Characterization of Substitution Mutations of eIF4G Gene Generated through Adenine Base Editors in Rice

Yaiphabi Kumam,Raveendran M,E Kokiladevi ,L Arul ,Rajadurai Chinnasamy Perumal ,S Varanavasiappan ,Chandrasekhar Gopalakrishnan ,K K Kumar ,S Manonmani ,D Sudhakar

doi:10.29321/maj.10.000544

Abstract

Adenine base editor (ABE) creates A to G transitions within its editing window. In the present study, an ABE was used to target a stretch of six amino acid residues, VLFPNL in translation initiation factor four gamma (eIF4G) gene of rice. Agrobacterium-mediated transformation of rice cultivar ASD16 resulted in T0 events with high mutation efficiency of 89.29 %. Substitution mutations of A > G occurred within the editing window of four to eight bases at A7 > G7 (74.67 %) and A4 > G4 (2.46 %). Non-canonical substitutions of G > C/A was also observed at G15 > C15 (9.29 %) and G8 > A8 (1.15 %). A total of 15 missense base substitution events affecting the target residue was identified. Taken together, the present study showed that ABEs create unexpected base substitutions besides efficient canonical editing of A > G in the rice genome

Highlights

Genome editing by CRISPR/Cas technology has never ceased to evolve, making it the most attractive tool of the 21st century
Molecular analysis by PCR for the presence of cas9 and hpt genes in the 112 independent events confirmed that all the mutants were positive for these genes (Fig. 2a and 2b)
Earlier reports by Macovei et al (2018) suggested that mutations affecting the above stretch of 11 amino acid residues can impart resistance against tungro disease

Summary

Introduction

Genome editing by CRISPR/Cas technology has never ceased to evolve, making it the most attractive tool of the 21st century. Base-editing technology, comprising of cytosine base editors (CBEs) and adenine base editors (ABEs) have refined CRISPR/Cas by performing irreversible substitutions of C٠G to T٠A (Komor et al, 2013) and A٠T to G٠C (Gaudelli et al, 2017) base pairs respectively These substitutions take place exclusively within the editing window and are highly precise in targeting human cells (Komor et al, 2013), mouse cells (Gaudelli et al, 2017), bacterial cells (Zheng et al, 2018) and crop species (Qin et al, 2020; Veillet et al, 2020; Wang et al, 2020: Wu et al, 2020)

Methods

Results

Conclusion