DeMell April, Mendoza Maria R, Scholthof Herman B
Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA.
Present address: Plant Biology, UC Davis, Davis, CA, USA.
PNAS Nexus. 2023 Dec 14;3(1):pgad436. doi: 10.1093/pnasnexus/pgad436. eCollection 2024 Jan.
A tomato bushy stunt virus (TBSV)-derived vector system was applied for the delivery of CRISPR/Cas9 gene editing materials, to facilitate rapid, transient assays of host-virus interactions involved in the RNA silencing pathway. Toward this, single guide RNAs designed to target key components of the virus-induced host RNA silencing pathway (, , ) were inserted into TBSV-based GFP-expressing viral vectors TBSV-GFP (TG) and its P19 defective mutant TGΔP19. This produced rapid, efficient, and specific gene editing in planta. Targeting , , or partially rescued the lack of GFP accumulation otherwise associated with TGΔP19. Since the rescue phenotypes are normally only observed in the presence of the P19 silencing suppressor, the results support that the DCL2, HEN1, and AGO2 proteins are involved in anti-TBSV RNA silencing. Additionally, we show that knockdown of the RNA silencing machinery increases cargo expression from a nonviral binary Cas9 vector. The TBSV-based gene editing technology described in this study can be adapted for transient heterologous expression, rapid gene function screens, and molecular interaction studies in many plant species considering the wide host range of TBSV. In summary, we demonstrate that a plant virus can be used to establish gene editing while simultaneously serving as an accumulation sensor for successful targeting of its homologous antiviral silencing machinery components.
一种源自番茄丛矮病毒（TBSV）的载体系统被用于递送CRISPR/Cas9基因编辑材料，以促进对RNA沉默途径中宿主-病毒相互作用的快速、瞬时检测。为此，将设计用于靶向病毒诱导的宿主RNA沉默途径关键组分（ 、 、 ）的单向导RNA插入基于TBSV的表达绿色荧光蛋白（GFP）的病毒载体TBSV-GFP（TG）及其P19缺陷型突变体TGΔP19中。这在植物中实现了快速、高效且特异性的基因编辑。靶向 、 或 部分挽救了否则与TGΔP19相关的GFP积累缺失。由于通常仅在存在P19沉默抑制子时才观察到挽救表型，结果支持DCL2、HEN1和AGO2蛋白参与抗TBSV RNA沉默。此外，我们表明RNA沉默机制的敲低增加了来自非病毒二元Cas9载体的货物表达。考虑到TBSV广泛的宿主范围，本研究中描述的基于TBSV的基因编辑技术可适用于许多植物物种的瞬时异源表达、快速基因功能筛选和分子相互作用研究。总之，我们证明一种植物病毒可用于建立基因编辑，同时作为其同源抗病毒沉默机制组分成功靶向的积累传感器。
