Journal: Current biology : CB

Article Title: Two MYB proteins in a self-organizing activator-inhibitor system produce spotted pigmentation patterns

doi: 10.1016/j.cub.2019.12.067

Figure Lengend Snippet: (A) RNAi of RTO in M. lewisii generates a range of anthocyanin spot patterns. (B) RNAi of MgRTO in M. guttatus recapitulates the rto-like phenotype. (C) Over-expression of RTO in M. lewisii abolishes anthocyanin production throughout the corolla. (D–G) Relative expression of NEGAN and RTO in M. lewisii RTO over-expression lines (D), M. lewisii RTO RNAi lines (E), M. guttatus RTO RNAi lines (F), and M. guttatus CRISPR/Cas9 mediated knockout lines (G). All relative transcript levels are measured by qRT-PCR, standardized to the corresponding wild-type (LF10 for M. lewisii, MAC for M. guttatus). Error bars represent 1 SD from three biological replicates. (H) BiFC assay shows that the wild-type RTO protein interacts with ANbHLH1, whereas the D>G amino acid replacement in the mutant rto protein abolishes or attenuates the interaction. See also Figures S2 and S4–S6 and Tables S5 and S6.

Article Snippet: S. pyogenes Cas9 nuclease , New England Biolabs , Cat#M0386S.

Techniques: Over Expression, Expressing, CRISPR, Knock-Out, Quantitative RT-PCR, Bimolecular Fluorescence Complementation Assay, Mutagenesis

Journal: Current biology : CB

Article Title: Two MYB proteins in a self-organizing activator-inhibitor system produce spotted pigmentation patterns

doi: 10.1016/j.cub.2019.12.067

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: S. pyogenes Cas9 nuclease , New England Biolabs , Cat#M0386S.

Techniques: Recombinant, Multiplex Assay, SYBR Green Assay, Sequencing, Generated, Clone Assay, Software

Journal: Cell Death & Disease

Article Title: Requirement for Serine-384 in Caspase-2 processing and activity

doi: 10.1038/s41419-020-03023-6

Figure Lengend Snippet: a The three-dimensional structure of catalytically active fragments of caspase-2 (1pyo.pdb) and potential phosphorylation sites (Ser-307, Ser-220, Thr-380, Ser-384). Predicted phosphorylation sites are highlighted by orange and red, the active center of caspase-2 is shown in green. The large catalytic p19 subunit is shown in blue, and the small catalytic subunit is shown in purple. b The reported phosphorylation sites of caspase-2 (green) and potential phosphorylation residues (red) are shown along with two critical caspase-2 active site residues: cysteine (Cys) and histidine (His). c The overexpression of caspase-2 WT and caspase-2 mutants in HEK293T cells. The processing of caspase-2 in HEK293T cells treated with 70 µM cisplatin for 18 h was analyzed by western blot. The bands corresponding to cleaved fragments of caspase-2 are marked as p37-caspase-2, p19-caspase-2. One representative experiment from three independent experiments is shown.

Article Snippet: CAOV-4 cells were transfected with Caspase-2 CRISPR/Cas9 KO plasmids using Lipofectamine™ LTX Reagent with PLUS™ transfection reagent (#15338100, ThermoFisher, USA) according to manufacturer’s protocol.

