Unprecedented Control of Genome Editing in Flies Promises Insight Into Human Development, Disease

UW-Madison researchers say fine control of genome editing in fruit flies promises to provide new
insights into embryonic development, nervous system function, and the understanding of human
disease. (Credit: Copyright Jeff Miller)

In an era of widespread genetic sequencing, the ability to edit and alter an organism's DNA is a powerful way to explore the information within and how it guides biological function.

A paper from the University of Wisconsin-Madison in the August issue of the journal Genetics takes genome editing to a new level in fruit flies, demonstrating a remarkable level of fine control and, importantly, the transmission of those engineered genetic changes across generations.

Both features are key for driving the utility and spread of an approach that promises to give researchers new insights into the basic workings of biological systems, including embryonic development, nervous system function, and the understanding of human disease.

"Genome engineering allows you to change gene function in a very targeted way, so you can probe function at a level of detail" that wasn't previously possible, says Melissa Harrison, an assistant professor of biomolecular chemistry in the UW-Madison School of Medicine and Public Health and one of the three senior authors of the new study.

Disrupting individual genes has long been used as a way to study their roles in biological function and disease. The new approach, based on molecules that drive a type of bacterial immune response, provides a technical advance that allows scientists to readily engineer genetic sequences in very detailed ways, including adding or removing short bits of DNA in chosen locations, introducing specific mutations, adding trackable tags, or changing the sequences that regulate when or where a gene is active.

The approach used in the new study, called the CRISPR RNA/Cas9 system, has developed unusually fast. First reported just one year ago by scientists at the Howard Hughes Medical Institute and University of California, Berkeley, it has already been applied to most traditional biological model systems, including yeast, zebrafish, mice, the nematode C. elegans, and human cells. The Wisconsin paper was the first to describe it in fruit flies and to show that the resulting genetic changes could be passed from one generation to the next.

"There was a need in the community to have a technique that you could use to generate targeted mutations," says Jill Wildonger, a UW-Madison assistant professor of biochemistry and another senior author of the paper. "The need was there and this was the technical advance that everyone had been waiting for."

"The reason this has progressed so quickly is that many researchers -- us included -- were working on other, more complicated, approaches to do exactly the same thing when this came out," adds genetics assistant professor Kate O'Connor-Giles, the third senior author. "This is invaluable for anyone wanting to study gene function in any organism and it is also likely to be transferable to the clinical realm and gene therapy."

The CRISPR RNA/Cas9 system directs a DNA-clipping enzyme called Cas9 to snip the DNA at a targeted sequence. This cut then stimulates the cell's existing DNA repair machinery to fill in the break while integrating the desired genetic tweaks. The process can be tailored to edit down to the level of a single base pair -- the rough equivalent of changing a single letter in a document with a word processor.

The broad applicability of the system is aided by a relatively simple design that can be customized through creation of a short RNA sequence to target a specific sequence in the genome to generate the desired changes. Previous genome editing methods have relied on making custom proteins, which is costly and slow.

"This is so readily transferable that it's highly likely to enable gene knockout and other genome modifications in any organism," including those that have not previously been used for laboratory work, says O'Connor-Giles. "It's going to turn non-model organisms into genetic model organisms."

That ease may also pay off in the clinic. "It can be very difficult and time-consuming to generate models to study all the gene variants associated with human diseases," says Wildonger. "With this genome editing approach, if we work in collaboration with a clinician to find [clinically relevant] mutations, we can rapidly translate these into a fruit fly model to see what's happening at the cellular and molecular level."

The work, led by genetics graduate student Scott Gratz, was the joint effort of three UW-Madison labs -- particularly notable, Harrison says, that each is in a different department and headed by a female assistant professor. "This has been an amazing collaboration," she says. "It wouldn't have worked if any one of us had tried it on our own."

Scientists Visualize How Cancer Chromosome Abnormalities Form in Living Cells

In new research, scientists have directly observed events that lead to formation of a chromosome abnormality that is often found in cancer cells. (Screenshot from a YouTube video, available at:http://www.youtube.com/watch?v=rPS49ZaeLag) (Credit: Image courtesy of National Cancer Institute (NCI) at NIH)

For the first time, scientists have directly observed events that lead to the formation of a chromosome abnormality that is often found in cancer cells. The abnormality, called a translocation, occurs when part of a chromosome breaks off and becomes attached to another chromosome.  The results of this study, conducted by scientists at the National Cancer Institute (NCI), part of the National Institutes of Health, appeared Aug. 9, 2013, in the journal Science. 

Chromosomes are thread-like structures inside cells that carry genes and function in heredity. Human chromosomes each contain a single piece of DNA, with the genes arranged in a linear fashion along its length.

For the first time, scientists have directly observed events that lead to formation of a chromosome abnormality that is often found in cancer cells. The results of this study--from senior author Tom Mistelli, Ph.D., lead author Vassilis Roukos, Ph.D., and colleagues at the National Cancer Institute--appears in the Aug 9, 2013 issue of the journal Science

.

