Journal: Viruses

Article Title: Validation of Candidate Host Cell Entry Factors for Bovine Herpes Virus Type-1 Based on a Genome-Wide CRISPR Knockout Screen

doi: 10.3390/v16020297

Figure Lengend Snippet: Multiplex CRISPRi against pro-viral candidate genes results in reduced viral titers. ( a ). CRISPR3i against a host gene in action by binding immediately downstream of TSS in tandem for synergistic gene repression. ( b ). The one-step cloning strategy to synthesize PiggyBac transposon vectors carrying three sgRNA expression cassettes in tandem to implement CRISPR3i by PB transposition. ( c ). Plaque formation assay results in cells with CRISPR3i expression intended to interfere with transcription of pro-viral genes involved in HS biosynthesis and other functions, as identified by the CRISPRko screen; CTRL represents virus titer from cells expressing three non-targeting sgRNAs (n >= 3). Results were ordered by p -value. ****: p < 0.0001; ***: p < 0.005; **: p < 0.01; *: p < 0.05; n.s. not significant with p > 0.05 based on one-way ANOVA followed by multiple comparisons between CTRL and CRISPR3i cells, error bars represent +/− 1 SD.

Article Snippet: The PiggyBac vector used to deliver CRISPRi, PB-U6g5_PGK_Puro2aBFP, was constructed by cutting out the segment containing the hU6 sgRNA expression cassette and the Puro2aBFP selection marker from pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W (gift from Kosuke Yusa, Addgene # 67974) and cloning it into a PiggyBac backbone.

Techniques: Multiplex Assay, Binding Assay, Cloning, Expressing, Plaque Formation Assay, Virus

Journal: Iranian Journal of Basic Medical Sciences

Article Title: CRISPR/Cas9, a new approach to successful knockdown of ABCB1/P-glycoprotein and reversal of chemosensitivity in human epithelial ovarian cancer cell line

doi: 10.22038/IJBMS.2017.25145.6230

Figure Lengend Snippet: The linear map of CRISPR/Cas9 vector (A) and the locations and sequences of the three sgRNAs targeting the fourth and fifth exons of ABCB1 gene (B)

Article Snippet: Three custom-designed sgRNA/Cas9 expression vectors, targeting the fourth and fifth exons of ABCB1 gene (Accession No NM_000927.3), were designed using CRISPR DESIGN ( http://crispr.mit.edu/ ) tool and were then synthesized by Guangzhou FulenGen Company (P R China).

Techniques: CRISPR, Plasmid Preparation

Journal: Iranian Journal of Basic Medical Sciences

Article Title: CRISPR/Cas9, a new approach to successful knockdown of ABCB1/P-glycoprotein and reversal of chemosensitivity in human epithelial ovarian cancer cell line

doi: 10.22038/IJBMS.2017.25145.6230

Figure Lengend Snippet: Real-time Q-PCR analysis of expression of ABCB1. The mRNA levels of ABCB1 were analyzed by real-time PCR, 72 hr after antibiotic selection. Expression levels of ABCB1 were normalized to an average of two reference genes, GAPDH and β-Actin. A2780/ADR cells transfected with CRISPR/Cas9 showed a remarkable decrease in ABCB1 expression level compared to the control and scramble groups; bars: SE (***, P< 0.001 )

Article Snippet: Three custom-designed sgRNA/Cas9 expression vectors, targeting the fourth and fifth exons of ABCB1 gene (Accession No NM_000927.3), were designed using CRISPR DESIGN ( http://crispr.mit.edu/ ) tool and were then synthesized by Guangzhou FulenGen Company (P R China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Selection, Transfection, CRISPR

