DNA Free CRISPR/DCAS9 Based Transcriptional Activation System for UGT76G1 Gene in Stevia rebaudiana Bertoni Protoplasts

Abstract

The UDP-glycosyltransferase 76G1 (UGT76G1) is responsible for the conversion of stevioside to rebaudioside A. Four single guide RNAs (sgRNAs) were designed from the UGT76G1 proximal promoter region of stevia by using the online-based tool, benchling. The dCas9 fused with VP64 as a transcriptional activation domain (TAD) was produced and purified for the formation of ribonucleoproteins (RNPs) by mixing with the in vitro transcribed sgRNAs. Protoplast yield was the highest from leaf mesophyll of in vitro grown stevia plantlets (3.16 × 106/g of FW) using ES5 (1.25% cellulase R-10 and 0.75% macerozyme R-10). The RNPs were delivered into the isolated protoplasts through the Polyethylene glycol (PEG)-mediated transfection method. The highest endogenous activation of the UGT76G1 gene was detected at 27.51-fold after 24 h of transfection with RNP30 consisting of CRISPR/dCas9-TAD with sgRNA30 and a similar activation level was obtained using RNP18, RNP33, and RNP34, produced using sgRNA18, sgRNA33, and sgRNA34, respectively. Activation of UGT76G1 by RNP18 led to a significant increase in the expression of the rate-limiting enzyme UGT85C2 by 2.37-fold and there was an increasing trend in the expression of UGT85C2 using RNP30, RNP33, and RNP34. Successful application of CRISPR/dCas9-TAD RNP in activating specific genes can avoid the negative integration effects of introduced DNA in the host genome.

Genome editing based on <i>in vitro</i>-assembled ribonucleoproteins in plants

“…1, PEG-mediated cell transfection) This method serves as a rapid means of assessing the gene editing efficiency of the Cas system. PEG-mediated protoplast gene editing has been successfully adapted to numerous plant species, including Arabidopsis (Yoo et al 2007, Woo et al 2015, rice (Woo et al 2015), apple (Malnoy et al 2016), wheat (Liang et al 2017), soybean (Kim et al 2017, potato (Andersson et al 2018, Carlsen et al 2022, cabbage (Murovec et al 2018), banana (Wu et al 2020), Benʼs tobacco (Wu et al 2023), Stevia rebaudiana (Ghose et al 2022), chili pepper (Kim et al 2020), andmaize (SantʼAna et al 2020).…”