Journal: Nature genetics

Article Title: Kmt1e regulates a large neuron-specific topological chromatin domain

doi: 10.1038/ng.3906

Figure Lengend Snippet: ( a ) cPcdh locus and surrounding sequences ~2Mb of mouse chr. 18, including TADs called (TADtree) and H3K9me3 tracks for KO and WT. Notice ‘shrinkage’ of broadly (>100–200kb) stretched ‘R1’ and ‘R2’ blocks of H3K9me3-tagged chromatin in KO neurons. ( b ) Overview on cell-type specific 3C-PCR, cropped agarose gels showing specific loop products for c Pcdh and B2m control. No lig=3C without DNA ligase, L=100bp DNA ladder. Dot graphs summarizing 3C-PCR (mean±S.E.M.; 1dot=1animal) cPcdh loop1,2,3 as indicated. All data normalized to B2m 3C, N (Loop1) =3/group, *P (Loop1) =0.05, Mann Whitney, one-tailed; N (Loop2) =4/group, *P (Loop2) =0.014,Mann Whitney, two-tailed. Loop defects in KO include A/R1(de novo CTCF peak A in R1)-HS16 and A/R1-B/R2 (de novo CTCF peak B in R2). In contrast, shorter-range Pcdha8 promoter-HS5 enhancer loop is maintained in KO neurons. Complete gels shown in . ( c ) Summary presentation of 3C-PCR. ( d ) dCas9-SunTag superactivation of HS16 cPcdh enhancer with U6-sgRNA cassette upstream of CK-dCas9-10xGCN4 epitope -BFP cassette, and CK-svFv-sGFP-VP64 cassette on separate vector. Representative FACS sort shows dually labeled BFP + GFP + NG108 cells. NC=negative control ( e ) RT-PCR quantification (mean±S.E.M.; 1dot=1cell culture or animal) of Pcdha3 , Pcdha8 , Pcdhb16 , Pcdhgb2 and Pcdhgb8 transcripts (black arrows in panel d mark genomic positions), normalized to Gapdh RNA. (Top) BFP + GFP + NG108 cells with (HS16/VP64) and without (VP64) sgRNA HS16 cassette. (Bottom) adult KO and WT PFC. N=4 VP64/3 hs16/vp64 (NG108 cells), *P ( Pcdha8 ) =0.0268, *P ( Pcdha11 ) =0.0437, *P ( Pcdhgb8 ) =0.0126, unpaired t test, one-tailed; N=6/group (mice), **P ( Pcdha3 ) =0.002, **P ( Pcdha8 ) =0.002, *P ( Pcdha11 ) =0.026, **P ( Pcdhgb8 ) =0.0022, Mann Whitney, two-tailed. See also for additional 3C-PCR loop quantifications.

Article Snippet: Stable NPC lines were validated via FACS using Cas9-AF488 antibody (Cell Signaling Technologies, 5uL/1×10 6 cells #34963S). sgRNA design and cloning: The sgRNAs were designed using the Optimized CRISPR Design tool (See URLs) at the genomic regions of interest.

Techniques: Control, MANN-WHITNEY, One-tailed Test, Two Tailed Test, Plasmid Preparation, Labeling, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: Science advances

Article Title: Codelivery of CRISPR-Cas9 and chlorin e6 for spatially controlled tumor-specific gene editing with synergistic drug effects.

doi: 10.1126/sciadv.abb4005

Figure Lengend Snippet: Fig. 1. Schematic of the preparation, delivery, and intracellular fate of NIR-sensitive and reducing agent–sensitive nanoparticles containing Nrf2-targeting Cas9/sgRNA and the antitumor photosensitizer Ce6. Nanoparticles are formed by (i) self-assembly of anionic micelles using nitrilotriacetic acid–disulfanediyldipropionate– polyethyleneglycol–b-polycaprolactone (NTA-SS-PEG-PCL) copolymer encapsulating Ce6, (ii) binding of His-tagged Cas9/sgRNA, and (iii) coating with cationic iRGD-modified copolymer. Nanoparticles bind tumor cells via integrin-iRGD binding and are internalized. Upon NIR irradiation, Ce6 generates reactive oxygen species (ROS), and nanoparticles are released from lysosomes into the cytoplasm, where a disulfide bond is reduced, releasing Cas9/sgRNA. Cas9/sgRNA targets the antioxidant gene Nrf2, enhancing tumor cell sensitivity to ROS synergistically with Ce6. PDT, photodynamic therapy.