Journal: Iranian Journal of Basic Medical Sciences

Article Title: CRISPR/Cas9, a new approach to successful knockdown of ABCB1/P-glycoprotein and reversal of chemosensitivity in human epithelial ovarian cancer cell line

doi: 10.22038/IJBMS.2017.25145.6230

Figure Lengend Snippet: Detection of indels generated by CRISPR/Cas9 using surveyor assay. Red arrowheads indicate predicted Cas9 cutting sites of ABCB1 PCR products., sgRNA-1: PCR product size: 510 bp, predicted cleavage size: 220 & 290 bp, sgRNA-2: PCR product size: 510 bp, predicted cleavage size: 170 & 340 bp, sgRNA-3: PCR product size: 952 bp: Predicted cleavage size: 335 & 617 bp, Control-1: 510 bp, Control-2: 952 bp

Article Snippet: Three custom-designed sgRNA/Cas9 expression vectors, targeting the fourth and fifth exons of ABCB1 gene (Accession No NM_000927.3), were designed using CRISPR DESIGN ( http://crispr.mit.edu/ ) tool and were then synthesized by Guangzhou FulenGen Company (P R China).

Techniques: Generated, CRISPR

Journal: Iranian Journal of Basic Medical Sciences

Article Title: CRISPR/Cas9, a new approach to successful knockdown of ABCB1/P-glycoprotein and reversal of chemosensitivity in human epithelial ovarian cancer cell line

doi: 10.22038/IJBMS.2017.25145.6230

Figure Lengend Snippet: Doxorubicin chemosensitivity. MTT assay was performed to evaluate A2780/ADR cells viability, transfected and untransfected cells (control), 48 hr post-treatment with increasing concentrations of doxorubicin. The optical density of each well was measured with a microplate spectrophotometer at 490 nm. The viability of cells transfected with CRISPR/Cas9 was dramatically decreased compared to the control and scrambled transfected cells. In all groups, the proliferation rate of the cells was decreased with increasing concentrations of doxorubicin. The greatest decrease in proliferation rate was observed in A2780/ADR cells transfected with CRISPR/Cas9 compared to other two groups. Each condition was repeated three times (***, P <0.001)

Article Snippet: Three custom-designed sgRNA/Cas9 expression vectors, targeting the fourth and fifth exons of ABCB1 gene (Accession No NM_000927.3), were designed using CRISPR DESIGN ( http://crispr.mit.edu/ ) tool and were then synthesized by Guangzhou FulenGen Company (P R China).

Techniques: MTT Assay, Transfection, Spectrophotometry, CRISPR

Journal: eLife

Article Title: The proteolysis of ZP proteins is essential to control cell membrane structure and integrity of developing tracheal tubes in Drosophila

doi: 10.7554/eLife.91079

Figure Lengend Snippet: ( A ) Generation of three independent pio mutant alleles which lack all relevant protein domains. Schema of CRISPR/Cas9-mediated mutagenesis to generate frame shift mutations in the 5' region of the pio ORF. Map of genomic pio region and of single guide RNA (sgRNA) target site (red letters) with PAM (green letters), ORF (black boxes), and UTRs (gray boxes). Alleles of pio with generated indels that cause ORF frame shifts in pio 17c , pio 5M , and pio 11R mutant alleles. Frame shifts result in the expression of truncated Pio proteins. Signal peptide (blue), ZP domain (green), transmembrane domain (yellow), frame shifted amino acid sequence (red), and protease cleavage sites (pcs) are indicated. SMART analysis of Pio (PB_FBpp0072219) supports previous findings by . N-terminal ZP module (66–351aa) and C-terminal transmembrane domain (TM, 410–432aa) are indicated. The anti-Pio antibody (kindly provided by the Affolter lab) recognizes a polypeptide stretch between 186 and 200aa within (arrow) the ZP module. Thus, anti-Pio antibody detects Pio also after Furin (predicted site at 355aa) and Np processing. ( B,C ) The pio 5m mutant stage 16 embryos show sinusoidal tube overexpansion phenotype with chitin staining and quantification reveals significantly increased dorsal trunk length (n=10 embryos). Bars represent ± SD and p-value from t-test is indicated with an asterisk (***, p<0.0001). ( D ) Orthogonal projection shows a bulge-like apical cell membrane deformation pio 5m mutant dorsal trunk at beginning of stage 17 and collapsed tube lumen. The Pio (red) and Uif (blue) antibodies stainings are shown together with chitin (green). Note that Pio staining is strongly reduced in pio 5m mutant stage 16 embryos when compared with control . ( E,F ) The pio 5m mutant stage 17 embryos show loose taenidial fold formation and airway gas-filling defects (n=147). Altogether, tracheal phenotypes of pio 5M and pio 17c alleles are similar. The tracheal phenotypes include the branch disintegration phenotype observed from pio 2R-16 point mutation allele. ( G ) Confocal Z-stack projections of pio 5M and pio 5M /+ heterozygous control embryos. In contrast to control, pio 5M mutants show strongly reduced Pio staining indicated in red (left) and as single channel in gray (middle panel). Yellow dashes mark the tracheal dorsal trunk. ( H ) Airyscan images of dorsal trunks. Tracheal Pio staining is strongly reduced in stage 16 and stage 17 in pio 17c and pio 5M mutant embryos. Scale bars represent 10 µm.