Techniques: Over Expression, Western Blot

Journal: Cell Death & Disease

Article Title: Requirement for Serine-384 in Caspase-2 processing and activity

doi: 10.1038/s41419-020-03023-6

Figure Lengend Snippet: a The processing of caspase-2 and caspase-3 and the appearance of PARP cleavage product p89 (cPARP) in HEK293T and CAOV-4-C2 −/− cells that transiently overexpress the caspase-2 WT and caspase-2 Ser-384Ala . Both cell lines were treated with 70 µM cisplatin for 18 h and caspase processing was analyzed by western blot. The bands corresponding to cleaved fragments of caspase-2, caspase-3, caspase-8 and PARP are marked as p37-caspase-2/p19-caspase-2, p19/17-caspase-3, p43/41-caspase-8 and cPARP, correspondingly. b The Ser-384Ala substitution led to a reduction of caspase-2 peptidase activity. Caspase-2 activity was measured using a substrate—Ac-VDVAD-AMC in lysates of HEK293T and CAOV-4-C2 −/− cells that were non-transfected or transfected with caspase-2 WT and caspase-2 Ser-384Ala . Both cell lines were treated with 70µM cisplatin for 18h and caspase activity was measured. The histograms demonstrate the slope of enzymatic activity curve. c The processing of caspase-2 and the cleavage of substrate Bid in HEK293T cells that transiently overexpress the caspase-2 WT and caspase-2 Ser-384Ala . Cells were treated with 70 µM cisplatin for 18 h and caspase processing and Bid cleavage was analyzed by western blot. The bands corresponding to cleaved fragments of caspase-2 are marked as p37-caspase-2/p19-caspase-2, and the truncated form of Bid is marked as tBid. The amount of full-length Bid and tBid forms were quantified by densitometry analysis and normalized on vinculin level. d The substitution Ser-384Ala in caspase-2 negatively regulated apoptotic cell death. HEK293T and CAOV-4-C2 −/− cells were transfected with caspase-2 WT and caspase-2 Ser-384Ala and treated for 18h with 70µM cisplatin. Cells were stained with the conjugate of annexin V-FITC (AnV) and propidium iodide (PI) and examined by flow cytometry. The “black” columns demonstrate the AnV+/PI− stained apoptotic population, while the “gray” columns indicate the AnV+/PI+ stained late apoptotic/necrotic cells. Results are presented as the mean of three distinct experiments ± SEM.

Article Snippet: CAOV-4 cells were transfected with Caspase-2 CRISPR/Cas9 KO plasmids using Lipofectamine™ LTX Reagent with PLUS™ transfection reagent (#15338100, ThermoFisher, USA) according to manufacturer’s protocol.

Techniques: Western Blot, Activity Assay, Transfection, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Requirement for Serine-384 in Caspase-2 processing and activity

doi: 10.1038/s41419-020-03023-6

Figure Lengend Snippet: a The processing of caspase-2 was analyzed by western blot. The bands corresponding to cleaved fragments of caspase-2 are marked as p37-caspase-2/p19-caspase-2. b Annotated spectra are shown with all of the fragments that matched an expected fragment from in silico digestion (in color). Below each spectrum is a table with calculated ion masses for each peptide; colored values match corresponding peaks in the spectrum.

Article Snippet: CAOV-4 cells were transfected with Caspase-2 CRISPR/Cas9 KO plasmids using Lipofectamine™ LTX Reagent with PLUS™ transfection reagent (#15338100, ThermoFisher, USA) according to manufacturer’s protocol.

Techniques: Western Blot, In Silico

Journal: Cell Death & Disease

Article Title: Requirement for Serine-384 in Caspase-2 processing and activity

doi: 10.1038/s41419-020-03023-6

Figure Lengend Snippet: a The processing of caspase-2 in HEK293T cells, which transiently overexpressed caspase-2 WT , caspase-2 Ser-384Asp , and caspase-2 Ser-384Ala (Ser-384Ala, Ser-384Asp), and were treated with 70 µM cisplatin for 18 h, was analyzed by western blot. The bands corresponding to cleaved fragments of caspase-2 are marked as p37-caspase-2/p19-caspase-2. b The detection of phosphorylated caspase-2 by Phos-tag SDS-PAGE in combination with western blot. The lysates from HEK293T cells with transiently overexpressed caspase-2 WT and caspase-2 Ser-384Ala were subjected to the analysis. The bands corresponding to caspase-2 and its predicted phosphoforms are shown.

Article Snippet: CAOV-4 cells were transfected with Caspase-2 CRISPR/Cas9 KO plasmids using Lipofectamine™ LTX Reagent with PLUS™ transfection reagent (#15338100, ThermoFisher, USA) according to manufacturer’s protocol.

