Journal: The CRISPR Journal

Article Title: Increased Efficiency for Biallelic Mutations of the CCR5 Gene by CRISPR-Cas9 Using Multiple Guide RNAs As a Novel Therapeutic Option for Human Immunodeficiency Virus

doi: 10.1089/crispr.2020.0019

Figure Lengend Snippet: Schematic diagrams of CCR5 and the targeting vectors. (A) Schematic representation of the CCR5 loci with exons 1–3 and the Δ32 mutation. The sgRNA target Sites I and II are indicated by violet and red bars, respectively; the PCR primers encompassing the sgRNA and the Δ32 mutation are identified with gold bars. (B) The sequence of the 558 bp CCR5 amplicon showing the target sites for sgRNA1 (violet) and sgRNA2 (red), the PAMs (blue), and the PCR primers (gold). The gray-highlighted text is transmembrane domains and the white text is intracellular/extracellular domains. The Δ32 mutation is gray strikethrough. (C) A schematic diagram of the CCR5 protein structure as the seven-transmembrane receptor. The target sites for sgRNA1 (violet) and sgRNA2 (red), the PAMs (blue), and the PCR primers (gold brackets) are identified. The gold arrows indicated the predicted cutting sites. (D) Schematic diagram of pX458, the Cas9/sgRNA expression vector. The GFP is expressed with the Cas9 as a 2A fusion product, which is cleaved. The sgRNA is expressed by the U6 promoter. (E) Schematic diagram of the lentiviral vector expressing two sgRNAs, Cas9, and GFP. The cis-acting lentiviral elements (CMVp, Ψ, RRE, cPPT, wPRE, PGKp, LTRs) are also labeled. GFP, green fluorescent protein, PAM, protospacer adjacent motif; PCR, polymerase chain reaction; sgRNA, single-guide RNA.

Article Snippet: The pSpCas9(BB)-2A-GFP plasmid (pX458), the multiple lentiviral expression (MuLE) Entry vectors for sgRNA expression pMuLE ENTR U6 stuffer sgRNA scaffold L1-L4 (#62128), the pMuLE ENTR U6 stuffer sgRNA scaffold R4-R3 (#62131), Cas9 expression pMuLE ENTR SV40-hCas9 L3-L2 (#62133), and destination vector pMuLE Lenti Deste eGFP (#62175) were obtained from Addgene (Watertown, MA).

Techniques: Mutagenesis, Sequencing, Amplification, Expressing, Plasmid Preparation, Labeling, Polymerase Chain Reaction

Journal: The CRISPR Journal

Article Title: Increased Efficiency for Biallelic Mutations of the CCR5 Gene by CRISPR-Cas9 Using Multiple Guide RNAs As a Novel Therapeutic Option for Human Immunodeficiency Virus

doi: 10.1089/crispr.2020.0019

Figure Lengend Snippet: Targeting CCR5 with an sgRNA using CRISPR-Cas9. (A) CCR5 gene editing with CRISPS-Cas9. The 558 bp PCR amplicon (black arrow) was generated from sorted ASCs after transfection with control (Ctrl) or sgRNA2. Surveyor assay (Sur) demonstrates cleavage of the amplicon into the expected 316 and 242 bp fragments (black arrow). The frequency of indels is 35%.60 (B) Sequencing results of TOPO cloned PCR amplicon generated with the Fwd and Rev CCR5 primers. The sequences (right column) were sorted from largest inserts (green letters) to the largest deletions (green dashes). The left column is the size of the indel. The center column is the frequency of each sequence. ASC, adipose-derived stem cell; indels, insertions or deletions; Fwd, forward; Rev, reverse.

Article Snippet: The pSpCas9(BB)-2A-GFP plasmid (pX458), the multiple lentiviral expression (MuLE) Entry vectors for sgRNA expression pMuLE ENTR U6 stuffer sgRNA scaffold L1-L4 (#62128), the pMuLE ENTR U6 stuffer sgRNA scaffold R4-R3 (#62131), Cas9 expression pMuLE ENTR SV40-hCas9 L3-L2 (#62133), and destination vector pMuLE Lenti Deste eGFP (#62175) were obtained from Addgene (Watertown, MA).