Article Snippet: Vectors for sgRNA expression were injected into nos-Cas9-3A embryos by BestGene.

Techniques: Mutagenesis, CRISPR, Generated, Expressing, Sequencing, Staining, Membrane, Control

Journal: eLife

Article Title: The proteolysis of ZP proteins is essential to control cell membrane structure and integrity of developing tracheal tubes in Drosophila

doi: 10.7554/eLife.91079

Figure Lengend Snippet: ( A ) The stage 17 pio loss-of-function embryos are able to clear chitin-matrix proteins from tracheal tube lumina, as indicated by Obst-A (red), Knk (red), and WGA (blue) stainings. Shown are confocal overview and higher magnification images and the corresponding orthogonal projections of the tube lumen (right hand). White arrows point to the apical cell membrane and yellow arrows to the tube lumen area from which orthogonal projections were generated. Scale bars represent 10 µm. ( B ) Schema of the 5’ region of the pio genomic locus together with the donor vector containing two homology arms (red) and mCherry encoding sequences followed by a 3xP3-DsRed marker gene flanked by PiggyBac transposon ends (yellow). The sgRNA recognition site (magenta), PAM (green), translated DNA (black boxes), and UTRs (gray boxes) are indicated before and after CRISPR/Cas9-mediated homology-directed repair. Signal peptides (blue), mCherry (red), ZP domains (green), Furin cleavage sites (pcs), and transmembrane domains are indicated below in Pio and mCherry::Pio proteins. ( C ) Confocal maximum intensity projections of anti-mCherry immunostainings of whole-mount embryos. mCherry::Pio shows a Pio-characteristic ectodermal expression pattern. ep: epidermis, fg: foregut, hg: hindgut, ps: posterior spiracle, ts: tracheal system, sg: salivary glands.

Article Snippet: Vectors for sgRNA expression were injected into nos-Cas9-3A embryos by BestGene.

Techniques: Membrane, Generated, Plasmid Preparation, Marker, CRISPR, Expressing

Journal: Nature Communications

Article Title: Transient expression of an adenine base editor corrects the Hutchinson-Gilford progeria syndrome mutation and improves the skin phenotype in mice

