Journal: bioRxiv

Article Title: Guide-specific loss of efficiency and off-target reduction with Cas9 variants

doi: 10.1101/2023.03.16.532856

Figure Lengend Snippet: (A, B) Left: scatter plot showing the categorization of sgRNAs into three groups: “Inefficient” (grey), “Both efficient” (blue), and “WT efficient only” (red), based on the screens with (A) HiFi and (B) LZ3. Right: the log odds ratios of nucleotide frequency between “Both efficient” and “WT efficient only” groups. The data were retrieved by combining all sgRNAs targeting essential genes in the EpiC and Tiling libraries. (C) Left: scatter plot showing the three sgRNA groups defined based on the indel rates in a published dataset using WT SpCas9 and the Cas9 variant HF1 . Right: the log odds ratios of nucleotide frequency between the groups of “Both efficient” and “WT efficient only”. (D) The KL divergence representing the significance of nucleotide difference between the “WT efficient only” and “Both efficient” groups at each position of the spacer. The KL divergence was averaged among the variants HiFi, LZ3, and HF1. (E) Upper: the point mutations (red asterisks) introduced to HiFi, LZ3 and HF1. Bottom: the structural representation of local interactions between the RNA/DNA heteroduplex and REC3 domain of WT SpCas9 (PDB: 7S4U). The positions 9-11 interacting with residues are marked in blue, the positions 15-17 interacting with residues are marked in red, and the residues mutated in any of the three Cas9 variants are marked with asterisks.

Article Snippet: Proteins were separated on 4-15% gradient gels (Bio-Rad) and transferred onto 0.45-μM PVDF membranes, followed by blocking with 5% non-fat milk and incubation with primary antibody against Cas9 (CST, #14697, 1:2,000), YAP1 (CST, #14074, 1:2,000), MTAP (CST, #4158, 1:2,000) or β-actin (Sigma, #A5316, 1:10,000).

Techniques: Variant Assay

Journal: bioRxiv

Article Title: Guide-specific loss of efficiency and off-target reduction with Cas9 variants

doi: 10.1101/2023.03.16.532856

Figure Lengend Snippet: (A) Schematic plot of the synthetic dual-target system for the evaluation of off-target effects for a specific sgRNA-target pair. (B) Boxplot comparing the off-on ratios of sgRNA-target pairs harboring 1-, 2-, or 3-mismatch (MM) among the screens with WT SpCas9, HiFi and LZ3. (C) Pairwise comparison of the off-target rates of sgRNA-target pairs among the screens with WT SpCas9, HiFi and LZ3. The consistency of off-target effects is measured in Pearson correlation. (D) Scatter plot comparing the off-on ratios of all the 1-MM sgRNA-target pairs between WT SpCas9 and the Cas9 variants (VT: average off-on ratio of HiFi and LZ3). The sgRNA-target pairs are assigned to three groups based on the off-target effects with WT SpCas9 and the degree of off-target reduction by the variants. (E) Boxplot showing the variant-mediated off-target reduction at each mismatch position measured as the ratio of off-target effects (off-on ratio) between the variants and WT SpCas9 (VT/WT ratio). The p -value is calculated using the two-tailed Manny-Whitney U test. (F) The median VT/WT ratios corresponding to different mismatch contexts between the sgRNA and the target DNA. (G) Comparison of VT/WT ratios at indicated percentile of expected energy barriers caused by the mismatches during R-loop formation. (H) Scatter plot showing the association between the energy barrier caused by a specific mismatch type and the median VT/WT ratio for that mismatch type. rXdY refers to mismatch type where the sgRNA sequence is X and the target DNA sequence is Y.

Article Snippet: Proteins were separated on 4-15% gradient gels (Bio-Rad) and transferred onto 0.45-μM PVDF membranes, followed by blocking with 5% non-fat milk and incubation with primary antibody against Cas9 (CST, #14697, 1:2,000), YAP1 (CST, #14074, 1:2,000), MTAP (CST, #4158, 1:2,000) or β-actin (Sigma, #A5316, 1:10,000).

