Fig. 2

Schematic diagrams of guide RNA (gRNA) targeting sites and CRISPR-Cas9 multiplexing constructs to target three genes encoding glucuronic acid transferases (GLCATs).A. Two sites were chosen to target GLCAT14A (A1 and A2), one site was chosen to target GLCAT14B (B1), and three sites were chosen to target GLCAT14C (C1, C2, and C3). Online software CRISPR-P 2.0 (http://crispr.hzau.cn/cgi-bin/CRISPR2/CRISPR) was used for designing all gRNAs. Pfam domain predictions: Pf02485 identified the glucuronosyltransferase (GLCAT) domain (http://www.sanger.ac.uk/Software/Pfam/). B. (1) Three gRNAs (A1, B1, and C1 in Figure 2A) were assembled head-to-tail with each gRNA regulated by an individual promoter and terminator to target the three GLCATs (GLCAT14A, GLCAT14B, and GLCAT14C). (2) Four gRNAs (A2, B1, C1, and C2 in Figure 2A) were assembled head-to-tail to target three GLCATs (GLCAT14A, GLCAT14B, and GLCAT14C). The two constructs [(1) and (2)] were cloned into the pHEE401E plasmid vector engineered by Wang et al., 2015b), which contains a maize codon-optimized Cas9 (zCas9) gene driven by an Arabidopsis egg-cell specific promoter (E.C1.1) fused with an egg-cell specific enhancer (E.C1.2)