Corporate Banner
Satellite Banner
Technology
Networks
Scientific Communities
 
Become a Member | Sign in
Home>News>This Article
  News
Return

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 2,500+ scientific posters on ePosters
  • More Than 3,800+ 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 TechnologyNetworks.com 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.

Related Content

First Gene that Causes Mitral Valve Prolapse Identified
An international research collaboration led by MGH investigators has identified the first gene in which mutations cause the common form of mitral valve prolapse, a heart valve disorder that affects almost 2.5 percent of the population.
Tuesday, August 11, 2015
Intracellular Microlasers Could Allow Precise Labeling of up to a Trillion Individual Cells
MGH investigators have induced structures incorporated within individual cells to produce laser light at wavelengths that differ based on the size, shape and composition of each microlaser, allowing precise labeling of individual cells.
Tuesday, August 04, 2015
Evo-Engineered CRISPR-Cas9s Hit More Gene-Editing Targets
Scientists have engineered a more effective CRISPR- Cas9 system paving the way for more advanced applications.
Wednesday, June 24, 2015
Reducing Chemotherapy Drug’s Cardiac Side Effects
Preventing doxorubicin-induced cardiotoxicity could improve chemotherapy outcomes, reduce heart failure risk.
Thursday, December 11, 2014
Direct Drug Screening Of Biopsies Could Overcome Resistance
Combining genetic with pharmacologic screening of tumors may enable truly individualized treatment regimens.
Monday, November 17, 2014
Culture System Replicates Course Of Alzheimer’s Disease
Three-dimensional system should significantly reduce time and costs of drug development.
Monday, October 20, 2014
Genetic Signals Reflect the Evolutionary Impact of Cholera
Study identifies regions of genome associated with cholera susceptibility in Bangladesh.
Monday, July 08, 2013
Mass. General, Duke Study Identifies Two Genes that Combine to Cause Rare Syndrome
Mutations in genes that regulate cellular metabolism found in families with ataxia, dementia and reproductive failure.
Monday, May 13, 2013
Phase 1 ALS Trial is First to Test Antisense Treatment of Neurodegenerative Disease
No serious adverse effects seem from central-nervous-system infusion of drug that blocks mutated protein.
Tuesday, April 09, 2013
Protein Controlling Glucose Metabolism also a Tumor Suppressor
A protein known to regulate how cells process glucose also appears to be a tumor suppressor, adding to the potential that therapies directed at cellular metabolism may help suppress tumor growth.
Tuesday, December 11, 2012
Detection, Analysis of 'Cell Dust' may Allow Diagnosis, Monitoring of Brain Cancer
System combining nanotechnology and NMR detects particles shed by brain tumors in bloodstream.
Thursday, November 15, 2012
Circulating Tumor Cells can Reveal Genetic Signature of Dangerous Lung Cancers
MGH-developed device promises improvements in targeted therapy, treatment monitoring.
Friday, July 04, 2008
Gene Variation may Elevate Risk of Liver Tumor in Patients with Cirrhosis
A single alteration in the epidermal growth factor (EFG) gene may greatly increase the risk of developing hepatocellular carcinoma, researchers say.
Tuesday, January 08, 2008
Scientific News
Health Risks of Saturated Fats Aggravated by Immune Response
Research shows that the presence of saturated fats resulted in monocytes migrating into the tissues of vital organs.
Changing the Biological Data Visualisation World
Scientists at TGAC, alongside European partners, have created a cutting-edge, open source community for the life sciences.
NIH Study Finds Calorie Restriction Lowers Some Risk Factors for Age-Related Diseases
Two-year trial did not produce expected metabolic changes, but influenced other life span markers.
Immunotherapy Agent Benefits Patients with Drug-Resistant Multiple Myeloma in First Human Trial
Daratumumab proved generally safe in patients, even at the highest doses.
Low-level Arsenic Exposure Before Birth Associated with Early Puberty in Female Mice
Study examine whether low-dose arsenic exposure could have similar health outcomes in humans.
Inciting an Immune Attack On Cancer Cells
A new minimally invasive vaccine that combines cancer cells and immune-enhancing factors could be used clinically to launch a destructive attack on tumors.
‘Mutation-Tracking’ Blood Test for Breast Cancer
Scientists have developed a blood test for breast cancer able to identify which patients will suffer a relapse after treatment, months before tumours are visible on hospital scans.
Cellular Contamination Pathway for Heavy Elements Identified
Berkeley Lab scientists find that an iron-binding protein can transport actinides into cells.
Intensity of Desert Storms May Affect Ocean Phytoplankton
MIT study finds phytoplankton are extremely sensitive to changing levels of desert dust.
Common ‘Heart Attack’ Blood Test May Predict Future Hypertension
Small rises in troponin levels may have value as markers for subclinical heart damage and high blood pressure.
Scroll Up
Scroll Down
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
2,500+ scientific and medical posters
A library of 2,500+ scientific videos on LabTube
3,800+ scientific videos
Close
Premium CrownJOIN TECHNOLOGY NETWORKS PREMIUM FREE!