Genomic and biochemical comparison of allelic triple-mutant lines derived from conventional breeding and multiplex gene editing

Authors: LIU, JUNQI - UNIVERSITY OF MINNESOTA; KUMAR, RITESH - UNIVERSITY OF MINNESOTA; GUNAPATI, SAMATHA - UNIVERSITY OF MINNESOTA; MULKEY, STEVEN - UNIVERSITY OF MINNESOTA; QIU, YINJIE - UNIVERSITY OF MINNESOTA; XIONG, YER - UNIVERSITY OF MINNESOTA; RAMASUBRAMANIAN, VISHNU - UNIVERSITY OF MINNESOTA; MICHNO, JEAN-MICHEL - UNIVERSITY OF MINNESOTA; AWASTHI, PRAVEEN - UNIVERSITY OF MINNESOTA; GALLAHER, DANIEL - UNIVERSITY OF MINNESOTA; NGUYEN, THI THAO - UNIVERSITY OF MISSOURI; KIM, WON-SEOK - UNIVERSITY OF MISSOURI; KRISHNAN, HARI; LORENZ, AARON - UNIVERSITY OF MINNESOTA; STUPAR, ROBERT - UNIVERSITY OF MINNESOTA

Submitted to: The Plant Genome
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/8/2025
Publication Date: 6/5/2025
Citation: Liu, J., Kumar, R., Gunapati, S., Mulkey, S., Qiu, Y., Xiong, Y., Ramasubramanian, V., Michno, J., Awasthi, P., Gallaher, D.D., Nguyen, T., Kim, W., Krishnan, H.B., Lorenz, A.J., Stupar, R.M. 2025. Genomic and biochemical comparison of allelic triple-mutant lines derived from conventional breeding and multiplex gene editing. The Plant Genome. 18(2). https://doi.org/10.1002/tpg2.70056.
DOI: https://doi.org/10.1002/tpg2.70056

Interpretive Summary: There are ongoing debates about how plant products developed using gene editing technologies (e.g., CRISPR) should be regulated. To address this issue, it is important to determine if plant products developed by conventional and gene editing technologies are functionally equivalent. In this study, we conducted an extensive biochemical and genetic characterization of triple-mutant plants developed using a gene editing method and compared them to those developed using a conventional backcross breeding strategy. Comparisons of genomic, biochemical, and field performance data indicated that the gene-edited triple mutants are functionally equivalent to the conventionally bred triple mutants, posing no greater risk to producers or consumers. The results of our study should assist regulatory agencies in approving the use of gene-edited plant products, allowing them to be treated as equivalents to traditional crops.

Technical Abstract: Multiplex gene editing allows for the simultaneous targeting and mutagenesis of multiple loci in a genome. This tool is particularly valuable for plant genetic improvement, as plant genomes often require mutations at multiple loci to confer useful and/or novel traits. However, the regulation of gene editing can vary depending on the number of loci targeted. In this study, we developed triple-mutant soybean (Glycine max (L.) Merrill) lines using different crop improvement strategies, including conventional backcross breeding of standing variant alleles and clustered regularly interspaced short palindromic repeats-based multiplex editing to introduce new alleles. The mutations were targeted to genes encoding seed antinutritional components, as previously described in a triple null soybean carrying knockout alleles for a Kunitz trypsin inhibitor, a soybean agglutinin, and the allergen P34 protein. The products developed from these respective genetic improvement pipelines were tested for differences between the triple-mutant lines and their parental lines. Analyses included genomics, seed proteomics, trypsin inhibition, seed protein digestibility, and harvestable yield of the different lines. We observed that both multiplex gene editing and conventional breeding approaches produced essentially equivalent products in comparison to their parental lines. We conclude that the multiplex gene editing strategy is not inherently riskier than conventional breeding for developing complex mutant lines of this type.