Generating stable, gene-edited plant lines using CRISPR-Cas9 typically involves a time-consuming process of outcrossing to eliminate CRISPR-Cas9-associated sequences and obtain transgene-free lines. This process is further complicated by the need for plant regeneration and selection steps, especially in species that are difficult to transform or have long generation times. This research aimed to overcome these challenges by developing a system that delivers Cas9 and gRNA transcripts from transgenic roots to wild-type shoots via grafting. The researchers hypothesized that the addition of TLS motifs to Cas9 and gRNA transcripts would facilitate their movement from the transgenic rootstock to the wild-type scion, leading to gene editing in the recipient tissue and the production of transgene-free offspring in a single generation. The successful implementation of such a system would significantly accelerate plant breeding programs and make gene editing accessible to a wider range of plant species.

Previous studies have demonstrated the mobility of certain RNA molecules across graft junctions in plants. The movement of mRNA molecules tagged with TLS motifs has been shown to occur from transgenic rootstocks to wild-type scions, as well as from host plants to parasitic *Cuscuta* plants. This prior work provided the foundation for the current study's hypothesis that similar mechanisms could be used to deliver CRISPR-Cas9 components for gene editing. Other approaches to generating transgene-free edited plants involve transient expression of Cas9/gRNA components using *Agrobacterium*-mediated transformation or plant virus-mediated gene editing. However, these methods often face limitations in accessibility and efficiency, particularly in recalcitrant plant species. The use of ribonucleoprotein (RNP) complexes for direct delivery of Cas9 protein and gRNA is also explored but can be expensive and complex.

The researchers designed gRNAs targeting the *NIA1* gene in *Arabidopsis thaliana*, which is involved in nitrate reduction, allowing for phenotypic identification of mutants. They also designed gRNAs targeting a 35S promoter::H2B-Venus::35S terminator::Basta construct. The Cas9 and gRNA transcripts were fused to two types of TLS motifs, TLS1 (tRNA^Met sequence) and TLS2 (tRNA^Met-ADT sequence). *Arabidopsis* lines expressing Cas9 (with and without TLS motifs) and the gRNAs (with and without TLS motifs) were generated using an estradiol-inducible promoter. Hypocotyl grafting was performed between transgenic rootstocks and wild-type scions. RT-PCR and genomic PCR were used to detect the presence of Cas9 and gRNA transcripts in the scions and to identify gene edits, respectively. The heritability of the edits was assessed by analyzing the progeny of grafted plants. Similar experiments were conducted using *Brassica rapa* as a scion to determine cross-species applicability. The researchers used RT-qPCR to quantify the levels of Cas9 transcripts in various tissues, assessing the efficiency of root-to-shoot transport. Phenotypic screening on ammonium-deficient medium and Sanger sequencing were employed to confirm the presence of homozygous *nial* mutants.

The addition of TLS motifs was crucial for the root-to-shoot movement of Cas9 and gRNA transcripts. Without TLS motifs, the transcripts were not detected in the scions. With TLS motifs, the transcripts were detected in both juvenile and adult plants, across various tissues including flowers and siliques. Gene editing was observed in the scions grafted onto transgenic rootstocks expressing Cas9-TLS and gRNA-TLS, with chlorotic *nial* mutant phenotypes observed in grafted plants grown on NH₄⁺-deficient medium. Genomic PCR and Sanger sequencing confirmed the presence of the expected *NIA1* genomic deletions in the wild-type scions. Notably, the edited *NIA1* gene was heritable, with the progeny of the grafted plants exhibiting the edited genome. The editing frequency was estimated at approximately 5-6 per 1,000 seeds, with a lower frequency of homozygous mutants. These findings were replicated using the artificial 35S promoter::H2B-Venus::35S terminator::Basta construct. The experiment also demonstrated the successful transport of the mobile CRISPR-Cas9 system to *Brassica rapa* scions, resulting in gene edits in this distantly related species, confirming the broad applicability of the system. The success rate of the grafting procedure was high. The observed editing efficiency, particularly of homozygous mutants, was likely underestimated due to limited sample sizes. The study demonstrated that approximately 1 out of 1000 root-produced Cas9-TLS transcripts were detectable in the shoots, suggesting a relatively high level of transfer efficacy. A notable finding was that despite lower transcript levels in siliques compared to flowers, siliques showed more genomic deletions, suggesting that meiocytes might be particularly receptive to the graft-mobile transcripts.

This study provides a significantly improved method for generating heritable, transgene-free genome edits in plants. The use of graft-mobile TLS-fused Cas9 and gRNA transcripts eliminates the need for laborious and time-consuming transgene removal steps, significantly accelerating the plant breeding process. The success of this method in both *Arabidopsis thaliana* and *Brassica rapa*, two distantly related species, highlights its broad potential applicability across various plant species. The finding that the method successfully generates heritable genome edits in a single generation represents a substantial advancement over existing techniques. The observed efficiency, while not extremely high, is comparable to other transgene-free editing methods, and could likely be further optimized. This approach offers a promising alternative to other methods, particularly in species that are difficult to transform or regenerate from protoplasts. The method also provides opportunities for exploring otherwise lethal mutations using the chimeric nature of the somatic edits in grafted wild-type scions.

This research presents a novel and efficient method for generating heritable, transgene-free genome edits in plants using graft-mobile CRISPR-Cas9 components fused with TLS motifs. The success of the method in both Arabidopsis and Brassica rapa demonstrates its broad applicability. The single-generation approach significantly reduces the time and effort required for producing transgene-free edited plants, offering a valuable tool for plant breeding and research. Future research could focus on optimizing the efficiency of the system, exploring additional TLS motifs or combinations thereof, and extending its application to a wider range of plant species, including monocots and economically important crops. The findings offer a significant contribution to plant biotechnology, advancing the field of genome editing.

While the method is highly efficient for generating heritable edits, the overall frequency of edits remains relatively low. The study primarily focused on deletions, and the efficiency for other types of edits might differ. The current method requires the generation of transgenic rootstocks, which may be a limiting factor in some settings. Although the grafting process showed high success rates, this might vary depending on the specific plant species and grafting technique used. The limited sample sizes for assessing editing efficiency in the progeny of grafted plants might lead to underestimation of actual editing frequency.