WRKY20 dual sgRNA approach.

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Target gene gRNA NameTarget sequence / PAM*Restriction Enzyme Young Seedling Albino (YSA)gYSAGCGCGCCACCTCGGCCGAAG / CGGSfiI Phytoene desaturase (PDS) gPDS-1†CGTCCAACCCATTCCTCTGC.

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WRKY20 dual sgRNA approach. WRKY20 dual sgRNA approach. A, genomic structure of WRKY20 and location of the sgRNAs. Dark green boxes designate exons; light green boxes, UTRs; solid lines, introns. B, PCR analysis of T1 lines. Leaf genomic DNA of 2 batches (A and B) of 20 chimeric T1 plants was PCR amplified. The expected size of the WT WRKY20 amplicon is indicated as well as the expected size of the deletion of 247 bp between Cas9 cut sites. Four continued T1 lines having one T-DNA locus are highlighted with green boxes. C, Cas9 PCR for the four continued lines in T2 generation. Putative Cas9 null-segregants are indicated with green boxes. D, Cas9 null-segregants were genotypes for WRKY20. The selected lines A15-8 (wrky2-1 wrky20-1) and B2-5 (wrky2-1 wrky20-2) are boxed. E, Sequence alignment of the simultaneously targeted loci for Col-0 and alleles wrky20-1 and wrky20-2. PAMs are highlighted, the Cas9 cut sites indicated with triangles. Deleted bases are indicated with dashed lines. The reading frame is marked. Laurens Pauwels et al. G3 2018;8:2603-2615 ©2018 by Genetics Society of America