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: 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