CRISPR-Cas9 Antibody

Western Blot: CRISPR-Cas9 Antibody [NBP2-52717] -

Western Blot: CRISPR-Cas9 Antibody [NBP2-52717] - The expression of CAS9 in hiPSCs & hESCs promotes DNA DSB damage. (A) The inducible expression of CAS9 promotes DNA DSB damage responses in hiPSCs after 2 µg/mL Doxy treatment. The relative levels of the phosphorylation of p53 & H2AX are indicated at the bottom. Consistent data were obtained from two independent experiments. (B) The impact of expression levels of CAS9 on DNA DSB damage in hESCs. At the same lentiviral titers, the expression levels of CAS9 in hESCs transduced by standard lentiviral vector are higher than those transduced by the inducible lentiviral vector after 2 µg/mL Doxy treatment. Much lower expression levels of CAS9 can also promote DNA DSB damage in hESCs after the treatment with lower dosages of Doxy. The relative levels of the phosphorylation of p53 & H2AX are indicated at the bottom Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/32170574), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: CRISPR-Cas9 Antibody [NBP2-52717] -

Western Blot: CRISPR-Cas9 Antibody [NBP2-52717] - dCAS9 & Cpf1 impair NHEJ & induce genetic mutations. (A) Co-immunoprecipitation assay confirmed the interaction between dCAS9 & KU86. (B) Comet assay analysis of DNA damage in hESCs expressing dCAS9 or treated with doxorubicin. CTL, human fibroblasts with lentiviral empty vector were treated with 2 µg/mL doxycycline for three days; Doxy, Dox, Doxy + Dox, human fibroblasts with lentiviral CAS9 inducible expression vector were treated with 2 µg/mL doxycycline for 3 days or 0.5 µmol/L Dox for 2 h or 2 µg/mL doxycycline for three days + 0.5 µmol/L Dox for 2 h, respectively. Tail length was analyzed using Image J software. Data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (C) The expression of dCAS9 induces mutation of endogenous HPRT gene. WT, WT hESCs; CTL, CAS9, dCAS9, hESCs with empty expression vector, CAS9 inducible expression vector. Cells with dCAS9 inducible expression vector were treated with 2 µg/mL doxycycline for 3 days before HAT treatment. n = 3. Data are presented as mean value ± SD. **P < 0.01, ***P < 0.001. (D) The expression of Cpf1 increased the levels of gamma H2AX. (E) Cpf1 interacts with KU86 as confirmed by Co-immunoprecipitation. Protein extract of Flag-tagged Cpf1 was immunoprecipitated with anti-Flag antibody & the presence of Cpf1 & KU86 in the immunoprecipitate was examined by Western blot Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/32170574), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: CRISPR-Cas9 Antibody [NBP2-52717] -

Western Blot: CRISPR-Cas9 Antibody [NBP2-52717] - Both CAS9 & XCAS9 impair NHEJ & induce genetic mutations. (A & B) Expression of CAS9 & XCAS9 impairs NHEJ. Traffic Light Reporter system was established in 293 cells harboring the CAS9 & XCAS9 inducible expression vectors. After the induction of CAS9 & XCAS9 expression with 2 µg/mL doxycycline for 3 days (left panel), the efficiency of NHEJ (mcherry) & HDR (GFP) was analyzed by flow cytometry (middle panel). Statistic analysis of the efficiency of NHEJ (left panel of A) & HDR (B) is presented. n = 3. Data are presented as mean values ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. ns, non-significant. (C) The expression of CAS9 in hESCs induces genomic mutations at the endogenous HPRT locus. After hESCs harboring CAS9 inducible expression vector were selected with HAT medium for 5 days, they were treated with 2 µg/mL doxycycline for CAS9 expression for various time periods, & subsequently, treated with 5 µg/mL 6-TG or mock treated for 4 days. Mutational rate is calculated as the ratio of colony number in 6-TG treated samples versus untreated controls. n = 3. Data are presented as mean values ± SD. ***P < 0.001. (D) XCAS9 interacts with KU86. Protein extracts from 293FT cells expressing Flag-tagged CAS9 or XCAS9 were immunoprecipitated with anti-Flag antibody. The immune precipitates were analyzed for the presence of CAS9, XCAS9 & KU86. The relative ratio of KU86 versus CAS9 or XCAS9 is indicated. (E) The expression of XCAS9 increases the number of gamma H2AX foci in hESCs. hESCs harboring XCAS9 inducible expression vector were treated with or without 2 µg/mL doxycycline for 3 days. n = 20. Scale bar, 10 µm. Data are presented as mean values ± SD. ***P < 0.01 Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/32170574), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: CRISPR-Cas9 Antibody [NBP2-52717] -

Western Blot: CRISPR-Cas9 Antibody [NBP2-52717] - The expression of CAS9 in human fibroblasts promotes DNA DSB damage & activates DNA damage response pathways. (A) The expression of CAS9 in human fibroblasts activates DNA damage responses. The expression of CAS9 was induced with 2 µg/mL Doxy treatment. The relative levels of phosphorylation of BRCA1, CHK1, CHK2 & p53 are indicated at the bottom. n = 3. Data are presented as mean value ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (B) The expression of CAS9 increased the number of gamma H2AX foci in human fibroblasts. CTL or CAS9, human fibroblasts with CAS9 inducible expression cassette plated on chamber slides were treated with or without 2 µg/mL doxycycline for 3 days. The expression of CAS9 & gamma H2AX foci was revealed by immunoflourescence analysis. Representative images are shown. Scale bar, 10 µm. Unpaired t test. n = 20. Data are presented as mean values ± SD. ***P < 0.001. (C) CAS9 induces DNA DSB damage in human fibroblasts. CTL, human fibroblasts with lentiviral empty vector were treated with 2 µg/mL doxycycline for three days; Doxy, Dox, Doxy + Dox, human fibroblasts with lentiviral CAS9 inducible expression vector were treated with 2 µg/mL doxycycline for 3 days or 0.5 µmol/L Dox for 2 h or 2 µg/mL doxycycline for three days + 0.5 µmol/L Dox for 2 h, respectively. Representative images are shown. n = 40. Unpaired t test. Data are presented as mean value ± SD. **P < 0.01 Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/32170574), licensed under a CC-BY license. Not internally tested by Novus Biologicals.