Large-scale genome modifications in Arabidopsis thaliana using TALEN and CRISPR/Cas9

Abstract

Due to the world becoming over populated, it is becoming increasingly difficult to supply food for everyone. The only believed solution to this issue is to find ways to make plants more productive so that less land will have to be used to grow more food in the future. Conventional breeding is too time consuming too keep up with a fast populating world and the amount of land that would be required to grow enough food is excessive. Luckily, plant research has been advancing rapidly and now we have a modern breeding technique called "Genome editing" with sequence-specific nucleases (SSNs). Genome editing already allows scientists to edit plant genomes for research and molecular breeding. Unfortunately, with all the tools provided for genome editing the ability to make large-scale genome modifications has been limited in plants. There have been attempts of large genome modifications in plants with some more successful than others. However, for the plant model, Arabidopsis thaliana, there have not been any reports of site-specific large T-DNA insertions using TALEN and few reports of Large chromosomal deletions using a CRISPR/Cas9 system. This study demonstrated the potential of a proof-of-concept for large T-DNA insertions mediated by TALEN and large chromosomal deletions mediated by CRISPR/Cas9 in a site-specific manner. A. thaliana wild type plants were transformed with genome editing T-DNA constructs and their seeds were screened using either hygromycin antibiotic or BASTA herbicide treatments. The surviving plants were genotyped through PCR and gel electrophoresis to analyze their genome modifications. The results suggest that there are candidate plants with successful large-scale genome editing in both projects. Results in the large chromosomal deletion project suggest that there are a small number of cells containing successful deletion that can produce enough BAR protein to provide the entire plant herbicide resistance. Identifying these candidate plants will help future projects to better understand SSN mutational inheritance to progeny and the probability of obtaining fully mutant plants.