Techniques: Variant Assay, Two Tailed Test, Sequencing

Journal: Scientific Reports

Article Title: CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death

doi: 10.1038/s41598-023-31141-6

Figure Lengend Snippet: CRISPR-Cas9 genetic screen identifies genes that attenuate Tun-mediated neurodegeneration. ( A ) Cas9 gene was inserted upstream of the endogenous GAPDH. ( B ) Cas9 expression was detected on d4 neurons. GAPDH and B-Actin are shown as controls. Original blots are presented in Supplementary Fig. . ( C ) Diagram showing the strategy for the screen. ( D ) Transduction efficiency of the three replicates. ( E ) Viability of Tun-treated samples (100 nM Tun for 7 days) was about 70%, as judged by quantifying the total DNA of the samples. ( F ) Editing efficiency of iNeurons-Cas9. ( G ) Gene hits ranked based on their p-value. Red line indicates p-value of 0.05. Data are shown as mean ± SEM, n = 3.

Article Snippet: PVDF membranes were incubated overnight with 1/1000 Cas9 (#14697, Cell Signalling) , 1/2000 B-Actin (#ab8227, Abcam) , 1/10,000 GAPDH (Abcam, ab128915) , 1/100 KAT2B (#sc-13124, Santa Cruz Biotechnology) , followed by 1 h incubation with HRP-conjugated secondary antibodies.

Techniques: CRISPR, Expressing, Transduction

Journal: World Journal of Gastroenterology

Article Title: Stress granules inhibit endoplasmic reticulum stress-mediated apoptosis during hypoxia-induced injury in acute liver failure

doi: 10.3748/wjg.v29.i8.1315

Figure Lengend Snippet: Changes of stress granules, endoplasmic reticulum stress and apoptosis levels with the change of hypoxia time. A and B: The level of apoptosis was detected by flow cytometry; C and D: The detection kits were used to evaluate the content of HIF-1α in homogenates, lactate dehydrogenase in cell supernatant; E: The expression of G3BP1 in cells was detected by immunofluorescence; F: The expression of TIA-1 in cells was detected by immunofluorescence; G: The histogram displays the amount of molecules expression; H-J: The protein expression levels of G3BP1, TIA-1, ATF4, CHOP, BAX, BCL2, c-cas3 and c-cas9 was detected by western blot. n = 3. Data was shown mean ± SD. a P < 0.05 vs 0 h group; b P < 0.05 vs 4 h group; c P < 0.05 vs 8 h group.

Article Snippet: Rabbit anti-human/ mouse cleaved caspase3 (c-cas3), c-cas9 antibodies were purchased from Cell Signaling Technology (Boston, United States).

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Western Blot

Journal: World Journal of Gastroenterology

Article Title: Stress granules inhibit endoplasmic reticulum stress-mediated apoptosis during hypoxia-induced injury in acute liver failure

doi: 10.3748/wjg.v29.i8.1315

Figure Lengend Snippet: Effects of stress granules agonist on endoplasmic reticulum stress and apoptosis in hepatocyte hypoxia model. A and B: The level of apoptosis was detected by flow cytometry; C and D: The detection kits were used to evaluate the content of HIF-1α in homogenates, lactate dehydrogenase in cell supernatant; E: The expression of G3BP1 in cells was detected by immunofluorescence; F: The expression of TIA-1 in cells was detected by immunofluorescence; G: The histogram displays the amount of molecules expression; H-J: The protein expression levels of G3BP1, TIA-1, ATF4, CHOP, BAX, BCL2, c-cas3 and c-cas9 was detected by western blot. n = 3. Data was shown mean ± SD. a P < 0.05 vs normal group; b P < 0.05 vs hypoxia group.

Article Snippet: Rabbit anti-human/ mouse cleaved caspase3 (c-cas3), c-cas9 antibodies were purchased from Cell Signaling Technology (Boston, United States).

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Western Blot

Journal: World Journal of Gastroenterology

Article Title: Stress granules inhibit endoplasmic reticulum stress-mediated apoptosis during hypoxia-induced injury in acute liver failure

doi: 10.3748/wjg.v29.i8.1315

Figure Lengend Snippet: Effect of stress granules on G3BP1 and endoplasmic reticulum stress related molecules in hepatocyte hypoxia model through endoplasmic reticulum stress. A: The expression of G3BP1 in cells was detected by immunofluorescence; B: The expression of TIA-1 in cells was detected by immunofluorescence; C: The histogram displays the amount of molecules expression; D-F: The protein expression levels of G3BP1, TIA-1, ATF4, CHOP, BAX, BCL2, c-cas3 and c-cas9 was detected by western blot. n = 3. a P < 0.05 vs normal group; b P < 0.05 vs hypoxia group; c P < 0.05 vs hypoxia + siRNA-ATF4.

