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

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.

CRISPR/Cas9-Based Gene Editing Using Egg Cell-Specific Promoters in Arabidopsis and Soybean

“…Nevertheless, CRISPR/Cas9 genome editing systems have been successfully utilized in soybean since the first demonstrations in 2015. It has been applied to create targeted mutations in hairy roots, somatic embryos and stable transgenic plants (Jacobs et al, 2015;Li et al, 2015Li et al, , 2019Michno et al, 2015;Sun et al, 2015;Du et al, 2016;Chilcoat et al, 2017;Cai et al, 2018Cai et al, , 2020Kanazashi et al, 2018;Al Amin et al, 2019;Bao et al, 2019;Campbell et al, 2019;Cheng et al, 2019;Do et al, 2019;Zhang et al, 2019;Michno et al, 2020;Wang et al, 2020;Wu et al, 2020). Various promoters have been deployed for expression of Cas9.…”

Similar Seed Composition Phenotypes Are Observed From CRISPR-Generated In-Frame and Knockout Alleles of a Soybean KASI Ortholog

“…Trimmomatic version 0.33 ( Bolger et al., 2014 ) was used for adapter removal, keeping a minimum read length of 40 bp and quality cutoff set to a phred score of 20. Read mapping to the soybean genome was conducted using bwa version 0.7.17 ( Li and Durbin, 2010 ) following the same parameters as Michno et al. (2020) .…”

Broadening the GMO risk assessment in the EU for genome editing technologies in agriculture

“…For example, in one study, the DNA template encoding CRISPR/Cas9 was not only detected at the target location in soybeans as intended, but also at other multiple, apparently random, genomic locations [135]. In another study, CRISPR/Cas sequences were found at multiple genomic sites showing microhomology at the transgene integration sites indicating that the integration of CRISPR/Cas sequences might not be completely random [136].…”