Fig. 1From: CRISPR-Cas9 multiplex genome editing of the hydroxyproline-O-galactosyltransferase gene family alters arabinogalactan-protein glycosylation and function in ArabidopsisSchematic illustration of gRNA target sites and the two CRISPR multiplexing constructs. a. Three gRNAs were chosen for each of the GALTs, which belong to the five-membered galactosyltransferase (GALT) gene family. gRNAs were labeled as 3–1, 3–2, and 3–3 for GALT3; 4–1, 4–2, and 4–3 for GALT4; 6–1, 6–2, and 6–3 for GALT6; these gRNAs were used in gene constructs shown in 1b. (Green and orange represent 5′-UTR and 3′-UTR regions, respectively; blue represents exons; black lines represent introns. Online software named CRISPR-P 2.0 (http://crispr.hzau.cn/cgi-bin/CRISPR2/CRISPR) was used to design all gRNAs. Pfam domain predictions: Pf01762 corresponds to the Galactosyltransferase (GALT) domain; Pf00337 corresponds to the Galactose-binding lectin (GALECTIN) domain. b-1. Five gRNAs (3–1, 3–2, 4–1, 6–1, and 6–2 shown in 1a) with each gRNA fused with a tRNA were cloned in a single polycistronic transcription unit to target GALT3, GALT4, and GALT6 simultaneously. b-2. Four gRNAs (3–3, 4–2, 4–3, and 6–3 in 1a) were assembled as four individual transcription units to target GALT3, GALT4, and GALT6. Both B-1 and B-2 were cloned in the pHEE401E vector, which contains a maize codon-optimized Cas9 gene (zCas9) driven by an Arabidopsis egg-cell specific promoter (E.C.1.1) fused with an egg-cell specific enhancer (E.C.1.2) [19]Back to article page