THIRD WORLD NETWORK BIOSAFETY INFORMATION SERVICE
Dear Friends and Colleagues
Genetic Engineering Technique Causes Unexpected Genome Damage
The bacterium Agrobacterium tumefaciens has been the workhorse in plant genetic engineering. Using a combination of new techniques, a recent study has now obtained a clearer picture of what happens when genes are spliced into the genomes of plants and animals.
The study found that inserting new genes into a plant using this bacterium as a shuttle creates major unintended effects in the genome. In addition to identifying multiple complete and partial genetically modified gene insertions, numerous large rearrangements of the plant genome were detected. Furthermore, epigenetic (gene regulatory) changes were also identified that could have a wide range of effects, from the silencing of the introduced GM gene to alterations in function of multiple host gene systems. These effects are likely to result in substantial alterations in overall gene expression and consequent changes in the biochemistry, composition and growth characteristics of the GM plants.
Noteworthy is that the findings won’t enable genetic engineers to prevent DNA damage in the first place, but only to more easily spot the lines in which they have caused the most unintended damage. In addition, increased efficiency in weeding out those GM plants with the most off-target genetic and/or epigenetic damage will not ensure good crop performance and food safety. This is because even small changes in gene function can bring about unpredictable major alterations in the biochemistry and hence composition of plants. Thus, generic testing for unexpected toxic effects from the GM process (both old-style transgenic and newer gene editing) is still needed.
With best wishes,
Third World Network
131 Jalan Macalister
Websites: http://www.twn.my/and http://www.biosafety-info.net/
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THE COMPLEX ARCHITECTURE AND EPIGENOMIC IMPACT OF PLANT T-DNA INSERTIONS
Florian Jupe, Angeline C. Rivkin, Todd P. Michael, et al.
18 January 2019
The bacterium Agrobacterium tumefaciens has been the workhorse in plant genome engineering. Customized replacement of native tumor-inducing (Ti) plasmid elements enabled insertion of a sequence of interest called Transfer-DNA (T-DNA) into any plant genome. Although these transfer mechanisms are well understood, detailed understanding of structure and epigenomic status of insertion events was limited by current technologies. Here we applied two single-molecule technologies and analyzed Arabidopsis thaliana lines from three widely used T-DNA insertion collections (SALK, SAIL and WISC). Optical maps for four randomly selected T-DNA lines revealed between one and seven insertions/rearrangements, and the length of individual insertions from 27 to 236 kilobases. De novo nanopore sequencing-based assemblies for two segregating lines partially resolved T-DNA structures and revealed multiple translocations and exchange of chromosome arm ends. For the current TAIR10 reference genome, nanopore contigs corrected 83% of non-centromeric misassemblies. The unprecedented contiguous nucleotide-level resolution enabled an in-depth study of the epigenome at T-DNA insertion sites. SALK_059379 line T-DNA insertions were enriched for 24nt small interfering RNAs (siRNA) and dense cytosine DNA methylation, resulting in transgene silencing via the RNA-directed DNA methylation pathway. In contrast, SAIL_232 line T-DNA insertions are predominantly targeted by 21/22nt siRNAs, with DNA methylation and silencing limited to a reporter, but not the resistance gene. Additionally, we profiled the H3K4me3, H3K27me3 and H2A.Z chromatin environments around T-DNA insertions using ChIP-seq in SALK_059379, SAIL_232 and five additional T-DNA lines. We discovered various effects ranging from complete loss of chromatin marks to the de novo incorporation of H2A.Z and trimethylation of H3K4 and H3K27 around the T-DNA integration sites. This study provides new insights into the structural impact of inserting foreign fragments into plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes.
DEMYSTIFYING GMOS: NEW RESEARCH SHOWS UNEXPECTED CHANGES IN PLANT DNA
By Scott Simonsen, Feb 11, 2019
Genetically modified organisms (GMOs) are one of the most contentious topics in science today. But a study from the Salk Institute, published last month in PLOS Genetics, may help clear up some of the confusion. Using a combination of techniques known as nanopore sequencing and optical mapping, researchers believe they have a clearer picture of what happens when genes are spliced into the genomes of plants and animals.
In particular, the study showed that scientists can determine to what extent surrounding areas of the host DNA have been affected by gene splicing, a point that is often a source of concern for those worried about the possible long-term impacts of GMO consumption.
How GMOs Are Created
To genetically modify a plant or animal, scientists first sequence the entire genome of the organism to determine which stretches of DNA have beneficial qualities. These would include traits that help the organism survive drought, produce higher nutrient densities, be less susceptible to insects or diseases, or be able to withstand certain pesticides, among many others. The DNA sequence containing desirable traits is then removed and implanted into the genome of the host organisms, thereby transferring the beneficial properties.
The most common method of doing this is by using the bacteria Agrobacterium tumefaciens. Several decades ago, it was discovered that when this bacteria caused crown gall tumors on tree trunks, some of the bacteria’s DNA was transferred into the DNA of the tree; the bacteria’s transfer DNA (T-DNA), a circular piece of DNA that can bind with other DNA sequences, was found to be scattered throughout the tree. Since then, researchers have used this bacteria’s T-DNA to help carry the desired genes into all kinds of organisms.
