Journal: Cells

Article Title: IL-3-Induced Immediate Expression of c- fos and c- jun Is Modulated by the IKK2-JNK Axis

doi: 10.3390/cells11091451

Figure Lengend Snippet: Effects of IKK1 and/or IKK2 KO on IL-3-induced expression of IEGs. ( A ) IKK1 and IKK2 protein levels in Ba/F3 parental cells (WT) and CRISPR/Cas9-mediated IKK1 KO, IKK2 KO, and IKK1/2 DKO cells. Whole-cell lysates were immunoblotted with the indicated Abs. A dagger denotes the detection of IKK2 by potential cross-reactivity of the anti-IKK1 Ab. ( B – D ) Relative levels of expression of c- fos ( B ), c- jun ( C ), and c- myc ( D ) mRNAs measured 20, 40, and 40 min, respectively, after IL-3 stimulation of Ba/F3 parental, IKK1 KO, IKK2 KO, and IKK1/2 DKO cells. * p < 0.05; n.s., not significant, as assessed by the Kruskal-Wallis test with the Steel-Dwass test. Each dot represents the result from independent experiments ( n = 5). Means are indicated by bars.

Article Snippet: To generate IKK1 or IKK2 single KO clones, 5 × 10 6 parental Ba/F3 cells were electroporated with 120 μg of a Cas9 expression plasmid (41815, Addgene), 120 μg of a sgRNA expression plasmid targeting IKK1 or IKK2 , and 1 μg of a plasmid encoding a puromycin resistant gene using the Gene Pulser Xcell (1652660J1, Bio-Rad, Richmond, CA, USA).

Techniques: Expressing, CRISPR

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: Scientific Reports

Article Title: The IL-17-IL-17RA axis is required to promote osteosarcoma progression in mice

doi: 10.1038/s41598-023-49016-1

Figure Lengend Snippet: Establishment of IL-17RA-deficient AX cells. ( a ) Two independent IL-17RA-deficient AX lines (KO1 and KO2) were generated by lentiviral transduction using the CRISPR/Cas9 system. AX cells transduced with scramble constructs served as controls (AX-SCR). IL-17RA expression on these AX cell lines, which harbor a GFP tag, was analyzed by flow cytometry. Solid or dotted lines in histograms represent AX cells stained with anti-IL-17RA or isotype control antibody, respectively. ( b ) IL-17RA-deficient and control AX cells (Parental AX cells and AX-SCR) were cultured 24 h with or without (vehicle) IL-17A (500 ng/ml) and the number of AX cells was analyzed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay measuring absorbance at 570 nm. Data represent mean absorbance ± s.d. (n = 6, * P < 0.05). ( c ) IL-17RA knockout (middle and bottom rows) or scramble-transduced AX cells (top row) were treated with or without (vehicle) IL-17A (500 ng/ml). After 24 h, total RNA was prepared, and expression of ALP , Oc or Runx2 relative to β-actin was analyzed by quantitative real-time PCR. Data represent mean indicated gene expression relative to β-actin ± s.d. (n = 6, * P < 0.05). ( d ) IL-17RA knockout (middle and right rows) or scramble-transduced AX cells (left rows) were treated with or without (vehicle) IL-17A (500 ng/ml). After 48 h, total protein was extracted, and ALP, Oc and Runx2 levels were analyzed by western blot. The graph shows the normalization of ALP, Oc and Runx2 levels to total Actin levels (* P < 0.05, n = 3).

Article Snippet: The lentiviral pLV[2CRISPR]-hCas9:T2A:Puro-U6 expression vector (VectorBuilder, Inc., Chicago, IL, USA) containing the sequence of murine IL-17RA gRNAs (two independent sequences) or scramble gRNA were used. gRNA sequences were as follows.