Article Snippet: Rabbit anti-human/ mouse cleaved caspase3 (c-cas3), c-cas9 antibodies were purchased from Cell Signaling Technology (Boston, United States).

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: World Journal of Gastroenterology

Article Title: Stress granules inhibit endoplasmic reticulum stress-mediated apoptosis during hypoxia-induced injury in acute liver failure

doi: 10.3748/wjg.v29.i8.1315

Figure Lengend Snippet: Effect of stress granules agonist on G3BP1, HIF-1α, lactate dehydrogenase and endoplasmic reticulum stress related molecules in acute liver failure mice liver. A: The expression of G3BP1 and TIA-1 in liver was detected by immunofluorescence; B: The histogram displays the amount of molecules expression; C and D: The detection kits were used to evaluate the content of HIF-1α in liver tissue homogenate, lactate dehydrogenase in serum; E-G: The protein expression levels of G3BP1, TIA-1, ATF4, CHOP, BAX, BCL2, c-cas3 and c-cas9 was detected by western blot. n = 10. Data was shown mean ± SD. a P < 0.05 vs normal group; b P < 0.05 vs model group.

Article Snippet: Rabbit anti-human/ mouse cleaved caspase3 (c-cas3), c-cas9 antibodies were purchased from Cell Signaling Technology (Boston, United States).

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: bioRxiv

Article Title: Targeting DNA2 Overcomes Metabolic Reprogramming in Multiple Myeloma

doi: 10.1101/2023.02.22.529457

Figure Lengend Snippet: (A) Schematic of the CRISPR/Cas9 screening. Stable Cas9 + JJN3 or Cas9 + KMS11 cells were transduced with a library of pooled sgRNAs targeting 196 genes involved in several DNA repair pathways. A portion of cells was collected as a reference sample after 48 hours of transduction. Cells were continuously cultured under puromycin selection and treated with NT or ILF2 ASOs for 3 weeks. ILF2 sensitizer genes were identified using deep sequencing of the sgRNA barcodes and the drugZ algorithm to assess differences in the representation of all sgRNAs between NT ASO– and ILF2 ASO–treated cells across the 3 independent sets of experiments. NGS, next-generation sequencing. (B) Ranking of the DNA repair genes whose sgRNAs were significantly depleted in ILF2 ASO–treated JJN3 cells as compared with NT ASO–treated cells. The inset shows genes on the top ranks (adjusted P <0.01). (C) Western blot analysis of DNA2 in whole-cell lysates (W), nuclei (N), and mitochondria (M) isolated from JJN3 cells. Vinculin, Lamin A, and COX IV were used as the loading controls for W, N, and M, respectively. (D) Kaplan–Meier plots of progression-free survival (PFS) according to DNA2 expression in MM PCs as evaluated by microarray analysis. Shown are the median progression-free survival durations of patients who were enrolled in the Arkansas Total Therapy 2 trial and received high-dose chemotherapy followed by stem cell transplantation (n=256; P= 0.0126). (E) Frequencies of apoptotic JJN3 cells after 3 weeks of exposure to NT or ILF2 ASOs followed by 48 hours of treatment with vehicle (Veh) or NSC at 2 μM. Data are expressed as the mean ± S.D. from one representative experiment performed in triplicate. Statistically significant differences were detected using one-way ANOVA (**** P <0.0001; *** P <0.001). (F) Representative Western blot analysis of ILF2, cleaved caspase 3, and γH2AX in JJN3 cells treated with NT or ILF2 ASOs for 3 weeks prior to receiving NT or ILF2 ASOs alone (Veh) or in combination with 1 μM NSC for 48 hours. Vinculin was used as a loading control. (G) Differences in the luciferase signal in NSG mice engrafted with ILF2 ASO–resistant GFP + Luc + JJN3 cells after receiving NT or ILF2 ASOs alone (NT or ILF2+Veh) or in combination with NSC every day for 7 days. Data are expressed as the mean bioluminescence activity relative to that of the NT ASOs+Veh group [Δ flux of luciferase signal (photons/second, p/s] ± S.D. for each mouse (NT ASOs+Veh, n=22; NT ASOs+NSC, n=15; ILF2 ASOs+Veh, n=19; ILF2 ASOs+NSC, n=11; n=3 independent experiments). Statistically significant differences were detected using one-way ANOVA (** P <0.01; * P <0.05).

