Bamboo mosaic virus-mediated transgene-free genome editing in bamboo

Abstract

Introduction

The common method of delivering CRISPR/Cas reagents for genome editing in plants involves Agrobacterium-mediated transformation or preassembled CRISPR/Cas9 ribonucleoprotein complex delivery (Woo et al., 2015; Toda et al., 2019; Ye et al., 2020). These methods require labor-intensive and time-consuming plant tissue culture processes (Huang et al., 2022). Unfortunately, most plants exhibit extremely low efficiency in callus induction and regeneration; these technical challenges greatly hinder the application of genome editing. Recent developments in plant RNA virus-based expression vectors (Ma et al., 2020; Chen et al., 2022) provide a convenient, efficient, and cost-effective way for DNA-free genome editing in plants, leveraging the fact that virus RNA does not integrate into the genome. However, the stability of virus vectors is negatively correlated with the size of the inserted foreign genes. Consequently, achieving efficient expression of Streptococcus pyogenes Cas9 (SpCas9, c. 4.2 kb) by virus-based vectors remains challenging. Most reported viruses capable of delivering Cas9 proteins are negative-strand RNA viruses (Ma et al., 2020; Liu et al., 2023; Zhao et al., 2024), with only a few positive-strand RNA viruses identified (Uranga et al., 2021; Lee et al., 2024). Thus, delivering virus-based sgRNA vectors to plants overexpressing Cas9 is the most commonly used strategy (Ali et al., 2015; Jiang et al., 2019; Li et al., 2021). However, it is difficult to use this method to generate a Cas9-free mutant by crossing with wild-type (WT) plants with long flowering cycles, such as bamboo (Ye et al., 2017). Bamboo mosaic virus (BaMV) has a typical flexible filamentous virion structure with the positive-sense single-stranded RNA genome (Hsu et al., 2018). The BaMV-mediated expression system can effectively drive the expression of large foreign gene fragments (Jin et al., 2023). For the first time, we developed a BAMV-mediated Cas protein and sgRNA delivery system in WT Nicotiana benthamiana and bamboo. This approach enables targeted gene editing in noninfected leaves or stems in bamboo without the need for Cas9-expressing transgenic lines, leveraging BaMV's large cargo ability to transport Cas9 proteins.