Fig. 1

Scheme for Agrobacterium-mediated in-leaf GFP tagged CRISPR/Cas9 mutation generation combined with FACS enrichment of GFP expressing protoplasts. a Guide RNA (gRNA) target sequence may be selected on the basis of in silico prediction analysis and the presence of a Restriction Enzyme (RE) recognition motif spanning the SpCas9 cleavage site (− 3 bp upstream of the protospacer adjacent motif (PAM) [34]) for fast RE-mediated mutation screening. Primers flanking the gRNA target for PCR mediated-mutation scoring are indicated. The deconstructed bean yellow dwarf virus (BeYDV) replicon is produced from the Agrobacterium tumefaciens delivered T-DNA, that contains the viral cis-acting Long (LIR) and Short Intergenic Regions (SIR) in a Long-Short-Long region (pLSL) arrangement, which together with the co-expressed trans acting Rep/RepA replication initiation proteins facilitate replicational release and Gemini Virus Replicon (GVR) circularization allowing joining of the two BeYDV replicon LIR elements within plant cell nuclei [17]. Abbreviations: Left and Right T-DNA border, LB & RB, Cauliflower mosaic virus 35S promoter, CMV35S, Arabidopsis thaliana U6 promotor, AtU6-Pro [35, 36], hygromycin phosphotransferase, HPT, Streptococcus pyogenes Cas9, SpCas9, nopaline synthase terminator, NOS, Nucleus Localization Signal, NLS, 2A self-cleaving sequence of foot-and-mouth disease virus (FMDV), 2A [37, 38], Agrobacterium tumefaciens, A. tumefaciens, Nicotiana benthamiana, N. benthamiana. The replicon constructs (a) are transformed into A. tumefaciens by electroporation, grown under selection overnight and re-suspended in infiltration buffer to a final total OD₆₀₀ of ca. 0.2 where after the abaxial side of young expanding leaves of 3–4 week old N. benthamiana plants are infiltrated with the agrobacterium strain carrying the construct of interest using a syringe and left for 2–4 days (b). Protoplasts are isolated (c) and subjected to florescence microscopy (for estimation of protoplast isolation and transformation efficiencies) and to Fluorescence Activated Cell Sorting (FACS) (d) of GFP (SpCas9-2A-GFP) expressing protoplasts for mutation enrichment. The target region on the genome is amplified by PCR (e) with mutations scored by the high throughput screening technique Indel Detection by Amplicon Analysis (IDAA) [39] (f), which allows for detection of down to 1 bp deletions and insertions (indels) and by restriction enzyme (RE) analysis (g), which monitors resistant mutated RE recognition/cleavage sites. Optionally, explants with stable PGE editing can be obtained by embedding the protoplasts in alginate, followed by callus induction and shoot regeneration as outlined in [40]. Protoplasts shown in (c) are presented as light-, fluorescent micrographs and overlay hereof