Virus induced gene editing using potyviral vectors in Cas12a expressing plants

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are revolutionizing precision genome editing and gene expression control in crop plants. While effective CRISPR-Cas applications traditionally rely on labor-intensive stable genetic transformation to deliver Cas nucleases and guide RNAs into plant cells, plant viruses have emerged as a faster and efficient alternative, a strategy known as virus-induced gene editing (VIGE). Cas12a, Class 2 Type V CRISPR nucleases, are an alternative to broadly used Cas9 for plant genome engineering. Both kind of nucleases offer precise editing, but some Cas12a unique features make them particularly well suited for VIGE. In this study, we first used a tobacco rattle virus vector to compare editing efficiency of various target sequences and CRISPR RNA (crRNA) architectures in Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a)-expressing Nicotiana benthamiana plants, evaluating results in infected tissues and seeds. Next, we developed a tobacco etch virus (genus Potyvirus)-derived vector efficiently delivering crRNAs throughout the plant. This approach enabled generation of plants with all four edited alleles in the allotetraploid N. benthamiana through in vitro regeneration from infected leaves, and to produce edited non-infected progeny, although at a very low frequency. We then demonstrated the successful application of the potyviral vector for VIGE in agronomically important crops, such as tomato or cultivated tobacco. Finally, we replicated this design using two other potyviral vectors, turnip mosaic virus, and lettuce mosaic virus. Given the conserved biological properties among potyviruses, we believe these findings are broadly applicable to the largest genus of plant RNA viruses, significantly expanding the host range of the VIGE technology.