Article Snippet: The primary antibodies anti-ILF2/NF45 (Santa Cruz, sc365068), anti-vinculin (Sigma, V9131), anti-γH2AX (Cell Signaling, 2577S), anti-cleaved caspase 3 (Cell Signaling, 966S), and anti-Cas9 (Cell Signaling, 14697S), in addition to secondary anti-mouse and anti-rabbit digital antibodies (Kindle Biosciences LLP), were used.

Techniques: CRISPR, Transduction, Cell Culture, Selection, Sequencing, Next-Generation Sequencing, Western Blot, Isolation, Expressing, Microarray, Transplantation Assay, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Targeting DNA2 Overcomes Metabolic Reprogramming in Multiple Myeloma

doi: 10.1101/2023.02.22.529457

Figure Lengend Snippet: (A) Western blot analysis of ILF2, γH2AX, cleaved caspase 3, and Cas9 in NT ASO– or ILF2 ASO–treated KMS11 (left) and JJN3 (right) cells after 3 weeks of culture. The 3 biological replicates from the experiment described in are shown (#1-3). Vinculin was used as a loading control. (B) Correlation of the sgRNAs’ gene-level log 2 fold changes in KMS11 (left) and JJN3 (right) cells among the 3 independent sets of experiments. (C) Density functions of gene-level log 2 fold changes (FC) of essential and non-essential genes in KMS11 (left) or JJN3 (right) samples collected after 3 weeks of NT (top) or ILF2 (bottom) ASO treatment. (D) Ranking of DNA repair genes whose sgRNAs were significantly depleted in ILF2 ASO–treated KMS11 cells as compared with NT ASO–treated cells. The inset shows genes on the top ranks (adjusted P <0.01). (E) Representative immunofluorescence images of DNA2 in JJN3 cells. Image was captured and processed using a Delta Vision OMX V4 Blaze Super-Resolution System. Green indicates DNA2; red, TOM20 (mitochondrial marker); and blue, DAPI. Scale bars represent 5 μm. (F) Violin plot of DNA2 expression in the PCs of newly diagnosed MM patients (n=543). Samples were divided into 2 groups (with or without the 1q21 amplification). The lines inside each violin plot define the 4 quartiles of DNA2 expression. Statistically significant differences were detected using a 2-tailed Student t-test (**** P <0.0001). (G) Kaplan–Meier plots of progression-free survival (PFS) according to DNA2 expression in MM PCs. Shown are the median progression-free survival durations of patients who received PIs alone (n=129; left; P= 0.0182); patients who received PIs in combination with other therapies (n=326; middle; P< 0.0001); and patients who received immunomodulatory drugs (n=37; right; P= 0.5682). (H) Frequencies of live JJN3 cells after treatment with NSC at the indicated concentrations for 24, 48, and 72 hours. Data from one representative experiment performed in triplicate are expressed as the mean frequencies ± S.D. of live cells among all cells at each timepoint. (I) Left, frequencies of apoptotic JJN3 cells after 3 weeks of NT ASO or ILF2 ASO exposure followed by 48 hours of treatment with vehicle (Veh) or the DNA2 inhibitor C5 (C5) at the indicated concentrations. Data are expressed as the mean ± S.D. from one representative experiment. Statistically significant differences were detected using one-way ANOVA (**** P < 0.0001; * P <0.05). Right, representative Western blot analysis of ILF2 and cleaved caspase 3 in JJN3 cells treated with NT or ILF2 ASOs alone or in combination with C5 at the indicated concentrations for 48 hours. Vinculin was used as a loading control. (J) Schematic of ASO and NSC treatments in MM xenografts. ILF2 ASO–resistant GFP + Luc + JJN3 cells (1 x 10 6 ) were injected into NSG mice. Ten days after transplantation, mice were injected with luciferin, and tumor burden was quantified using the IVIS Spectrum bioluminescence imaging system. Mice were randomized into 4 groups based on tumor burden on day 0. Mice were injected with NT or ILF2 ASOs alone (25 mg/kg) or in combination with NSC (10 mg/kg) every day for 7 days. Tumor burden was evaluated by bioluminescence imaging on days 0 and 7.

