Comparison of Different Systems for Expressing Multiple gRNAs.

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Comparison of Different Systems for Expressing Multiple gRNAs. Comparison of Different Systems for Expressing Multiple gRNAs. (A) Structure of six constructs for expressing gRNA9 and gRNA10, which both target the tomato ARF8A gene. CmYLCV, Cestrum yellow leaf curling virus promoter; Csy4, 20-bp Csy4 hairpin; tRNA, 77-bp pre-tRNAGly gene; rb, 15-bp ribozyme cleavage site; ribozyme, 58-bp synthetic ribozyme; AtU6, Arabidopsis U6 promoter; At7SL, Arabidopsis 7SL promoter. (B) Possible editing outcomes due to expression of both gRNAs. Sequences targeted by gRNA9 and gRNA10 are shown on the top. Cleavage sites are indicated by arrows. (C) Overall mutation frequencies as determined by deep sequencing. Error bars represent se of three replicates (pools of protoplasts). Statistical significance was determined by Tukey’s test. *P < 0.05 and ***P < 0.001. Tomáš Čermák et al. Plant Cell 2017;29:1196-1217 ©2017 by American Society of Plant Biologists