A trait that is often changed in rice so resulting rice plants have a smaller size and a higher yield is caused by changes in the so-called semi-dwarf 1 (SD1) gene. This gene is modified in various rice elite lines in the present study with CRISPR/Cas9 and examined in detail in subsequent generations for unintended changes in the genome. This shows that there are unintentional changes in the genome (i.e. in addition to the intended changes to the SD1 gene) in the various elite lines after application of CRISPR/Cas9:
- Major rearrangements such as deletions or insertions of the genome occur in the vicinity of the target sequence. Such unintended on-target effects that have already been observed in human cells and have not yet been described in plants (e.g. original publications: Adikusuma F, Piltz S, Corbett MA, Turvey M, McColl SR, Helbig KJ, Beard MR, Hughes J, Pomerantz RT, Thomas PQ (2018) Large deletions induced by Cas9 cleavage. Nature 560 (7717): E8-E9. doi: 10.1038 / s41586-018-0380-z and Kosicki M, Tomberg K, Bradley A (2018) Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 36 (8): 765-771. doi: 10.1038 / nbt.4192)
- In addition, so-called off-target effects were found in some genome-edited lines, in which the CRISPR/Cas9 gene scissors caused unintended changes in the genome. However, such off-target effects were analyzed in only 5 areas of the genome that are very similar to the actual target sequence. Comment: A genome-wide analysis of the genome using whole genome sequencing methods could reveal further off-target effects in genomic regions that are less similar to the target sequence.
However, it cannot be conclusively determined whether the off-target effects found in the study were clearly caused by the gene scissors or were triggered during the work in the cell culture. - The components of the CRISPR/Cas9 system were introduced into the cells with the help of Agrobacterium tumefaciens so that the DNA encoding the gene scissors can be incorporated into the genome of the rice plants. Parts of the vector plasmid were also incorporated into the genome of the rice plants and were detectable in the second generation after the application of genome editing. This result confirms the authors in their statement that early molecular investigations should be used to identify such additionally integrated DNA elements in order to cross them out in subsequent generations.
Additionally to the investigation of the unwanted changes in the genome of the genome-edited rice plants, various SD-1 alleles (alleles are different variants of a gene) that resulted from genome editing were investigated. It was shown that in only one genome-edited elite line, the changes of the SD1 gene led to both the size of the rice plants being reduced and the yield being increased, thus containing the desired traits. In this line, neither the DNA encoding the gene scissors nor other additional DNA sequences were present in the genome. The study shows that unwanted changes occur frequently in rice plants and can be detected at an early stage by means of molecular studies.