Techniques: Western Blot, SDS Page

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Molecular and functional correction of a deep intronic splicing mutation in CFTR by CRISPR-Cas9 gene editing

doi: 10.1016/j.omtm.2023.101140

Figure Lengend Snippet: Targeted excision of the deep intronic mutation (A) CFBE3849 cells were transfected four times at 48-h intervals with Cas9 RNPs with U3D1 gRNAs then PCR DNA sequences analyzed for indels in CFTR intron 22 by ICE. Representative ICE analyses of individual samples for, (B) indels, and, (C) larger, targeted excision events.

Article Snippet: RNP complexes were formed with gRNA and Truecut Cas9 protein v2 (ThermoFisher Scientific) at a weight ratio of 1:4 gRNA:Cas9 in OptiMEM (Life Technologies) and incubated for 5 min for the complex to form.

Techniques: Mutagenesis, Transfection

Journal: bioRxiv

Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

doi: 10.1101/231035

Figure Lengend Snippet: ( a ) Schematic of Cas9 fused to the HUH endonuclease PCV with a covalently attached ssODN. ( b ) SDS-PAGE of Cas9 variants reacted with an Alexa 488 fluorescently labelled ssDNA containing the PCV recognition sequence. The top panel is the coomassie stained gel, and the bottom panel is the identical fluorescently imaged gel. PCV is fused to either the carboxyl (Cas9-PCV) or amino (PCV-Cas9) terminus of Cas9. Cas9-PCV(Y96F) represents catalytically inactive PCV(Y96F) fused to Cas9. (C) SDS-PAGE gel shift assay of Cas9 reacting with two ssDNA templates containing the PCV recognition sequence of differing lengths in a 1:1 ssDNA:Cas9 molar ratio.

Article Snippet: Streptococcus pyogenes Cas9 was amplified out of the plasmid pET15_SP-Cas9 (a gift from Niels Geijsen, Addgene plasmid #62731) and inserted in pTD68_SUMO-PCV2 at the BamHI site using Infusion cloning (Clontech) to create C-terminally fused Cas9-PCV.

Techniques: SDS Page, Sequencing, Staining, Electrophoretic Mobility Shift Assay

Journal: bioRxiv

Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

doi: 10.1101/231035

Figure Lengend Snippet: ( a ) Schematic of split luciferase insertion. The C-terminus of NanoLuc nanoluciferase (HiBiT) is encoded on the 200bp ssODN along with the 5’ PCV recognition sequence and targeted to the 3’ end of GAPDH . ( b ) Assaying luminescence using different Cas9 variants when inserting HiBiT into GAPDH in HEK-293T cells. PCV is fused to either the amino (PCV-Cas9) or carboxyl (Cas9-PCV) terminus of Cas9. Transfections were performed with ssODN lacking the PCV recognition sequence (PCV- ssODN) or ssODN containing the PCV recognition sequence (PCV+ ssODN). Units are displayed in relative light units (RLU) normalized to Cas9. ( c ) The calculated fold change from (b) between the PCV-ssODN and PCV+ ssODN is shown for each variant. ( d ) Targeting the GAPDH locus in U2-OS cells. ( e ) Targeting a locus in vinculin in HEK-293T cells using an ssODN containing the PCV recognition sequence. ( f ) Fold change in RLU compared to Cas9 when varying the amount of RNP (equimolar ssODN). All graphs represent data from one of multiple independent experiments exhibiting similar results. Data are shown as mean +/− SD (n=3). Significance calculated using 2-tailed Student’s t-test: ** P < 0.01, *** P < 0.001, ns = no significance (P >0.05).

Article Snippet: Streptococcus pyogenes Cas9 was amplified out of the plasmid pET15_SP-Cas9 (a gift from Niels Geijsen, Addgene plasmid #62731) and inserted in pTD68_SUMO-PCV2 at the BamHI site using Infusion cloning (Clontech) to create C-terminally fused Cas9-PCV.

Techniques: Luciferase, Sequencing, Transfection, Variant Assay

Journal: bioRxiv

Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

doi: 10.1101/231035

Figure Lengend Snippet: ( a ) HEK-293T cells stably expressing a mutant mCherry-GFP reporter are edited by HDR through a frameshift correction, restoring mCherry activity. ( b ) Representative microscopy images of fluorescent reporter editing. ( c ) The percent of mCherry positive cells determined by flow cytometry at two different RNP concentrations using an ssODN containing the PCV recognition sequence ( d ) RNP transfections at 3 pmol in the presence or absence of ssODN. Data are shown as mean +/− SD (n=3). For (c) and (d), the statistical significance of %mCherry positive cells between PCV-fusions of Cas9 and Cas9 alone was <0.001, calculated using 2-tailed Student’s t-test.

Article Snippet: Streptococcus pyogenes Cas9 was amplified out of the plasmid pET15_SP-Cas9 (a gift from Niels Geijsen, Addgene plasmid #62731) and inserted in pTD68_SUMO-PCV2 at the BamHI site using Infusion cloning (Clontech) to create C-terminally fused Cas9-PCV.

Techniques: Stable Transfection, Expressing, Mutagenesis, Activity Assay, Microscopy, Flow Cytometry, Sequencing, Transfection

Journal: Nature

Article Title: Targeting the SHOC2–RAS interaction in RAS-mutant cancers

doi: 10.1038/s41586-025-08931-1

Figure Lengend Snippet: a , Schematic describing the Ba/F3 model of oncogene addiction. Ba/F3 relies on IL-3–JAK–STAT signalling for survival unless engineered to express oncogenes such as RAS . Mut, mutation. b , Effect on cell viability for the indicated Ba/F3 isogenic models after treatment for 72 h with the KRAS(G12C) inhibitor adagrasib or the KRAS(G12D) inhibitor MRTX1133. DMSO, dimethyl sulfoxide. c , Genetic screen diagram: Ba/F3 stably engineered to co-express Cas9 and different NRAS and KRAS mutants were transduced with a mouse genome-wide sgRNA library, antibiotic selected, expanded and subjected to genomic DNA isolation and next-generation sequencing (NGS) analysis to identify sensitizer and rescuer genes. d , PCA bi-plot representing PC1 ( x axis) and PC2 ( y axis) from the 250 most variable genes in the screen. Coloured dots represent PCA scores for each condition. Arrows represent loadings for genes driving the PCA distribution, coloured according to the contribution to the eigenvectors. Selected genes are highlighted with a black dot. e , Scatter plot representing hits from differential representation analysis between screens in Q61 lines and G12 lines. The x axis represents scoring and the y axis represents statistics (see the ‘CRISPR screen analysis’ section in the ). Non-significant genes (absolute Q < 2 and RSA > –2) are represented as a density plot to avoid overplotting. f , Dot–plot representing screening results of selected hits. Colours represent Q scoring and dot sizes represent statistical significance (RSA). g , Schematic illustrating the RAS signalling cellular context for the indicated RAS G12 and Q61 hits. h , Effect on cell viability for the indicated Ba/F3 isogenic models following 72 h treatment with inhibitors for SHP2, SOS1 and IGFR. The dotted lines indicate 50% of growth inhibition. i , Effect on cell viability of the indicated Ba/F3 isogenic models following transduction with sgRNAs against SHOC2 , sgSHOC2 or non-targeting sgNT. Data in b , h and i are mean ± s.d. of n = 3 biologically independent samples. Images in a , c and g created using BioRender (Galli, G., 2025): a, https://BioRender.com/c48w611 ; c , https://BioRender.com/m41c915 ; g , https://BioRender.com/q07k743 .

Article Snippet: SHOC2 (53600), Phospho-c-Raf Ser259 (9421), GFP (2956), phospho-p44/42 MAPK (Erk1/2), Thr202/Tyr204 (4370), p44/42 MAPK (Erk1/2) (9102), phospho-MEK1/2 Ser217/221 (9154), phospho-Akt Thr308 (9275), phospho-Akt Ser473 (9271), phospho-S6 ribosomal protein Ser235/236 (2211), phospho-S6 ribosomal protein Ser240/244 (Ref. 2215), phospho-IGF-I receptor β Tyr1131 (3021), IGF-I receptor β (9750), Cas9 HRP conjugate (97982), HRP-linked mouse IgG (7076) and HRP-linked rabbit IgG (7074) antibodies were purchased from Cell Signaling Technology.

