Journal: Molecular Therapy. Nucleic Acids

Article Title: A dual conditional CRISPR-Cas9 system to activate gene editing and reduce off-target effects in human stem cells

doi: 10.1016/j.omtn.2022.04.013

Figure Lengend Snippet: Dual conditional CRISPR-Cas9 system reduces off-target effects (A) H9 cells were used to generate pooled clonal cells with several CRISPR-Cas9 systems: our dual conditional Cas9 system (Dual), doxycycline-inducible Cas9 system (Dox), Shield1-regulated Cas9 system (Shield1), constitutive Cas9-expressing system (Const), and Cas9 ribonucleoprotein delivery system (RNP). For the inducible systems, the cells were treated with doxycycline and/or Shield1 for 3 days to knock out p53 , EMX1 , HBB , or FANCF gene. After DNA extraction, 500- to 1,000-bp sequences that comprise the knockout site were amplified by PCR and subsequent TIDE assay was performed. The data are expressed as indel frequency (%) (0% as no editing and 100% near-complete editing); p values were calculated by comparison with the uninduced dual conditional system (−/−). (B) SW-sgGFP cells (iPSC-Cas9-sgGFP) that were seeded in six-well plates were treated with or without doxycycline and/or Shield1 ligand. The cells were collected at 24 h, and Wes assay was performed. Dual control with doxycycline and Shield1 eliminates background Cas9 expression. (C) Dual control of Cas9 expression is measured by p53 promoter reporter assay. p53 promoter reporter plasmid was transfected in SW-sgp53 cells (iPSC-Cas9-sg p53 ) after 3 days of doxycycline and Shield1 treatment. Doxycycline and Shield1 were treated throughout the experiment to induce p53 gene knockout. Twenty-four hours after p53 promoter reporter plasmid transfection, 0.4 μg/mL doxorubicin was added to induce p53 expression; 48 h after transfection, the cells were measured for luciferase activity. SW-sgGFP (iPSC-Cas9-sgGFP), SW-Cas9 clones that express sgRNA targeting GFP gene; SW-sg p53 (iPSC-Cas9-sg p53 ), SW-Cas9 clones that express sgRNA targeting p53 gene. Data are shown as mean values ±SEM of triplicate experiments; ns, not significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: The following antibodies were purchased commercially: anti-Cas9 (Cat#: NBP2-36440; Novus Biologicals, Centennial, CO, USA), anti-p53 (Cat#: NBP2-29453, Novus Biologicals), anti-PTEN (Cat#: AF847-SP, Novus Biologicals), anti-APC (Cat#: NB100-91662SS, Novus Biologicals), and anti-actin (Cat#: ab8227, Abcam, Cambridge, UK).

Techniques: CRISPR, Expressing, Knock-Out, DNA Extraction, Amplification, Comparison, Control, Reporter Assay, Plasmid Preparation, Transfection, Gene Knockout, Luciferase, Activity Assay, Clone Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: A dual conditional CRISPR-Cas9 system to activate gene editing and reduce off-target effects in human stem cells

doi: 10.1016/j.omtn.2022.04.013

Figure Lengend Snippet: Dual conditional CRISPR-Cas9 system is efficient for gene knockout (A) Using H9-Cas9 clones that express sgRNAs targeting p53 , PTEN , and APC genes, T7 endonuclease assay was performed to test knockout efficiency. Each H9-Cas9 clone was treated with doxycycline and Shield1 for 5 days to knock out p53 , PTEN , and APC , respectively. After DNA extraction, 500- to 1,000-bp sequences that comprise the knockout site were amplified by PCR, and subsequent T7 endonuclease assay was performed. (B) TIDE assay was also performed for the same cell clones. The cells were treated with doxycycline and Shield1 for 10 days followed by DNA extraction and PCR amplification for the sequences including the knockout site. (C) For the same cell clones, doxycycline and Shield1 were added for 5 or 10 days continuously. Then, Wes assay was performed after protein extraction. Wes assay showed Cas9 protein levels with or without CRISPR-Cas9-induced knockout. The assays were repeated with triplicate experiments.

Article Snippet: The following antibodies were purchased commercially: anti-Cas9 (Cat#: NBP2-36440; Novus Biologicals, Centennial, CO, USA), anti-p53 (Cat#: NBP2-29453, Novus Biologicals), anti-PTEN (Cat#: AF847-SP, Novus Biologicals), anti-APC (Cat#: NB100-91662SS, Novus Biologicals), and anti-actin (Cat#: ab8227, Abcam, Cambridge, UK).

Techniques: CRISPR, Gene Knockout, Clone Assay, Knock-Out, DNA Extraction, Amplification, Protein Extraction

Journal: Molecular Therapy. Nucleic Acids

Article Title: A dual conditional CRISPR-Cas9 system to activate gene editing and reduce off-target effects in human stem cells

doi: 10.1016/j.omtn.2022.04.013

Figure Lengend Snippet: Conditional CRISPR-Cas9 system and human stem cell hepatocyte differentiation (A) Diagram of conditional CRISPR-Cas9 system with various promoter systems. (B) H9-Cas9 clones that were replaced with EF1α and human albumin ( hALB ) promoters were differentiated into hepatocyte-like cells. Level of sgRNA targeting p53 gene was measured by RT-qPCR during hepatocyte differentiation. (C) CMV, EF1α, and hALB promoter activities in the conditional CRISPR-Cas9 system, as shown by the Nanoluciferase activities, were compared during hepatocyte differentiation of SW-Cas9 (iPSC) and H9-Cas9 (ESC) clones. Data are shown as mean values ±SEM of triplicate experiments; ns, not significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: The following antibodies were purchased commercially: anti-Cas9 (Cat#: NBP2-36440; Novus Biologicals, Centennial, CO, USA), anti-p53 (Cat#: NBP2-29453, Novus Biologicals), anti-PTEN (Cat#: AF847-SP, Novus Biologicals), anti-APC (Cat#: NB100-91662SS, Novus Biologicals), and anti-actin (Cat#: ab8227, Abcam, Cambridge, UK).

Techniques: CRISPR, Clone Assay, Quantitative RT-PCR

Journal: Investigative Ophthalmology & Visual Science

Article Title: Neuroprotection of SRT2104 in Murine Ischemia/Reperfusion Injury Through the Enhancement of Sirt1-Mediated Deacetylation

doi: 10.1167/iovs.64.4.31

Figure Lengend Snippet: Primary Antibody List

Article Snippet: p53 , Mouse , NOVUS BIOLOGICALS , nbp2-29453 , WB , 1:200.

Techniques: