Design and Preliminary Evaluation of sgRNA for Targeted Mutation in the β-(1,2)-Xylosyltransferase (XylT) Gene of Lemna minor Toward Engineering the N-Glycosylation Pathway

Shojaei Baghini, Sadegh; Esfahani, Kasra; Arezoumandi, Mahdi; Taghipour, Elham; Salmanian, Ali Hatef

doi:10.22034/jpr.2026.8660.3421

Design and Preliminary Evaluation of sgRNA for Targeted Mutation in the β-(1,2)-Xylosyltransferase (XylT) Gene of Lemna minor Toward Engineering the N-Glycosylation Pathway

Articles in Press

Document Type : Research Paper

Authors

Sadegh Shojaei Baghini ¹

Kasra Esfahani ¹

Mahdi Arezoumandi ¹

Elham Taghipour ¹

Ali Hatef Salmanian ²

¹ National Institute of Genetic Engineering and Biotechnology (NIGEB), Department of Agricultural Biotechnology, Plant Bioproducts Department, Tehran, Iran.

² Faculty member/ NIGEB

10.22034/jpr.2026.8660.3421

Abstract

Duckweed (Lemna minor L.) has emerged as an efficient platform for recombinant protein production due to its advantageous characteristics, including rapid growth, a biomass doubling time of approximately 1–2 days, and high protein content. However, the presence of non-human sugars such as α-(1,3)-fucose and β-(1,2)-xylose in the structure of N-glycans presents a major challenge for the therapeutic application of these proteins. In this study, with the aim of humanizing the glycosylation pattern in L. minor, the CRISPR/Cas9 genome editing system was used to introduce targeted mutations in the β-(1,2)-xylosyltransferase (XylT) gene. The regeneration of transgenic plants under hygromycin selection resulted in 30 independent transgenic lines. Sequence analysis of 10 randomly selected lines confirmed mutations in the XylT gene. The results showed that only 2 lines (20%) carried heterozygous mutations (mutation in a single allele), while 8 out of 10 lines (80%) displayed a chimeric pattern. These findings demonstrate the potential of CRISPR/Cas9 for targeted editing of the XylT gene in L. minor. As the first report of applying this technology in native Iranian duckweed, this study highlights the feasibility of manipulating genes involved in the N-glycosylation pathway in this plant system. However, the high frequency of chimerism observed in transgenic lines underscores a key limitation and emphasizes the need for further optimization of genome editing conditions in duckweed.

Keywords

CRISPR/Cas9

Lemna minor

N-glycosylation

β-(1

2)-xylosyltransferase

Subjects