Abstract
Plant genomes have undergone multiple rounds of whole-genome duplication (WGD) throughout their evolutionary history. As a result, many species, including Arabidopsis thaliana, retain duplicated genomic segments, or syntenic regions, which harbor large numbers of paralogous genes preserved from these ancient WGD events. We deleted four large, duplicated blocks, ranging from ~115 kb to ~684 kb using Staphylococcus aureus Cas9 to explore the effects of knocking out these blocks in Arabidopsis. Large deletions like these remain rare, especially in small and gene-dense plant genomes. Deletions were subsequently verified using whole-genome sequencing, which revealed minimal off-target effects. The number of deleted genes ranged from 16 to 60, and transposable elements ranged from 4 to 112 among the four deleted blocks. Two deletion lines showed distinct phenotypes resulting from the loss of many genes, while two others displayed no obvious defects, including for flowering time or hypocotyl elongation. Moreover, RNA-sequencing revealed that expression compensation, where deletions of paralogous genes lead to the upregulation of intact paralogues, was not a general response to the deleted regions under the conditions tested. Thus, it is possible to obtain viable plants when deleting large fragments that may be redundant or that contain nonessential genes. These results demonstrate that large chromosomal deletions can be used as a tool for genome engineering approaches, such as genome minimization in plants and allele replacement using homology-directed repair and other precision editing methods. Targeted deletions of large chromosome fragments will be a valuable tool for research and biotechnology applications.