Techniques: Mutagenesis, Stable Transfection, Transduction, Genome Wide, DNA Extraction, Next-Generation Sequencing, CRISPR, Inhibition

Journal: Blood Advances

Article Title: Gene editing rescue of a novel MPL mutant associated with congenital amegakaryocytic thrombocytopenia

doi: 10.1182/bloodadvances.2016002915

Figure Lengend Snippet: Gene editing or autophagic delivery of mutant Mpl to the cell surface rescue receptor function in vitro. (A) Transient overexpression of GRASP55 tagged with a V5 epitope results in accumulation of the lower-molecular-weight core-glycosylated form of Mpl regardless of WT or mutant status. Receptors are shown to be signaling competent on the basis of phosphorylation of key signaling proteins in the Jak/STAT and PI3K pathways in response to Tpo. (B-C) XTT-II proliferation assays performed on UT-7 or Ba/F3 cell lines expressing WT or mutant MplmNG and selected for growth in the presence of Tpo (panel C, solid lines) or eltrombopag (Elt) (panel C, dotted lines). CRISPR-Cas9–edited cells that were reverse-engineered to restore the WT sequences in MPL exon 5 from the mutated W272R sequence (labeled Mpl W272R>WT) are represented by blue open circles. UT-7 cells were edited by using the D10A Cas9 mutant and 2 single gRNAs in a double nickase approach. A classical WT Cas9 approach (ie, coupled to a unique single gRNA) was used to edit Ba/F3 cells. **P < .005; ***P < .0001.

Article Snippet: Cas9 protein (50 ng; New England Biolabs) was mixed with 250 ng of in vitro–transcribed gRNA (either #1WT or #1WR) and incubated at 37°C for 5 minutes in a PCR device to form RNPs.

Techniques: Mutagenesis, In Vitro, Over Expression, Molecular Weight, Expressing, CRISPR, Sequencing, Labeling

Journal: Blood Advances

Article Title: Gene editing rescue of a novel MPL mutant associated with congenital amegakaryocytic thrombocytopenia

doi: 10.1182/bloodadvances.2016002915

Figure Lengend Snippet: Functional rescue strategies for Mpl mutants. Schematic summary of the 2 rescue approaches used to restore Mpl function: (1) overexpression of GRASP55 to force immature Mpl receptor expression at the cell surface using unconventional autophagy-dependent secretion and (2) CRISPR-Cas9 gene editing to convert mutated Mpl DNA sequence to WT sequence. sgRNA, single-guide RNA.

Article Snippet: Cas9 protein (50 ng; New England Biolabs) was mixed with 250 ng of in vitro–transcribed gRNA (either #1WT or #1WR) and incubated at 37°C for 5 minutes in a PCR device to form RNPs.

Techniques: Functional Assay, Over Expression, Expressing, CRISPR, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B

