Figure 1

CRISPR/Cas mediated gene manipulation.

(a) The pCAG-EGxxFP plasmid contains 5′ and 3′ EGFP fragments that share 482 bp under a ubiquitous CAG promoter. A 500-bp genomic fragment containing the sgRNA target sequence was placed between the EGFP fragments of the pCAG-EGxxFP plasmid. The resulting target plasmid was co-transfected with pX330 plasmids expressing the sgRNA and hCas9 into HEK293T cells. Once the target sequence was digested by sgRNA guided CAS9 endonuclease, homology dependent repair (HR: homologous recombination, or SSA: single-strand annealing) took place and reconstituted the EGFP expression cassette. MCS; multi cloning site. (b) The DSB efficiency was validated with the pCAG-EGxxFP system by observing EGFP fluorescence 48 hrs after the transfection (scale bar: 200 μm). The percentages of EGFP-positive cells are indicated. (c) Schematic representation of the positions of each sgRNA and primer to check the CRISPR/Cas mediated mutations (left side of (c)). Electrophoresis of the PCR products from each of the pX330 plasmid-injected mice (the right side of (c)). At least four PCR products (yellow arrowheads) were larger than expected (WT: red arrowheads) in the Peg10-ORF2-sgRNA-injected mice. One and two PCR products (yellow arrowheads) were larger than Cxx1a WT and Cxx1b WT, respectively, in the Cxx1a/b-sgRNA-injected pups. Three PCR products (yellow arrowheads) were larger than 1041 bp (Rgag1 WT) in the Rgag1-sgRNA-injected pups.