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Breeding Non-GMO Plants With Next-Generation Chloroplast Gene-Editing

Researchers have developed a new technique to improve the diversity of crop species without affecting the nuclear DNA of future seeds.

For decades now, breeding better crops has been possible with the help of genetic engineering, but compared to the age-old technique of traditional plant breeding, this relatively “new” approach can be considered an unfamiliar concept. Unfortunately, “unfamiliar” is not a word most people would like to associate with their food.

Despite the benefits of genetically modified crops, from reduced need for pesticides to enhanced nutrient composition and food quality, many consumers are reluctant to purchase products with “GMO” food labels. Due to various controversies surrounding the effects and safety of genetically modified foods, many have steered away from these “artificial” plants.

Noticing the slow acceptance of genetically modified foods, geneticists at the University of Tokyo have proposed a new strategy of genetically engineering crops that work around the technicalities of “GMO.” Rather than modifying and permanently altering the nuclear DNA of plants, which would affect future generations of crops, they have chosen to edit the DNA of plant chloroplasts and successfully produced the same results without leaving any of the genetic engineering technology behind to be inherited by future seeds. Moreover, this new technology can also help to grow new and more robust crops.

Previously, scientists have attempted to make genetic modifications on chloroplast genomes. However, to produce any noticeable effect on the plant’s offspring in a way that does not alter the nuclear DNA, scientists needed to perform the same genetic modifications in every copy of the chloroplast DNA – a tedious and laborious effort. This is because every plant cell can contain multiple chloroplasts, each with its own circular DNA that is maintained separately from the nuclear DNA. Furthermore, early techniques could only insert new DNA fragments into chloroplasts with the addition of extra genetic tags or markers. In other words, the experiment would leave genetic footprints behind.

“Now we’ve got a way to modify chloroplast genes specifically and measure their potential to make a good plant,” said Associate Professor Shin-ichi Arimura, who led the group that performed the research.

In their proof-of-concept work, Arimura and colleagues have successfully improved upon past techniques and made seamless genome editing possible. The objective of their study was to create uniform, inheritable modifications to only specific parts of the chloroplast DNA without making any permanent alterations to the nuclear DNA or leave genome editing tools behind.

Their journey began with an existing gene toolkit called TALENs. The early versions of TALENs simply comprised of a DNA-recognising and -cutting protein. However, in the last few years, TALENs has been upgraded to become a highly versatile technology with customisable DNA recognition sequences and an enzyme that replaces GC pairs in the DNA code to AT pairs. This new enzyme is significantly more precise compared to the previous DNA cutting enzyme that could only cut or insert whole genes.

Arimura’s team harnessed the powers of the improved TALEN and incorporated an extra “chloroplast-targeting” component. This combination became the team’s finalised version called ptpTALECDs. However, to use ptpTALECDs, the researchers would need to build a matching left and right pair of ptpTALECDs in the bacteria for every genome edit to be made. This process is incredibly complicated because the pairs of large TALENs proteins and the chloroplast-targeting signals must be expressed simultaneously as a single unit from the nuclear DNA.

“Building the ptpTALECDs was an extremely laborious process, but we have a very dedicated master’s degree student who did almost all the work, Issei Nakazato,” said Arimura. Nakazato is the first author of the research publication.

After designing the ptpTALECDs DNA sequence, the researchers then inserted the DNA into model plants Arabidopsis thaliana. Upon entering the plants’ nuclei, the plant cells can begin to produce ptpTALECDs in the same way other proteins are produced. The chloroplast-targeting sequence would ensure that the translated ptpTALECDs proteins are shuttled out of the nucleus and into the chloroplast, where they are expected to edit every chloroplast genome they encounter.

All first-generation plants subjected to ptpTALECDs are considered genetically modified organisms since their nuclear DNA is permanently altered to contain the ptpTALECD sequence and produce the ptpTALECDs protein. However, some second-generation plants grown by reproducing these genetically modified plants through self-fertilisation or with non-modified plants would not be considered genetically modified organisms. This is because some of these second-generation seeds would not inherit the ptpTALECD sequence, and only inherit the modified chloroplasts from the ovules. In such a case, they would not be considered genetically modified organisms since their nuclear DNA contains none of the ptpTALECDs’ genetic engineering machinery.

“Chloroplast DNA encodes less than one per cent of the total genetic material in a plant, but it has a very important effect on photosynthesis, and therefore the health of the plant. Hopefully, this method will be useful in fundamental research and applied agriculture,” explained Arimura.

With their highly versatile ptpTALECDs, the team is optimistic that various crop species can now be altered in a straightforward manner. To date, Arimura’s team has successfully demonstrated the system’s power by editing three chloroplast genes and examining the expected outcomes in the offspring plants. In the near future, their novel method is expected to help breed better crops that are more easily accepted by both farmers and consumers. [APBN]

Source: Nakazato et al. (2021). Targeted base editing in the plastid genome of Arabidopsis thaliana. Nature Plants, 7, 906-913.