Fig. 4

Improving CRISPR/Cas9 gene editing efficiency by interference with RNA-silencing pathway gene expression. a A schematic diagram of the CRISPR/Cas9 system containing an RNA interference construct. BPNLS, bipartite nuclear localization signal; Sense, sense fragment of targeted gene; PDK, PDK intron from pHellsgate; Antisense, antisense fragment of targeted gene. b–f T1 transgenic plants transformed with pRNAi-Cas9-sgRTT4 containing an empty RNA interference construct (RNAi-CK) (b), an AGO1 5′UTR RNAi construct (c), an AGO1 CDS RNAi construct (d), a DCL1 3′UTR RNAi construct (e) and a DCL1 CDS RNAi construct (f). Bar = 1 cm. g Histogram showing the frequency of WT, chimeras, and mutants in T1 populations transformed with different pRNAi-Cas9-sgRTT4 constructs. The numbers indicates % of mutation = (chimera + mutant)/total (h). Relative integrity of the TT4 gene in RNAi-CK, RNAi-AGO1 CDS and RNAi-DCL1 CDST1 plants. Eight individual T1 plants were analyzed for each construct by real-time PCR. i, j Relative expression of AGO1 (i) and Cas9 (j) in Col-0, RNAi-CK, and RNAi-AGO1 CDS T1 plants determined by quantitative RT-PCR. k, l Relative expression of DCL1 (k) and Cas9 (l) in Col-0, RNAi-CK, and RNAi-DCL1 CDS T1 plants determined by quantitative RT-PCR. Each column in (h–l) indicates means ± SD of three technical replicates. The values of Col-0 samples were arbitrarily designated as 1. *Significant difference at p < 0.05 under two-tailed Student’s t-test. **Significant difference at p < 0.01 under two-tailed Student’s t test. “p > 0.05” indicates no significant difference