Journal: eLife

Article Title: Erythrocyte CD55 mediates the internalization of Plasmodium falciparum parasites

doi: 10.7554/eLife.61516

Figure Lengend Snippet:

Article Snippet: Software, algorithm , Two sgRNAs (Guide RNA design) , Designed using GPP web portal Broad Institute , , .

Techniques: Blocking Assay, Control, Generated, Affinity Purification, Growth Assay, Clinical Proteomics, Solvent, Software, Sequencing, Modification, Synthesized, Recombinant, Purification, Invasion Assay, SYBR Green Assay, Staining

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: Development (Cambridge, England)

Article Title: Deciphering the role of cis - regulatory elements and TFAP2C in the activation of zygotic Sox2 expression in mouse preimplantation embryos

doi: 10.1242/dev.204626

Figure Lengend Snippet: Disruption of TFAP2C motifs within the proximal promoter impairs Sox2 expression. (A) Schematic illustrating the location of two TFAP2C binding motifs in the Sox2 proximal promoter (PP). The red arrows indicate the location of the sgRNA binding sites. The two sgRNAs are predicted to delete a ∼65 bp fragment containing the TFAP2C motifs. (B) Real-time qPCR analysis of Sox2 transcripts in control (Cas9 alone) and Sox2 PP motif-edited embryos at the morula stage (E3.25). A total of three biological replicates were used, with 20 pooled morulae per replicate. Values are presented as mean±s.d. * P <0.05 (paired Student's t -test). (C) Confocal IF analysis of SOX2 expression in control and Sox2 PP motif-edited embryos at the late morula stage (E3.75). Two representative embryos are shown. Type 1 embryos exhibited partial SOX2 expression, whereas type 2 embryos exhibited a complete loss of SOX2. A total of three biological replicates were used, with seven embryos per control and treatment groups. Nuclei were counterstained with DAPI (blue). An equivalent confocal optical section near the equator of each embryo was used for comparisons. Scale bar: 20 µm. (D) Quantitation of the total cell numbers and SOX2-positive cell numbers in control versus Sox2 PP motif-edited embryos at the late morula stage. Values are presented as mean±s.d. ** P <0.01, *** P <0.001 (paired Student's t -test). (E) Analysis of the SOX2-positive to total cell number ratios in control versus Sox2 PP motif-edited embryos. Values are presented as mean±s.d. *** P <0.001 (paired Student's t -test).

Article Snippet: CRISPR custom-designed sgRNAs and SpCas9 2NLS nuclease protein were purchased from Synthego.

Techniques: Disruption, Expressing, Binding Assay, Control, Quantitation Assay

Journal: eLife

Article Title: High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion

doi: 10.7554/eLife.64911

Figure Lengend Snippet: ( A ) Schematic of the genomic structure around the targeting sites. Short vertical purple lines indicate the site positions and whether the targeted sequences are at the upper (purple lines above the DNA schematic) or lower (purple lines below the DNA schematic) DNA strand. All scale bars are 250 bp. ( B ) Schematic donor plasmid designs used in this manuscript. Triangles illustrate the indicated single guide RNA (sgRNA) targeting sites with orientation. Note that CRISPR-mediated insertion of exon (CRISPIE) allows for the inclusion of multiple cutting sites, as indicated, which provides flexibility for excising the donor DNA or the possibility to erase the inserted module at a later time. The phases of the exon modules (i.e., the reading frames) are labeled. The junctional intron (100 bp) and exon (~10 bp) sequences are taken from intron 17 of mouse Camk2a and its surrounding exons, with the exception that the 5’ intron (250 bp)/exon junction of b5 is taken from intron 21 and exon 22 of VCL , b6 does not involve any intron/exon junction (targeting coding sequences, or CDS), and the 3’ intron (250 bp)/exon junction of b8 is taken from exon 18 and intron 19 of mouse PSD-95 . The donors of b1–b5 must be released from the parental plasmid by an additional sgRNA/ Streptococcus pyogenes Cas9 (SpCas9) (not depicted here) that cuts one of the indicated flanking sgRNA targeting sites. b6–b10 can be excised using the DRS-2 sgRNA from the same plasmid in the presence of SpCas9. ( C ) Schematic adeno-associated virus (AAV) for use with SpCas9-expressing cells/animals.

Article Snippet: Software, algorithm , GPP sgRNA designer , BROAD Institute , https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design , .