doi: 10.1038/s41467-022-30800-y

Figure Lengend Snippet: a The HGPS-causing point mutation c.1824C > T (p. G608G) was corrected by ABEmax-VQR that recognizes the PAM variant 5´-NGA-3´ (orange). The crRNA part of the sgRNA is positioning the c.1824C > T mutation at the protospacer position A6 (red) in reference to the PAM sequence. b Targeted deep-seq. of the protospacer region shows high mutation (A6, red) editing and low bystander (A10, purple) editing in transfected HGPS patient B-lymphoblasts (#1–2, ABE to sgRNA ratio 1:1, #3–4 ABE to sgRNA ratio 3:1, untreated HGPS ctrl #1-#2). c Targeted deep-seq. validation of potential off-target loci shows no increase in off-target mutation frequency in comparison to untreated controls (shown; 10 off-target loci, full analysis in Supplementary Fig. & Fig. , n = 2 independent experiments). d , e Comparison of the c.1824C allelic fraction as measured by targeted deep-seq. analysis and by a LMNA c.1824C > T ddPCR assay (red, c.1824 A:T; blue, c.1824 G:C; grey, c.1820 A:T; purple, 1820G:C). f Progerin and lamin A transcripts analyzed by ddPCR-absolute quantification (ddPCR-ABS), untreated samples are depicted in red, ABE treated samples are depicted in blue. g Detection of progerin levels by western blot in ABE-transfected HGPS patient B-lymphoblasts (+ABE; #3, #4, ABEmax-VQR: sgRNA mass ratio 1:1 and #5, #6 ABEmax-VQR: sgRNA mass ratio 3:1), untreated HGPS patient B-lymphoblasts (HGPS #1 and #2) and unaffected sibling control B-lymphoblasts (ctrl) repeated with n = 2 independent experiments with similar results. Data are presented as mean values. Source data for ( d – g ) are provided as a Source Data file.

Article Snippet: Mammalian pCMV-ABEmax-VQR-P2A-GFP and LMNA G608G targeting sgRNA expression vectors were send to Vectalys by Flash Therapeutics and cloned into an RNA transfer plasmid containing a U6 promotor sequence for the sgRNA and an EF1α promotor sequence for the ABEmax-VQR (RLP.U6.sg1hLMNA.opt.PP7.EF1.ABEmax-VQR.MS2).

Techniques: Mutagenesis, Variant Assay, Sequencing, Transfection, Western Blot

Journal: Nature Communications

Article Title: Transient expression of an adenine base editor corrects the Hutchinson-Gilford progeria syndrome mutation and improves the skin phenotype in mice

doi: 10.1038/s41467-022-30800-y

Figure Lengend Snippet: a Description of the human tetop-LA G608G+ minigene containing the common c.1824C > T point mutation in exon 11 of LMNA . In the presence of doxycycline (DOX) the transcription is inhibited. Bi-transgenic animals K5tTA + , tetop-LA G608G+ express the HGPS c.1824C > T mutation . b Base editing of the c.1824T point mutation in humanized HGPS mice with keratinocyte-specific expression of progerin. HGPS mice were ID injected with either LF-ABE or saline solution at the age of 21 days (P21) for two consecutive days. Treated skin was then analyzed two days (2d) or four weeks (4wks) post-LF-ABE injection. c Targeted deep-seq. analysis of the protospacer region shows c.1824 A:T to G:C mutation correction ranging from 2.9% to 5.8% and no bystander editing in HGPS mouse skin (#1–#4, n = 4) at 4wks post-injection with 4 × 10 10 total LF-ABE vps. We could detect a very low formation of indels within the protospacer region in ABE treated HGPS skin. d Frequencies of the c.1824C allele fraction in HGPS mouse skin obtained by ddPCR-RED 2d after LF-ABE treatment with either 1 × 10 10 or 4 × 10 10 total vps ( n = 3 and n = 8, biologically independent samples respectively), or 4 wks post LF-ABE (4 × 10 10 total vps, n = 10 biologically independent samples) or saline injection ( n = 4 biologically independent samples). e , f Progerin transcript quantification by in situ hybridization two days post-injection with 4 × 10 10 total vps of the LF-ABE ( n = 3 biologically independent samples) shows a significant reduction of progerin transcripts across the IFE ( p = 0.0131). The first cell layer on top of the white line is defined as the basal skin layer (BL). g , h LF-ABE treatment of HGPS mice with a developed skin phenotype at the age of 47 days (P47), showed a c.1824T mutation correction frequency of 3.8% ( n = 3 biologically independent samples) two days post-injection. Scale bar: d = 10 μm. Saline-treated samples are depicted in red, LF-ABE treated samples are depicted in blue. For ( d , f , h ) data are presented as mean values +/− SEM. f P value was calculated by two-tailed unpaired t test, 95% CI. Source data are provided as a Source Data file for ( d , f , h ).

Article Snippet: Mammalian pCMV-ABEmax-VQR-P2A-GFP and LMNA G608G targeting sgRNA expression vectors were send to Vectalys by Flash Therapeutics and cloned into an RNA transfer plasmid containing a U6 promotor sequence for the sgRNA and an EF1α promotor sequence for the ABEmax-VQR (RLP.U6.sg1hLMNA.opt.PP7.EF1.ABEmax-VQR.MS2).

Techniques: Mutagenesis, Transgenic Assay, Expressing, Injection, In Situ Hybridization, Two Tailed Test

Journal: Cell Death & Disease

Article Title: TMEM116 is required for lung cancer cell motility and metastasis through PDK1 signaling pathway

doi: 10.1038/s41419-021-04369-1

Figure Lengend Snippet: A The expression level of TMEM116 in mouse embryonic lungs were examined by RT-PCR and western blot analyses. B Immuno-fluorescence (IF) staining analysis showed TMEM116 expression in E14.5, PN1 and adult mouse lungs. C Double IF staining analysis of TMEM116 and p63, β-tubulin, CC10, PGP9.5, T1-α, and Spc respectively. D IF staining analysis of TMEM116 and GFP in Dermo1-cre; ROSA mTmG mice. Representative images from three independent experiments are shown above. Scale bar: 50 μm.

Article Snippet: SgRNA expression vectors against human TMEM116 were obtained from Cyagen Biosciences Inc and subcloned into pCRISPR-W2 vector.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Fluorescence, Staining

Journal: Cell Death & Disease

Article Title: TMEM116 is required for lung cancer cell motility and metastasis through PDK1 signaling pathway

doi: 10.1038/s41419-021-04369-1

Figure Lengend Snippet: A Double IF staining analysis of TMEM116 and CC10, β-tubulin, and αSMA respectively in human LUAD tissues and adjacent tissues. B Double IF staining of TMEM116 and CC10, β-tubulin, and αSMA respectively in human LUSC tissues and adjacent tissues. C The expression of TMEM116 in the 16HBE, A549, H1299, H441 cells by western blot analysis. D H&E staining analysis of BaP-induced A/J mouse lung tissue. E Double IF staining analysis of TMEM116 and CC10, β-tubulin, and αSMA respectively in A/J mouse lung cancer tissues and normal tissues adjacent to carcinoma. Representative images from three independent experiments are shown above. Scale bar: 200 μm.

Article Snippet: SgRNA expression vectors against human TMEM116 were obtained from Cyagen Biosciences Inc and subcloned into pCRISPR-W2 vector.

Techniques: Staining, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: TMEM116 is required for lung cancer cell motility and metastasis through PDK1 signaling pathway

doi: 10.1038/s41419-021-04369-1

Figure Lengend Snippet: A TMEM116 KD and control cells were subjected to colony formation assays. B TMEM116 KD and control cells were subjected to colony morphology assays. Scale bar: 500 μm. C TMEM116 KD and control cells were subjected to CCK-8 proliferation assay. D TMEM116 KD and control cells were subjected to wound-healing assay. Scale bar: 1000μm. Representative images from over 30 non-overlapping fields at each time point are shown. E TMEM116 KD and control cells were subjected to Transwell migration and invasion assays. Scale bar: 1000 μm. The bars represent the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Representative images from three independent experiments are shown above.

Article Snippet: SgRNA expression vectors against human TMEM116 were obtained from Cyagen Biosciences Inc and subcloned into pCRISPR-W2 vector.

