ptd68 vector  (Addgene inc)

Journal: Communications Biology

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

doi: 10.1038/s42003-018-0054-2

Figure Lengend Snippet: Selective covalent attachment of ssDNA to Cas9-PCV. 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 labeled 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: For the fluorescent oligonucleotide reactions, 1.5 pmol of Alexa 488-conjugated ssDNA (IDT) was incubated with 1.5 pmol Cas9-PCV in the above conditions and separated by SDS-PAGE.

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

Journal: Communications Biology

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

doi: 10.1038/s42003-018-0054-2

Figure Lengend Snippet: Covalent attachment of ssODN to PCV fusions of Cas9 enhances insertion of a peptide tag. a Schematic of split luciferase insertion. The C-terminus of nanoluciferase (HiBiT) is encoded on the 200 bp 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 Targeting the GAPDH locus in U2-OS cells. d Targeting a locus in vinculin in HEK-293T cells using an ssODN containing the PCV recognition sequence. e Fold change in RLU compared to Cas9 when varying the RNP concentration with equimolar ssODN. f HDR frequency and HDR/indel ratio from deep sequencing of the GAPDH locus. Graphs represent data from one of multiple independent experiments exhibiting similar results using distinct samples. The data with error bars are shown as mean+/− SD ( n = 3). Significance calculated using 2-tailed Student’s t test: ** P < 0.01, *** P < 0.001

Article Snippet: For the fluorescent oligonucleotide reactions, 1.5 pmol of Alexa 488-conjugated ssDNA (IDT) was incubated with 1.5 pmol Cas9-PCV in the above conditions and separated by SDS-PAGE.

Techniques: Luciferase, Sequencing, Transfection, Concentration Assay

Journal: Communications Biology

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

doi: 10.1038/s42003-018-0054-2

Figure Lengend Snippet: Covalent tethering enhances HDR efficiency in a fluorescent reporter cell line. 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. Scale bars are 50 µm. c The percent of mCherry positive cells determined by flow cytometry at two different RNP concentrations using a 175 bp 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: For the fluorescent oligonucleotide reactions, 1.5 pmol of Alexa 488-conjugated ssDNA (IDT) was incubated with 1.5 pmol Cas9-PCV in the above conditions and separated by SDS-PAGE.

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

Journal: bioRxiv

Article Title: Advanced Peptide Nanoparticles Enable Robust and Efficient delivery of gene editors across cell types

doi: 10.1101/2024.11.27.624305

Figure Lengend Snippet: a Graphical illustration of the hPep particles, a schematic showing the Stoplight reporter system, and a general analysis method. b GFP% percentage after treatment with Cas9 RNP:hPep (200 ng Cas9 per 96-well, HEK293T SL cells) with increasing MR. n=4 independent experiments ± SD. Gel mobility assay to determine at what MR the RNP no longer migrates through the gel due to charge neutralization or particle size. c Screening of additives to enhance nanoparticle efficiency using hPep3 and hPep4 (25 ng of Cas9 per 96-well, HEK293T SL cells). The particles were formed in HBG diluted with the additive to a final concentration between 1.25-15 w/v%. Mean value of n=3 independent experiments ± SD. d Effect of serum on transfection efficiency of hPep3-CPP in PVA-PEG buffer (x-axis indicates conc. of Cas9 RNP added to HEK293T SL cells). One day after the transfection, serum was added to 10% in the 0% FBS condition. Mean value of n=3 independent experiments ± SD. e Testing of RNP:hPep3 compatibility with different storage methods (10/25/50 ng Cas9 per well. HEK293T SL cells). Mean value of n=3 independent experiments ± SD.

Article Snippet: The His-NLS-Cre recombinase (herein called Cre), PCV-Cas9, Adenine base editor 8e (herein called ABE8e), and Prime editor 2 (herein called PE2) (Addgene plasmids: 62730, 123643, 161788, 132775 respectively), were expressed by Escherichia coli (BL21 (DE3) T1R pRARE2) upon induction at an optical density of 3 utilizing IPTG.

