Discovering “CRISPR” methods for genetic recombination

Screen Shot 2014-03-27 at 9.54.19 PM

By Olivia Zhu

In a lecture to an overflowing auditorium in the Bryan Research Building on March 27th, Dr. Jennifer Doudna, of the University of California, Berkeley, unraveled her story of research into CRISPRs, or “clustered regularly interspaced short palindromic repeats.” Dr. Doudna specializes in RNA; she started her project on CRISPRs seven years ago, when CRISPRs were denounced as no more than junk.

The CRISPR method includes a modifiable RNA sequence whose function is to recognize target sequences on DNA. The RNA also includes a target sequence that induces cleavage by the associated protein, CAS9. CAS9 introduces double-stranded breaks and represents an exciting improvement over the previous, less efficient collection of nine proteins used to cleave DNA; the breaks make room for insertion of new genes. The CRISPR-CAS9 system has inserted genes into a wide range of organisms, including bacteria, yeast, nematode worms, fruit flies, plants, fish, mice, and even human cells.

Jennifer Doudna

Jennifer Doudna of UC Berkeley and the Howard Hughes Medical Institute

While researchers are actively investigating the possibility of using CRISPR technology to alter genes, Doudna said the mechanism behind CRISPR gene editing remains unclear. For example, it seems extraordinary that the CRISPR-CAS9 system can locate and unwind specific DNA sequences in human cells, as the DNA there is highly condensed around histones and methylated.

Doudna’s lab is working to understand the details of the CRISPR process. One current hypothesis includes the idea that there is a spring mechanism that allows the CAS9 protein to effectively cleave DNA strands.

Nevertheless, CRISPR technology has been instrumental in allowing more precise and efficient genetic modification. What we once considered junk has spurred substantial advances across various fields of science.

Stem Cells Might Tell Us Why Chimps Can’t Blush

Guest post by graduate student Sheena Faherty

Clint the Chimpanzee

Clint the chimpanzee was the first member of Pan troglodytes to have his DNA sequenced. Thanks, dude. (Photo from Yerkes National Primate Research Center.)

Clint the chimpanzee is at it again.

The first chimpanzee to have his genome sequenced in 2005 has now made another mammoth contribution to science, this time with his stem cells.

Using these stem cells, Greg Wray, professor in Biology and Evolutionary Anthropology and his former Ph.D. student, Lisa Pfefferle, recently published an article detailing an exciting new genomic tool that provides a sneak peek into how fundamental differences at the genetic level can lead to drastic differences we see at the outward level between humans and chimpanzees.

This fascinating new approach is based on a specific type of adult stem cells, known as adipose derived stromal cells (ASC). The beauty of ASCs is that they can be manipulated to morph into different types of mature cells. These cells can then be poked, prodded, and scrutinized under the microscope as a means to delve into fundamental questions regarding the molecular basis of human origins.

This work adds a powerful new tool to the field of comparative primate genomics. The goal is to discover the source of traits that set humans apart from other animals, like spoken language or the sole ability to blush when embarrassed.

By comparing humans with our closest genetic cousin, the chimpanzee, we can begin to uncover qualities unique to both humans and chimpanzees. These discoveries might lie within the genome.

Lisa Pfefferle developed a new technique, based on Clint's stem cells, to get at human-chimp differences. (Photo courtesy of Lisa Pfefferle.)

Lisa Pfefferle developed a new technique, based on Clint’s stem cells, to get at human-chimp differences. (Photo courtesy of Lisa Pfefferle.)

In a beautifully designed experiment, Wray and Pfefferle obtained a precious stock of Clint’s frozen ASCs, manipulated them into fat cells, known as adipocytes, and then compared his adipocytes with three different populations of human ASCs. (Clint, a resident of the Yerkes National Primate Research Center in Georgia, died at age 24 a few months before his genome was published.)

Using next-generation sequencing approaches, the researchers were then able to compare over 10,000 genes between human and chimpanzee. The results of this comparison show central differences within the set of genes that may be contributing to the obvious dissimilarities between humans and chimpanzees.

For example, genes controlling the development and function of the immune system were significantly higher in chimpanzees than in humans. It is well documented that chimpanzees are able to heal wounds faster than humans. This may be why.

In contrast, genes involved in the cell cycle and DNA processing, important for passing on genetic information and repairing DNA damage within cells, were expressed at a higher level in humans.

This novel approach of using ASCs in a controlled laboratory setting will undoubtedly be a valuable complement to existing studies on comparative primate genomics.