However, the problem with this method is its lack of precision. That is, when this process occurs, researchers are not sure exactly what happens. Recent advancements in DNA sequencing techniques led some scientists to suspect that the structure and chemistry of the host DNA might be changed more than originally thought due to unknown interactions with the T-DNA, as well as the amount and length of the T-DNA transferred into the host.
Those designing and selling genetically modified products merely test the new organism for the desired traits and, if they are present, the process is considered a success.
Nanopore Sequencing and Optical Mapping
Originally created in the mid 1990s, nanopore sequencing is considered one of the most effective methods of detecting genetic changes on a molecular level. It works by placing two tiny electrodes near a nano-sized hole in a membrane filled with an electrolyte. When a strand of DNA is sent through this hole, the different bases that make up this molecule create unique variations in the electric current, which can be detected and analyzed. This allows researchers to know in great detail the structure of the molecule that just passed through the hole.
Optical mapping is a technique that creates a high-resolution map of a genome by severing a strand of DNA at specific sites with restriction enzymes, creating a unique fingerprint. That is, restriction enzymes digest specific sequences of DNA, separating the strand into various fragments, the distribution of which is inevitably different from other strands.
While neither of these methods is entirely new, the Salk Institute team combined them, creating a picture with an unprecedented level of detail. They employed a new nanopore long-read DNA sequencing technique, which made assembling the picture of a complete genome much easier because it extends the size of the data that can be collected, reducing the complexity of assembling the pieces. They also created optical genome maps by using the Bionano Genomics Irys system, which they demonstrated can achieve levels of resolution on the scale of a single molecule.
The team found that one insertion attempt could result in as many as seven unintended insertions or manipulations of the host’s genome. Some of these were up to ten times larger than intended, resulting in large segments of the host’s DNA being damaged or relocated. Furthermore, the incoming DNA was sometimes found to be out of place, cut in half, or out of sequence.
To GMO Or Not to GMO?
What these new results mean for the GMO debate is open to interpretation. Whatever side you might be on, this research demonstrates there’s more happening on the molecular scale than we originally thought.
Feeding the world’s future population is not only going to involve genetically modified foods, it’s going to require them. Current agricultural yields are not nearly high enough for the projected 9.7 billion people of 2050 to live on.
So what comes next to help determine whether GMOs are the way to go, and how to make sure they’re safe? More research.
NEW RESEARCH CONFIRMS GM CAUSES MASSIVE OFF-TARGET DAMAGE TO PLANT GENOMES
28 January 2019
A new open-access paper by researchers at the Salk Institute in the US confirms that the GM transformation process in plants is extraordinarily damaging at a genetic and epigenetic level. The researchers found that inserting new genes into a plant using the bacterium Agrobacterium tumefaciens as a shuttle creates major unintended effects in the genome. The authors studied four different GM lines of the standard laboratory model plant Arabidopsis.
The GMO lobby promotes GM methods, especially the new gene editing methods, using the metaphors of scissors or a scalpel to imply that these methods are precise and targeted. But based on the evidence, we suggest an alternative more accurate metaphor – that of a chainsaw in the hands of a young child. Hence our banner image for this article.
In addition to identifying multiple complete and partial GM gene insertions, numerous large rearrangements of the plant genome were detected. Furthermore, epigenetic (gene regulatory) changes were also identified that could have a wide range of effects, from the silencing of the introduced GM gene to alterations in function of multiple host gene systems.
The authors of the new paper do not discuss the consequences of this damage at a genetic and epigenetic level of the genome. However, it will result in substantial alterations in overall gene expression and consequent changes in the biochemistry, composition and growth characteristics of the GM plants.
New gene editing techniques, which rely on tools such as CRISPR, often involve the use of Agrobacterium in order to deliver this system into plant cells and in some cases to insert new genetic material into the genome. Thus, new gene editing GM techniques will not solve the problems highlighted by this new study.
In addition, the tissue culture process that is an obligatory part of all GM processes, including gene editing, is already known to cause mutations on a vast scale. So the implications of the new paper from the Salk researchers are that the tissue culture-induced mutations will be piled on top of the damage caused by the Agrobacterium-mediated GM transformation process.
It’s noteworthy how the Salk’s press release bends over backwards, forwards, and sideways to try to put a positive spin on the new paper’s findings. Titled, “New technologies enable better-than-ever details on genetically modified plants”, the press release says the results “offer new ways to more effectively minimize potential off-target effects”.
In reality, however, the paper only describes new DNA mapping and sequencing techniques that can better identify the true extent and nature of the damage created by the genetic manipulation, enabling genetic engineers to more efficiently discard the most badly damaged GM plant lines. As one of the researchers says, “Current methods require screening of hundreds of transgenic lines to find good performing ones, such as those without extra insertions, so this technology could provide a more efficient approach.”
In other words, the new findings won’t enable genetic engineers to prevent DNA damage in the first place, but only to more easily spot the lines in which they have caused the most unintended damage.
However, increased efficiency in weeding out those GM plants with the most off-target genetic and/or epigenetic damage will not ensure good crop performance and food safety. This is because even small changes in gene function can bring about unpredictable major alterations in the biochemistry and hence composition of plants. Thus, generic testing for unexpected toxic effects from the GM process (both old-style transgenic and newer gene editing) will still be needed.