Am2222

Total RNA was extracted from ∼30 mg seedlings in 600 μl of extraction buffer (0.1 M glycine–NaOH, pH 9.0, 100 mM NaCl, 10 mM EDTA, 2% SDS) and extracted with equal volumes of phenol–chloroform (pH 4.3) and chloroform, precipitated with ethanol and resuspended in sterile water. For qRT-PCR assays, 5 μg total RNA was DNase treated (Ambion AM2222, www.thermofisher.com), precipitated in ethanol and resuspended in sterile water. One microgram of DNase-treated total RNA and random primer was used for the first-strand cDNA reaction (NEB, E6300S, www.neb.com). qPCRs were done using qPCR Master Mix (NEB, M3003S, www.neb.com). qPCR reactions were run in a Light Cycler 96 (Roche) real-time PCR machine. At least three biological samples were assessed in each experiment and standard error bars shown. P-values were calculated using unpaired two-tailed Student’s t-test to assess the significance of differences; t-test comparison of samples is shown where considered relevant. For primers, please see Supplementary Table S1.

Total RNA was extracted from infiltrated leaves using RNeasy Plant Mini Kit (Qiagen) and the yield was quantified using a nanodrop. A total of 1 μg RNA from control (NT gRNAs) and experimental samples were used for DNase I treatment (Ambion, AM2222) followed by reverse transcription using a poly-dT primer and the Superscript III First Strand cDNA Synthesis System for RT–PCR (Invitrogen). Quantitative PCR was performed on Quant studio 3 Real-Time PCR System from Applied Biosystems using iTaq PowerUP^TM SYBR Green pre-formulated 2x master mix (Applied Biosystems). Relative expression levels based on fold changes were calculated using the ddCT method. Cycle 3 GFP mRNA expression levels from the TRBO-GFP replicon were normalized against transcripts of the tobacco PP2A, then to compare gRNAs across experiments, mRNA expression levels within a batch of plants are normalized relative to the mean GFP levels in experiments with non-viral targeting controls (dCas13d with no gRNA in Figure 2D and Cas13d with gNT in Figure 2E) performed at the same time as the other experimental conditions. The samples were performed in three biological replicates.