Figure 1
From: Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants
Schematic illustrating the CRISPR/Cas9 vectors using truncated gRNAs, target sites in the Arabidopsis genes and mutation detection.
(a) Schematic structure of the CRISPR/Cas9 vectors used in this study. The plant codon-optimized Cas9 with 3 × FLAG and 2 × NLSs was inserted in front of the 2A peptide fused with GBBSD2 under the control of the 2 × CaMV 35S promoter. The tru-gRNA can be inserted into the Bsa I site and the AtU6-1 promoter was used in the expression cassette. Cas9 with a single NLS was also used to assess the effect of NLS number on mutation efficiency. The vectors were named pEgP526-2A-GFBSD2 and pEgP526-2A-GFBSD2 (Sp_SN), respectively. (b) Target sequences for mutagenesis with tru-RNA-guided genome editing in the Arabidopsis genome. The selected target sequences (17–18 b) for each gene are shown in black boxes and the PAM sequences are light blue boxes. Arrows; translational start sites (+1), Gray boxes; exons. (c) GFP fluorescence in the T1 transgenic plants of the OST2-CRISPR2 (pEgP526-2A-GFBSD2). Bar = 100 μm. (d) Cel-1 analysis of T1 transgenic plants of GL1-CRISPR1 and OST2-CRISPR2 (pEgP526-2A-GFBSD2) and PCR-RFLP analysis of ABI4-CRISPR (pEgP526-2A-GFBSD2) with high GFP fluorescence compared with the wild-type control. Black arrows indicate the PCR products for wild-type sequence and yellow stars show the mutated bands digested with Cel-1 nuclease (GL1-CRISPR1 and OST2-CRISPR2) or undigested in PCR-RFLP (ABI4-CRISPR). L1, L2; individual T1 lines for transgenic plants. (e) Cel-1 analysis of T1 plants of OST2-CRISPR2 transformed with the single NLS-Cas9 cassette {pEgP526-2A-GFBSD2 (Sp_SN)}. (f) Heteroduplex mobility assay (HMA) T1 plants transformed with the CRISPR/Cas9. Multiple heteroduplex peaks (blue triangles) were detected in PCR amplicons from each of the CRISPR/Cas9-transformed plants, whereas a single peak was detected from each wild-type control (black triangle).