CITATION: Pfefferle, LW and Wray GA. Insights From a Chimpanzee Adipose Stromal Cell Population: Opportunities for Adult Stem Cells to Expand Primate Functional Genomics. October 2013: 1–18, doi:10.1093/gbe/evt148

Duke Students Travel to D.C. to Present Findings to FDA

By Nonie Arora

Duke students outside the FDA. Evelyna Kliassov, Ryan Gimple, Jenae Logan, Hiruni Amarasekara, Biqi Zhang, Selina Chen, and Akash Shah. Credit: Huntington Willard.

Duke students outside the FDA. Evelyna Kliassov, Ryan Gimple, Jenae Logan, Hiruni Amarasekara, Biqi Zhang, Selina Chen and Akash Shah. Credit: Huntington Willard

Last month, Duke seniors presented findings on noninvasive prenatal testing at the Food and Drug Administration (FDA) in Washington D.C.

The students explained to government officials that noninvasive prenatal testing requires only a blood sample from a pregnant woman. Current tests, such as amniocentesis, involve extracting cells from the placenta or fluid surrounding the fetus.

Instead, with the new technology labs genetically sequence fetal, cell-free DNA from in the mother’s blood to test for certain disorders. The method can detect when a fetus does not have the normal number of chromosomes. Specifically, it can detect abnormalities in chromosomes 13, 18 and 21, which can lead to disorders such as Down’s Syndrome.

The technology can also identify some fetal, sex-linked disorders and certain single-gene mutations. It is reliable after seven weeks of pregnancy, the students reported.

The presentation was a final project of the Genome Sciences & Policy capstone course, which leads to students earning a certificate in the field.

The students said Duke geneticist Hunt Willard and Dr. Robert Cook-Deegan, the professors for the course, chose noninvasive prenatal testing as the capstone topic because it is a new and rapidly growing field.

“Our professors wanted us to have a feel for what it’s like to research technology while it’s happening, while decisions are being made about whether it’s accurate and reliable,” said Biqi Zhang, one of the students in the class.

To investigate the scientific basis for noninvasive prenatal testing, its challenges, the active stakeholders and associated ethical considerations, these students interviewed individuals involved with different aspects of the technology.

“We had to go out and connect with many well-established professionals in related fields. It was exciting to develop skills that you normally don’t inside the classroom,” said Selina Chen, another student in the course.

“We had the opportunity to contact researchers and CEOs of companies to gain a comprehensive understanding of the technology,” Zhang added.

Evelyna Kliassov presenting on cost-effectiveness of noninvasive prenatal testing to the FDA. Credit: Huntington Willard.

Evelyna Kliassov presenting on cost-effectiveness of noninvasive prenatal testing to the FDA. Credit: Huntington Willard.

The students said that the technology can and will fundamentally alter prenatal medicine. Throughout the semester, they have gained a nuanced understanding of its complexities and the viewpoints of many different stakeholders involved, from technology startup CEOs to primary care physicians.

“It was most exciting being able to go into the real world and see how this technology is being clinically implemented,” said capstone student Ryan Gimple.

“Traveling to the FDA was definitely nerve-wracking, for me at least,” capstone student Hiruni Amarasekara said. “We wanted to present a comprehensive report of the technology so that they could use this information in their decision making process on whether to recommend the test in the future. It was hard to tell what the FDA was thinking as we were presenting our information.”

The FDA has not yet stated a position on use of noninvasive prenatal testing.

Hope for Understanding Ourselves Goes to the Dogs

By Ashley Yeager

Brian Hare and Evan MacLean, co-directors of Duke's Canine Cognition Center, play with Lilu, a labradoodle. Credit: Ashley Yeager, Duke.

Brian Hare and Evan MacLean, co-directors of Duke’s Canine Cognition Center, play with Lilu, a labradoodle. Credit: Ashley Yeager, Duke.

Lilu, a beautiful brown poodle-labradoodle mix, couldn’t sit still. Scents of pizza and peanut butter dog treats and the sights of new people easily distracted her.

The ADD behavior could be one trait that made her fail out of service-dog training.

“Six out of every ten dogs wash out of service training. But it’s hard right now for scientists to understand why,” said Duke evolutionary anthropologist Evan MacLean, co-director of the university’s Canine Cognition Center.

He, along with biological anthropologist Brian Hare and geneticist Misha Angrist spoke about ‘Genes, Brains and Games’ in man’s best friend as part of the Science and Society Journal Club on April 26.

MacLean and Hare explained that dogs have taken on many jobs in human society, acting as everything from pets, to our eyes and ears to being like coal-mine canaries searching for hidden bombs and missing people.

“Dog vocations require different sets of cognitive skills,” MacLean said. He studies military dogs, looking for traits that make them more suited for service tasks than pets like Lilu.

MacLean would ultimately like to identify the genetic components that underlie the characteristics suited for each type of job that a dog might do.

Scientists are interested in correlating dogs’ cognitive traits to their associated genes because the animals are “the most exquisite example of artificial selection,” Angrist said.

