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 the often devastating disorders caused by these tiny changes, replicating these changes in the lab in order to study them in human stem cells has proven challenging. But now, scientists at the UC San Francisco-affiliatedGladstone 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.

Bruce Conklin, MD

Led by Gladstone investigator and professor in the UCSF School of Medicine,Bruce Conklin, MD, the research team describes in an issue ofNature 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 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 one-in-a-thousand, 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 in a cell 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 cells even 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, saidConklin. Our high-efficiency, high-fidelity method could very well be the basis for the next phase of human genetics research.

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