Large Scale Deletion and Rebalancing within the k1C Kafirin Family in Sorghum

Authors

Tyler W. Ferris, University of Nebraska-LincolnFollow

First Advisor

David R. Holding

Committee Members

Scott Sattler, Devin Rose, Tom Clemente, James Schnable

Date of this Version

8-2025

Document Type

Thesis

Citation

A thesis presented to the faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree of Master of Science

Major: Agronomy

Under the supervision of Professor David R. Holding

Lincoln, Nebraska, August 2025

Comments

Copyright 2025, Tyler W. Ferris. Used by permission

Abstract

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 because of the abundance of proline-rich kafirins that constitute >70% of proteins that 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 (SbiRTX430.05G198600, SbiRTX430.05G198800, SbiRTX430.05G202500, SbiRTX430.05G202600, SbiRTX430.05G202700, SbiRTX430.05G202800, and SbiRTX430.05G203000) we observed no significant decrease in k1C expression or compensatory increase in non-kafirins. 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 seed protein in one line. Protein bodies were observed to be more irregularly shaped with seeds retaining near wild-type levels of vitreous endosperm. RNA IsoSeq was performed to assess the expression of the k1C family and capture differentially-expressed non-kafirin genes. We observed that five k1C members had biologically significant expression with only two k1C members not deleted with 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 regions like the k1C subfamily to compensate the effects of CRISPR-induced multigene deletions.

Advisor: David R. Holding