Journal: EMBO Reports

Article Title: Targeting monocytic Occludin impairs transendothelial migration and HIV neuroinvasion

doi: 10.1038/s44319-024-00190-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The initial plasmid backbone used was the pLenti-spCas9-T2A-Crimson-P2A-puro (kindly provided by Dr. Feng Zheng) and derived versions of this plasmid were also generated to express mNeonGreen (Addgene #122183), TagRFP-T (Addgene #122200) or iRFP670 (Addgene # 122182) fluorescent proteins.

Techniques: Isolation, Recombinant, Sequencing, Knock-Out, DNA Ligation, Transfection, Cell Viability Assay, Blocking Assay, Protease Inhibitor, Virus, Software, Electroporation, Imaging, Polymer, Microscopy, Flow Cytometry, Membrane

Journal: Nature Communications

Article Title: Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

doi: 10.1038/s41467-020-16542-9

Figure Lengend Snippet: a Schematic representation of dOTS. A dRNA (green) with perfect complementarity for an off-target site directs Cas9 binding but not cleavage, protecting the site. b Indel frequencies and specificity ratios (on-target/off-target indel frequency ratios) at the FANCF -sgRNA2 on-target site and OT1 24 h after transfection of HEK-293T cells with Cas9, sgRNA, and FANCF -sgRNA2 OT1 dRNA1 or a nontargeting control dRNA (dNT) that does not target genomic DNA. For conditions without dRNA, an equivalent amount of pMAX-GFP was substituted. Means of n = 3 biological replicates depicted by solid lines. c Normalized specificity ratios, computed as the specificity ratio of the best dRNA condition (Supplementary Table ) divided by the specificity ratio of the sgRNA only condition for 19 guide/off-target pairs tested in HEK-293T cells. Points depict the mean of n = 3 biological replicates, error bars show the standard error of the mean. d Indel frequencies and specificity ratios at the FANCF -sgRNA2 on-target site and OT1 24 h after transfection in U2OS cells, and e Elf1 embryonic stem cells. Control samples to the right of the x-axis break were performed separately. iCas9 denotes stable integration of Cas9 under the control of a doxycycline-inducible promoter. Means of n = 3 cell culture replicates depicted by solid lines. OT off-target. Source data are available in the Source Data file.

Article Snippet: High-specificity variants of Cas9 — eSpCas9(1.1) (gift from F. Zheng, Broad Institiute; Addgene plasmid 71814) and VP12 (‘SpCas9-HF1’, gift from K. Joung, Massachusetts General Hospital; Addgene plasmid 72247) were subcloned into pcDNA5/FRT/TO backbone (ThermoFisher).

Techniques: Binding Assay, Transfection, Control, Cell Culture

Journal: Nature Communications

Article Title: Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

doi: 10.1038/s41467-020-16542-9

Figure Lengend Snippet: Representative gels of in vitro cleavage of PCR products containing either a FANCF -sgRNA2 OT1 or b the FANCF -sgRNA2 on-target site with either 150 or 450 fmoles of Cas9 FANCF -sgRNA2 RNP in the presence of variable amounts of the Cas9 FANCF- sgRNA2 OT1 dRNA1 complex. Fraction of uncut DNA determined by gel densitometry. Means of n = 3 replicates depicted by solid lines. For uncropped gels, see Supplementary Fig. . Source data are available in the Source Data file.

Article Snippet: High-specificity variants of Cas9 — eSpCas9(1.1) (gift from F. Zheng, Broad Institiute; Addgene plasmid 71814) and VP12 (‘SpCas9-HF1’, gift from K. Joung, Massachusetts General Hospital; Addgene plasmid 72247) were subcloned into pcDNA5/FRT/TO backbone (ThermoFisher).