doi: 10.1016/j.omtn.2018.12.008

Figure Lengend Snippet: Construction of Non-viral Homology-Directed System for Correcting Mutated Canine Coagulation Factor IX (A) Schematic outline of the cFIX target locus. The top panel shows the cFIX gene, including the UTRs (black bars), introns (bright gray bars), and exons (light gray bars). Nucleotide and amino acid sequences of wild-type cFIX (WT cFIX) and a major disease causing cFIX mutation (G1477A, mutated cFIX) are shown (white letters in dark square). The bottom panel shows the mutated donor sequence (cFIXmod) used in this study (mutations are marked with an asterisk), which results in the identical amino acid sequence as WT cFIX. The gray horizontal bar schematically shows the guide RNA (gRNA)-binding site used in this study (gRNA-CRISPR/Cas9). Differences in the donor and mutated cFIX sequences are marked with stars. (B) Schematic outline of the DNA sequences contained in the Tet-on-inducible CRISPR/Cas9 for cutting the cFIX-mutated strand and the donor DNA that was transfected as PCR product (cFIXmod). NLS, nuclear localization signal; PA, polyadenylation signal; TREG3, TRE3G promoter; EF1a, human elongation factor-1 alpha promoter; gRNA, guide RNA; Tet-on, tetracycline-controlled transcriptional activation. (C) CRISPR/Cas9 nuclease activity measured by T7E1 assay after transfection with the CRISPR/Cas9-encoding plasmid and the donor DNA. MW, molecular-weight size marker; NC, negative control referring to the mixture of untreated Huh7-cFIXmut, PLC/PRF/5-cFIXmut, and Hep3B-cFIXmut cells; 1, CRISPR/Cas9-treated Huh7-cFIXmut cells; 2, CRISPR/Cas9-treated PLC/PRF/5-cFIXmut cells; 3, CRISPR/Cas9-treated Hep3B-cFIXmut cells. (D) CRISPR/Cas9 nuclease off-target analysis of the top 5 predicted off-target sites. These were the histone deacetylase 7 gene (HDAC7), DNA-packaging protein Histone H3 (H3K27), the centrosomal protein of 192 kDa (CEP192), and the Zinc and Ring Finger 2 gene (ZNRF2), which were analyzed in Huh7-cFIXmut, PLC/PRF/5-cFIXmut, and Hep3B-cFIXmut cells by T7E1 assay after transfection with the CRISPR/Cas9-encoding plasmid.

Article Snippet: The final vector was constructed by PCR amplifying the gRNA expression cassette and cloning into the pShV-TRE3G-Cas9-EF1α-Tet-ON3G plasmid via the restriction enzyme NheI-HF (New England Biolabs).

Techniques: Coagulation, Mutagenesis, Sequencing, Binding Assay, CRISPR, Transfection, Activation Assay, Activity Assay, Plasmid Preparation, Molecular Weight, Marker, Negative Control, Histone Deacetylase Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B

doi: 10.1016/j.omtn.2018.12.008

Figure Lengend Snippet: Design of Donor DNA Sequences for the Two-Vector and the Single-Vector Systems (A) Donor DNA used for the two-vector system. A PCR product of 208 bp (cFIXmod) covering the mutated cFIX location was used. (B) Schematic diagram of single vectors containing the Tet-on-inducible CRISPR/Cas9 for cutting the cFIX-mutated strand and the donor DNA. Three molecular setups of single vectors, which delivered all components for HDR, were designed: plasmid 1 (P1), plasmid 2 (P2), and plasmid 3 (P3). For P1 and P2, the donor is flanked by the gRNA recognition sequence. Note that the gRNA recognition sequences in P1 are in the opposite orientation while the gRNA recognition sequences of P2 flank the donor DNA in the identical orientation. The donor contained in plasmid P3 lacks the gRNA recognition sequence. NLS, nuclear localization signal; PA, polyadenylation signal; TREG3, TRE3G promoter; EF1a, human elongation factor-1 alpha promoter; gRNA, guide RNA; Tet-on, tetracycline-controlled transcriptional activator.

Article Snippet: The final vector was constructed by PCR amplifying the gRNA expression cassette and cloning into the pShV-TRE3G-Cas9-EF1α-Tet-ON3G plasmid via the restriction enzyme NheI-HF (New England Biolabs).

Techniques: Plasmid Preparation, CRISPR, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B

doi: 10.1016/j.omtn.2018.12.008

Figure Lengend Snippet: Gene Correction of Mutated cFIX through Naked DNA (A) The principle of the amplification-refractory mutation system qPCR (ARMS-qPCR) assay. Two qPCRs of each sample are performed. Primers of the reference PCR (Re-F,-R) amplify the upstream area of the HR region. The forward detection primer (Det-F) binds the upstream area of the HR region, and the reverse primer (Det-R) specifically binds to the HR cassette. (B–D) Naked DNA-mediated HDR events in mutated cFIX stable cell lines measured via ARMS-qPCR. Mod1%, Cas9 + donor, Cas9, Donor, and cell-cFIXmut display controls containing 1% modified template, cells transfected with CRISPR/Cas9 and optimized donor sequence, cells transfected with CRISPR/Cas9, cells transfected with optimized donor sequence, and cFIX stable cell lines Huh7-cFIXmut (B), PLC-PRF-5-cFIXmut (C), and Hep3B-cFICmut (D). (E) Indels measured by T7E1 assay after transfection with the non-viral homology-directed repair plasmids at the endogenous hFIX locus and the uncorrected cFIX transgene in Huh7-cFIXmut cells. MW, molecular weight markers; NC, negative control, PLC-cFIXmut or Hep3B-cFIXmut without treatment. P1, P2, P3, and Cas9 + donor display cells treated with plasmid 1, plasmid 2, plasmid 3, or Cas9 + donor. Data points represent SEM of three independent experiments performed in triplicates. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The final vector was constructed by PCR amplifying the gRNA expression cassette and cloning into the pShV-TRE3G-Cas9-EF1α-Tet-ON3G plasmid via the restriction enzyme NheI-HF (New England Biolabs).

