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Category Archives: Transhuman News

New method identifies genome-wide off-target cleavage sites of CRISPR-Cas nucleases

Posted: December 17, 2014 at 3:43 pm

PUBLIC RELEASE DATE:

16-Dec-2014

Contact: Sue McGreevey smcgreevey@partners.org 617-724-2764 Massachusetts General Hospital @MassGeneralNews

Massachusetts General Hospital (MGH) investigators have developed a method of detecting, across the entire genome of human cells, unwanted DNA breaks induced by use of the popular gene-editing tools called CRISPR-Cas RNA-guided nucleases (RGNs). Members of the same team that first described these off-target effects in human cells describe their new platform, called Genome-wide Unbiased Indentification of DSBs Evaluated by Sequencing (GUIDE-seq), in a report being published online in Nature Biotechnology.

"GUIDE-seq is the first genome-wide method of sensitively detecting off-target DNA breaks induced by CRISPR-Cas nucleases that does not start with the assumption that these off-target sites resemble the targeted sites," says J. Keith Joung, MD, PhD, associate chief for Research in the MGH Department of Pathology and senior author of the report. "This capability, which did not exist before, is critically important for the evaluation of any clinical use of CRISPR-Cas RGNs."

Used to cut through a double strand of DNA in order to introduce genetic changes, CRISPR-Cas RGNs combine a bacterial gene-cutting enzyme called Cas9 with a short RNA segment that matches and binds to the target DNA sequence. In a 2013 Nature Biotechnology paper, Joung and his colleagues reported finding that CRISPR-Cas RGNs could also induce double-strand breaks (DSBs) at sites with significant differences from the target site, including mismatches of as many as five nucleotides. Since such off-target mutations could potentially lead to adverse effects, including cancer, the ability to identify and eventually minimize unwanted DSBs would be essential to the safe clinical use of these RGNs, the authors note.

The method they developed involves use of short, double-stranded oligonucleotides that are taken up by DSBs in a cell's DNA, acting as markers of off-target breaks caused by the use of CRISPR-Cas. Those tags allow the identification and subsequent sequencing of those genomic regions, pinpointing the location of off-target mutations. Experiments with GUIDE-seq showed it was sensitive enough to detect off-target sites at which CRISPR RGNs induced unwanted mutations of a gene that occur with a frequency of as little as 0.1 percent in a population of cells. These experiments also revealed that, since many such mutations took place at sites quite dissimilar from the targeted site, no easy rules would predict the number or location of off-target DSBs.

Two existing tools designed to predict off-target mutations by analysis of the target sequence were much less effective than GUIDE-seq in predicting confirmed off-target sites and also misidentified sites that did not prove to have been cut by the enzyme. Comparing GUIDE-seq with a tool called ChIP-seq - which identifies sites where proteins bind to a DNA strand - confirmed that ChIP-seq does not provide a robust method for identifying CRISPR-Cas-induced DSBs.

GUIDE-seq was also able to identify breakpoint hotspots in control cell lines that were not induced to express the CRISPR RGNs. "Various papers have described fragile genomic sites in human cells before," Joung notes, "but this method may be the first to identify these sites without the addition of drugs that enhance the occurrence of such breaks. We also were surprised to find those breaks occurred largely at different sites in the two cell lines used in this study. The ability to capture these RGN-independent breaks suggests that GUIDE-seq could be a useful tool for studying and monitoring DNA repair in living cells."

In addition, GUIDE-seq was able to verify that an MGH-developed approach for improving the accuracy of CRISPR-Cas by shortening the guiding RNA segment reduced the number of DSBs throughout the genome. Joung also expects that GUIDE-seq will be useful in identifying off-target breaks induced by other gene-editing tools. Along with pursuing that possibility, he notes the importance of investigating the incidence and detection of off-target mutations in human cells not altered to create cell lines - a process that transforms them into immortalized cancer cells. Understanding the range and number of off-target mutations in untransformed cells will give a better picture of how CRISPR-Cas RGNs and other tools would function in clinical applications.

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New method identifies genome-wide off-target cleavage sites of CRISPR-Cas nucleases

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Microbiome may have shaped early human populations

Posted: at 3:43 pm

PUBLIC RELEASE DATE:

16-Dec-2014

Contact: David Salisbury david.salisbury@vanderbilt.edu 615-343-6803 Vanderbilt University @vanderbiltu

We humans have an exceptional age structure compared to other animals: Our children remain dependent on their parents for an unusually long period and our elderly live an extremely long time after they have stopped procreating.

