Engineering Mobile RNA Systems for Plant Genome Editing Applications

CRISPR-based genome-editing technologies have emerged as precise and efficient methods for crop improvement. However, current genome editing approaches are often limited by their reliance on tissue culture and stable transgene expression of CRISPR/Cas, restricting scalability and regulatory flexibility. Mobile RNAs capable of moving into the shoot apical meristem offer a promising solution for tissue culture-free and transgene-free genome editing. This can be achieved, for example, by grafting wild-type shoots onto broadly compatible transgenic donor rootstocks expressing mobile CRISPR/Cas components generated via in planta transformation, or by circumventing viral exclusion from meristems to enable heritable genome editing via RNA virus–mediated delivery. To engineer and evaluate mobile RNA systems, transgenic rootstocks expressing single-guide RNAs (sgRNAs) were generated via isopentenyl transferase (ipt)- mediated in planta transformation. Four mobile RNA binary constructs were designed, including sgRNAs fused to tRNAMetΔDT or FLOWERING LOCUS T (FT), as well as sgRNAs tethered to the mobile proteins FT or LeT6. Level 1 modules were assembled using Gibson and Golden Gate methods and subsequently combined into transferred DNA (T-DNA) backbone to generate Level 2 mobile sgRNA binary vectors. These constructs, along with a control lacking a mobility motif, were transformed into Agrobacterium tumefaciens GV3101. Transient expression assays of all vectors in Nicotiana benthamiana leaves confirmed the successful expression of the AmCyan reporter. De novo transgenic chimeric tomato shoots exhibiting AmCyan signal were subsequently generated via ipt-mediated in planta transformation. The chimeric shoots and shoots exhibiting uniform reporter expressions potentially generated from ongoing experiments will serve as rootstocks for grafting with Cas9-expressing scions. These experiments will enable the assessment of sgRNA mobility via reverse transcription-polymerase chain reaction (RT-PCR), the evaluation of target-editing efficiency, and the determination of the heritable transmission of edits in progeny. Collectively, this work establishes a foundation for fully transgene-free and tissue-culture-independent genome-editing systems, advancing both fundamental research and applied crop-improvement strategies.