Techniques: Generated, Transduction, CRISPR, Construct, Expressing, Flow Cytometry, Staining, Cell Culture, Knock-Out, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cell reports

Article Title: Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair.

doi: 10.1016/j.celrep.2016.01.019

Figure Lengend Snippet: Figure 2. Cell-Cycle-Tailored Expression of hCas9-hGem(1/110) (A–D) Representative western blots showing steady-state levels of hCas9 and hCas9-hGem(1/110) in HEK293T (A) and A549 (C) cells 72 hr after transfection with 500 ng (HEK293T) or 2 mg (A549) expression vector each. ACTB serves as loading control. (B and D) Real-time qPCR analysis of hCas9 transcript levels in HEK293T (B) and A549 (D) cells 72 hr after transfection. Shown is the mean ± SEM of three independent experiments. IB, immunoblot; rel., relative; norm., normalized. *p < 0.05; **p < 0.01. (E) Representative western blot showing expression of hCas9 variants and endogenous Geminin in HEK293T after nocodazole treatment (200 ng/ml; 22 hr). (F) Representative western blot showing cell-cycle-resolved expression of hCas9 variants and endogenous Geminin in HEK293T. Vinculin and Ponceau S are shown to verify equal protein loading. All experiments were performed in biological replicates (n = 3). See also Figure S1.

Article Snippet: The hCas9 expression plasmid (pX330) (Cong et al., 2013; Ran et al., 2013) was acquired from Addgene (plasmid #42230).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Cell reports

Article Title: Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair.

doi: 10.1016/j.celrep.2016.01.019

Figure Lengend Snippet: Figure 3. hCas9-hGem(1/110) Enhances HDR at a Monoallelic Reporter Gene Locus (A) Overview of the engineered single-copy EGFP locus in the HEK293T reporter cell line. (B) Percent EGFP-positive cells analyzed 72 hr after transfection with indicated amounts of pX330 variants and repair plasmid. (C) Relative (rel.) rates of HDR. Rate of hCas9 set to 1. (D) Representative western blot showing expression of hCas9 variants in HEK293T 72 hr after transfection with the indicated plasmid amounts. IB, immunoblot. All experiments were performed in biological replicates (n R 3). Data indicate the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The hCas9 expression plasmid (pX330) (Cong et al., 2013; Ran et al., 2013) was acquired from Addgene (plasmid #42230).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: Cell reports

Article Title: Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair.

doi: 10.1016/j.celrep.2016.01.019

Figure Lengend Snippet: Figure 4. hCas9-hGem(1/110) Enhances Exogenous Donor-Template-Mediated HDR at an Endogenous Non-coding RNA Locus (A) Schematic overview of the endogenous MALAT1 non-coding RNA locus. The target region of the designed sgMALAT1 is highlighted (blue line). Cleavage occurs between the TATA box and transcriptional start site (red arrow). (legend continued on next page)

Article Snippet: The hCas9 expression plasmid (pX330) (Cong et al., 2013; Ran et al., 2013) was acquired from Addgene (plasmid #42230).

Techniques:

Journal: Cell reports

Article Title: Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair.

doi: 10.1016/j.celrep.2016.01.019

Figure Lengend Snippet: Figure 5. Transient G2/M Cell-Cycle Arrest Further Increases HDR (A) Timeline of cell treatments. HEK293T cells were transfected with pX330 and repair donor plasmids (1 mg each). Nocodazole (200 ng/ml) was added to transiently arrest cells in the G2/M stahe. After 21 hr, medium was changed, and cells continued proliferation for an additional 48 hr. (B) HDR frequency in HEK293T cells (with or without nocodazole) analyzed 100 hr after transfection of 1 mg pX330 and 1 mg MALAT1 repair donor plasmid. (C and D) Absolute (abs.) percentage (C) and relative (rel.) rate (D) of HDR at the endogenous MALAT1 locus. Relative rate of hCas9 set to 1. All experiments were performed in biological replicates (n = 4). Data indicate the mean ± SEM. *p < 0.05; **p < 0.01. See also Figure S2.

Article Snippet: The hCas9 expression plasmid (pX330) (Cong et al., 2013; Ran et al., 2013) was acquired from Addgene (plasmid #42230).

Techniques: Transfection, Plasmid Preparation, Relative Rate