Journal: International Journal of Molecular Sciences

Article Title: A CRISPR/Cas9-Based System with Controllable Auto-Excision Feature Serving Cisgenic Plant Breeding and Beyond

doi: 10.3390/ijms23105597

Figure Lengend Snippet: Transgenic components were removed in the final product of Arabidopsis thaliana (L.) Heyhn. transformed by pHHCGR− Hsp18.2 /pHHCGS− Hsp18.2 vector. ( A ) Screening for T1 lines carrying single copy insert (SCI) of pHHCGR− Hsp18.2 or pHHCGS− Hsp18.2 by ddPCR. ( B ) In transgenic T1 lines with SCI, the presence or absence of PCR products of Cas9p (1082 bp), Bar (422 bp), and eGFP (749 bp) showed distinct results in the same plant before and after editing. ( C ) In transgenic T1 lines with SCI, confocal images showed distinct results of eGFP expression in the same plant before and after editing. Bright, bright-field microscopy; PI, propidium iodide, fluorescent dye staining nuclei due to intercalating binding with DNA, excitation wavelength (535 nm) and emission wavelength (617 nm) on confocal microscope; eGFP, eGFP protein mainly accumulated in the nuclear region due to an SV40 NLS added before the eGFP coding region, excitation wavelength (488 nm) and emission wavelength (509 nm) on the confocal microscope. Scale bar, 15 µm. ( D ) Sequencing results of PCR products showing the junction sequence after editing and repair via non-homologous end-joining (NHEJ). BE/AE, before/after editing; TR, truncated region; |, cutting site. Three-base PAM sequence highlighted in green, mismatched bases indicated with red font color. ( E ) Selection for T2 lines carrying double inserts of the target gene by ddPCR.

Article Snippet: The ddPCR reactions and data analysis were performed with the QX200 Droplet Digital PCR System (Bio-Rad, Hercules, CA, USA; QX Manager Software Standard Edition v1.2) following the manufacturer’s instructions.

Techniques: Transgenic Assay, Transformation Assay, Plasmid Preparation, Expressing, Microscopy, Staining, Binding Assay, Sequencing, Non-Homologous End Joining, Selection

Journal: International Journal of Molecular Sciences

Article Title: A CRISPR/Cas9-Based System with Controllable Auto-Excision Feature Serving Cisgenic Plant Breeding and Beyond

doi: 10.3390/ijms23105597

Figure Lengend Snippet: Confirmation of target genes in T2 progenies of selected Arabidopsis thaliana (L.) Heyhn. T1 lines carrying single copy insert of pHHCGS- Hsp18.2 .

Article Snippet: The ddPCR reactions and data analysis were performed with the QX200 Droplet Digital PCR System (Bio-Rad, Hercules, CA, USA; QX Manager Software Standard Edition v1.2) following the manufacturer’s instructions.

Techniques: