Shotgun vs. scalpel

When Holger Puchta wants to illustrate the enormous genetic potential of a single plant species, he refers to Brassica oleracea, better known in horticulture as wild cabbage. This is because the herbaceous wild plant with the yellow flowers is the ancestor of a whole range of vegetables that have provided varying degrees of pleasure on the plates of generations of children. White cabbage and kale, cauliflower and Brussels sprouts, broccoli and kohlrabi all descend from the inconspicuous cruciferous plant.

"It's amazing what classical breeding has teased out of this one species," says Holger Puchta. But all that took time. A lot of time. The breeding history of wild cabbage goes way back to ancient times, and probably beyond. "We obviously don't have centuries or even decades if we want to counter the consequences of climate change and continue to feed a growing world population," explains the molecular biologist. "We need agricultural crops that are as salt, heat and diseaseresistant as possible, high-yielding, and flavorful. And we need them fast. The standard tools of classical breeding and mutagenesis work too slowly. With molecular scissors, we can now do it within one or two plant generations, within months."

Holger Puchta counters reservations about this technology with a practical look back at the past: "You have to realize how breeding was done before gene shears and how it still is. Mutagenesis has been common and legal practice for about 70 years. In this process, radioactive irradiation creates 10,000 uncontrolled mutations in a plant. By chance, a desired result then sometimes emerges." In contrast, molecular scissors create a single mutation in a very specific gene in a very targeted way. "So we have mutagenesis and thousands of uncontrolled mutations on one side, and molecular scissors and a single targeted change on the other. It’s like having a shotgun and a scalpel. The latter is faster, safer and highly specific," says Puchta.

Since 2012, a very specific gene-scissors methodology has been used worldwide for this purpose: CRISPR/Cas, the development of which earned scientists Emmanuelle Charpentier and Jennifer Doudna the 2020 Nobel Prize in Chemistry. "Through CRISPR/Cas, we are currently experiencing the biggest revolution in 30 years in biology, medicine and agriculture, because the method is extremely flexible and accurate," says Holger Puchta. At the Karlsruhe Institute of Technology (KIT), he has significantly expanded the possible applications of CRISPR/Cas and, for the first time, lifted it from the level of individual genes to the level of the chromosome.

Genes are arranged linearly on chromosomes. For a long time, CRISPR/Cas was only used to change individual genes on a chromosome. A few years ago, however, Puchta and his team succeeded in exchanging entire parts of chromosomes and thus recombining the genes. Thanks to this breakthrough from Karlsruhe, it is now possible to, among other things, separate good properties of a plant from bad ones. If, for example, in species X a gene for a bitter substance that makes the plant's fruit taste bad is located directly next to a desirable gene that makes it resistant to a virus, both genes will always be inherited together because of their immediate proximity on the chromosome. Therefore in the normal generation sequence, all plants with resistance will always also be bitter. With Puchta's technique, the chromosome part with the virus resistance can now be separated, transferred to another chromosome without bitter and then inherited separately.

Also using CRISPR/Cas, Holger Puchta's research group succeeded in solving a similar problem. If genes for positive traits are located far apart on a chromosome, they are usually separated from each other during inheritance and are thus gradually lost during breeding. Using molecular scissors, KIT researchers managed to cut out most of a chromosome, turn it 180 degrees, and reinsert it. "In this way, we shut down almost a complete chromosome and make it virtually invisible for gene exchange with other chromosomes," explains Holger Puchta. "The positive properties on the inverted section are thus fixed, and will in the future always be inherited together as a package, and will therefore be preserved."