Targeted insertion of regulatory elements enables translational enhancement in rice

Abstract

In-locus editing of agronomically-important genes to optimize their spatiotemporal expression is becoming an important breeding approach. Compared to intensive studies on mRNA transcription, manipulating protein translation by genome editing has not been well exploited. Here, we found that precise knock-in of a regulating element into the 5’UTR of a target gene could efficiently increase its protein abundance in rice. We firstly screened a translational enhancer (AMVE) from alfalfa mosaic virus using protoplast-based luciferase assays with an 8.5-folds enhancement. Then the chemically modified donor of AMVE was synthesized and targeted inserted into the 5’UTRs of two genes (WRKY71 and SKC1) using CRISPR/Cas9. Following the in-locus AMVE knock-in, we observed up to a 2.8-fold increase in the amount of WRKY71 protein. Notably, editing of SKC1, a sodium transporter, significantly increased salt tolerance in T2 seedlings, indicating the expected regulation of AMVE knock-in. These data demonstrated the feasibility of such in-locus editing to enhance protein expression, providing a new approach to manipulating protein translation for crop breeding.

Untranslated yet indispensable—UTRs act as key regulators in the environmental control of gene expression

“…Other UTR modifications include the editing of splice sites in the WAXY 5' UTR to improve rice grain quality ( Zeng et al , 2020 ) and efforts to implement artificial riboswitches to control gene expression in planta ( Bocobza and Aharoni, 2014 ). The cis -regulatory elements of viruses have also been employed to alter gene expression in plants; the Omega element from tobacco mosaic virus has been widely used to boost translation of transgenes ( Gallie, 2002 ), while recently a targeted knock-in of a translational enhancer from alfalfa mosaic virus has been used to enhance salt tolerance in rice ( Shen et al , 2023 ). The approaches discussed above aimed at constitutive changes in gene expression, and evidence that genetic modification of UTRs can tailor such changes to specific environmental conditions has yet to be obtained.…”

Advanced metabolic engineering strategies for increasing artemisinin yield in Artemisia annua L.

“…Zhou et al have obtained gene knocking-out A. annua lines using the CRISPR/Cas9 system; however, the genome editing efficiency needs to be further improved [ 88 ]. In addition to knocking-out, in-locus editing such as targeted insertion of regulatory elements enables transcriptional and translational enhancement [ 180 , 181 ]. We believe that the application of the recently developed plant biotechnologies would facilitate the germplasm innovation of high-artemisinin producing A. annua plants.…”

STOP1 and STOP1-like proteins, key transcription factors to cope with acid soil syndrome

“…Genome editing is a powerful tool for improving crop varieties. By knocking in cis-elements or high-throughput editing at the STOP1 promoter region ( Shen et al., 2023 ; Tian et al., 2023 ), STOP1 expression can be environmentally induced or constitutively enhanced. The STOP1 protein also can be stabilized by point substitution through base editing of phosphorylation sites ( Tian et al., 2022 ).…”