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  • 99
    New England Biolabs rna guided dna endonuclease expression cassettes
    Rna Guided Dna Endonuclease Expression Cassettes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna guided dna endonuclease expression cassettes/product/New England Biolabs
    Average 99 stars, based on 8 article reviews
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    99
    Thermo Fisher crispr associated protein 9 rna guided dna endonuclease cas9
    Crispr Associated Protein 9 Rna Guided Dna Endonuclease Cas9, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore single guide rna sgrna oligos
    In vivo validation of the role of Lfng in metastasis. To validate the role of Lfng in metastasis, two independent Lfng targeting experiments were performed in B16‐F0 cells: one using a single <t>gRNA</t> to introduce a single base pair insertion (A, C and D) and another using two gRNAs to induce a 4.8‐kb deletion (B, E and F). (A) Sanger sequence trace of the targeted region in clone <t>g2d1</t> carrying a homozygous 1‐bp insertion. (B) Image showing from top to bottom, the expected junction sequence after the deletion caused by the targeting of Lfng using two gRNAs, the expected reference sequence and the Sanger sequence traces observed and assembled with SeqMan Pro (Lasergene) against the expected reference sequence. The expected junction sequence separated by a single base insertion can be observed . Experimental metastasis assays using control and Lfng ‐deficient cell lines (tail‐vein‐injected into wild‐type female mice (symbols representing individual mice with horizontal bar at the mean ± SD and statistics performed using a Mann–Whitney test; data shown are representative of two independent experiments)). Photographs are representative images of the lungs from mice injected with control and Lfng ‐deficient cell lines. Plasmid rescue showing that introduction of the Lfng cDNA reverts the metastatic phenotype of L1 cells ( L1‐Lfng ; Lfng‐transfected cells, L1‐PB , vector‐only controls) (G and H). (G) A western blot with an anti‐Flag antibody shows restoration of Lfng expression (clone L1‐Lfng ). An anti‐vinculin antibody was used as a loading control. These results are representative of three independent experiments. (H) Experimental metastasis assays using control L1‐PB cells and Lfng‐transfected cells. Please note experiments in e and h were performed with 5 × 10 5 and 4 × 10 5 cells, respectively, hence the different metastasis counts.
    Single Guide Rna Sgrna Oligos, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    TaKaRa dna directed rna guided endonuclease rgen system
    In vivo validation of the role of Lfng in metastasis. To validate the role of Lfng in metastasis, two independent Lfng targeting experiments were performed in B16‐F0 cells: one using a single <t>gRNA</t> to introduce a single base pair insertion (A, C and D) and another using two gRNAs to induce a 4.8‐kb deletion (B, E and F). (A) Sanger sequence trace of the targeted region in clone <t>g2d1</t> carrying a homozygous 1‐bp insertion. (B) Image showing from top to bottom, the expected junction sequence after the deletion caused by the targeting of Lfng using two gRNAs, the expected reference sequence and the Sanger sequence traces observed and assembled with SeqMan Pro (Lasergene) against the expected reference sequence. The expected junction sequence separated by a single base insertion can be observed . Experimental metastasis assays using control and Lfng ‐deficient cell lines (tail‐vein‐injected into wild‐type female mice (symbols representing individual mice with horizontal bar at the mean ± SD and statistics performed using a Mann–Whitney test; data shown are representative of two independent experiments)). Photographs are representative images of the lungs from mice injected with control and Lfng ‐deficient cell lines. Plasmid rescue showing that introduction of the Lfng cDNA reverts the metastatic phenotype of L1 cells ( L1‐Lfng ; Lfng‐transfected cells, L1‐PB , vector‐only controls) (G and H). (G) A western blot with an anti‐Flag antibody shows restoration of Lfng expression (clone L1‐Lfng ). An anti‐vinculin antibody was used as a loading control. These results are representative of three independent experiments. (H) Experimental metastasis assays using control L1‐PB cells and Lfng‐transfected cells. Please note experiments in e and h were performed with 5 × 10 5 and 4 × 10 5 cells, respectively, hence the different metastasis counts.
    Dna Directed Rna Guided Endonuclease Rgen System, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Bioneer Corporation chimeric dna rna guides
    Mechanism underlying the enhanced Cpf1 editing efficiency and specificity of the combination of chimeric <t>DNA-RNA</t> guide and SpCas9 nickase. (A) Schematics of the chimeric DNA-RNA-guided editing with Cpf1. Off-target DNA cleavage is dramatically decreased by chimeric (cr)RNA guided Cpf1 binding because of the higher mismatch sensitivity. The red arrowhead indicates the cleavage site for double-strand breaks. (B) Enhanced genome editing with a combination of SpCas9 nickase (D10A) and chimeric DNA-RNA guided Cpf1. DNA supercoiling generates a tension that inhibits the chimeric (cr)RNA guided Cpf1 cleavage activity. Nickase relaxed supercoiling enhances chimeric (cr)RNA guided Cpf1 DNA cleavage activity on genomic DNA. The small and large red arrowhead indicates the cleavage site for nick by SpCas9 nickase (D10A) and enhanced double-strand break by Cpf1. Red color in the (cr)RNA indicates DNA substitution. PAM sequences (NGG for SpCas9 nickase and TTTN for AsCpf1) are shown in blue color and mismatched sequence to target sequence are shown in orange color, respectively.
    Chimeric Dna Rna Guides, supplied by Bioneer Corporation, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ATUM dna 2 0 grna
    Mechanism underlying the enhanced Cpf1 editing efficiency and specificity of the combination of chimeric <t>DNA-RNA</t> guide and SpCas9 nickase. (A) Schematics of the chimeric DNA-RNA-guided editing with Cpf1. Off-target DNA cleavage is dramatically decreased by chimeric (cr)RNA guided Cpf1 binding because of the higher mismatch sensitivity. The red arrowhead indicates the cleavage site for double-strand breaks. (B) Enhanced genome editing with a combination of SpCas9 nickase (D10A) and chimeric DNA-RNA guided Cpf1. DNA supercoiling generates a tension that inhibits the chimeric (cr)RNA guided Cpf1 cleavage activity. Nickase relaxed supercoiling enhances chimeric (cr)RNA guided Cpf1 DNA cleavage activity on genomic DNA. The small and large red arrowhead indicates the cleavage site for nick by SpCas9 nickase (D10A) and enhanced double-strand break by Cpf1. Red color in the (cr)RNA indicates DNA substitution. PAM sequences (NGG for SpCas9 nickase and TTTN for AsCpf1) are shown in blue color and mismatched sequence to target sequence are shown in orange color, respectively.
    Dna 2 0 Grna, supplied by ATUM, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc chimeric grna dna template
    Mechanism underlying the enhanced Cpf1 editing efficiency and specificity of the combination of chimeric <t>DNA-RNA</t> guide and SpCas9 nickase. (A) Schematics of the chimeric DNA-RNA-guided editing with Cpf1. Off-target DNA cleavage is dramatically decreased by chimeric (cr)RNA guided Cpf1 binding because of the higher mismatch sensitivity. The red arrowhead indicates the cleavage site for double-strand breaks. (B) Enhanced genome editing with a combination of SpCas9 nickase (D10A) and chimeric DNA-RNA guided Cpf1. DNA supercoiling generates a tension that inhibits the chimeric (cr)RNA guided Cpf1 cleavage activity. Nickase relaxed supercoiling enhances chimeric (cr)RNA guided Cpf1 DNA cleavage activity on genomic DNA. The small and large red arrowhead indicates the cleavage site for nick by SpCas9 nickase (D10A) and enhanced double-strand break by Cpf1. Red color in the (cr)RNA indicates DNA substitution. PAM sequences (NGG for SpCas9 nickase and TTTN for AsCpf1) are shown in blue color and mismatched sequence to target sequence are shown in orange color, respectively.
    Chimeric Grna Dna Template, supplied by Addgene inc, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc grna expression dna
    Genome-editing activity of the TALENs and CRISPR/Cas9 systems targeting HIV LTR. (A) LTR-driven GFP expression after TF with CRISPR/Cas9 or TALENs expressing plasmid <t>DNA.</t> Jurkat cells were infected with an LTIG lentiviral vector. Five days after infection, the GFP positive cells were sorted (Jurkat/LTIG) and co-transfected with T5 <t>gRNA-expressing</t> and hCas9-expressing DNA or LTR TALEN-L and -R expressing DNA. The level of GFP expression was analyzed by flow cytometry 5 days after TF. (B) Sequence analysis of TALENs targeting sites. The DNA sequences of the TAR and adjacent regions of LTR are indicated. Nineteen sequences were obtained from Jurkat/LTIG cells, which were transfected twice with TAR TALEN-LR. The WT reference sequence is shown at the top. The target sequences of TAR TALENs and T5 gRNA are indicated in orange and green, respectively. The putative cleavage site of T5 CRISPR is indicated with an orange arrowhead. (C) Genome-editing activity of TAR TALENs and T5 CRISPR in c19 cells, latently transduced with an LTIG lentiviral vector. The level of GFP expression after 48 hours of TNF-α stimulation is shown. The error bars in B and D show standard deviations (n = 3).
    Grna Expression Dna, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    NanoVector b self assembled dna guided rna nanovector
    Characterization of the <t>RNA</t> nanoparticle/doxorubicin physical conjugates (a) Construct of Endo28-3WJ-sph1 RNA nanoparticle with predicted chelating sites for doxorubicin indicated by red stars. (b) Native PAGE showing the assembly of Endo28-3WJ-sph1 nanoparticles. Lane 1 is 100bp <t>DNA</t> ladder (Thermo Scientific). (c) Fluorescence spectra of doxorubicin solution (1.4μM) with increasing concentration of RNA nanoparticles. Insert: A hill plot for the nanoparticle titration (K d = 140nM, 0.1 equivalent of the RNA nanoparticles). (d) The percentage of doxorubicin released from the nanoparticle doxorubicin conjugates at 37°C. The doxorubicin concentration was quantified by its fluorescence intensity in release medium.
    B Self Assembled Dna Guided Rna Nanovector, supplied by NanoVector, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology crispr cas9 mediated genomic dna mutation crispr guide rna grna sequence design
    <t>CRISPR-Cas9-mediated</t> frameshift mutations of CXCR7 resulted in the onset of cellular senescence. ( a ) X-gal assay for SA-β-galactosidase (SA-gal) activity in C4-2B CXCR7 frameshift mutant cells. Positive SA-gal expression is indicated by dark blue coloration to the cells. Upper-left panel: C4-2B cells with wild-type (WT) CXCR7 expression untreated (UT) (negative control), Lower-left panel: WT C4-2B cells treated with 10 µM H 2 O 2 (positive control). Upper-middle, lower middle, and upper-right panels: representative images in CXCR7-CRISPR-mutated cells. Lower-right panel: Sanger sequencing chromatogram of the <t>CXCR7-gRNA</t> target site illustrating overlapping unique CXCR7 InDel mutations. ( b ) X-gal assay in LNCaP CXCR7 frameshift mutant cells and controls (UT or H 2 O 2 treated).
    Crispr Cas9 Mediated Genomic Dna Mutation Crispr Guide Rna Grna Sequence Design, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore cas9 nuclease endotoxin free plasmid dna expressing single guide rna endotoxin free plasmid dna
    <t>CRISPR-Cas9-mediated</t> frameshift mutations of CXCR7 resulted in the onset of cellular senescence. ( a ) X-gal assay for SA-β-galactosidase (SA-gal) activity in C4-2B CXCR7 frameshift mutant cells. Positive SA-gal expression is indicated by dark blue coloration to the cells. Upper-left panel: C4-2B cells with wild-type (WT) CXCR7 expression untreated (UT) (negative control), Lower-left panel: WT C4-2B cells treated with 10 µM H 2 O 2 (positive control). Upper-middle, lower middle, and upper-right panels: representative images in CXCR7-CRISPR-mutated cells. Lower-right panel: Sanger sequencing chromatogram of the <t>CXCR7-gRNA</t> target site illustrating overlapping unique CXCR7 InDel mutations. ( b ) X-gal assay in LNCaP CXCR7 frameshift mutant cells and controls (UT or H 2 O 2 treated).
    Cas9 Nuclease Endotoxin Free Plasmid Dna Expressing Single Guide Rna Endotoxin Free Plasmid Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher u6 grna geneart dna string
    <t>CRISPR-Cas9-mediated</t> frameshift mutations of CXCR7 resulted in the onset of cellular senescence. ( a ) X-gal assay for SA-β-galactosidase (SA-gal) activity in C4-2B CXCR7 frameshift mutant cells. Positive SA-gal expression is indicated by dark blue coloration to the cells. Upper-left panel: C4-2B cells with wild-type (WT) CXCR7 expression untreated (UT) (negative control), Lower-left panel: WT C4-2B cells treated with 10 µM H 2 O 2 (positive control). Upper-middle, lower middle, and upper-right panels: representative images in CXCR7-CRISPR-mutated cells. Lower-right panel: Sanger sequencing chromatogram of the <t>CXCR7-gRNA</t> target site illustrating overlapping unique CXCR7 InDel mutations. ( b ) X-gal assay in LNCaP CXCR7 frameshift mutant cells and controls (UT or H 2 O 2 treated).
