Mutation detection using Agrobacterium-mediated transient expression in N. benthamiana.
a Schematic illustrations showing the locations of gRNAs, PAM positions, and primers in the genes, NbPDS, PIP2-1-mCherry and YFP targeted for genome editing. b Resolvase assay for detecting mutation in NbPDS and mCherry targeted by the gRNA constructs pDe-Cas9-D10A-gNbPDS and pDe-Cas9-D10A-gmCherry. High fidelity PCR was performed with primers NbPDS-F and NbPDS-R for the NbPDS target, and mCherry-F and mCherry-R for the mCherry target (panel A and Table 1) using template DNA isolated from leaf spots on N. benthamiana infiltrated with Agrobacterium tumefaciens carrying the indicated CRISPR–Cas9 constructs; N. benthamiana was stably transformed with the PIP2-1-mCherry gene, which served as a transgene target for the gmCherry gRNA. Amplicons were subjected to Resolvase assays (Guide-it™ Mutation Detection Kit, Cat#631443, Clontech) and reactions were run on a 1.5% agarose gels. Digested fragments, which result from gRNA-induced mutations, are indicated by *, and their sizes are ~ 570 and ~ 150 bp for NbPDS, and ~ 277 and ~ 150 bp for mCherry. Undigested fragments are 727 bp for NbPDS and 427 bp for mCherry. Lower panel shows a representative N. benthamiana leaf infiltrated in three independent sites with the indicated CRISPR–Cas9 constructs. c Surveyor (CEL II)—nuclease assay for detecting mutation in a transiently transformed YFP gene carried on pGWB415-35S::HA-YFP plasmid. PCR was performed with forward primer (35SpromF) and reverse primer (EYFPStopXhoR) (panel A and Table 1) using template DNA from Wild Type N. benthamiana co-transformed with pGWB415-35S::HA-YFP along with either pDe-Cas9-D10A-gYFP or pDe-Cas9-D10A-gNbPDS (negative control). Surveyor (CEL II)—nuclease assay was performed with amplicons and reactions were run on 1.5% agarose gels (mismatch-specific Surveyor nuclease, Surveyor® Mutation Detection Kit; Cat#706025, IDTdna.com). gRNA-induced mutations are revealed by the digested ~ 700 and ~ 400 bp fragments, marked by *; the undigested fragment is 1067 bp, which is shown in both lanes. Lower panel shows a representative N. benthamiana leaf infiltrated in three independent sites with the indicated CRISPR–Cas9 constructs