Category Archives: Genetic Engineering

PUBLIC RELEASE DATE:

30-Mar-2014

Contact: Sarah McDonnell s_mcd@mit.edu 617-253-8923 Massachusetts Institute of Technology

CAMBRIDGE, MA -- Using a new gene-editing system based on bacterial proteins, MIT researchers have cured mice of a rare liver disorder caused by a single genetic mutation.

The findings, described in the March 30 issue of Nature Biotechnology, offer the first evidence that this gene-editing technique, known as CRISPR, can reverse disease symptoms in living animals. CRISPR, which offers an easy way to snip out mutated DNA and replace it with the correct sequence, holds potential for treating many genetic disorders, according to the research team.

"What's exciting about this approach is that we can actually correct a defective gene in a living adult animal," says Daniel Anderson, the Samuel A. Goldblith Associate Professor of Chemical Engineering at MIT, a member of the Koch Institute for Integrative Cancer Research, and the senior author of the paper.

The recently developed CRISPR system relies on cellular machinery that bacteria use to defend themselves from viral infection. Researchers have copied this cellular system to create gene-editing complexes that include a DNA-cutting enzyme called Cas9 bound to a short RNA guide strand that is programmed to bind to a specific genome sequence, telling Cas9 where to make its cut.

At the same time, the researchers also deliver a DNA template strand. When the cell repairs the damage produced by Cas9, it copies from the template, introducing new genetic material into the genome. Scientists envision that this kind of genome editing could one day help treat diseases such as hemophilia, Huntington's disease, and others that are caused by single mutations.

Scientists have developed other gene-editing systems based on DNA-slicing enzymes, also known as nucleases, but those complexes can be expensive and difficult to assemble.

"The CRISPR system is very easy to configure and customize," says Anderson, who is also a member of MIT's Institute for Medical Engineering and Science. He adds that other systems "can potentially be used in a similar way to the CRISPR system, but with those it is much harder to make a nuclease that's specific to your target of interest."

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14 hours ago Figure 1:Synechocystis cyanobacteria could become factories of bioplastic production. Credit: Kiminori Toyooka, RIKEN Center for Sustainable Resource Science

The production of plastics using biological systems such as bacteria could lead to the sustainable manufacture of biodegradable and biocompatible plastics using carbon from the atmosphere. So far, however, it has proved exceedingly difficult to increase the yields of bioplastics to industrially viable levels. Takashi Osanai, Masami Yokota Hirai and colleagues from the RIKEN Center for Sustainable Resource Science have now engineered a cyanobacterium strain that produces triple the normal yield of the bioplastic polyhydroxybutyrate (PHB).

The species of cyanobacterium known as Synechocystis (Fig. 1) starts to produce PHB when nutrients such as nitrogen become scarce. This metabolic adaptation helps the cyanobacteria survive under low resource conditions. However, the organisms do not naturally produce sufficient yields of PHB for commercial applications.

To boost the levels of PHB produced by the organism, the research team created a strain of Synechocystis with higher than normal expression levels of Rre37, a regulatory protein known to be involved in sugar metabolism during times of nitrogen starvation. Genetic and metabolic analyses showed that Rre37 facilitates the conversion of glycogen, a sugar storage molecule, into PHB. "In Rre37, we found a novel regulator activating bioplastic production in cyanobacteria," says Osanai.

The same team previously identified another protein, SigE, involved in bioplastic production. Similar to the results with Rre37, the researchers found that overexpression of SigE, which contributes to the initiation of RNA synthesis, led to more PHB accumulation under nitrogen-limited conditions.

Their latest Synechocystis strain expressed elevated levels of both Rre37 and SigE. Gene expression analysis revealed that Rre37 and SigE each activate different pairs of genes involved in PHB biosynthesis. Extraction of PHB from the cyanobacteria showed that the bioplastic concentrations were even greater in the strain with bolstered Rre37 and SigE activity compared to those with only one overexpressed protein or in the unaltered 'wild-type' strain. "By the double overexpression of Rre37 and SigE, PHB levels increased by three times compared to the wild type," says Osanai. "However, even further increase in bioplastic production is required for commercial applications," he notes.

In addition to helping transform glycogen into PHB, the researchers documented an important new role played by Rre37 in nitrogen metabolism. Levels of aspartate, a type of amino acid, increased in the Rre37-overexpressing strain of Synechocystis. Compiled transcriptome and metabolome data point to a new metabolic cycle that is something of a hybrid between the well-known Kreb's and urea cycles, which describe common biochemical reactions in the body.

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More information: Osanai, T., Oikawa, A., Numata, K., Kuwahara, A., Iijima, H., Doi, Y., Saito, K. & Hirai, M. "Pathway-level acceleration of glycogen catabolism by response regulator Rre37 in the cyanobacterium Synechocystis sp. PCC 6803." Plant Physiologyadvance online publication, 12 February 2014 (DOI: 10.1104/pp.113.232025). http://dx.doi.org/10.1104/pp.113.232025

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