Engineering The Human Genome One Letter At A Time

Image Caption: Beating-heart cells derived from iPS cells are shown. A single DNA base-pair of the PRKAG2 gene was edited using the method developed by Drs. Miyaoka and Conklin. Credit: Luke Judge/Gladstone Institutes

Anne D. Holden, PhD Gladstone Institutes

Gladstones innovative technique in stem cells to boost scientists ability to study and potentially cure genetic disease

Sometimes biology is cruel. Sometimes simply a one-letter change in the human genetic code is the difference between health and a deadly disease. But even though doctors and scientists have long studied disorders caused by these tiny changes, replicating them to study in human stem cells has proven challenging. But now, scientists at the Gladstone Institutes have found a way to efficiently edit the human genome one letter at a time not only boosting researchers ability to model human disease, but also paving the way for therapies that cure disease by fixing these so-called bugs in a patients genetic code.

Led by Gladstone Investigator Bruce Conklin, MD, the research team describes in the latest issue of Nature Methods how they have solved one of science and medicines most pressing problems: how to efficiently and accurately capture rare genetic mutations that cause disease as well as how to fix them. This pioneering technique highlights the type of out-of-the-box thinking that is often critical for scientific success.

Advances in human genetics have led to the discovery of hundreds of genetic changes linked to disease, but until now weve lacked an efficient means of studying them, explained Dr. Conklin. To meet this challenge, we must have the capability to engineer the human genome, one letter at a time, with tools that are efficient, robust and accurate. And the method that we outline in our study does just that.

One of the major challenges preventing researchers from efficiently generating and studying these genetic diseases is that they can exist at frequencies as low as 1%, making the task of finding and studying them labor-intensive.

For our method to work, we needed to find a way to efficiently identify a single mutation among hundreds of normal, healthy cells, explained Gladstone Research Scientist Yuichiro Miyaoka, PhD, the papers lead author. So we designed a special fluorescent probe that would distinguish the mutated sequence from the original sequences. We were then able to sort through both sets of sequences and detect mutant cellseven when they made up as little one in every thousand cells. This is a level of sensitivity more than one hundred times greater than traditional methods.

The team then applied these new methods to induced pluripotent stem cells, or iPS cells. These cells, derived from the skin cells of human patients, have the same genetic makeup including any potential disease-causing mutations as the patient. In this case, the research team first used a highly advanced gene-editing technique called TALENs to introduce a specific mutation into the genome. Some gene-editing techniques, while effective at modifying the genetic code, involve the use of genetic markers that then leave a scar on the newly edited genome. These scars can then affect subsequent generations of cells, complicating future analysis. Although TALENs, and other similarly advanced tools, are able to make a clean, scarless single letter edits, these edits are very rare, so that new technique from the Conklin lab is needed.

Our method provides a novel way to capture and amplify specific mutations that are normally exceedingly rare, said Dr. Conklin. Our high-efficiency, high-fidelity method could very well be the basis for the next phase of human genetics research.

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Engineering The Human Genome One Letter At A Time