Generation of new ß-conglycinin-deficient soybean lines by editing the lincRNA lincCG1 using the CRISPR/Cas9 system

Authors: SONG, BO - Northeast Agricultural University; LUO, TINGTING - Northeast Agricultural University; FAN, YUANHANG - Northeast Agricultural University; LI, MING - Northeast Agricultural University; QIU, ZHENDONG - Heilongjiang Academy Of Agricultural Sciences; TIAN, YUSU - Northeast Agricultural University; SHANG, YUZHUO - Northeast Agricultural University; MA, CHONGXUAN - Northeast Agricultural University; LIU, CHANG - Northeast Agricultural University; CAO, QINGQIAN - Northeast Agricultural University; PENG, YUHAN - Northeast Agricultural University; XU, PENGFEI - Northeast Agricultural University; Krishnan, Hari; WANG, ZHENHUI - Jilin Agricultural University; ZHANG, SHUZHEN - Northeast Agricultural University; LIU, SHANSHAN - Northeast Agricultural University

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/14/2024
Publication Date: 7/3/2024
Citation: Song, B., Luo, T., Fan, Y., Li, M., Qiu, Z., Tian, Y., Shang, Y., Ma, C., Liu, C., Cao, Q., Peng, Y., Xu, P., Krishnan, H.B., Wang, Z., Zhang, S., Liu, S. 2024. Generation of new ß-conglycinin-deficient soybean lines by editing the lincRNA lincCG1 using the CRISPR/Cas9 system. Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acs.jafc.4c02269.
DOI: https://doi.org/10.1021/acs.jafc.4c02269

Interpretive Summary: One of the major deterrents to using soybean as food and feed is the presence of the anti-nutritional factors. Among them, beta-conglycinin an abundant seed protein has been identified as a potential allergen. Producing hypoallergenic soybean or soy food products with minimal amounts of allergens is an effective approach to managing food allergies and increasing soy food safety. Long non-coding RNAs (lncRNAs) are a group of RNA molecules that do not encode proteins; they regulate gene expression via diverse mechanisms. In this study, we have cloned the full-length lincCG1 sequence and edited it by using the CRISPR/Cas9 system. Loss-of-function lincCG1 mutants led to lack of the allergic beta-conglycinin, higher content of protein, sulfur-containing amino acids, and free arginine. Our study demonstrates that allergen-free soybean lines can be created by editing lincCG1 sequences utilizing the CRISPR/Cas9 system. The results of our study will enhance the profitability of soybean farmers and promote the wider use of allergen-free soybeans in food and feed industry.

Technical Abstract: Soybean ß-conglycinin is a major allergen that adversely affects the nutritional properties of soybean. Soybean deficient in ß-conglycinin is associated with low allergenicity and high nutritional value. Long intergenic noncoding RNAs (lincRNAs) regulate gene expression and are considered important regulators of essential biological processes. Despite increasing knowledge of the functions of lincRNAs, relatively little is known about the effects of lincRNAs on the accumulation of soybean ß-conglycinin. The current study presents the identification of a lincRNA lincCG1 that was mapped to the intergenic noncoding region of the ß-conglycinin a-subunit locus. The full-length lincCG1 sequence was cloned and found to regulate the expression of soybean seed storage protein (SSP) genes via both cis- and trans-acting regulatory mechanisms. Loss-of-function lincCG1 mutations generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system led to the deficiency of the allergenic a'-, a-, and ß-subunits of soybean ß-conglycinin as well as higher content of proteins, sulfur-containing amino acids, and free arginine. The dominant null allele LincCG1, and consequently, the ß-conglycinin-deficient phenotype associated with the lincCG1-gene-edited line was stably inherited by the progenies in a Mendelian fashion. The dominant null allele LincCG1 may therefore be exploited for engineering/developing novel hypoallergenic soybean varieties. Furthermore, Cas9-free and ß-conglycinin-deficient homozygous mutant lines were obtained in the T1 generation. This study is the first to employ the CRISPR/Cas9 technology for editing a lincRNA gene associated with the soybean allergenic protein ß-conglycinin. Moreover, this study reveals that lincCG1 plays a crucial role in regulating the expression of the ß-conglycinin subunit gene cluster, besides highlighting the efficiency of employing the CRISPR/Cas9 system for modulating lincRNAs, and thereby regulating soybean seed components.