title: Activity and specificity of TRV-mediated gene editing in plants.
abstract: Plant trait engineering requires efficient targeted genome-editing technologies. Clustered regularly interspaced palindromic repeats (CRISPRs)/ CRISPR associated (Cas) type II system is used for targeted genome-editing applications across eukaryotic species including plants. Delivery of genome engineering reagents and recovery of mutants remain challenging tasks for in planta applications. Recently, we reported the development of Tobacco rattle virus (TRV)-mediated genome editing in Nicotiana benthamiana. TRV infects the growing points and possesses small genome size; which facilitate cloning, multiplexing, and agroinfections. Here, we report on the persistent activity and specificity of the TRV-mediated CRISPR/Cas9 system for targeted modification of the Nicotiana benthamiana genome. Our data reveal the persistence of the TRV- mediated Cas9 activity for up to 30 d post-agroinefection. Further, our data indicate that TRV-mediated genome editing exhibited no off-target activities at potential off-targets indicating the precision of the system for plant genome engineering. Taken together, our data establish the feasibility and exciting possibilities of using virus-mediated CRISPR/Cas9 for targeted engineering of plant genomes.
authors.affiliation: a Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, King Abdullah University of Science and Technology ; Thuwal , Kingdom of Saudi Arabia.
authors.forename: Zahir
authors.initials: Z
authors.lastname: Ali
authors.affiliation: a Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, King Abdullah University of Science and Technology ; Thuwal , Kingdom of Saudi Arabia.
authors.forename: Aala
authors.initials: A
authors.lastname: Abul-Faraj
authors.affiliation: a Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, King Abdullah University of Science and Technology ; Thuwal , Kingdom of Saudi Arabia.
authors.forename: Marek
authors.initials: M
authors.lastname: Piatek
authors.affiliation: a Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, King Abdullah University of Science and Technology ; Thuwal , Kingdom of Saudi Arabia.
authors.forename: Magdy M
authors.initials: MM
authors.lastname: Mahfouz
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D018744:DNA, Plant; D017343:Genes, Plant; D005818:Genetic Engineering / Q000379:methods*; D018745:Genome, Plant; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010942:Plant Viruses; D017393:RNA Editing; D012328:RNA Viruses; D012367:RNA, Viral; D014026:Nicotiana / Q000235:genetics* / Q000821:virology
chemical_list: D018744:DNA, Plant; D012367:RNA, Viral
keywords: CRISPR/Cas9 system; TRV; plant genome engineering; synthetic site-specific nucleases (SSNs); viral-mediated genome editing