By Seth Palmer
May 28, 2015
Bastian Minkenberg takes a leaf tissue sample from a rice plant. Credit: Seth Palmer.
The CRISPR/Cas9 genome-editing system is seen by some as perhaps the most important biotechnology discovery of the century, but even it has its limitations. Employing a DNA-cutting protein (Cas9) and so-called guide RNA (gRNA), the CRISPR/Cas9 system allows scientists to specifically target and modify almost any DNA site in an organism's genome, but with a bottleneck in the number of sites that can be modified simultaneously – until now.
The Yang Lab's recent breakthrough is the introduction of a synthetic gene made from guide RNA and transfer RNA repeats (polycistronic tRNA-gRNA gene or PTG) which, together with Cas9, effectively cuts multiple genomic sites simultaneously for mutation or other modifications – thereby improving the multiplexing capability of the CRISPR/Cas9 system with high efficiencies of between 80 and 100%.
From cells to plants
My job,” explains Bastian Minkenberg, “was basically to take our research from the bench and make a genetically modified plant out of it.”
He uses a modified pathogenic bacterium (Agrobacterium tumefaciens) to transfer a DNA piece carrying the CRISPR/Cas9 genome-editing device into undifferentiated rice callus cells which can then be grown into mature plants.
Minkenberg folds a leaf sample before sealing it in a vial for analysis. Credit: Seth Palmer.
Although the Lab's work was receiving funding from the National Science Foundation's Plant Genome Research Program, more money was needed to produce the required genome-edited plants, so Minkenberg applied for an additional $5,000 of funding through the Huck Institutes' Graduate Research Dissertation Award.
“Our project was trying to produce a library of mutants,” Minkenberg says, “which involves production of a lot of plants. We knew we needed extra funding to make that happen. The award gave us the money to pay for materials to do the tissue culture and transformation experiments for producing transgenic rice plants, and now I have several hundreds of plants that I need to extract DNA samples from, amplify the target site, and sequence it to see if there are intended mutations.”
Minkenberg places a leaf sample in a vial for DNA extraction. Credit: Seth Palmer.
Refining the results
“With agrobacterium-mediated transformation,” Minkenberg explains, “the genome-editing device is integrated into the plant's genome to cause the intended mutation, but if we self-pollinate the plants we are able to remove the transgene. So the resulting plants are transgene-free, but contain the specific mutation we induced.”
Through Mendelian inheritance, roughly a quarter of the resulting offspring will lose the T-DNA transgene completely, so “about one in four plants will have two copies of the transgene,” Minkenberg says. “Two in four will have only one copy, and the one remaining will have no copy at all and that's what we want – a transgene-free , genetically modified rice plant.”
The researchers expect the transgene-free nature of these plants to reduce GMO-related regulatory burden and hope that it will also alleviate public concerns about the safety of GMO crops.
Looking forward, Minkenberg will also collect seed stock for future experiments to analyze what effect the mutation had on the plant.
“Our genomic targets are critical for biotic and abiotic stress,” he says, “so we'll look specifically at the effects of the mutation on rice disease resistance and drought tolerance.”
A leaf sample from a rice plant, ready for DNA extraction. Credit: Seth Palmer.
The entire process – starting with producing the rice callus, then genetically modifying it, growing mature plants, and harvesting their seed – takes about half a year.
“If you start with immature seed,” Minkenberg explains, “after four weeks you'll have a callus that you can mix with the Agrobacterium solution. Then, about four weeks later, I'll transfer the calluses that survived our selection process to a regeneration medium where they'll start to grow shoots, and after four more weeks I'll transfer those shoots to a root-induction medium so they can grow roots. After one more week, I can put them into soil. So that's a total of a little over three months to that point, and then the plants need to grow and set seed, so that will take maybe three more months – about six months total, now. Our improved genome-editing technique, however, is so efficient that the only remaining step is to self-pollinate the plants and analyze the progeny for the T-DNA loss and for the inherited mutation. This process is much faster than a traditional plant breeding approach that requires extensive backcrossing and scoring of a very high number of progeny plants.”
“In the future,” he says, “our Lab's technique could help to accelerate precision breeding of transgene-free crops with improved agronomic traits such as high yield, better quality, stress tolerance, and disease resistance. I think these are really exciting results, and it's thanks in part to the Huck award that we're able to continue these experiments in such a tough funding climate.”