Article Snippet: After tumor sections were fixed in 4% paraformaldehyde overnight, the levels of luciferase protein and Cas9 were measured using rabbit polyclonal anti-firefly luciferase antibody (Abcam, Cambridge, UK) and mouse monoclonal anti-Cas9 antibody (Cell Signaling Technology, Danvers, MA, USA) with CLSM.

Techniques: Binding Assay, Modification, Irradiation

Journal: Science advances

Article Title: Codelivery of CRISPR-Cas9 and chlorin e6 for spatially controlled tumor-specific gene editing with synergistic drug effects.

doi: 10.1126/sciadv.abb4005

Figure Lengend Snippet: Fig. 2. Characterization, tumor cell uptake, and in vivo biodistribution of nanoparticles. (A) SDS-PAGE of Cas9- and Ce6-containing nanoparticles (CC-NPs) prepared at different NTA/Cas9 ratios. (B) Cas9 loading efficiency (LE) and loading content (LC) in CC-NPs. (C) Size and zeta potential of CC-NPs. (D) Size and zeta potential of iRGD- PD–coated CC-NPs (T-CC-NPs) using different amounts of iRGD-PD. Data are shown as means ± SD (n = 3). (E) Transmission electronic microscopy (TEM) images of NTA- Ce6-NPs (self-assembled from NTA-SS-PEG-PCL and Ce6), CC-NPs, and T-CC-NPs. Scale bars, 200 nm. Cas9/sgRNA complex was prepared at a molar ratio of 1. (F) TEM image of T-Au-NP (His-tagged Au nanoparticles conjugated to NTA-NP and then coated with iRGD). (G) Transfection efficiency of CNE-2 cells with T-CC-NPs or NT-CC-NPs. Mean ± SD (n = 3). ***P < 0.001. (H) Confocal laser scanning microscopy (CLSM) of single CNE-2 cells transfected with T-CC-NPs or NT-CC-NPs. Nuclei (blue) were stained using Hoechst 33324; lysosomes (purple) were labeled with LysoTracker Red; Ce6 emitted red fluorescence. Scale bar, 10 m. (I) Flow cytometry of cells in (B) and (C). (J) Biodistribution of T-CD-NPs and NT-CD-NPs [containing the dye 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) rather than Ce6] in CNE-2 xeno- graft mice. (K) Quantification of DiR fluorescence in (E) (mean ± SD, n = 3). eGFP protein was fused with Cas9 to its tumor cell uptake. MFI, mean fluorescence intensity.

Article Snippet: After tumor sections were fixed in 4% paraformaldehyde overnight, the levels of luciferase protein and Cas9 were measured using rabbit polyclonal anti-firefly luciferase antibody (Abcam, Cambridge, UK) and mouse monoclonal anti-Cas9 antibody (Cell Signaling Technology, Danvers, MA, USA) with CLSM.

Techniques: In Vivo, SDS Page, Zeta Potential Analyzer, Transmission Assay, Microscopy, Transfection, Confocal Laser Scanning Microscopy, Staining, Labeling, Fluorescence, Flow Cytometry

Journal: Science advances

Article Title: Codelivery of CRISPR-Cas9 and chlorin e6 for spatially controlled tumor-specific gene editing with synergistic drug effects.

