“…At least three biological replicates should be conducted to determine the optimal sgRNA. (C) A binary vector containing the expression cassettes of dCas9-TV and the optimal sgRNA is constructed for Agrobacterium-mediated transformation of Arabidopsis and rice (Oryza sativa ;Kramer & Coen, 2001), agarose gel electrophoresis (see Current Protocols article; Voytas, 2000), Sanger DNA sequencing (see Current Protocols article; Slatko, Albright, Tabor, & Ju, 1999), plasmid DNA extraction by maxiprep (see annotation to step 33), isolation of Arabidopsis mesophyll protoplasts (Yoo, Cho, & Sheen, 2007; see Current Protocols article; Li & Zhang, 2014) or rice leaf sheath protoplasts (Zhang et al, 2011b), electroporation (see Current Protocols article; Seidman, Struhl, Sheen, & Jessen, 2001), colony PCR (see Current Protocols article; Woodman, 2008), stable transformation of Arabidopsis (Clough & Bent, 1998) or rice (Hiei, Ohta, Komari, & Kumashiro, 1994), and transformant selection for Arabidopsis (Clough and Bent, 1998;Zhang, Henriques, Lin, Niu, & Chua, 2006) or rice (Hiei et al, 1994;Hiei & Komari, 2008) NOTE: Experiments involving PCR and RNA require extremely careful technique to prevent contamination and RNA degradation.…”