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Home | Pregnancy Timeline | News Alerts |News Archive Aug 15, 2013

 

transcription

Standard model of how mRNA is created within the nucleus.

Image credit: Wikipedia





WHO Child Growth Charts

 

 

 

New gene repair technique advances regenerative medicine

Using human pluripotent stem cells and DNA-cutting protein from meningitis bacteria, researchers from the Morgridge Institute for Research and Northwestern University have created an efficient way to target and repair defective genes.

Writing in the Proceedings of the National Academy of Sciences, the team reports that the novel technique is much simpler than previous methods and establishes the groundwork for major advances in regenerative medicine, drug screening and biomedical research.

Zhonggang Hou of the Morgridge Institute’s regenerative biology team and Yan Zhang of Northwestern University served as first authors on the study; James Thomson, director of regenerative biology at the Morgridge Institute, and Erik Sontheimer, professor of molecular biosciences at Northwestern University, served as principal investigators.


“With this system, there is the potential to repair any genetic defect, including those responsible for some forms of breast cancer, Parkinson’s and other diseases. The fact that it can be applied to human pluripotent stem cells opens the door for meaningful therapeutic applications.”

Zhonggang Hou, PhD, Morgridge Institute’s regenerative biology team


Zhang said the Northwestern University team focused on Neisseria meningitidis bacteria because it is a good source of the Cas9 protein needed for precisely cleaving damaged sections of DNA.


“We are able to guide this protein with different types of small RNA molecules, allowing us to carefully remove, replace or correct problem genes. This represents a step forward from other recent technologies built upon proteins such as zinc finger nucleases and TALENs.”

Yan Zhang, PhD, Northwestern University, one of two first authors

Previous gene correction methods required engineered proteins to help with the cutting. Hou says scientists can now synthesize RNA with the new process in as little as one to three days – compared with the weeks or months needed to engineer suitable proteins.


Thomson, who also serves as the James Kress Professor of Embryonic Stem Cell Biology at the University of Wisconsin–Madison, a John D. MacArthur professor at UW–Madison’s School of Medicine and Public Health and a professor in the department of molecular, cellular and developmental biology at the University of California, Santa Barbara, says the discovery holds many practical applications.


“With this system, there is the potential to repair any genetic defect, including those responsible for some forms of breast cancer, Parkinson’s and other diseases.”

Zhonggang Hou, PhD, Morgridge Institute’s regenerative biology team


“Human pluripotent stem cells can proliferate indefinitely and they give rise to virtually all human cell types, making them invaluable for regenerative medicine, drug screening and biomedical research,” Thomson says. “Our collaboration with the Northwestern team has taken us further toward realizing the full potential of these cells because we can now manipulate their genomes in a precise, efficient manner.”

Sontheimer, who serves as the Soretta and Henry Shapiro Research Professor of Molecular Biology with Northwestern’s department of molecular biosciences, Center for Genetic Medicine and the Robert H. Lurie Comprehensive Cancer Center of Northwestern University, says the team’s results also offer hopeful signs about the safety of the technique.

“A major concern with previous methods involved inadvertent or off-target cleaving, raising issues about the potential impact in regenerative medicine applications,” he said. “Beyond overcoming the safety obstacles, the system’s ease of use will make what was once considered a difficult project into a routine laboratory technique, catalyzing future research.”

Abstract
Genome engineering in human pluripotent stem cells (hPSCs) holds great promise for biomedical research and regenerative medicine. Recently, an RNA-guided, DNA-cleaving interference pathway from bacteria [the type II clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) pathway] has been adapted for use in eukaryotic cells, greatly facilitating genome editing. Only two CRISPR-Cas systems (from Streptococcus pyogenes and Streptococcus thermophilus), each with their own distinct targeting requirements and limitations, have been developed for genome editing thus far. Furthermore, limited information exists about homology-directed repair (HDR)-mediated gene targeting using long donor DNA templates in hPSCs with these systems. Here, using a distinct CRISPR-Cas system from Neisseria meningitidis, we demonstrate efficient targeting of an endogenous gene in three hPSC lines using HDR. The Cas9 RNA-guided endonuclease from N. meningitidis (NmCas9) recognizes a 5′-NNNNGATT-3′ protospacer adjacent motif (PAM) different from those recognized by Cas9 proteins from S. pyogenes and S. thermophilus (SpCas9 and StCas9, respectively). Similar to SpCas9, NmCas9 is able to use a single-guide RNA (sgRNA) to direct its activity. Because of its distinct protospacer adjacent motif, the N. meningitidis CRISPR-Cas machinery increases the sequence contexts amenable to RNA-directed genome editing.

Also contributing to the study, which was supported by funding from sources including the National Institutes of Health, the Wynn Foundation and the Morgridge Institute for Research, were Nicholas Propson, Sara Howden and Li-Fang Chu from the Morgridge Institute for Research.

Original press release: http://www.news.wisc.edu/22021