Article Snippet: The primary antibodies anti-ILF2/NF45 (Santa Cruz, sc365068), anti-vinculin (Sigma, V9131), anti-γH2AX (Cell Signaling, 2577S), anti-cleaved caspase 3 (Cell Signaling, 966S), and anti-Cas9 (Cell Signaling, 14697S), in addition to secondary anti-mouse and anti-rabbit digital antibodies (Kindle Biosciences LLP), were used.

Techniques: Western Blot, Immunofluorescence, Marker, Expressing, Amplification, Injection, Transplantation Assay, Imaging

Journal: Cell reports

Article Title: Macrophage NFATC2 mediates angiogenic signaling during mycobacterial infection

doi: 10.1016/j.celrep.2022.111817

Figure Lengend Snippet: (A) Immunofluorescence staining of THP-1 macrophages in the presence or absence of γ Mtb to identify potential NFAT isoforms of interest in human cells. NFATC2 is most robustly expressed and inducibly nuclear of these isoforms in macrophages. NFATC1 and NFATC3 are comparatively less expressed and less extensively translocated by 8 h post exposure. NFATC4 is very lowly expressed initially but, like all the isoforms, appears to be upregulated at the protein level after γ Mtb exposure. Initial staining from a single experiment, with NFATC2 validation from three biological replicates in (D–G). (B) Magnified images showing robust VEGFA expression in cells with nuclear NFATC2 compared with cells with nuclear NFATC1, NFATC3, and NFATC4, which lack the strong correspondence between NFAT nuclear localization and VEGFA induction, which is only seen with NFATC2 staining. (C) Blinded quantitation of the relationship between each NFAT isoform and the induction of VEGFA. We calculated the subset of cells expressing both VEGFA and demonstrating obvious NFAT nuclear localization and normalized to the total number of cells expressing VEGFA in that field. Initial staining from a single experiment, with validation in (D–G). Total number of cells counted = 5,859. Statistics from ANOVA with Tukey honest significant differences test. (D) THP-1 cells transduced with Cas9-expressing lentiviruses targeting either NFATC2 ( and ) or safe-targeting loci were selected with puromycin and then treated with γ Mtb or vehicle. Safe-targeting-transduced THP-1 cells robustly responded to γ Mtb with VEGFA production at 8 h post exposure, but fewer NFATC2-transduced cells produce VEGFA after stimulation and at lower staining intensity. Representative of three biological replicates. (E) Magnified images showing high VEGFA induction in γ Mtb -treated NFATC2-targeted THP-1 cells compared with γ Mtb -treated safe targeting (ST) THP-1 cells. The ST cells show robust VEGFA induction and NFAT nuclear translocation while NFATC2 cells show diminished VEGFA induction and disordered nuclear translocation and occasional lack of NFATC2 staining entirely; the antibody used to detect NFATC2 is N-terminal to the sgRNA sites, so residual expression is likely captured by the antibody. Representative of three biological replicates. (F) Blinded quantitation of the percentage of VEGFA + cells within entire images for (D and E). Each data point represents the percentage of VEGFA + cells in the field; 15 total fields were counted for each groups (5 from each of 3 independent experiments). Total number of cells counted = 5,029. Statistics from ANOVA with Tukey honest significant differences test. (G) Blinded quantitation of the percentage of VEGFA + and nuclear-localized NFAT out of the total number of cells with NFAT nuclear localization within entire images for (D) and (E). Fifteen total fields were counted for each groups (5 from each of 3 independent experiments). Total number of cells counted = 5,029. Statistics from ANOVA with Tukey honest significant differences test. (H) Blinded quantitation of the percentage of VEGFA + and nuclear-localized NFAT of the total number of cells in the images for (D) and (E). Fifteen total fields were counted for each group (5 from each of 3 independent experiments). Total number of cells counted = 5,029. Statistics from ANOVA with Tukey honest significant differences test.

Article Snippet: monoclonal mouse anti-Cas9 antibody , Cell Signaling , Cat# 7A9-3A3.

Techniques: Immunofluorescence, Staining, Expressing, Quantitation Assay, Transduction, Translocation Assay

Journal: Cell reports

Article Title: Macrophage NFATC2 mediates angiogenic signaling during mycobacterial infection

doi: 10.1016/j.celrep.2022.111817

Figure Lengend Snippet:

Article Snippet: monoclonal mouse anti-Cas9 antibody , Cell Signaling , Cat# 7A9-3A3.

