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.

Originally posted here:
Engineering The Human Genome One Letter At A Time

Over the last decade, as DNA-sequencing technology has grown ever faster and cheaper, our understanding of the human genome has increased accordingly. Yet scientists have until recently remained largely ham-fisted when theyve tried to directly modify genes in a living cell. Take sickle-cell anemia, for example. A debilitating and often deadly disease, it is caused by a mutation in just one of a patients three billion DNA base pairs. Even though this genetic error is simple and well studied, researchers are helpless to correct it and halt its devastating effects.

Now there is hope in the form of new genome-engineering tools, particularly one called CRISPR. This technology could allow researchers to perform microsurgery on genes, precisely and easily changing a DNA sequence at exact locations on a chromosome. Along with a technique called TALENs, invented several years ago, and a slightly older predecessor based on molecules called zinc finger nucleases, CRISPR could make gene therapies more broadly applicable, providing remedies for simple genetic disorders like sickle-cell anemia and eventually even leading to cures for more complex diseases involving multiple genes. Most conventional gene therapies crudely place new genetic material at a random location in the cell and can only add a gene. In contrast, CRISPR and the other new tools also give scientists a precise way to delete and edit specific bits of DNAeven by changing a single base pair. This means they can rewrite the human genome at will.

It is likely to be at least several years before such efforts can be developed into human therapeutics, but a growing number of academic researchers have seen some preliminary success with experiments involving sickle-cell anemia, HIV, and cystic fibrosis (see table below). One is Gang Bao, a bioengineering researcher at the Georgia Institute of Technology, who has already used CRISPR to correct the sickle-cell mutation in human cells grown in a dish. Bao and his team started the work in 2008 using zinc finger nucleases. When TALENs came out, his group switched quickly, says Bao, and then it began using CRISPR when that tool became available. While he has ambitions to eventually work on a variety of diseases, Bao says it makes sense to start with sickle-cell anemia. If we pick a disease to treat using genome editing, we should start with something relatively simple, he says. A disease caused by a single mutation, in a single gene, that involves only a single cell type.

In little more than a year, CRISPR has begun reinventing genetic research.

Bao has an idea of how such a treatment would work. Currently, physicians are able to cure a small percentage of sickle-cell patients by finding a human donor whose bone marrow is an immunological match; surgeons can then replace some of the patients bone marrow stem cells with donated ones. But such donors must be precisely matched with the patient, and even then, immune rejectiona potentially deadly problemis a serious risk. Baos cure would avoid all this. After harvesting blood cell precursors called hematopoietic stem cells from the bone marrow of a sickle-cell patient, scientists would use CRISPR to correct the defective gene. Then the gene-corrected stem cells would be returned to the patient, producing healthy red blood cells to replace the sickle cells. Even if we can replace 50 percent, a patient will feel much better, says Bao. If we replace 70 percent, the patient will be cured.

Though genome editing with CRISPR is just a little over a year old, it is already reinventing genetic research. In particular, it gives scientists the ability to quickly and simultaneously make multiple genetic changes to a cell. Many human illnesses, including heart disease, diabetes, and assorted neurological conditions, are affected by numerous variants in both disease genes and normal genes. Teasing out this complexity with animal models has been a slow and tedious process. For many questions in biology, we want to know how different genes interact, and for this we need to introduce mutations into multiple genes, says Rudolf Jaenisch, a biologist at the Whitehead Institute in Cambridge Massachusetts. But, says Jaenisch, using conventional tools to create a mouse with a single mutation can take up to a year. If a scientist wants an animal with multiple mutations, the genetic changes must be made sequentially, and the timeline for one experiment can extend into years. In contrast, Jaenisch and his colleagues, including MIT researcher Feng Zhang (a 2013 member of our list of 35 innovators under 35), reported last spring that CRISPR had allowed them to create a strain of mice with multiple mutations in three weeks.

Genome GPS

The biotechnology industry was born in 1973, when Herbert Boyer and Stanley Cohen inserted foreign DNA that they had manipulated in the lab into bacteria. Within a few years, Boyer had cofounded Genentech, and the company had begun using E. coli modified with a human gene to manufacture insulin for diabetics. In 1974, Jaenisch, then at the Salk Institute for Biological Studies in San Diego, created the first transgenic mouse by using viruses to spike the animals genome with a bit of DNA from another species. In these and other early examples of genetic engineering, however, researchers were limited to techniques that inserted the foreign DNA into the cell at random. All they could do was hope for the best.

It took more than two decades before molecular biologists became adept at efficiently changing specific genes in animal genomes. Dana Carroll of the University of Utah recognized that zinc finger nucleases, engineered proteins reported by colleagues at Johns Hopkins University in 1996, could be used as a programmable gene-targeting tool. One end of the protein can be designed to recognize a particular DNA sequence; the other end cuts DNA. When a cell then naturally repairs those cuts, it can patch its genome by copying from supplied foreign DNA. While the technology finally enabled scientists to confidently make changes where they want to on a chromosome, its difficult to use. Every modification requires the researcher to engineer a new protein tailored to the targeted sequencea difficult, time-consuming task that, because the proteins are finicky, doesnt always work.

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Genome Surgery