Techniques: CRISPR, Amplification, Generated, Transfection, Control, Sequencing, Clone Assay, Derivative Assay

Journal: The CRISPR Journal

Article Title: Increased Efficiency for Biallelic Mutations of the CCR5 Gene by CRISPR-Cas9 Using Multiple Guide RNAs As a Novel Therapeutic Option for Human Immunodeficiency Virus

doi: 10.1089/crispr.2020.0019

Figure Lengend Snippet: Targeting CCR5 with two guide RNAs using a tetracistronic lentiviral vector expressing CRISPR-Cas9. (A) CCR5 gene editing with CRISPS/Cas9. Surveyor assay (Sur) or standard PCR product (PCR) with the 558 bp amplicon generated from ASCs transduced with the dual-guide (sgRNA1 and sgRNA2) lentiviral vector or control (Ctrl). Arrows indicate the WT allele (558 bp), the deleted allele (∼500 bp) with the ∼60 bp deletion, and the expected 302 bp and 256/242 bp fragments from editing sgRNA1 and sgRNA1/2, respectively. (B) Sequencing results of TOPO cloned PCR amplicon generated with the Fwd and Rev CCR5 primer pairs. The sequences (right column) were sorted from largest inserts (green letters) to the largest deletions (dashes). The left column is the size of the indel. The center column is the frequency of each sequence.

Article Snippet: The pSpCas9(BB)-2A-GFP plasmid (pX458), the multiple lentiviral expression (MuLE) Entry vectors for sgRNA expression pMuLE ENTR U6 stuffer sgRNA scaffold L1-L4 (#62128), the pMuLE ENTR U6 stuffer sgRNA scaffold R4-R3 (#62131), Cas9 expression pMuLE ENTR SV40-hCas9 L3-L2 (#62133), and destination vector pMuLE Lenti Deste eGFP (#62175) were obtained from Addgene (Watertown, MA).

Techniques: Plasmid Preparation, Expressing, CRISPR, Amplification, Generated, Transduction, Control, Sequencing, Clone Assay

Journal: Communications Biology

Article Title: Tau affects P53 function and cell fate during the DNA damage response

doi: 10.1038/s42003-020-0975-4

Figure Lengend Snippet: a , b Parental (wt) or 232P (Tau-KO) cells treated 30 min without (basal) or with 60 μM etoposide and recovered for 6 h in the absence (eto) or presence of 10 µg/mL KU-55933 and/or 5 µg/mL nutlin-3. a Mean intensity ± sem of single-cell nuclear P53 or MDM2 (DAPI mask, ImageJ) shown as fold of basal conditions, n > 100 cells/condition distributed over five images. b Percent clCasp3-positive cells shown as mean ± SD of five images (basal) or 15 images (treatments), n > 500 cells/condition. Non-parametric independent samples test and Kruskal–Wallis pairwise comparison between cell lines (in bold) or for treatment for each cell line (in vertical). c Western bot analysis of P53 in parental (wt) or 232 P (Tau-KO) cells at basal conditions, after 30 min 60 μM etoposide and 4 h recovery without or with 10 μM MG132. GAPDH served as loading control. d Parental (wt) or 232P (Tau-KO) cells pre-treated for 30 min with 60 μM etoposide followed by 4 h with 10 μM MG132, were incubated with 25 μM of cycloheximide (CHX) for the indicated chase times. Single-cell nuclear P53 or nuclear MDM2 (DAPI mask, ImageJ) shown as fold of wt cells at basal conditions. Mean intensity ± sem of n > 100 cells/condition distributed over five images. Independent measures ordinary two-way ANOVA, source of variation for cell lines (bold), multiple Bonferroni pairwise comparisons of treatment for each line (in italic). e Parental (wt) or (Tau-KO) 232P cells treated for 30 min with 60 μM etoposide and 6 h recovery analyzed with a 90 kDa MDM2 rabbit antibody (green and middle panel with GAPDH as loading control) or a 60 and 90 kDa MDM2 mouse antibody (red and bottom panel).

Article Snippet: The cell lysates for P53 immune precipitation were cleared by centrifugation at 20,000 g per 10 min. For immune blots , we used 0.2 μg/mL Tau13 (sc-21796, Santa Cruz), 0.18 μg/mL GAPDH (ab181602, Abcam), 0.4 μg/mL P53 DO-1 (sc-126, Santa Cruz), 4 μg/mL P53 Pab 1801 (sc-98, Santa Cruz), 0.5 μg/mL pS 15 -P53 (ab223868, Abcam), 0.1 μg/mL rabbit D1V2Z MDM2 (#86934, Cell Signaling), 0.2 μg/mL mouse SMP14 MDM2 (sc965, Santa Cruz), 0.05 μg/mL clAsp 175 -Caspase3 (#9661, Cell Signaling), or 0.2 μg/mL p21 (sc53870, Santa Cruz).

