alt-r hdr donor oligos  (Integrated DNA Technologies)

Journal: Human Molecular Genetics

Article Title: An enhancer variant associated with breast cancer susceptibility in Black women regulates TNFSF10 expression and antitumor immunity in triple-negative breast cancer

doi: 10.1093/hmg/ddac168

Figure Lengend Snippet: Regulation of TNFSF10 expression by rs31074711 Alleles. ( A ) Depicted is CRISPR-Cas9 genome editing of rs13074711 from TC genotype to CC genotype through HDR. DNA sequencing chromatograms showed the shift from T to C, after genome editing in HCC1806 cells. ( B ) qRT-PCR analysis of TNFSF10 mRNA in HCC1806 cells with TC or CC genotype of rs31074711 that were treated with IFN-β for 1 day. Results were normalized to RNA18S and fold induction is shown relative to the untreated control with TC genotype. ( C ) Western blot analysis of TNFSF10 protein expression in HCC1806 cells with TC or CC genotype 1-day post IFN-β treatment. Tublin was used as an endogenous loading control. ( D ) HCC1806 cells with TC or CC genotypes were treated with IFN-β and subjected to the IncuCyte Live-Cell Imaging System. Apoptosis was quantified using green fluorescent signals from caspase-3/7–positive apoptotic cells normalized to cell density. All data represent the mean and SD of n = 3–6 biological replicates and are representative of at least two independent experiments. * * * P < 0.001.

Article Snippet: For genome editing, gRNAs were designed to target the TNFSF10 enhancer region containing SNP rs13074711, which were incubated with S.p.Cas9 Nuclease V3 and transfected into TNBC cells, along with Alt-R homology-directed repair (HDR) donor oligos (IDT) for homologous recombination.

Techniques: Expressing, CRISPR, DNA Sequencing, Quantitative RT-PCR, Western Blot, Live Cell Imaging