Integration, abundance, and transmission of mutations and transgenes in a series of CRISPR/Cas9 soybean lines

Jean-Michel Michno, Kamaldeep Virdi, Adrian O. Stec,  Junqi Liu, Xiaobo Wang, Yer Xiong & Robert M. Stupar

BMC Biotechnology volume 20, Article number: 10 (2020)

Abstract

Background

As with many plant species, current genome editing strategies in soybean are initiated by stably transforming a gene that encodes an engineered nuclease into the genome. Expression of the transgene results in a double-stranded break and repair at the targeted locus, oftentimes resulting in mutation(s) at the intended site. As soybean is a self-pollinating species with 20 chromosome pairs, the transgene(s) in the T0 plant are generally expected to be unlinked to the targeted mutation(s), and the transgene(s)/mutation(s) should independently assort into the T1 generation, resulting in Mendellian combinations of transgene presence/absence and allelic states within the segregating family. This prediction, however, is not always consistent with observed results.

Results

In this study, we investigated inheritance patterns among three different CRISPR/Cas9 transgenes and their respective induced mutations in segregating soybean families. Next-generation resequencing of four T0 plants and four T1 progeny plants, followed by broader assessments of the segregating families, revealed both expected and unexpected patterns of inheritance among the different lineages. These unexpected patterns included: (1) A family in which T0 transgenes and mutations were not transmitted to progeny; (2) A family with four unlinked transgene insertions, including two respectively located at paralogous CRISPR target break sites; (3) A family in which mutations were observed and transmitted, but without evidence of transgene integration nor transmission.

Conclusions

Genome resequencing provides high-resolution of transgene integration structures and gene editing events. Segregation patterns of these events can be complicated by several potential mechanisms. This includes, but is not limited to, plant chimeras, multiple unlinked transgene integrations, editing of intended and paralogous targets, linkage between the transgene integration and target site, and transient expression of the editing reagents without transgene integration into the host genome.

See https://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-020-00604-3

Figure 1: Transformation vectors used in whole-plant transformations. Plant expression cassettes were integrated into the host soybean genome through Agrobacterium-based whole-plant transformation methods using destination vectors listed on the right hand side. Promoters are blue, Cas9 endonucleases are orange, plant-selectable markers are green, and guide RNAs are pink. Different shading within each color group indicates different variants for each sequence class (e.g., the GmUbi and 35S promoters are different shades of blue)