Techniques: In Vitro

Journal: Nature Communications

Article Title: Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

doi: 10.1038/s41467-020-16542-9

Figure Lengend Snippet: a Editing of FANCF- sgRNA2 on-target and OT1 sites using chemically inducible Cas9 (ciCas9) from 0 to 16 h after activation with A115. Nontargeting dRNA is a 14-base control dRNA targeting a non-endogenous site. NT = non-transfected control. Points depict the mean of n = 3 biological replicates. Error bars show the standard error of the mean. b Indel frequencies and specificity ratios at VEGFA sgRNA3 on-target and OT2 sites in cells transfected with plasmids encoding Cas9 and varying ratios of VEGFA sgRNA3 and dRNA2. dRNA untreated cells were transfected with Cas9 and a 1:1 VEGFA sgRNA3:GFP plasmid ratio. Error bars depict s.e.m. ( n = 3 cell culture replicates). OT off-target. Source data are available in the Source Data file.

Article Snippet: High-specificity variants of Cas9 — eSpCas9(1.1) (gift from F. Zheng, Broad Institiute; Addgene plasmid 71814) and VP12 (‘SpCas9-HF1’, gift from K. Joung, Massachusetts General Hospital; Addgene plasmid 72247) were subcloned into pcDNA5/FRT/TO backbone (ThermoFisher).

Techniques: Activation Assay, Control, Transfection, Plasmid Preparation, Cell Culture

Journal: Nature Communications

Article Title: Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

doi: 10.1038/s41467-020-16542-9

Figure Lengend Snippet: Indel frequencies and specificity ratios 24 h after transfection with a plasmids encoding WT Cas9, a dRNA targeting VEGFA sgRNA3 OT2 (dRNA2) and a truncated guide VEGFA tru-sgRNA3, or b High-specificity variants of Cas9 and a dRNA targeting FANCF -sgRNA2 OT1 (dRNA1). Wild-type Cas9 (WT), eSpCas9 (E), SpCas9-HF1 (HF1), HypaCas9 (Hypa). Means of n = 3 cell culture replicates depicted by solid lines. OT off-target. Source data are available in the Source Data file.

Article Snippet: High-specificity variants of Cas9 — eSpCas9(1.1) (gift from F. Zheng, Broad Institiute; Addgene plasmid 71814) and VP12 (‘SpCas9-HF1’, gift from K. Joung, Massachusetts General Hospital; Addgene plasmid 72247) were subcloned into pcDNA5/FRT/TO backbone (ThermoFisher).

Techniques: Transfection, Cell Culture

Journal: Nature Communications

Article Title: Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

doi: 10.1038/s41467-020-16542-9

Figure Lengend Snippet: a Schematic depicting dReCS and alignment of BFP, GFP, sgRNA, and dRNA sequences. dRNA (green) exhibiting perfect complementarity for the repaired site directs Cas9 binding but not cleavage, protecting the repaired site. Single base change to generate GFP from BFP is displayed in green with affected codon indicated by gray box. PAM sequences are underlined. Black arrow indicates best dRNA, as determined by maximal improvement in HDR yield. b Indels and homology-directed repair (HDR) as assessed by flow cytometry, where indels lead to a loss of BFP signal, and HDR leads to a loss of BFP and gain of GFP signal. c HDR as a percentage of total Cas9 edits observed. Means of n = 3 cell culture replicates depicted by solid lines. dRNA BFP sgRNA1 dRNA3 (see Supplementary Fig. , Supplementary Data Set ). Source data are available in the Source Data file.

Article Snippet: High-specificity variants of Cas9 — eSpCas9(1.1) (gift from F. Zheng, Broad Institiute; Addgene plasmid 71814) and VP12 (‘SpCas9-HF1’, gift from K. Joung, Massachusetts General Hospital; Addgene plasmid 72247) were subcloned into pcDNA5/FRT/TO backbone (ThermoFisher).

Techniques: Binding Assay, Flow Cytometry, Cell Culture

Journal: Cell reports

Article Title: CPG15/Neuritin Mimics Experience in Selecting Excitatory Synapses for Stabilization by Facilitating PSD95 Recruitment

doi: 10.1016/j.celrep.2019.07.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Finally, CPG15 between the EcoRI and XmaI sites was replaced with Synaptophysin-TdTomato , which was amplified from the plasmid Ai34 (Addgene plasmid# 34881 , a gift from Hongkui Zheng) with primers containing MfeI and XmaI sites, to generate pCAG-Synaptophysin-TdTomato-IRES2-OHT-Cre .

Techniques: Virus, Recombinant, Software