    U6 Grna Geneart Dna String, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Integrated DNA Technologies grna transcript dna oligos
    <t>CRISPR-Cas9-mediated</t> frameshift mutations of CXCR7 resulted in the onset of cellular senescence. ( a ) X-gal assay for SA-β-galactosidase (SA-gal) activity in C4-2B CXCR7 frameshift mutant cells. Positive SA-gal expression is indicated by dark blue coloration to the cells. Upper-left panel: C4-2B cells with wild-type (WT) CXCR7 expression untreated (UT) (negative control), Lower-left panel: WT C4-2B cells treated with 10 µM H 2 O 2 (positive control). Upper-middle, lower middle, and upper-right panels: representative images in CXCR7-CRISPR-mutated cells. Lower-right panel: Sanger sequencing chromatogram of the <t>CXCR7-gRNA</t> target site illustrating overlapping unique CXCR7 InDel mutations. ( b ) X-gal assay in LNCaP CXCR7 frameshift mutant cells and controls (UT or H 2 O 2 treated).
    Grna Transcript Dna Oligos, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    ATUM dna 2 0 s grna designer
    <t>CRISPR-Cas9-mediated</t> frameshift mutations of CXCR7 resulted in the onset of cellular senescence. ( a ) X-gal assay for SA-β-galactosidase (SA-gal) activity in C4-2B CXCR7 frameshift mutant cells. Positive SA-gal expression is indicated by dark blue coloration to the cells. Upper-left panel: C4-2B cells with wild-type (WT) CXCR7 expression untreated (UT) (negative control), Lower-left panel: WT C4-2B cells treated with 10 µM H 2 O 2 (positive control). Upper-middle, lower middle, and upper-right panels: representative images in CXCR7-CRISPR-mutated cells. Lower-right panel: Sanger sequencing chromatogram of the <t>CXCR7-gRNA</t> target site illustrating overlapping unique CXCR7 InDel mutations. ( b ) X-gal assay in LNCaP CXCR7 frameshift mutant cells and controls (UT or H 2 O 2 treated).
    Dna 2 0 S Grna Designer, supplied by ATUM, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bio-Rad cas9 grna plasmid dna
    <t>CRISPR/Cas9-mediated</t> gene targeting in mouse iPSCs. ( a ) Schematic diagram showing the site of Arg1 gene modification using CRISPR/Cas9. The <t>gRNA</t> was designed to target a 20 nt region in intron 6 of Arg1 . ( b ) Cas9:gRNA off-target analysis. A table showing the output from CRISPR design tool. The on-site target is given at the top, followed by off-target sequences organized in decreasing Cas9-cleavage probability. ( c ) Detection of Cas9:gRNA-mediated on-target cleavage of Arg1 by the Surveyor nuclease assay. The cleavage products were shown as extra bands (149 bp + 197 bp) indicated by the arrows. Mutation frequency (indel) was calculated by measuring the band intensities. ( d ) Sequencing data from TOPO-cloned PCR amplicons of CRISPR/Cas9-modified genomic <t>DNA</t> showing a few examples of NHEJ-mediated indel mutations at the desired location. gRNA target site is in bold. Deletions are shown in dashes. Nucleotide substitution is shown in lowercase. The net change in length caused by each indel mutation is to the right of each sequence.
    Cas9 Grna Plasmid Dna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher grna geneart dna string
    <t>CRISPR/Cas9-mediated</t> gene targeting in mouse iPSCs. ( a ) Schematic diagram showing the site of Arg1 gene modification using CRISPR/Cas9. The <t>gRNA</t> was designed to target a 20 nt region in intron 6 of Arg1 . ( b ) Cas9:gRNA off-target analysis. A table showing the output from CRISPR design tool. The on-site target is given at the top, followed by off-target sequences organized in decreasing Cas9-cleavage probability. ( c ) Detection of Cas9:gRNA-mediated on-target cleavage of Arg1 by the Surveyor nuclease assay. The cleavage products were shown as extra bands (149 bp + 197 bp) indicated by the arrows. Mutation frequency (indel) was calculated by measuring the band intensities. ( d ) Sequencing data from TOPO-cloned PCR amplicons of CRISPR/Cas9-modified genomic <t>DNA</t> showing a few examples of NHEJ-mediated indel mutations at the desired location. gRNA target site is in bold. Deletions are shown in dashes. Nucleotide substitution is shown in lowercase. The net change in length caused by each indel mutation is to the right of each sequence.
