Journal: iScience

Article Title: Mitochondrial succinate dehydrogenase function is essential for sperm motility and male fertility

doi: 10.1016/j.isci.2022.105573

Figure Lengend Snippet:

Article Snippet: Cas9 nuclease included a C-terminal SV40-NLS and 6xHis tag, and was expressed from the plasmid pHO4d-Cas9 (Addgene plasmid #67881, a gift from Michael Nonet ), and produced by Protein Production and Characterisation at Sydney Analytical (University of Sydney). crRNAs were designed using the IDT Custom Alt-R® CRISPR-Cas9 guide RNA design tool https://sg.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM . crRNAs were selected based on proximity to the edit site, presence of 3′ GG(NGG) or G(NGG), and IDT on-target and off-target score.

Techniques: Virus, Clone Assay, Recombinant, Sample Prep, Gene Knockout, CRISPR, Derivative Assay, Sequencing, Plasmid Preparation, Software

Journal: Science Advances

Article Title: Senataxin and DNA-PKcs redundantly promote non-homologous end joining repair of DNA double strand breaks during V(D)J recombination

doi: 10.1126/sciadv.ads5272

Figure Lengend Snippet: ( A ) Schematic of the retroviral V(D)J recombination substrate pMG-INV. The open and filled triangles represent the RSSs. The retroviral LTRs, CEs, and SEs upon RAG cleavage and CJs and SJs after NHEJ-mediated repair are indicated. The anti-sense GFP cDNA is inverted to the sense orientation upon completion of the recombination and its expression driven by the retroviral LTR (green). ( B ) Schematic diagram of a genome-wide CRISPR-Cas9 screen in WT abl pre-B cells for identifying genes important for V(D)J recombination upon DNA-PKcs inhibition with NU7441. ( C ) Fold enrichments of selected gRNAs from screens conducted in DMSO and NU7441-treated WT abl pre-B cells (both also treated with imatinib). The fold enrichment is calculated as the ratio of the normalized read counts of a gRNA from GFP − cells over that from GFP + cells. Asterisk denotes (*) five gRNAs to each gene. ( D ) Flow cytometric analysis for GFP expression in WT and three independently isolated Setx −/− abl pre-B cell lines with pMG-INV and treated with imatinib (imat.) in the presence or absence of the DNA-PKcs kinase inhibitor NU7441 for the indicated times. The percentages of GFP + cells are indicated in the top left corners of the histograms.

Article Snippet: For each screen, ~180 × 10 6 cells were transduced with a lentiviral gRNA library containing 90,230 gRNAs targeting 18,424 mouse genes (Addgene, #67988) at a 40 to 50% transduction efficiency, as determined by the percentage of blue fluorescent protein (BFP)–positive cells after transduction.

Techniques: Retroviral, Expressing, Genome Wide, CRISPR, Inhibition, Isolation