This paper examines where exactly CRISPR/Cas constructs are integrated into the genome of soy plants and how these, together with the intended changes resulting from their mode of action, are passed on to the following generation(s).
Interestingly, it is not only about whether and how the CRISPR gene scissors cut in the genome, but also where in the genome the DNA encoding the CRISPR gene scissors (here defined as the CRISPR/Cas construct) is integrated. The CRISPR/Cas construct is firstly incorporated into the genome of the plants so that the gene scissors and the guide RNA (gRNA), i.e. the guide of the gene scissors, can be formed at all. The CRISPR/Cas construct is introduced into the genome of soybean plants using the root bacterium Agrobacterium tumefaciens. This so-called Agrobacterium transformation is a common work step in the laboratory for introducing synthetically produced DNA constructs into plant cells and is one of the older genetic engineering methods. Using whole genome sequencing methods, the authors investigated the exact regions where the CRISPR/Cas construct was integrated. The regions in the genome where the construct was integrated are partly similar to one another in their DNA sequences, which is also known as microhomology.
It appears that the integration of the CRISPR/Cas construct is not completely arbitrary. In addition, there are several ways in which the CRISPR/Cas construct is integrated in the genome: It can be inserted once, several times, at the actual target sequence or at other locations in the genome. The CRISPR/Cas construct can then be removed from the genome of the plants by subsequent backcrossings.
According to the results of this paper, the integration of the CRISPR/Cas construct cannot be predicted and can occur in different copy numbers. In addition, the findings of the paper show that it is important to sequence the entire genome of the genome-edited plants (by whole-genome sequencing) in order to trace the inheritance of the CRISPR/Cas constructs. Outcrossing of multiple integrations of the CRISPR/Cas constructs can otherwise be incomplete.