Figure 5

Schematic representation of two plasmids used for the evaluation of CRISPR reagents. (A) CRISPR plasmid pTF6005²⁸ that carries a Cas9 expression cassette under the control of maize ubiquitin promoter and Cauliflower Mosaic Virus (CaMV) 35S terminator (T35S); OsPDS gRNA1 is regulated by OsU6 promoter; hygromycin resistance gene (hpt II) is driven by 2 × CaMV 35S promoter (P35S) and terminated by T35S. RB, T-DNA right border; LB, T-DNA left border; Sp^R, spectinomycin resistance gene; ColE1 ori, high copy number origin of replication for E. coli; pVS1, origin of replication from plasmid VS1 for Agrobacterium. (B) The reporter plasmid pKL2187 has genes for the red fluorescent protein tdTomato and the green fluorescent protein ZsGreen1. Transcription of the tdTomato gene is driven by a 2X P35S and terminated by an Agrobacterium nopaline synthase terminator (Tnos). The encoded tdTomato protein has an SV40 nuclear localization signal at the N terminus. Transcription of the ZsGreen1 gene is driven by a 2X P35S promoter and terminated by a potato protease inhibitor II terminator (TpinII). The translation start codon is preceded by a TMV Ω translational enhancer and is immediately followed by the target sequence of the OsPDS-gRNA1 expressed from pTF6005. The open reading frame for the flexible peptide linker 2X (GGGGS) and ZsGreen1 is out-of-frame by 1-bp with the start codon and is not translated; however, indel mutations at the gRNA target site can bring the ZsGreen1 gene in-frame and restore green fluorescence. Amp^R, ampicillin resistance gene. The plasmid pKL2188 is identical to pKL2187 except that the ZsGreen1 gene is in-frame with the start codon. Blue letters, gRNA target sequence; underscored red letter, PAM sequence. (C) Three guide RNAs tested for editing efficiency. All gRNAs were driven by OsU6 promoter in the CRISPR plasmid shown in (A). gRNA1, on-target gRNA; gRNA2 and gRNA3, artificial off-target gRNAs with one or two mismatches (white letters in black boxes).