Techniques: Comparison, Western Blot, Control, Incubation

Journal: Communications Biology

Article Title: Tau affects P53 function and cell fate during the DNA damage response

doi: 10.1038/s42003-020-0975-4

Figure Lengend Snippet: Cell lysates of SH-SY5Y treated with 10 μM of MG132 to stabilize P53 expression, without (ctrl) or with (eto) a 30 min pre-treatment with 60 μM etoposide, were subjected to immune precipitation of endogenous P53 with a rabbit antibody (P53) or with a rabbit GFP antibody as negative IP control (GFP). Western blot analysis for co-precipitation of MDM2 or Tau with the respective mouse antibodies as indicated. The blots on the top show the analysis of the starting material (cell lysates), those on the bottom the immunoprecipitation (IP). The P53 blots are entirely shown, whereas for MDM2 and Tau, the blots were cut between the 55 kDa and the 95 kDa protein size markers and analyzed separately.

Article Snippet: The cell lysates for P53 immune precipitation were cleared by centrifugation at 20,000 g per 10 min. For immune blots , we used 0.2 μg/mL Tau13 (sc-21796, Santa Cruz), 0.18 μg/mL GAPDH (ab181602, Abcam), 0.4 μg/mL P53 DO-1 (sc-126, Santa Cruz), 4 μg/mL P53 Pab 1801 (sc-98, Santa Cruz), 0.5 μg/mL pS 15 -P53 (ab223868, Abcam), 0.1 μg/mL rabbit D1V2Z MDM2 (#86934, Cell Signaling), 0.2 μg/mL mouse SMP14 MDM2 (sc965, Santa Cruz), 0.05 μg/mL clAsp 175 -Caspase3 (#9661, Cell Signaling), or 0.2 μg/mL p21 (sc53870, Santa Cruz).

Techniques: Expressing, Control, Western Blot, Immunoprecipitation

Journal: Communications Biology

Article Title: Tau affects P53 function and cell fate during the DNA damage response

doi: 10.1038/s42003-020-0975-4

Figure Lengend Snippet: PCR Primers (all specific for homo sapiens mRNAs).

Article Snippet: The cell lysates for P53 immune precipitation were cleared by centrifugation at 20,000 g per 10 min. For immune blots , we used 0.2 μg/mL Tau13 (sc-21796, Santa Cruz), 0.18 μg/mL GAPDH (ab181602, Abcam), 0.4 μg/mL P53 DO-1 (sc-126, Santa Cruz), 4 μg/mL P53 Pab 1801 (sc-98, Santa Cruz), 0.5 μg/mL pS 15 -P53 (ab223868, Abcam), 0.1 μg/mL rabbit D1V2Z MDM2 (#86934, Cell Signaling), 0.2 μg/mL mouse SMP14 MDM2 (sc965, Santa Cruz), 0.05 μg/mL clAsp 175 -Caspase3 (#9661, Cell Signaling), or 0.2 μg/mL p21 (sc53870, Santa Cruz).

Techniques:

Journal: Frontiers in Oncology

Article Title: Use of phage display biopanning as a tool to design CAR-T cells against glioma stem cells

doi: 10.3389/fonc.2023.1124272

Figure Lengend Snippet: Dual peptide CAR-T cell design. (A) Diagram of the CAR construct designs. The E-28t28z and E-8t28z constructs use CD28 and CD8 as the hinge/transmembrane domains respectively, while both constructs employed CD28 for the co-stimulatory domain. The E-28t28z-tCD34 and E-8t28z-tCD34 constructs are identical to the E-28t28z and E-8t28z constructs but feature co-expression of truncated CD34 as an extracellular marker. (B) Diagram of a CAR-T cell with the different dual peptide-based constructs as the antigen-recognition domain. (C) CD34+ expression analysis by flow cytometry allows for a built-in quantification of transduction efficiency. (D) ELISA results displaying IFN-gamma secretion from CAR- or mock-transduced (GFP) T cells co-cultured overnight against T387 GSCs or cell-free media, **** = p < 0.0001. Values shown in the bar chart represent the average of two independent experiments. (E) CD3ζ western blotting with GAPDH as a housekeeping gene, used for additional support to confirm the presence or lack of the CAR following transduction. LTR, Long terminal repeat; S, signal peptide; P2A, Porcine teschovirus-1 2A self-cleaving peptide; tCD34, Truncated CD34.

Article Snippet: Membranes were incubated with 0.2 μg/mL mouse anti-human CD3ζ (Santa Cruz Biotechnology, SC-166275, Dallas, TX) and 0.04 μg/mL rabbit anti-human glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology, SC-25778, Dallas, TX).

Techniques: Construct, Expressing, Marker, Flow Cytometry, Transduction, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot