Fig. 6
From: CRISPR guides induce gene silencing in plants in the absence of Cas
GIGS is also evident for sgRNA guides designed for Cas9. a Schematic of guide designs targeting PDS transcript for Cas13 s-guide 2, and Cas9 sgRNA 2. Each guide contains 28 nt antisense to the PDS transcript (sequence shown). The Cas9 sgRNA control contained 50% mismatch sequence to the PDS transcript (sgRNA 2 [50% mm]). Mismatch nucleotides are colored red, while shared nucleotides between the three guides are black. The Cas13 crRNA contains the 37-bp direct repeat (DR) sequence at the 5′ end. The Cas9 sgRNA contains the 78 bp trans-activating crRNA (tracrRNA, depicted as a line) at the 3′ end. b Representative images of leaves following TRV systemic delivery of single-guide 2 (s-guide 2) targeting PDS, and a Cas9 designed sgRNA designed to contain the same 28 bp targeting PDS as in s-guide 2. An sgRNA 2 control contained the sequence in sgRNA 2, but with 50% mismatches to the PDS transcript (sgRNA 2[50%mm]). Control sgRNA containing non-targeting guide sequence (NT-sgRNA). Photobleaching is seen in the asPDS sample, while interveinal yellowing is visible in the samples expressing s-guide 2 and sgRNA 2. c SPAD meter readings from photobleached leaf samples as described for b. Data collected from a total of six independent leaves from two experiments. d Quantification of the PDS transcript using qPCR for the same samples as measured in c. Data standardized to an endogenous transcript and normalized to TRV expressing NT-sgRNA. e Schematic of the modified sgRNA 2 targeting PDS where 8 nt were removed from the 5′ or 3′ end respectively, indicated by dashes in a red box. f Representative images of leaves following TRV systemic delivery of the indicated guides. g SPAD meter readings from photobleached leaf samples. h Quantification of the PDS transcript using qPCR for the same samples as measured in g. Statistical comparisons were made between NT-sgRNA and each treatment using a one-sided Mann-Whitney U test with Benjamini-Hochberg (BH) multiple testing correction. Samples with p values less than 0.05 (*) and 0.01 (**) are indicated. n.s., non-significant difference (p > 0.05)