doi: 10.1126/sciadv.abb4005

Figure Lengend Snippet: Fig. 3. Mechanism and characterization of lysosomal escape of nanoparticles and cytoplasmic release of Cas9/sgRNA. (A) Schematic of NIR-triggered generation of ROS by Ce6 and lysosomal escape of nanoparticles, followed by release of Cas9/sgRNA in the reducing environment of the cytoplasm. (B) CLSM images of intracellular distribution of nanoparticles under NIR irradiation. T-Cas9/NIR, T-CC/NIR, and NT-CC/NIR indicate T-Cas9-NPs, T-CC-NPs, and NT-CC-NPs with NIR irradiation, respectively. Scale bar, 10 m. (C) Fluorescence intensities in cells receiving T-Cas9/NIR or T-CC/NIR along the dotted yellow arrows in (B). (D) Flow cytometry and (E) CLSM imaging of ROS levels in cells receiving various treatments. Scale bar, 50 m. (F) Schematic of glutathione (reduced form) (GSH)–responsive Cas9 RNP release in the cyto- plasm. (G) SDS-PAGE of T-CC-NPs in PBS (pH 7.4) with or without 10 mM GSH (mimicking cytoplasmic conditions) and T-In-CC (reducing agent–insensitive nanoparticles) in PBS (pH 7.4) with 10 mM GSH for various times and then centrifuged with ultrafiltration to remove released Cas9/sgRNA. (H) Cas9 RNP release profile of T-CC-NPs in PBS (pH 7.4) with or without 10 mM GSH and T-In-CC-NPs in PBS (pH 7.4) with 10 mM GSH (mean ± SD, n = 3). (I) TEM images and (J) dynamic light scattering (DLS) analysis of T-CC-NPs with or without 10 mM GSH. Scale bar, 100 nm.

Article Snippet: After tumor sections were fixed in 4% paraformaldehyde overnight, the levels of luciferase protein and Cas9 were measured using rabbit polyclonal anti-firefly luciferase antibody (Abcam, Cambridge, UK) and mouse monoclonal anti-Cas9 antibody (Cell Signaling Technology, Danvers, MA, USA) with CLSM.

Techniques: Irradiation, Fluorescence, Flow Cytometry, Imaging, SDS Page

Journal: Science advances

Article Title: Codelivery of CRISPR-Cas9 and chlorin e6 for spatially controlled tumor-specific gene editing with synergistic drug effects.

doi: 10.1126/sciadv.abb4005

Figure Lengend Snippet: Fig. 4. Gene editing efficiency in vitro and in vivo. (A) CLSM images of U2OS-eGFP cells receiving nanoparticles under NIR irradiation. The U2OS-eGFP cell line contains a single copy of a reporter gene that constitutively expresses destabilized eGFP. Scale bar, 100 m. (B) eGFP-negative cells quantified using flow cytometry. (C) Genomic cleavage detection (GCD) analysis of eGFP gene disruption in U2OS-eGFP cells treated with nanoparticles. (D) Bioluminescence and fluorescence imaging of luciferase (Luc) and red fluorescence protein (RFP) in mice bearing CNE-2-Luc-RFP recombinant cells at time points after tail vein administration of nanoparticles under NIR irradiation. (E) Quantitation of fluorescence signals of tumor tissues in (D). Curved green and red arrows indicate changes of Luc bioluminescence and RFP fluorescence, respectively. Data are means ± SD (n = 3). **P < 0.01 and ***P < 0.001. (F) Western blot and (G) immunohistochemistry analyses of luciferase and (H) immunofluorescence analyses of Cas9 and Ce6 in tumor tissue sections from CNE-2 xenograft mice receiving various nanoparticles. Luciferase proteins were stained brown. Scale bars, 50 m.

Article Snippet: After tumor sections were fixed in 4% paraformaldehyde overnight, the levels of luciferase protein and Cas9 were measured using rabbit polyclonal anti-firefly luciferase antibody (Abcam, Cambridge, UK) and mouse monoclonal anti-Cas9 antibody (Cell Signaling Technology, Danvers, MA, USA) with CLSM.

Techniques: In Vitro, In Vivo, Irradiation, Flow Cytometry, Disruption, Fluorescence, Imaging, Luciferase, Recombinant, Quantitation Assay, Western Blot, Immunohistochemistry, Immunofluorescence, Staining

Journal: Science advances

Article Title: Codelivery of CRISPR-Cas9 and chlorin e6 for spatially controlled tumor-specific gene editing with synergistic drug effects.