    Grna Geneart Dna String, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs grna sequences
    <t>CRISPR/Cas9-mediated</t> gene targeting in mouse iPSCs. ( a ) Schematic diagram showing the site of Arg1 gene modification using CRISPR/Cas9. The <t>gRNA</t> was designed to target a 20 nt region in intron 6 of Arg1 . ( b ) Cas9:gRNA off-target analysis. A table showing the output from CRISPR design tool. The on-site target is given at the top, followed by off-target sequences organized in decreasing Cas9-cleavage probability. ( c ) Detection of Cas9:gRNA-mediated on-target cleavage of Arg1 by the Surveyor nuclease assay. The cleavage products were shown as extra bands (149 bp + 197 bp) indicated by the arrows. Mutation frequency (indel) was calculated by measuring the band intensities. ( d ) Sequencing data from TOPO-cloned PCR amplicons of CRISPR/Cas9-modified genomic <t>DNA</t> showing a few examples of NHEJ-mediated indel mutations at the desired location. gRNA target site is in bold. Deletions are shown in dashes. Nucleotide substitution is shown in lowercase. The net change in length caused by each indel mutation is to the right of each sequence.
    Grna Sequences, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher synthetic 233 bp double stranded dna dsdna grna construct
    <t>CRISPR/Cas9-mediated</t> gene targeting in mouse iPSCs. ( a ) Schematic diagram showing the site of Arg1 gene modification using CRISPR/Cas9. The <t>gRNA</t> was designed to target a 20 nt region in intron 6 of Arg1 . ( b ) Cas9:gRNA off-target analysis. A table showing the output from CRISPR design tool. The on-site target is given at the top, followed by off-target sequences organized in decreasing Cas9-cleavage probability. ( c ) Detection of Cas9:gRNA-mediated on-target cleavage of Arg1 by the Surveyor nuclease assay. The cleavage products were shown as extra bands (149 bp + 197 bp) indicated by the arrows. Mutation frequency (indel) was calculated by measuring the band intensities. ( d ) Sequencing data from TOPO-cloned PCR amplicons of CRISPR/Cas9-modified genomic <t>DNA</t> showing a few examples of NHEJ-mediated indel mutations at the desired location. gRNA target site is in bold. Deletions are shown in dashes. Nucleotide substitution is shown in lowercase. The net change in length caused by each indel mutation is to the right of each sequence.
    Synthetic 233 Bp Double Stranded Dna Dsdna Grna Construct, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    In vivo validation of the role of Lfng in metastasis. To validate the role of Lfng in metastasis, two independent Lfng targeting experiments were performed in B16‐F0 cells: one using a single gRNA to introduce a single base pair insertion (A, C and D) and another using two gRNAs to induce a 4.8‐kb deletion (B, E and F). (A) Sanger sequence trace of the targeted region in clone g2d1 carrying a homozygous 1‐bp insertion. (B) Image showing from top to bottom, the expected junction sequence after the deletion caused by the targeting of Lfng using two gRNAs, the expected reference sequence and the Sanger sequence traces observed and assembled with SeqMan Pro (Lasergene) against the expected reference sequence. The expected junction sequence separated by a single base insertion can be observed . Experimental metastasis assays using control and Lfng ‐deficient cell lines (tail‐vein‐injected into wild‐type female mice (symbols representing individual mice with horizontal bar at the mean ± SD and statistics performed using a Mann–Whitney test; data shown are representative of two independent experiments)). Photographs are representative images of the lungs from mice injected with control and Lfng ‐deficient cell lines. Plasmid rescue showing that introduction of the Lfng cDNA reverts the metastatic phenotype of L1 cells ( L1‐Lfng ; Lfng‐transfected cells, L1‐PB , vector‐only controls) (G and H). (G) A western blot with an anti‐Flag antibody shows restoration of Lfng expression (clone L1‐Lfng ). An anti‐vinculin antibody was used as a loading control. These results are representative of three independent experiments. (H) Experimental metastasis assays using control L1‐PB cells and Lfng‐transfected cells. Please note experiments in e and h were performed with 5 × 10 5 and 4 × 10 5 cells, respectively, hence the different metastasis counts.