Techniques: Plasmid Preparation, CRISPR, Labeling, Virus, Expressing

Journal: eLife

Article Title: High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion

doi: 10.7554/eLife.64911

Figure Lengend Snippet: ( A ) The conceptual design of the CRISPIE strategy showing that, although insertions/deletions (INDELs) and inverted insertion events may occur at the DNA level during editing, only wild-type mRNAs or mRNAs with the desired precise insertion are produced. Orange boxes: endogenous exon sequences; black lines: endogenous intron sequences; blue lines and green boxes: intron and exon sequences, respectively, of the designer donor module with white arrows showing the correct orientation of the reading frame; red stars: INDELs. ( B ) Schematics of the single guide RNA/ Streptococcus pyogenes Cas9 (sgRNA/SpCas9) plasmid (upper) and the donor plasmid (lower) that are used for panels D–G . Purple triangles: the sgRNA-targeted location and orientation, spl.: splicing; acptr: acceptor. See for design details. ( C ) Schematic of the targeted intron of ACTB with the purple triangle showing the sgRNA targeting site and orientation. Orange and gray boxes: exonic sequences that do and do not encode protein sequences, respectively. ( D ) Representative two-photon (2 p) images of live U2OS cells with the indicated transfection. Note that the mRuby3 (magenta) expression levels were highly variable across cells due to the variability in plasmid transfection; however, the green label intensities were comparable across cells, as expected for the expression from an endogenous locus. ( E ) Labeling efficiency (green cell counts over red cell counts) for ACTB in U2OS cells. n = 8 field of views (FOVs), two independent transfections. ( F ) Representative confocal (Airyscan) and super-resolution structured illumination microscopy (SIM) images of live U2OS cells expressing CRISPIEd β-actin. ( G ) Representative time-lapse confocal images of live U2OS cells showing the dynamics of actin ruffles (arrows). The dashed yellow line outlines the cell morphology at time zero. ( H ) Cell growth curves of β-actin-CRISPIEd cells and untransfected cells. n = 6 FOVs. Figure 1—source data 1. Numeric data for .

Article Snippet: Software, algorithm , GPP sgRNA designer , BROAD Institute , https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design , .

Techniques: Produced, Plasmid Preparation, Transfection, Expressing, Labeling, Microscopy

Journal: eLife

Article Title: High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion

doi: 10.7554/eLife.64911

Figure Lengend Snippet: ( A ) Schematic positions of the tested editing sites in the intron 1 and exon 2 of ACTB that are targeted in panels B and C . The asterisk marks the site used for ACTB in subsequent experiments. ( B ) Comparison of the editing efficiency of five different intronic locations. For comparison under identical conditions, a generic donor excised using an independent single guide RNA (sgRNA) (DRS-2) was used . n = 18 FOVs (field of views) from two independent transfections. ( C ) Representative images and quantification of successful editing rates at the intronic location i4 vs. the only two possible exonic locations for N-terminally labeled ACTB , each using their specifically designed donors. n = 18 FOVs from two independent transfections. Figure 3—source data 1. Numeric data for .

Article Snippet: Software, algorithm , GPP sgRNA designer , BROAD Institute , https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design , .

Techniques: Comparison, Transfection, Labeling

Journal: eLife

Article Title: High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion

doi: 10.7554/eLife.64911

Figure Lengend Snippet: ( A ) Schematic of the experimental design for erasing CRISPIE labels. DRS-1 single guide RNA (sgRNA) targeting sequences were used to flank the exon/intron module inside the other two sgRNA sites for excising the donor from the initial insertion. Later introduction of DRS-1 sgRNA and Streptococcus pyogenes Cas9 (SpCas9) can excise the inserted CRISPIE module. Note that after the insertion and erasure, both the original sgRNA targeting site and the DRS-1 site will be destroyed. ( B ) Representative images of a β-actin-CRISPIEd U2OS cell clone (left), which was transfected with the transfection marker mRuby3 (magenta in upper panels) and SpCas9 together with DRS-1 (middle), or with a control sgRNA that targeted mouse, but not human β-actin (right). Asterisks in the lower panel mark the transfected (i.e., mRuby-positive) cells. ( C ) Quantification of the erasure efficiency among the transfected cells, as identified by mRuby3 expression. n (field of views [FOVs]/transfections)=11/3 for DRS-1, and 9/3 for control sgRNA. Figure 6—source data 1. Numeric data for .

Article Snippet: Software, algorithm , GPP sgRNA designer , BROAD Institute , https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design , .

Techniques: Transfection, Marker, Control, Expressing

Journal: eLife

Article Title: High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion

doi: 10.7554/eLife.64911

Figure Lengend Snippet:

Article Snippet: Software, algorithm , GPP sgRNA designer , BROAD Institute , https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design , .

Techniques: Recombinant, Plasmid Preparation, Synthesized, Sequencing, Amplification, Cell Culture, Reverse Transcription, Software