Journal: Genes

Article Title: Genetic Deletion of DNAJB3 Using CRISPR-Cas9, Produced Discordant Phenotypes.

doi: 10.3390/genes14101857

Figure Lengend Snippet: Figure 2. Genotyping strategy. (A) Gel image of genotyping PCR products: the band with 366 bp indicates the Wild type (Wt) genotype, while bands shorter than 366 bp show deletions in the targeted region of the DNAjb3 gene, thus indicating a knockout genotype. The 100 bp DNA ladder (New England BioLabs, Ipswich, MA) was used in the gel image (1000 bp band in the ladder is marked). Numbers on top of the lines indicate strain/KO number, of which we focused on KO 30, 44 and 47. (B) The deletions were confirmed by sequencing of the PCR products in three KO lines #30, 44, 47: targeted sequences are marked by red; deleted sequences within the targeted region are underlined and italic; the initiation code ATG is marked by green; and a binding signal for Cas9 nuclease (TGG as a PAM sequence) is marked by light blue. (C) The deletions in DNA sequences (upper rows) resulted in changes in amino acid sequences (lower rows) in three KO lines: the sequence of sgRNA is shown in light blue; the target sequence in DNAJB3 in WT genotype is shown in red; and deleted sequences in all three lines are marked by dotted lines.

Article Snippet: Briefly, three pairs of optimal sgRNA oligos were designed by scanning the target sequence close to start codon with CRISPR Design (http://crispr.mit.edu/, ac- cessed on 9 July 2017) and cloned into Cas9-expression vector pX459-v2 (Addgene); the target sequence was used for the Dnajb3 gene (Gene ID: MGI:1306822).

Techniques: Knock-Out, Sequencing, Binding Assay

Journal: Genes

Article Title: Genetic Deletion of DNAJB3 Using CRISPR-Cas9, Produced Discordant Phenotypes.

doi: 10.3390/genes14101857

Figure Lengend Snippet: Figure 3. Validation of absence of DNAJB3 in protein samples from testes of knockout strains. (A) Western results demonstrated control protein bands for Tubulin at (52 kDa) for all 3 lines including 30, 44, 47 and WT. (B) DNAJB3 band was only seen in WT lane but not in KOs.

Article Snippet: Briefly, three pairs of optimal sgRNA oligos were designed by scanning the target sequence close to start codon with CRISPR Design (http://crispr.mit.edu/, ac- cessed on 9 July 2017) and cloned into Cas9-expression vector pX459-v2 (Addgene); the target sequence was used for the Dnajb3 gene (Gene ID: MGI:1306822).

Techniques: Biomarker Discovery, Knock-Out, Western Blot, Control