1. Volume 8, Issue 8, Pages 1288-1291 (August 2015)
Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System 
Zahir Ali, Aala Abul-faraj, Lixin Li, Neha Ghosh, Marek Piatek, Ali Mahjoub, Mustapha Aouida, Agnieszka Piatek, Nicholas J. Baltes, Daniel F. Voytas, Savithramma Dinesh-Kumar, Magdy M. Mahfouz 
Molecular Plant 
Volume 8, Issue 8, Pages (August 2015)
DOI: /j.molp
Copyright © 2015 The Author Terms and Conditions

2. Figure 1 TRV-Mediated Genome Editing in N. benthamiana.
(A) Schematic representation of TRV RNA1 and RNA2 genome organization and modification for targeted genome editing. RNA1 in the Agrobacterium binary vector system: LB (left border), 2Xp35S (2X CaMV 35S promoter), RdRNAP (134/194 kDa RNA-dependent RNA polymerase), MP (movement protein), 16k (cysteine rich protein), Rz (self-cleaving ribozyme), Tnos (nopaline synthase terminator), RB (right border). RNA2 in the Agrobacterium binary vector system: LB, p35S, CP (coat protein), Rz, Tnos, and RB. In RNA2, the gRNA was cloned under the pea early browning virus (PEBV) promoter (pPEBV::gRNA).
(B) Experimental scheme of the TRV-mediated genome editing. A 20-nucleotide target sequence preceding the PAM sequence can be cloned, in the gRNA backbone under the PEBV promoter, in the RNA2 genome. Agrobacterium cultures carrying the engineered TRV RNA2 genome, conferring user-selected sequence specificity, and the RNA1 genome are co-infiltrated into leaves of N. benthamiana overexpressing Cas9 (Cas9-OE) via agroinfection. Alternatively, multiple targets can be simultaneously edited by mixing RNA2 cultures that confer sequence specificities for multiple targets with RNA1 culture. After agroinfection, plants can be analyzed for the presence of the targeted modification; plant leaf discs carrying modified genomes can be either regenerated to recover mutant plants or the seed progeny can be screened for the presence of the modification, thereby bypassing the need for tissue culture. The virus-mediated genome editing in this report was performed in a N. benthamiana transgenic line overexpressing Cas9.
(C) T7EI-based mutation detection analysis of PDS gene in inoculated and systemic leaves of N. benthamiana. Targeted mutagenesis was detected in inoculated (lane 2) and systemic (lane 3) leaves co-infiltrated with RNA1 and RNA2 carrying a pPEBV::PDS.gRNA compared with vector control (lane 1).
(D) Multiplex editing of PDS and PCNA genes: DNA samples were isolated from the leaves of N. benthamiana Cas9 overexpression plants co-infiltrated with RNA2.pPEBV::PDS.gRNA, RNA2.pPEBV::PCNA.gRNA, and RNA1 cultures. PCR products of the 797-bp fragment of the PDS gene and 1098-bp fragment of the PCNA gene were subjected to T7EI mutation detection analysis. The PCNA mutagenesis is shown in lanes 3 and 4, the PDS mutagenesis in lanes 7 and 8, and vector controls in lanes 1 and 5. Mutation efficiency (as a percentage) was calculated using ImageJ software ( Arrowheads indicate restriction products.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions