Journal: Science translational medicine

Article Title: Precise genomic editing of pathogenic mutations in RBM20 rescues dilated cardiomyopathy

doi: 10.1126/scitranslmed.ade1633

Figure Lengend Snippet: (A) ABE correction of the R636Q mutation using sgRNA (blue) and ABEmax-VRQR-SpCas9. On-target: A6 (red). Bystander: A14 and A20 (green). Silent: A4, A13, and A19 (brown). (B) Percentage of adenine-to-guanine editing determined by deep sequencing of cDNA from hearts of R636Q/R636Q mice (6 weeks after ABE correction). Data are expressed as means ± SEM (n = 3). (C) Fractional shortening at 4 and 8 weeks after ABE correction in WT, R636Q/+, R636Q/R636Q, and corrected mice. Data are expressed as means ± SEM (n = 6 per group). Two-way ANOVA with Tukey’s multiple comparisons test was performed. ****P < 0.0001. (D) H&E staining of four-chamber hearts (12 weeks after ABE correction). Scale bar, 1 mm. LV, left ventricle. (E) Kaplan-Meier survival curve of all groups (n = 16 per group). Log-rank (Mantel-Cox) test was performed. ****P < 0.0001 for R636Q/R636Q versus other groups. (F) Immunohistochemistry showing the translocation of RBM20 in CMs of WT, R636Q/+, R636Q/R636Q, and corrected mice (12 weeks after ABE correction). cTnT (red), RBM20 (green), and DAPI (blue). Scale bar, 10 μm. (G) Quantification of RBM20 localization before and after correction in R636Q/R636Q mice. Data are expressed as means ± SEM (n = 4 per genotype). Two-way ANOVA with Bonferroni’s multiple comparisons test was performed. ****P < 0.0001.

Article Snippet: The N-terminal and C-terminal regions of ABEmax-VRQR-SpCas9 were extracted from CMV_Npu-ABEmax N-terminal (Addgene plasmid no. 137173) ( 52 ) and hu6 HGPS sgRNA expression and ABE7.10max VRQR C-terminal AAV vectors (Addgene plasmid no. 154430) ( 28 ) from D. Liu’s laboratory, respectively.

Techniques: Mutagenesis, Sequencing, Staining, Immunohistochemistry, Translocation Assay

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