    Journal: Molecular Oncology

    Article Title: Comparative genomics reveals that loss of lunatic fringe (LFNG) promotes melanoma metastasis

    doi: 10.1002/1878-0261.12161

    Figure Lengend Snippet: In vivo validation of the role of Lfng in metastasis. To validate the role of Lfng in metastasis, two independent Lfng targeting experiments were performed in B16‐F0 cells: one using a single gRNA to introduce a single base pair insertion (A, C and D) and another using two gRNAs to induce a 4.8‐kb deletion (B, E and F). (A) Sanger sequence trace of the targeted region in clone g2d1 carrying a homozygous 1‐bp insertion. (B) Image showing from top to bottom, the expected junction sequence after the deletion caused by the targeting of Lfng using two gRNAs, the expected reference sequence and the Sanger sequence traces observed and assembled with SeqMan Pro (Lasergene) against the expected reference sequence. The expected junction sequence separated by a single base insertion can be observed . Experimental metastasis assays using control and Lfng ‐deficient cell lines (tail‐vein‐injected into wild‐type female mice (symbols representing individual mice with horizontal bar at the mean ± SD and statistics performed using a Mann–Whitney test; data shown are representative of two independent experiments)). Photographs are representative images of the lungs from mice injected with control and Lfng ‐deficient cell lines. Plasmid rescue showing that introduction of the Lfng cDNA reverts the metastatic phenotype of L1 cells ( L1‐Lfng ; Lfng‐transfected cells, L1‐PB , vector‐only controls) (G and H). (G) A western blot with an anti‐Flag antibody shows restoration of Lfng expression (clone L1‐Lfng ). An anti‐vinculin antibody was used as a loading control. These results are representative of three independent experiments. (H) Experimental metastasis assays using control L1‐PB cells and Lfng‐transfected cells. Please note experiments in e and h were performed with 5 × 10 5 and 4 × 10 5 cells, respectively, hence the different metastasis counts.