Techniques: Recombinant, Cell Culture, Gas Phase Electrophoretic Molecular Mobility Analysis, Enzyme-linked Immunosorbent Assay, Derivative Assay, Software, Real-time Polymerase Chain Reaction

Journal: Genes

Article Title: A Tet-Inducible CRISPR Platform for High-Fidelity Editing of Human Pluripotent Stem Cells

doi: 10.3390/genes13122363

Figure Lengend Snippet: Development of Tet-inducible high-fidelity HF-iCas9 expressing cell lines. A platform for gene editing was devised by generating HF-iCas9 hPSC lines that incorporated a ( A ) plasmid carrying a U6 promoter for transient expression of an AAVS1 guide RNA (gRNA) and eSpCas9, and plasmids for biallelic integration of Tet-inducible eSpCas9 and constitutive reverse tetracycline-controlled trans-activator (rtTA) both at separate alleles at the AAVS1 SHS. A promoter-less splice acceptor (SA) site linked to the p2A sequence allows for the integration of the cassette within the intron of AAVS1 reducing chances of random integration. ( B ) A diagram highlighting the 3G Tet-On system for inducible HF-iCas9 that is activated upon binding of rtTA/dox to a tetracycline response element (TRE). ( C ) Assessment of the influence of dox concentration on cell proliferation. ( D1 , D2 ) Normalized relative (top) and absolute (bottom) expression of Cas9 measured by qRT-PCR after 0 to 3 days of dox (1 μg/mL). At day 0, the expression of Cas9 was undetectable. ( E – H ) Immunostaining against Cas9 in PSCs grown in the absence ( E , F ) or presence ( G , H ) of dox. Nuclei visualized with DAPI alone ( E , G ) or together with Cas9 ( F , H ). One-way ANOVA was used to test statistical significance and for multiple comparison tests, a Holm-Šídák’s multiple comparison was used with an α cutoff of 0.05. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant. Scale bar ( E – H ) = 200 µm. HA = Homology arm.

Article Snippet: A mouse Cas9-specific monoclonal antibody (clone 7A9-3A3 #14697; Cell Signaling Technology, Danvers, MN, USA) was diluted at 1:800 in PBS containing 2% horse serum and 0.1% Triton X-100 and incubated overnight at 4 °C.

Techniques: Expressing, Plasmid Preparation, Sequencing, Binding Assay, Concentration Assay, Quantitative RT-PCR, Immunostaining

Journal: Genes

Article Title: A Tet-Inducible CRISPR Platform for High-Fidelity Editing of Human Pluripotent Stem Cells

doi: 10.3390/genes13122363

Figure Lengend Snippet: Donor plasmid optimization for gene-editing by homology-directed repair. ( A ) Different DNA tagging sequences (DTS) were inserted into the SOX2-GFP donor plasmids. ( B ) The role of SV40 and HRE DTS sequences on SOX2-GFP integration was assessed by measuring the number of GFP+ cells relative to DAPI+ total cells. ( C ) Three different guide RNA scaffolds based on Ran et al. (2013) (V1), Dang et al. (2015) (V2) and Chen et al. (2013) (V3) illustrate the variation in the first stem-loop structure that interacts with Cas9. ( D ) Quantification of GFP+ cells after editing with SOX2-GFP donor plasmids harboring HRE and SV40 DTSs and the V1-3 scaffolds. ( E ) Illustration of SOX2-GFP donor fragment double cutter (dc) linearization by the introduction of SOX2 targeting sites at the distal ends of the homology arms. ( F ) Quantification of the effect of donor fragment linearization on integration in the presence of SV40 or HRE DTS fragments. ( G ) Illustration of a standard parental plasmid (PP) and a minicircle plasmid (MC) with bacterial sequence elements removed. ( H ) Agarose gel electrophoresis of Kpn1 digested PP and MC plasmids in the absence (−) or presence (+) of arabinose. ( I ) Comparison between PP and MC plasmids for SOX2-GFP integration. One-way ANOVA was used to test statistical significance and for multiple comparison tests, a Dunnett’s multiple comparison was used with an α cutoff of 0.05. * p < 0.05, ns = not significant.

Article Snippet: A mouse Cas9-specific monoclonal antibody (clone 7A9-3A3 #14697; Cell Signaling Technology, Danvers, MN, USA) was diluted at 1:800 in PBS containing 2% horse serum and 0.1% Triton X-100 and incubated overnight at 4 °C.

Techniques: Plasmid Preparation, Sequencing, Agarose Gel Electrophoresis