by Staff Writers
Boston MA (SPX) Apr 29, 2014
A next-generation genome editing system developed by Massachusetts General Hospital (MGH) investigators substantially decreases the risk of producing unwanted, off-target gene mutations. In a paper receiving online publication in Nature Biotechnology, the researchers report a new CRISPR-based RNA-guided nuclease technology that uses two guide RNAs, significantly reducing the chance of cutting through DNA strands at mismatched sites.
"This system combines the ease of use of the widely adopted CRISPR/Cas system with a dimerization-dependent nuclease activity that confers higher specificity of action," says J. Keith Joung, MD, PhD, associate chief for Research in the MGH Department of Pathology and senior author of the report.
"Higher specificity will be essential for any future clinical use of these nucleases, and the new class of proteins we describe could provide an important option for therapeutic genome editing."
Engineered CRISPR-Cas nucleases - genome-editing tools that combine a short RNA segment matching its DNA target with a DNA-cutting enzyme called Cas9 - have been the subject of much investigation since their initial development in 2012.
Easier to use than the earlier ZFN (zinc finger nuclease) and TALEN (transcription activator-like effector nuclease) systems, they have successfully induced genomic changes in several animal models systems and in human cells.
But in a previous Nature Biotechnology paper published in June 2013, Joung's team reported that CRISPR-Cas nucleases could produce additional mutations in human cells, even at sites that differed from the DNA target by as much as five nucleotides.
To address this situation, the investigators developed a new platform in which the targeting function of Cas9 was fused to a nuclease derived from a well-characterized enzyme called Fokl, which only functions when two copies of the molecule are paired, a relationship called dimerization.
This change essentially doubled the length of DNA that must be recognized for cleavage by these new CRISPR RNA-guided Fokl nucleases (RFNs), significantly increasing the precision of genome editing in human cells. Importantly, Joung and his colleagues also demonstrated that these new RFNs are as effective at on-target modification as existing Cas9 nucleases that target a shorter DNA sequence.
"By doubling the length of the recognized DNA sequence, we have developed a new class of genome -editing tools with substantially improved fidelity compared with existing wild-type Cas9 nucleases and nickases (enzymes that cleave a single DNA strand)," says Joung, an associate professor of Pathology at Harvard Medical School.
The research team also has developed software enabling users to identify potential target sites for these RFNs and incorporated that capability into ZiFiT Targeter, a software package freely available here.
Massachusetts General Hospital
The Clone Age - Cloning, Stem Cells, Space Medicine
|The content herein, unless otherwise known to be public domain, are Copyright 1995-2014 - Space Media Network. All websites are published in Australia and are solely subject to Australian law and governed by Fair Use principals for news reporting and research purposes. AFP, UPI and IANS news wire stories are copyright Agence France-Presse, United Press International and Indo-Asia News Service. ESA news reports are copyright European Space Agency. All NASA sourced material is public domain. Additional copyrights may apply in whole or part to other bona fide parties. Advertising does not imply endorsement, agreement or approval of any opinions, statements or information provided by Space Media Network on any Web page published or hosted by Space Media Network. Privacy Statement All images and articles appearing on Space Media Network have been edited or digitally altered in some way. Any requests to remove copyright material will be acted upon in a timely and appropriate manner. Any attempt to extort money from Space Media Network will be ignored and reported to Australian Law Enforcement Agencies as a potential case of financial fraud involving the use of a telephonic carriage device or postal service.|