    Article Snippet: 2.12 Lfng disruption using a single gRNA (g2d1 clone generation) Oligos with the Lfng gRNA sequence (Sigma‐Aldrich Corp, St. Louis, MO, USA) were cloned into the vector PX459 (Addgene #48139) following the Zhang laboratory protocol (Ran et al ., ).

    Techniques: In Vivo, Introduce, Sequencing, Injection, Mouse Assay, MANN-WHITNEY, Plasmid Preparation, Transfection, Western Blot, Expressing

    Mechanism underlying the enhanced Cpf1 editing efficiency and specificity of the combination of chimeric DNA-RNA guide and SpCas9 nickase. (A) Schematics of the chimeric DNA-RNA-guided editing with Cpf1. Off-target DNA cleavage is dramatically decreased by chimeric (cr)RNA guided Cpf1 binding because of the higher mismatch sensitivity. The red arrowhead indicates the cleavage site for double-strand breaks. (B) Enhanced genome editing with a combination of SpCas9 nickase (D10A) and chimeric DNA-RNA guided Cpf1. DNA supercoiling generates a tension that inhibits the chimeric (cr)RNA guided Cpf1 cleavage activity. Nickase relaxed supercoiling enhances chimeric (cr)RNA guided Cpf1 DNA cleavage activity on genomic DNA. The small and large red arrowhead indicates the cleavage site for nick by SpCas9 nickase (D10A) and enhanced double-strand break by Cpf1. Red color in the (cr)RNA indicates DNA substitution. PAM sequences (NGG for SpCas9 nickase and TTTN for AsCpf1) are shown in blue color and mismatched sequence to target sequence are shown in orange color, respectively.

    Journal: bioRxiv

    Article Title: Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

    doi: 10.1101/2020.02.04.933614

    Figure Lengend Snippet: Mechanism underlying the enhanced Cpf1 editing efficiency and specificity of the combination of chimeric DNA-RNA guide and SpCas9 nickase. (A) Schematics of the chimeric DNA-RNA-guided editing with Cpf1. Off-target DNA cleavage is dramatically decreased by chimeric (cr)RNA guided Cpf1 binding because of the higher mismatch sensitivity. The red arrowhead indicates the cleavage site for double-strand breaks. (B) Enhanced genome editing with a combination of SpCas9 nickase (D10A) and chimeric DNA-RNA guided Cpf1. DNA supercoiling generates a tension that inhibits the chimeric (cr)RNA guided Cpf1 cleavage activity. Nickase relaxed supercoiling enhances chimeric (cr)RNA guided Cpf1 DNA cleavage activity on genomic DNA. The small and large red arrowhead indicates the cleavage site for nick by SpCas9 nickase (D10A) and enhanced double-strand break by Cpf1. Red color in the (cr)RNA indicates DNA substitution. PAM sequences (NGG for SpCas9 nickase and TTTN for AsCpf1) are shown in blue color and mismatched sequence to target sequence are shown in orange color, respectively.

    Article Snippet: Chimeric DNA-RNA guides (bioneer) were batch synthesized according to the target sequences in each target gene (Table S1) .