doi: 10.1126/sciadv.abb4005

Figure Lengend Snippet: Fig. 5. Antitumor activity and spatial specificity of combined photodynamic therapy and Nrf2 gene editing. (A) Flow cytometry analysis of cell apoptosis. A1, dead cells; A2, late apoptotic cells; A3, normal cells; and A4, early apoptotic cells. (B) Tumor growth in CEN-2 xenograft tumor-bearing mice after tail vein injection of nanoparticles with or without NIR irradiation. Data are means ± SD (n = 6). **P < 0.01 and ***P < 0.001. Black arrows in (C) Black arrows in (B) indicate intravenous injection times. (C) Survival of CEN-2 xenograft mice in (B). Mice were euthanized when tumor volume reached 2000 mm3. (D) Hematoxylin and eosin (H&E)–stained and terminal deoxynucleotidyl transferase– mediated deoxyuridine triphosphate nick end labeling (TUNEL)–stained tumor tissue sections from CEN-2 xenograft mice receiving various treatments. (E) H&E staining of tissue sections from major organs. (F) ROS and (G) Nrf2 protein levels in tumor and liver tissues form CEN-2 xenograft tumor-bearing mice receiving T-CC/NIR or T-Cas9/NIR. NIR irra- diation was only applied on tumor tissues. Nuclei were stained blue using 4′,6-diamidino-2-phenylindole (DAPI); ROS and Nrf2 protein were labeled green and red, respectively. Scale bars, 50 (D and E) and 100 m (F and G). Doses of Ce6 and Cas9/sgRNA were 1 and 1.5 mg/kg, respectively. Cas9/sgRNA complex was prepared at a molar ratio of 1.

Article Snippet: After tumor sections were fixed in 4% paraformaldehyde overnight, the levels of luciferase protein and Cas9 were measured using rabbit polyclonal anti-firefly luciferase antibody (Abcam, Cambridge, UK) and mouse monoclonal anti-Cas9 antibody (Cell Signaling Technology, Danvers, MA, USA) with CLSM.

Techniques: Activity Assay, Flow Cytometry, Injection, Irradiation, Staining, End Labeling, TUNEL Assay, Labeling

Journal: Science advances

Article Title: Codelivery of CRISPR-Cas9 and chlorin e6 for spatially controlled tumor-specific gene editing with synergistic drug effects.

doi: 10.1126/sciadv.abb4005

Figure Lengend Snippet: Fig. 6. Mechanism of combined therapy of Ce6 photodynamic therapy and Nrf2 gene editing. (A) Schematic of synergistic effects of CRISPR-Cas9 gene editing and NIR-triggered ROS release. (B) Flow cytometry of ROS levels and (C) immunohistochemistry analyses of Cas9 and Nrf2 proteins in tumor tissue sections from CNE-2 xeno- graft mice receiving nanoparticles. Scale bar, 50 m. Cas9 and Nrf2 protein were stained brown, respectively. Deep sequencing analysis of indel percentage in (D) tumor tissue and (E) liver tissue from CNE-2 xenograft mice treated with T-CC/NIR. bp, base pair. (F) Comparison of on- and off-target of gene editing in tumor and liver tissues. (G) GCD analysis of tumor cells receiving various treatments. (H) Western blot and (I) immunofluorescence analyses of Ki67 (red), apoptotic protein caspase-3 (red), angiogenesis factors vascular endothelial growth factor–A (VEGF-A; green) and hypoxia-inducible factor 1 (HIF; green), and vascular endothelial cell biomarker CD31 (red) in tumor tissue sections from CNE-2 xenograft mice treated with nanoparticles. Nuclei were stained blue. Scale bar, 100 m. T-CC-NPs, NT-CC-NPs, and T-Cas9-NPs were prepared at an NTA/Cas9 ratio of 8 and a dose of 1.5 mg/kg Cas9 RNP.

Article Snippet: After tumor sections were fixed in 4% paraformaldehyde overnight, the levels of luciferase protein and Cas9 were measured using rabbit polyclonal anti-firefly luciferase antibody (Abcam, Cambridge, UK) and mouse monoclonal anti-Cas9 antibody (Cell Signaling Technology, Danvers, MA, USA) with CLSM.

Techniques: CRISPR, Flow Cytometry, Immunohistochemistry, Staining, Sequencing, Comparison, Western Blot, Immunofluorescence, Biomarker Discovery