Translocations are very rare events, and the scientists’ ability to visualize their occurrence in real time was made possible by recently available technology at NCI that enables investigators to observe changes in thousands of cells over long time periods. “Our ability to see this fundamental process in cancer formation was possible only because of access to revolutionary imaging technology,” said the study’s senior author, Tom Misteli, Ph.D., Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, NCI.Chromosome translocations have been found in almost all cancer cells, and it has long been known that translocations can play a role in cancer development. However, despite many years of research, just exactly how translocations form in a cell has remained a mystery. To better understand this process, the researchers created an experimental system in which they induced, in a controlled fashion, breaks in the DNA of different chromosomes in living cells. Using sophisticated imaging technology, they were then able to watch as the broken ends of the chromosomes were reattached correctly or incorrectly inside the cells.

The scientists involved with this study were able to demonstrate that translocations can occur within hours of DNA breaks and that their formation is independent of when the breaks happen during the cell division cycle. Cells have built-in repair mechanisms that can fix most DNA breaks, but translocations occasionally occur.

To explore the role of DNA repair in translocation formation, the researchers inhibited key components of the DNA damage response machinery within cells and monitored the effects on the repair of DNA breaks and translocation formation. They found that inhibition of one component of DNA damage response machinery, a protein called DNAPK-kinase, increased the occurrence of translocations almost 10-fold. The scientists also determined that translocations formed preferentially between pre-positioned genes.

“These observations have allowed us to formulate a time and space framework for elucidating the mechanisms involved in the formation of chromosome translocations,” said Vassilis Roukos, Ph.D., NCI, and lead scientist of the study. 

“We can now finally begin to really probe how these fundamental features of cancer cells form,” Misteli added.

This research was supported by the Intramural Research Program of the NCI’s Center for Cancer Research.

Article Source : National Cancer Institute (NCI) at NIH.
By Science and Universe ( Nandan)

The when and where of the Y: Research on Y chromosomes uncovers new clues about human ancestry

Abstract image of X and Y chromosomes. (Credit: iStockphoto)

First full-chromosome sequencing effort shows most recent male & female common ancestors lived around the same time

More than 7 billion people live on this planet – members of a  single species that originated in one place and migrated all over the Earth over tens of thousands of years.

But even though we all trace our family lineage to a few common ancestors, scientists still don’t know exactly when and how those few ancestors started to give rise to the incredible diversity of today’s population.

A brand-new finding, made using advanced analysis of DNA from all over the world, sheds new light on this mystery. By studying the DNA sequence of Y chromosomes of men from many different populations, scientists have determined that their male most recent common ancestor (MRCA) lived sometime between 120,000 and 156,000 years ago.

It’s the first time the human ancestry has been traced back through the male line by sequencing the DNA of many entire Y chromosomes.

And, it agrees reasonably well with previous findings about our female most recent common ancestor, made by studying DNA carried down through the human race’s female line. Such studies used DNA from mitochrondria -- structures inside cells – and placed that time of the most recent common ancestor between 99,000 and 148,000 years ago. That agreement makes the new finding especially significant:

The research was done by a team of scientists from Stanford University, the University of Michigan Medical School, Stony Brook University, and their colleagues, and is published in the journalScience.

The team hopes their work will lead to further research on Y chromosomes as vehicles for studying human history – and tracing male lineages back to the common “Adam” ancestors.

The new paper details how the team was able to make reliable measurements of the sequence variation along the Y chromosome – which is handed down only from father to son without exchanging, or recombining, genetic material with other chromosomes. Jeffrey Kidd, Ph.D., an assistant professor in U-M's departments ofHuman Genetics andComputational Medicine & Bioinformatics who worked on the new study, notes that only recently has it become possible to sequence Y chromosomes, because of technical limitations of previous approaches.

Kidd notes that this initial paper on Y chromosome sequence diversity provides important first evidence that the male most recent common ancestor did not live more recently than the female most recent common ancestor.

“We’re interested in understanding the historical relationships between many different human populations, and the migration patterns that have led to the peopling of the world,” he says. “We hope that others will make use of this approach and sequence additional chromosomes of interest that are related to the peopling of specific places.”

The study involved Y chromosomes obtained through the Human Genome Diversity Project, and from other sources. It included chromosomes from 69 men in several populations in sub-Saharan Africa, and from Siberia, Cambodia, Pakistan, Algeria and Mexico.

The great migrations of our ancestors out of Africa, across Asia and Europe and into the Americas all helped shape today’s populations – as did more recent forces related to colonialism and ever-growing global mobility.

Genetic studies such as this one may help anthropologists understand those migrations – and their timing – even better by giving them a genetic “clock” to use when studying today’s humans, or potentially DNA extracted from ancient bones. It may also help scientists understand the great genetic diversity seen across Africa, and the evolution process that led to modern humans.

The reconciliation of the timing of ancestors some might call “Adam” and “Eve”, however, may be this study’s most important immediate implication. 

“This has been a conundrum in human genetics for a long time,” said Carlos D. Bustamante, Ph.D., a professor of genetics at Stanford and senior author of the study. “Previous research has indicated that the male MRCA lived much more recently than the female MRCA. But now our research shows that there’s no discrepancy. In fact, if anything, the Y chromosome may be a bit older.”

Article source : University of Michigan Health System.

By Science and Universe