Could the microscopic fellow travelers that consider the human body to be their home - collectively known as the microbiome - have played an active role in shaping and maintaining this unusual aspect of human nature?

That is the speculative proposition advanced by Martin Blaser, professor of medicine and microbiology at NYU's Langone Medical Center, and supported by mathematical models produced by Glenn Webb, professor of mathematics at Vanderbilt University. They present their argument in a paper titled, "Host demise as a beneficial function of indigenous microbiota in human hosts," published online today in mBio, the journal of the American Society for Microbiology.

Scientists have known for a long time that every species of plant and animal acts as host for a distinctive collection of microorganisms. The human microbiome consists of about 100 trillion microbial cells, outnumbering the much larger human cells by about 10 to 1. Until recently they thought that the influence these microscopic communities have on their hosts was extremely limited. But recent research has found that their influence extends well beyond aiding digestion and producing bodily odors; they also aid brain development, reproduction and defense against infection. Taken together, the new evidence has led to the hologenomic theory of evolution, which proposes that the object of Darwin's natural selection is not just the individual organism as he proposed, but the organism plus its associated microbial community.

Blaser got the idea for the impact of microbes on human age structure from his lifetime research on Helicobacter pylori, a bacterium found in the stomach of more than 50 percent of the world's population.

H. pylori co-exists peacefully in people's stomachs for most of their lives. It even has some beneficial effects. In 1996, for example, Blaser discovered that it may help regulate levels of stomach acid. However, H. pylori is also a major cause of stomach cancer, a risk that increases with age.

"I began thinking that a real symbiont is an organism that keeps you alive when you are young and kills you when you are old. That's not particularly good for you, but it's good for the species," Blaser said.

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Microbiome may have shaped early human populations

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Commensal bacteria were critical shapers of early human populations

Posted: at 3:43 pm

PUBLIC RELEASE DATE:

16-Dec-2014

Contact: Garth Hogan ghogan@asmusa.org American Society for Microbiology @ASMnewsroom

WASHINGTON, DC--December 16, 2014--Using mathematical modeling, researchers at New York and Vanderbilt universities have shown that commensal bacteria that cause problems later in life most likely played a key role in stabilizing early human populations. The finding, published in mBio, the online open-access journal of the American Society for Microbiology, offers an explanation as to why humans co-evolved with microbes that can cause or contribute to cancer, inflammation, and degenerative diseases of aging.

The work sprung from a fundamental question in biology about senescence, or aging past the point of reproduction. "Nature has a central problem--it must have a way to remove old individuals, whether fish or trees or people," says Martin Blaser, microbiologist at New York University Langone Medical Center in New York City. "Resources are always limited. And young guys are ultimately competing with older ones."

In most species, individuals die shortly after the reproductive phase. But humans are weird--we have an extra long senescence phase. Blaser began to think about the problem from the symbiotic microbe's point of view and he came up with a hypothesis: "The great symbionts keep us alive when we are young, then after reproductive age, they start to kill us." They are part of the biological clock of aging.

In other words, he hypothesized that evolution selected for microbes that keep the whole community of hosts healthy, even if that comes with a cost to an individual host's health.

Modeling of early human population dynamics could tell him if he was on the right track. Blaser worked together with his collaborator Glenn Webb, professor of mathematics at Vanderbilt University in Nashville, to define a mathematical model of an early human population, giving it characteristics similar to a time 500-100,000 years ago, when the human population consisted of sparse, isolated communities.

Webb came up with a non-linear differential equation to describe the variables involved, their rates of change over time, and the relationship between those rates. "It can reveal something that's not quite appreciated or intuitive, because it sorts out relationships changing in time," even with many variables, such as age-dependent fertility rates and mortality rates, changing simultaneously, explains Webb.

Using this baseline model, the team could tweak the conditions to see what happened to the population dynamics. For example, they increased the fertility rate from roughly six children per female to a dozen, proposing that this might be one way for populations to overcome the burden of senescence, by boosting juvenile numbers. Instead, they were surprised to see that this created wild oscillations in total population size over time--an unstable scenario.

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