Journal: Biochemical and biophysical research communications

Article Title: Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system.

doi: 10.1016/j.bbrc.2014.01.141

Figure Lengend Snippet: Fig. 1. Generation of region-deleted mice by the offset-nicking method. (A) Schematic illustration of the Rosa26 gene structure and sequences around the target loci. The target sequences, PAM domain and PCR primer loci are indicated by arrows, underlining and white arrowheads, respectively. Red arrowheads show the break sites by Cas9 nickase and the distances between two break sites are indicated. (B) PCR analysis using tail tip DNAs of pups obtained from zygotes injected with Cas9D10A mRNA and 4 kinds of gRNAs with a wild-type pup. PCR products of the wild type or small-deletion and region-deletion are indicated. (C) An example of a genome sequence in a region-deleted pup. The waveform data of a DNA sequencer obtained from a PCR fragment of tail tip DNA and the predicted sequence are shown with target positions. (D) Mutation efficiency and states of target sites in the obtained pups. ⁄4 pups might have biallelic region deletion and 1 pup is monoallelic region deletion. ⁄⁄1 pup has a region deletion allele and double deletion allele with mosaicism, and the other pup has a double deletion allele and single deletion allele.

Article Snippet: According to the sequence of Cas9D10A [9], Cas9 nickase was constructed by mutation PCR of pCAG-T3-hCAS9-pA plasmid vector (Addgene plasmid 48625) [6] using the primers (Forward primer, 50-AGTACTCCATTGGGCTCGccATCGGCACAAAC, and Reverse primer, 50-CGAGCCCAATGGAGTACTTCTTGTCGGCTGC).

Techniques: Injection, Sequencing, Mutagenesis

Journal: Biochemical and biophysical research communications

Article Title: Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system.

doi: 10.1016/j.bbrc.2014.01.141

Figure Lengend Snippet: Fig. 2. Generation of knock-in mice by the offset-nicking method. (A) Schematic illustration of the Hprt gene structures, sequences around the target loci and ssODN template structure. The Target sequences, PAM domain and PCR primer loci are indicated by arrows, underlining and white arrowheads, respectively. The red arrowheads, blue arrowhead and blue box show break sites by Cas9 nickase, the FLAG-sequence insertion site and the FLAG-sequence in the ssODN template, respectively. The distances between two break sites are indicated. (B) An example of a genome sequence of a knock-in pup. A waveform data of a DNA sequencer obtained from PCR fragment of tail tip DNA and predicted sequence are shown with target positions, FLAG sequence and start codon. (C) Genome sequences around the target sites of pups obtained from the second experiment, in which the offset-nicking method was used to generate knock-in mice. The hyphens denote deleted nucleotides. F: female; M: male; KI: Knock-in.

Article Snippet: According to the sequence of Cas9D10A [9], Cas9 nickase was constructed by mutation PCR of pCAG-T3-hCAS9-pA plasmid vector (Addgene plasmid 48625) [6] using the primers (Forward primer, 50-AGTACTCCATTGGGCTCGccATCGGCACAAAC, and Reverse primer, 50-CGAGCCCAATGGAGTACTTCTTGTCGGCTGC).

Techniques: Knock-In, Sequencing