Abstract
BackgroundAs 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.ResultsIn 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.ConclusionsGenome 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.
Highlights
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
WPT608 included two guide RNA (gRNA) targeting gene model Glyma.16G209100. One of these gRNAs had a perfect match to the target site on Glyma.16G209100 and nearly a perfect match to its paralog gene model Glyma.09G159900
The other gRNA for gene model Glyma.16G209100 failed to result in mutations and is not further discussed below
Summary
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. 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, is not always consistent with observed results. Reagent platforms include zinc-finger nucleases and TAL effector nucleases, which can each be engineered as proteins that recognize and create DSBs at specific DNA sequences. These platforms have been used to modify genes in numerous different organisms, including plant species [1,2,3,4,5,6].
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