Techniques: Amplification, Mutagenesis, Stable Transfection, Modification, Transfection, CRISPR, Sequencing, Molecular Weight, Negative Control, Plasmid Preparation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B

doi: 10.1016/j.omtn.2018.12.008

Figure Lengend Snippet: Gene Correction of Mutated cFIX through the Viral Vectors HCAdV5-Cas9 and ssAAV2-cFIX (A) Schematic outline of HCAdV5-Cas9 and ssAAV2-cFIX genomes. ITR, inverted terminal repeat; NLS, nuclear localization signal; PA, polyadenylation signal; TREG3, TRE3G promoter; EF1a, human elongation factor-1 alpha promoter; CAG, cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter; gRNA, guide RNA; Tet-on, tetracycline-controlled transcriptional activation. (B) HCAdV5-Cas9 nuclease activity measured by T7E1 assay. MW, molecular-weight size marker; ND, no detection.

Article Snippet: The final vector was constructed by PCR amplifying the gRNA expression cassette and cloning into the pShV-TRE3G-Cas9-EF1α-Tet-ON3G plasmid via the restriction enzyme NheI-HF (New England Biolabs).

Techniques: Activation Assay, Activity Assay, Molecular Weight, Marker

Journal: Molecular Therapy. Nucleic Acids

Article Title: Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B

doi: 10.1016/j.omtn.2018.12.008

Figure Lengend Snippet: Genotypic and Phenotypic Corrections in Huh7-cFIXmut Cells after Co-infection with the Gene Correction Vectors HCAdV5-Cas9-cFIX or HCAdV5-Cas9 and ssAAV2-cFIX (A) Schematic outline of the experimental setup. Cellular genomic DNA was extracted at 72 h post-transduction for ARMS-qPCR assay, and cell media were collected at 48, 96, and 144 h post-transduction for the ELISA. (B) HCAdV5-Cas9-cFIX- or HCAdV5-Cas9- and ssAAV2-cFIX-mediated HDR events in mutated cFIX stable cells measured via ARMS-qPCR. Mod1%, controls containing 1% modified template; single vector, Huh7-cFIXmut cells transduced with HCAdV5-Cas9-cFIX; AdV + AAV (MOI 200), AdV + AAV (MOI 400),and AdV + AAV (MOI 600), Huh7-cFIXmut cells transduced with HCAdV5-Cas9 and ssAAV2-cFIX of different MOIs; Huh7-cFIXmut cells, mutated cFIX stable Huh7 cells; HCAdV5-Cas9, Huh7-cFIXmut cells transduced with HCAdV5-Cas9. (C) ELISA of cFIX concentration in the supernatant of Huh7-cFIXmut cells infected with HCAdV5-Cas9-cFIX or HCAdV-Cas9 and MOIs 200, 400, and 600 for ssAAV-cFIX. Data points represent SEM of three independent experiments performed in triplicates. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The final vector was constructed by PCR amplifying the gRNA expression cassette and cloning into the pShV-TRE3G-Cas9-EF1α-Tet-ON3G plasmid via the restriction enzyme NheI-HF (New England Biolabs).

Techniques: Infection, Transduction, Enzyme-linked Immunosorbent Assay, Modification, Plasmid Preparation, Concentration Assay