Techniques: Neutralization, Concentration Assay, Transfection

Journal: bioRxiv

Article Title: Advanced Peptide Nanoparticles Enable Robust and Efficient delivery of gene editors across cell types

doi: 10.1101/2024.11.27.624305

Figure Lengend Snippet: a Covalent attachment of a 60 nt oligo to PCV-Cas9 (53 nt after PCV binding). Particles were formed in DMEM/PVA-PEG buffer at increasing MR (15 ng protein per 96-well, HEK293T cells). Mean value of n=3 independent experiments ± SD. b Cre was complexed with hPep3 in DMEM/PVA-PEG buffer at increasing MR and added to MSC and HeLa cells harboring the TL reporter system in increasing concentration of Cre. Mean value of n=3 independent experiments ± SD. c HDR efficiency using a co-complexed ssDNA template. Particles were formed in DMEM/PVA-PEG (MR of 1:125) with increasing doses (1, 2, 4 nM) added to HEK293T harboring the SL3 system. Oligos were designed with homologous arms of equal length on each side of the expected DNA double-strand break site. Mean value of n=3 independent experiments ± SD.

Article Snippet: The His-NLS-Cre recombinase (herein called Cre), PCV-Cas9, Adenine base editor 8e (herein called ABE8e), and Prime editor 2 (herein called PE2) (Addgene plasmids: 62730, 123643, 161788, 132775 respectively), were expressed by Escherichia coli (BL21 (DE3) T1R pRARE2) upon induction at an optical density of 3 utilizing IPTG.

Techniques: Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: Advanced Peptide Nanoparticles Enable Robust and Efficient delivery of gene editors across cell types

doi: 10.1101/2024.11.27.624305

Figure Lengend Snippet: a &b Editing rates after treatment with various doses of RNP:hPep3 (1:125 MR, HEK293T) or RNP:RNAiMAX. The treatment was performed simultaneously on 6 different plates, with each plate analyzed by flow cytometry at different time points. The RNP:hPep3 treatment resulted in detectable GFP at 4 h of treatment, reaching the maximum after 24 h. Editing with RNAiMAX was first detected at 8 h, increasing each day until day 3. The short-time points are magnified in the top left corner of a & b. Mean value of n=3 independent experiments ± SD. c & d A Huh7 mCherry-GAL9 reporter cell line was used to evaluate endosomal rupture. A rapid occurrence of rupture events after adding RNP:hPep3 formulated in DMEM/PVA-PEG was observed. n=3 independent experiments ± SD. The rupture events were observed to cluster in the perinuclear area. e A media change was implemented 30 min or 2h after adding the Cas9:hPep3. Editing rates were determined based on the activation of the GFP reporter (left y-axis in green), and cytotoxicity was measured using a WST-1 assay (right y-axis in blue) of identically treated cells. A 2-h incubation resulted in editing equivalent to a 3-day incubation, while cytotoxicity was almost eliminated at all tested doses. Mean value of n=3 independent experiments ± SD.

Article Snippet: The His-NLS-Cre recombinase (herein called Cre), PCV-Cas9, Adenine base editor 8e (herein called ABE8e), and Prime editor 2 (herein called PE2) (Addgene plasmids: 62730, 123643, 161788, 132775 respectively), were expressed by Escherichia coli (BL21 (DE3) T1R pRARE2) upon induction at an optical density of 3 utilizing IPTG.

Techniques: Flow Cytometry, Activation Assay, WST-1 Assay, Incubation

Journal: bioRxiv

Article Title: Advanced Peptide Nanoparticles Enable Robust and Efficient delivery of gene editors across cell types

doi: 10.1101/2024.11.27.624305

Figure Lengend Snippet: a Gel electrophoresis of Atto550 tagged gRNA or Cas9 RNP in different buffers. The RNP was unable to migrate through the gel when incubated with PVA-PEG. b FE-SEM of isolated silica particles from the PVA-EPG solution. Individual particle size appears to range from 20-30 nm. Each tick in the scale bar equals 50 nm. ( c ) Effect of removal of silica on transfection efficiency of HEK293T SL cells treated with hPep3-RNP. Mean value of n=3 independent experiments ± SD. d Atto550 tagged RNP was formed under different conditions fractioned by centrifugation, with % of Atto550 signal measured in each fraction. Mean value of n=3 independent experiments ± SD. e Silica isolated from the PVA-PEG buffer by centrifugation was added to different buffers, with the addition to DMEM/PVA-PEG Pure recapturing the efficiency of RNP-CPP formulated in normal DMEM/PVA-PEG when added to HEK293T SL cells. Note: After dilution into the working buffer, the PVA-PEG contains 0.015% silica. Mean value of n=3 independent experiments ± SD. f Envisioned particle formation and structure. Results indicated that the protein adsorbed onto the silica surface, with the peptide interacting with both species. g ) Commercial 22 nm Silica beads were added to DMEM and DMEM/PVA-PEG Pure in increasing amounts compared to the PVA-PEG-derived concentration. Adding 1x, 0.015% w/v%, the concentration compared to PVA-PEG into PVA-PEG Pure resulted in a similar efficiency as normal PVA-PEG when transfecting HEK293T SL cells. Mean value of n=3 independent experiments ± SD.