Techniques: Control, CCK-8 Assay, Proliferation Assay, Wound Healing Assay, Migration

Journal: Cell Death & Disease

Article Title: TMEM116 is required for lung cancer cell motility and metastasis through PDK1 signaling pathway

doi: 10.1038/s41419-021-04369-1

Figure Lengend Snippet: A After tail vein injection by TMEM116 KD or control cells, metastatic nodules on lung surface were analyzed. Scale bar: 2 mm. B The xenografts tumorigenesis were analyzed by H&E staining. Scale bar: 200 μm. After subcutaneous injection into the flanks, tumor size ( C ), tumor volume ( D ), and tumor weight ( E ) were measured and analyzed. * P < 0.05, ** P < 0.01, *** P < 0.001. Representative images from three independent experiments are shown above.

Article Snippet: SgRNA expression vectors against human TMEM116 were obtained from Cyagen Biosciences Inc and subcloned into pCRISPR-W2 vector.

Techniques: Injection, Control, Staining

Journal: Cell Death & Disease

Article Title: TMEM116 is required for lung cancer cell motility and metastasis through PDK1 signaling pathway

doi: 10.1038/s41419-021-04369-1

Figure Lengend Snippet: A Western blot analysis of p110α, PDK1, p-AKT, AKT, p-FOXO3A, FOXO3A in control and TMEM116 KD A549 cells. B TMEM116 KD and control cells expressing p63 and E-cadherin were subjected to western blot analyses. C Real-time PCR analysis of E-Cad , p63 , TAp63 , ΔNp63 , DSP and PAR3 in control and TMEM116 KD cells. D TMEM116 KD , TMEM116 KD -PS48, control-PS48 and control cells expressing PDK1, p-AKT, AKT, p-FOXO3A, FOXO3A, p63, E-Cad were subjected to western blot analyses. Data represent the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Representative images from three independent experiments are shown above.

Article Snippet: SgRNA expression vectors against human TMEM116 were obtained from Cyagen Biosciences Inc and subcloned into pCRISPR-W2 vector.

Techniques: Western Blot, Control, Expressing, Real-time Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: TMEM116 is required for lung cancer cell motility and metastasis through PDK1 signaling pathway

doi: 10.1038/s41419-021-04369-1

Figure Lengend Snippet: A Control, control-PS48, TMEM116 KD and TMEM116 KD -PS48 cells were subjected to Transwell migration and invasion assays. More than three fields of cells in the lower chambers were counted. Scale bar: 1000 μm. B Control, control-PS48, TMEM116 KD and TMEM116 KD -PS48 cells were subjected to wound-healing assay. Scale bar: 1000 μm. Representative images from over 30 non-overlapping fields at each time point are shown. C Control, control-PS48, TMEM116 KD and TMEM116 KD -PS48 cells were subjected to colony formation assay. D Control, control-PS48, TMEM116 KD and TMEM116 KD -PS48 cells were subjected to colony morphology analyses. Scale bar: 500μm. E Control, control-PS48, TMEM116 KD and TMEM116 KD -PS48 cells were subjected to CCK8 assays at 0, 1, 3, 5 days. The bars represent the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. n = 6 mice. Representative images from three independent experiments are shown above.

Article Snippet: SgRNA expression vectors against human TMEM116 were obtained from Cyagen Biosciences Inc and subcloned into pCRISPR-W2 vector.

Techniques: Control, Migration, Wound Healing Assay, Colony Assay

Journal: Cell Death & Disease

Article Title: TMEM116 is required for lung cancer cell motility and metastasis through PDK1 signaling pathway

doi: 10.1038/s41419-021-04369-1

Figure Lengend Snippet: TMEM116 is required for lung cancer cell growth, motility and invasion. PDK1 signaling via AKT/FOXO3A/TAp63 is thought to be involved in this progression.

Article Snippet: SgRNA expression vectors against human TMEM116 were obtained from Cyagen Biosciences Inc and subcloned into pCRISPR-W2 vector.

Techniques: