Multiplexed Mutagenesis Using the Csy4 System in Tomato Protoplasts.

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Multiplexed Mutagenesis Using the Csy4 System in Tomato Protoplasts. Multiplexed Mutagenesis Using the Csy4 System in Tomato Protoplasts. (A) Maps of six tomato genes targeted for simultaneous deletion using 12 gRNAs. gRNA sites are shown as black arrows. Arrowheads represent primers used to detect the deletions. Lengths of PCR products from the wild-type locus are shown on the right; lengths of the deletion products are in parentheses. (B) Deletions of expected length were detected in each of the six genes by PCR. Blue and black arrowheads mark the unmodified and deletion products, respectively. Wild-type DNA from nontransformed cells was used as template. (C) DNA sequences of representative deletion products. The sequence of the unmodified locus for each of the six genes is shown on the top. gRNA target sites are underlined and PAM sequences are in red. Tomáš Čermák et al. Plant Cell 2017;29:1196-1217 ©2017 by American Society of Plant Biologists