Genome Editing by Grafting

Abstract

Grafting is the process of joining parts of two plants, allowing the exchange of molecules such as small RNAs (including microRNAs and small interfering RNAs), messenger RNAs, and proteins between the rootstock and the scion. Genome editing by grafting exploits RNAs, such as tRNA-like sequences (TLS motifs), to deliver the components (RNA) of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system from transgenic rootstock to wild-type scion. The complex Cas9 protein and sgRNA-TLS produced in the scion perform the desired modification without the integration of foreign DNA in the plant genome, resulting in heritable transgene-free genome editing. In this review, we examine the current state of the art of this innovation and how it helps address regulatory problems, improves crop recovery and selection, exceeds the usage of viral vectors, and may reduce potential off-target effects. We also discuss the promise of genome editing by grafting for plants recalcitrant to in vitro culture and for agamic-propagated species that must maintain heterozygosity for plant productivity, fruit quality, and adaptation. Furthermore, we explore the limitations of this technique, including variable efficiency, graft incompatibility among genotypes, and challenges in large-scale application, while highlighting its considerable potential for further improvement and future broader applications for crop breeding.

Keywords: CRISPR/Cas; genome editing; grafting; mobile RNA; tRNA-like sequences; transgene-free plants.

Figure 1

Using mobile CRISPR-RNA, inter- or intra-specific grafting between sunflower scion and transgenic rootstock. Mobile Cas9 and sgRNA RNAs fused to tRNA-like sequence (TLS) motifs moved from the root to the wild-type shoot. The CRISPR complex induced targeted genome editing (GE) in the distal parts and reproductive organs of the grafted scion. Red arrows correspond to the TLS (tRNA^Met)-Cas9 mRNA, while green arrows indicate TLS (tRNA^Met)-sgRNA. Transgene-free mutant seeds (light brown) and consequently mutant seedlings (light green) are obtained.

Figure 2

GEG in grapevine. Vectors expressing Cas9-TLS and sgRNA-TLS are cloned and transferred into an easily transformed grapevine genotype. After in vitro culture, the transgenic rootstock is grafted onto a wild-type elite cultivar scion. The CRISPR-RNA components are translated and transduced in the distal parts. The transgene-free stock is rooting, producing the identical elite cultivar scion but with the new desirable characters.