Barley stripe mosaic virus-mediated somatic and heritable gene editing in barley (Hordeum vulgare L.)

Abstract

Genome editing strategies in barley (Hordeum vulgare L.) typically rely on Agrobacterium-mediated genetic transformation for the delivery of required genetic reagents involving tissue culture techniques. These approaches are genotype-dependent, time-consuming, and labor-intensive, which hampers rapid genome editing in barley. More recently, plant RNA viruses have been engineered to transiently express short guide RNAs facilitating CRISPR/Cas9-based targeted genome editing in plants that constitutively express Cas9. Here, we explored virus-induced genome editing (VIGE) based on barley stripe mosaic virus (BSMV) in Cas9-transgenic barley. Somatic and heritable editing in the ALBOSTRIANS gene (CMF7) resulting in albino/variegated chloroplast-defective barley mutants is shown. In addition, somatic editing in meiosis-related candidate genes in barley encoding ASY1 (an axis-localized HORMA domain protein), MUS81 (a DNA structure-selective endonuclease), and ZYP1 (a transverse filament protein of the synaptonemal complex) was achieved. Hence, the presented VIGE approach using BSMV enables rapid somatic and also heritable targeted gene editing in barley.

A comprehensive review of in planta stable transformation strategies

“…agroinfiltration, agroinjection, agrospray, agrodrench, and rub inoculation) [235,236]; however, heritable mutations are challenging to generate due to the seclusion of viruses from the meristematic cells of the shoot apical meristem but have been reported on rare occasions (e.g. Tobacco rattle virus [234] and Barley stripe mosaic virus [237]). To obtain a greater efficiency at generating heritable genome editing events, Ellison et al [238] fused gRNA sequences to mobile FLOWERING LOCUS T (FT) sequences and cloned them into a Tobacco rattle virus vector (Fig.…”