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ORIGINAL RESEARCH article

Front. Plant Sci.

Sec. Plant Biotechnology

Volume 16 - 2025 | doi: 10.3389/fpls.2025.1686027

This article is part of the Research TopicGene Editing for Biofortification: Innovations and ApplicationsView all 4 articles

Large scale deletion and rebalancing within the k1C kafirin family in sorghum

Provisionally accepted
  • 1University of Nebraska-Lincoln, Lincoln, United States
  • 2University of Missouri, Columbia, United States
  • 3Shandon Agricultural University, Shandon, China

The final, formatted version of the article will be published soon.

Sorghum bicolor (L.) Monech (sorghum) is cultivated as food for humans and livestock and is valued for its low input requirements. However, sorghum grain protein is deficient in the essential amino acid lysine and has poor protein digestibility. This is because of the highly abundant proline-rich kafirins that constitute >70% of proteins and form low-digestibility protein bodies. To reduce kafirin expression in the endosperm and elicit wholesale proteome rebalancing to increase non-kafirin proteins, a single-guide RNA (sgRNA) Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) construct was previously used to target members of the highly-repetitive alpha-kafirin family, k1C in RTx430 sorghum. The current study was conceived to evaluate nutritional and biophysical characteristics of two sorghum lines with k1C family deletions compared to unedited RTx430. Despite confirming a ~400 kb deletion in the k1C family of both edited lines encompassing seven active genes, we observed no significant decrease in k1C expression or compensatory increase in non-kafirin proteins. We observed a significant increase in protein-bound amino acids in both k1C-deleted lines and in both edited lines relative to unedited RTx430 which increased total seed protein in one line. Protein bodies were observed to be more irregularly shaped with seeds retaining wild-type levels of vitreous endosperm. RNA IsoSeq was performed to measure k1C family gene expression and capture differentially-expressed non-kafirin genes. We observed that five k1C members had biologically significant expression with only two of the residual, non-deleted k1C genes having elevated expression in lines with the deletion. The results of this study demonstrate the ability of CRISPR/Cas9 editing of kafirins to elicit changes to the amino acid profile of sorghum and increase protein. Moreover, the results demonstrate an extreme example of the ability of highly repetitive genome regions such as the k1C subfamily to compensate the effects of CRISPR-induced multigene deletions.

Keywords: proteome rebalancing, prolamin, kafirin, Protein Bodies, Lysine, seed protein, Grain Quality

Received: 14 Aug 2025; Accepted: 03 Oct 2025.

Copyright: © 2025 Ferris, Hurst, Yobi, Alartouri, Li, Angelovici, Clemente and Holding. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: David Richard Holding, dholding2@unl.edu

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