmed to introduce several genetic changes at the same time.

However, the possibility that CRISPR-Cas RGNs might cause additional, unwanted genetic changes has been largely unexplored, so Joung's team set out to investigate the occurrence of "off-target" mutations in human cells expressing CRISPR-Cas RGNs.  Since the interaction between the guiding RNA segment and the target DNA relies on only 20 nucleotides, they hypothesized that the RNA might also recognize DNA segments that differed from the target by a few nucleotides.

Although previous studies had found that a single-nucleotide mismatch could prevent the action of some CRISPR-Cas RGNs, the MGH team's experiments in human cell lines found multiple instances in which mismatches of as many as five nucleotides did not prevent cleavage of an off-target DNA segment.  They also found that the rates of mutation at off-target sites could be as high or even higher than at the targeted site, something that has not been observed with off-target mutations associated with ZFNs or TALENs.

"Our results don't mean that RGNs cannot be important research tools, but they do mean that researchers need to account for these potentially confounding effects in their experiments.  They also suggest that the existing RGN platform may not be ready for therapeutic applications," says Joung, who is an associate professor of Pathology at Harvard Medical School.  "We are now working on ways to reduce these off-target effects, along with methods to identify all potential off-target sites of any given RGN in human cells so that we can assess whether any second-generation RGN platforms that are developed will be actually more precise on a genome-wide scale.  I am optimistic that we can further engineer this system to achieve greater specificity so that it might be used for therapy of human diseases."

Further Information
Access to this exclusive content is for Technology Networks Premium members only.

Join Technology Networks Premium for free access to:

  • Exclusive articles
  • Presentations from international conferences
  • Over 2,800+ scientific posters on ePosters
  • More than 4,000+ scientific videos on LabTube
  • 35 community eNewsletters

Sign In

Forgotten your details? Click Here
If you are not a member you can join here

*Please note: By logging into you agree to accept the use of cookies. To find out more about the cookies we use and how to delete them, see our privacy policy.

Scientific News
Ancient Viral Molecules Essential for Human Development
Genetic material from ancient viral infections is critical to human development, according to researchers at the Stanford University School of Medicine.
CRI Identifies Emergency Blood-formation Response
Researchers report that when tissue damage occurs, an emergency blood-formation system activates.
New Way to Force Stem Cells to Become Bone Cells
Potential therapies based on this discovery could help people heal bone injuries or set hardware, such as replacement knees and hips.
Dead Bacteria to Kill Colorectal Cancer
Scientists from Nanyang Technological University (NTU Singapore) have successfully used dead bacteria to kill colorectal cancer cells.
Promise of Newborn Stem Cells to Revolutionize Clinical Practice
In this article Shweta Sharma, PhD, discusses the potential of an Umbilical Cord Blood bank as an untapped source of samples for research and clinical trials.
The Life Story of Stem Cells
A model analyses the development of stem cell numbers in the human body.
Novel Stem Cell Line Avoids Risk of Introducing Transplanted Tumors
Progenitor cells might eventually be used to repair or rebuild damaged or destroyed organs.
Advancing Genome Editing of Blood Stem Cells
Genome editing techniques for blood stem cells just got better, thanks to a team of researchers at USC and Sangamo BioSciences.
Molecule Proves Key to Brain Repair After Stroke
Scientists found that a molecule known as growth and differentiation factor 10 (GDF10) plays a key role in repair mechanisms following stroke.
Towards Patient-Specific Drug Screening
A new breakthrough by the 3D stem cell printing team at Heriot-Watt could pave the way to individually tailored drug testing regimes, both reducing the need for animal testing and ensuring that patients receive drugs which are most effective for their individual needs.
Skyscraper Banner

Skyscraper Banner
Go to LabTube
Go to eposters
Access to the latest scientific news
Exclusive articles
Upload and share your posters on ePosters
Latest presentations and webinars
View a library of 1,800+ scientific and medical posters
2,800+ scientific and medical posters
A library of 2,500+ scientific videos on LabTube
4,000+ scientific videos