Article Snippet: The His-NLS-Cre recombinase (herein called Cre), PCV-Cas9, Adenine base editor 8e (herein called ABE8e), and Prime editor 2 (herein called PE2) (Addgene plasmids: 62730, 123643, 161788, 132775 respectively), were expressed by Escherichia coli (BL21 (DE3) T1R pRARE2) upon induction at an optical density of 3 utilizing IPTG.

Techniques: Nucleic Acid Electrophoresis, Incubation, Isolation, Transfection, Centrifugation, Derivative Assay, Concentration Assay

Journal: Communications Biology

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

doi: 10.1038/s42003-018-0054-2

Figure Lengend Snippet: Selective covalent attachment of ssDNA to Cas9-PCV. 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 labeled 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, Labeling, Sequencing, Staining, Electrophoretic Mobility Shift Assay

Journal: Communications Biology

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

doi: 10.1038/s42003-018-0054-2

Figure Lengend Snippet: Covalent attachment of ssODN to PCV fusions of Cas9 enhances insertion of a peptide tag. a Schematic of split luciferase insertion. The C-terminus of nanoluciferase (HiBiT) is encoded on the 200 bp 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 Targeting the GAPDH locus in U2-OS cells. d Targeting a locus in vinculin in HEK-293T cells using an ssODN containing the PCV recognition sequence. e Fold change in RLU compared to Cas9 when varying the RNP concentration with equimolar ssODN. f HDR frequency and HDR/indel ratio from deep sequencing of the GAPDH locus. Graphs represent data from one of multiple independent experiments exhibiting similar results using distinct samples. The data with error bars are shown as mean+/− SD ( n = 3). Significance calculated using 2-tailed Student’s t test: ** P < 0.01, *** P < 0.001

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, Concentration Assay

Journal: Communications Biology

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

doi: 10.1038/s42003-018-0054-2

Figure Lengend Snippet: Covalent tethering enhances HDR efficiency in a fluorescent reporter cell line. 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. Scale bars are 50 µm. c The percent of mCherry positive cells determined by flow cytometry at two different RNP concentrations using a 175 bp 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: Science signaling

Article Title: Phagocytosed photoreceptor outer segments activate mTORC1 in the retinal pigment epithelium

doi: 10.1126/scisignal.aag3315

Figure Lengend Snippet: (A) POS-induced mTORC1 activation in ARPE-19 cells transfected with either KLC1 small interfering RNA (siRNA) or control scrambled (Scr) siRNA. S6 phosphorylation and KLC1 knockdown were analyzed by Western blots. Quantification data are means from five independent experiments (mean ± SEM). *P<0.05 (Kruskal-Wallis test and Dunn’s multiple comparisons test). (B) and (C) Co- immunostaining of rhodopsin and myosin 6 (Myo6) in POS-treated ARPE-19 cells and RPE/choroid whole mounts, respectively. Images are representative of three independent experiments. Scale bar: 5 μm. (D) Quantitation of the data in B and C. Twenty fields with an area of 50 ϗ 50 μm2 were analyzed. (E) Depletion of Myo6 in ARPE-19 cells expressing Cas9 endonuclease and transfected with gRNA targeting Myo6. Blots are representative of three independent experiments. (F) Degradation of POS in cells depleted of Myo6 by CRISPR/Cas9. Quantification data are means from six independent experiments (mean ± SEM). *P<0.05 (Kruskal-Wallis test and Dunn’s multiple comparisons test). (G) POS-induced mTORC1 activation in cells depleted of Myo6 by CRISPR/Cas9. Quantification data are means from five independent experiments (mean ± SEM). ***P<0.001 (one-way ANOVA and Tukey- Kramer Multiple comparsions Test). (H) Amino Acid (AA)-induced mTORC1 activation in cells depleted of Myo6 by CRISPR/Cas9. Data are means of five independent experiments (mean ± SEM). ***P<0.001 (one-way ANOVA and Tukey-Kramer Multiple comparsions Test).

Article Snippet: Cas9 virus (lentiCas9-Blast) was purchased from Addgene.

Techniques: Activation Assay, Transfection, Small Interfering RNA, Western Blot, Immunostaining, Quantitation Assay, Expressing, CRISPR