In Portuguese water dogs, for example, just six substitutions in individual DNA bases of the dogs explain variations in body size. In humans, nearly every gene could factor into height. It’s the same challenge that makes understanding human cognition and intelligence difficult at the genetic level.

Of course, defining cognition and intelligence at the conceptual level isn’t so clear cut either. “It’s so hard for people, journalists and the general public, to understand multiple intelligences,” Hare said.

He explained that at a basic level, cognition is the ability to make inferences, and that when we think of intelligence we think of IQ and standardized tests. These tests, however, measure only one type of intelligence. They don’t measure the ability to empathize, to verbalize a new idea or to put two completely separate ideas together to form a new one, which are other, important facets of intelligence, or really multiple intelligences.

At the Canine Cognition Center, and through the citizen science website Dognition, Hare and MacLean use standardized tests to study the variation in dogs’ intelligence. The tests, unlike the SAT or ACT, “cast a wide net across skills sets dogs could use for different vocations,” Hare said.

Dogs like Lilu, he added, are “really the hope of the world” for understanding cognition.

Not your typical spring break

By Nonie Arora

Students in front of Eisenhower Executive Office Building, Credit: Bob Cook-Deegan

Students in front of Eisenhower Executive Office Building, Credit: Bob Cook-Deegan

Seventeen Duke students had a taste of science policy over spring break. We traveled to Washington D.C. to meet with influential scientists and policy makers from a variety of different institutions, from the Genetic Alliance to the Office of Science and Technology Policy of the White House.

The trip clarified for many of us what science policy is like in action, and the winding paths that guide people to this career.

The students contributed to a trip blog, on which they discuss experiences such as seeing Bo Obama, the First Dog (!), outside the White House and “sipping the kool-aid” of genome science at the National Human Genome Research Institute.

The trip was sponsored by Focus and the Institute for Genome Sciences & Policy under the direction of professor Bob Cook-Deegan.

Designing Microbial “Factories” Rationally

By Pranali Dalvi

Using microbes to manufacture chemicals is starting to be cheaper and greener than traditional chemistry. And their feedstock is sugar, not oil.

Source: 2010 Agricultural Biotechnology International Conference

On Friday, Dr. Michael Lynch spoke to an engaged audience about how microbes have ushered in a new era in metabolic and genetic engineering. Lynch is the co-founder and CSO of OPX Biotechnologies, a Colorado-based company that makes bio-based chemicals and fuels from microbes. OPXBIO microbes produce fatty acids from hydrogen and carbon dioxide. In turn, the fatty acids are used to make cleaners, detergents, jet fuel, and diesel.

Lynch said it’s easier to understand the genetic circuits and enzymatic pathways of microbes, thanks to  much cheaper DNA sequencing. What we still lack though, is an understanding of how to rationally design complex biological systems – likely because we fail to recognize the interplay among an organism’s genotype, phenotype, and environment.

It’s a complex set of factors that go into making phenotypic traits such as color, size, or shape.

“In an industrial setting [phenotypes] are equivalent to metabolism or higher production of the product of interest,” Lynch said. “In a clinical setting, [phenotypes] could be virulence or pathogenesis.”

One approach to understanding how phenotypes are controlled has been through functional genomics.

Let’s say we take a population of wildtype microorganisms and introduce genetic modifications in a controlled way. Next, we selectively screen for the phenotype of interest and compare the sequence of this phenotype to the wildtype to pinpoint the genetic mutations that made the difference.

Comparing phenotypes one at a time is inefficient, though. Lynch wanted to find a way to speed up this process.

“We wanted a process or technology or toolkit that evaluates all of your genes in parallel in a single experiment for the phenotype of interest,” Lynch explained.

Lynch found his inspiration in microbial biofilms, extracellular polysaccharide matrices that grow quickly.

OPXBIO’s Efficiency Directed Genome Engineering (EDGE) technology platform, Source:

Lynch’s studies revealed that microbial cultures grown in enriched media made biofilms, while those in minimal media did not. In a process known as destructional mutagenesis, Lynch and his colleagues then knocked out biofilm-making genes to identify what genes cause the biofilm phenotype in enriched medium but prevent it in minimal medium.

Lynch saw the individual microbial systems as factories that he can genetically modify to produce chemical compounds in biofilms – specifically, 3-hydroxypropionic acid – that can be chemically converted to commercially relevant compounds such as acrylic.

Scientists at OPXBIO have cracked the code for making acrylic from sugar.  They give sugar feedstocks to genetically modified bacteria, whose enzymes convert the sugar into acrylic molecules. Acrylic has broad commercial applications in paints, adhesives, diapers, detergents, and even fuel – a $10 billion global market.