    Techniques: Binding Assay, Activity Assay, Sequencing

    Target DNA cleavage by Cpf1 using a chimeric DNA-RNA guide in which the seed region of the (cr)RNA is replaced with DNA. (A) Comparative analysis of the target ( DNMT1 : orange) DNA cleavage efficiency using various (cr)RNAs harboring serial multiple or single DNA substitutions in the seed region close to the PAM (TTTN). (B) Comparative analysis of the target ( CCR5 : green) DNA cleavage efficiency. AsCpf1 (cr)RNA was serially replaced with single DNA nucleotides from the PAM. The RNA portion of the (cr)RNA is shown in blue, and the DNA portion is shown in red (‘D’ indicates a DNA and the number of substituted DNA nucleotides is indicated). All the cleavage efficiency was calculated from agarose gel separated band intensity (cleaved fragment intensity (%) / total fragment intensity (%)) and normalized to wild-type (cr)RNA (crRNA 1 or crRNA 51). Data are shown as means ± s.e.m. from three independent experiments. P -values are calculated using a two-tailed Student’s t-test (ns: not significant, P*:

    Journal: bioRxiv

    Article Title: Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

    doi: 10.1101/2020.02.04.933614

    Figure Lengend Snippet: Target DNA cleavage by Cpf1 using a chimeric DNA-RNA guide in which the seed region of the (cr)RNA is replaced with DNA. (A) Comparative analysis of the target ( DNMT1 : orange) DNA cleavage efficiency using various (cr)RNAs harboring serial multiple or single DNA substitutions in the seed region close to the PAM (TTTN). (B) Comparative analysis of the target ( CCR5 : green) DNA cleavage efficiency. AsCpf1 (cr)RNA was serially replaced with single DNA nucleotides from the PAM. The RNA portion of the (cr)RNA is shown in blue, and the DNA portion is shown in red (‘D’ indicates a DNA and the number of substituted DNA nucleotides is indicated). All the cleavage efficiency was calculated from agarose gel separated band intensity (cleaved fragment intensity (%) / total fragment intensity (%)) and normalized to wild-type (cr)RNA (crRNA 1 or crRNA 51). Data are shown as means ± s.e.m. from three independent experiments. P -values are calculated using a two-tailed Student’s t-test (ns: not significant, P*:

    Article Snippet: Chimeric DNA-RNA guides (bioneer) were batch synthesized according to the target sequences in each target gene (Table S1) .

    Techniques: Agarose Gel Electrophoresis, Two Tailed Test

    Cpf1 editing efficiency and specificity are enhanced by combination of a chimeric DNA-RNA guide and SpCas9 nickase. (A) Intracellular DNMT1 gene editing in HEK293FT cell using 3′-end DNA-substituted chimeric DNA-RNA-guided Cpf1 and SpCas9 (D10A) nickase. The RNA portion of the Cpf1 (cr)RNA is shown in blue, and the substituted DNA portion is shown in red. X axis indicates the relative indel frequency (%). Only (cr)RNA - Cpf1 treated (dark blue) and combination with dCas9 (blue) or nCas9 (pink) treated samples were indicated, respectively. dCas9 and nCas9 indicates deactivated Cas9 (D10A, H840A) and nickase Cas9 (D10A), respectively. Indel ratio (%) is calculated by targeted amplicon sequencing from DNMT1 site in HEK293FT cells (indel frequency (%) = mutant DNA read number / total DNA read number) and normalized to wild-type (cr)RNA (crRNA 1 for DNMT1 ). Data are shown as means ± s.e.m. from three independent experiments. P -values are calculated using a two-tailed Student’s t-test (ns: not significant, P*:

    Journal: bioRxiv

    Article Title: Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

    doi: 10.1101/2020.02.04.933614

    Figure Lengend Snippet: Cpf1 editing efficiency and specificity are enhanced by combination of a chimeric DNA-RNA guide and SpCas9 nickase. (A) Intracellular DNMT1 gene editing in HEK293FT cell using 3′-end DNA-substituted chimeric DNA-RNA-guided Cpf1 and SpCas9 (D10A) nickase. The RNA portion of the Cpf1 (cr)RNA is shown in blue, and the substituted DNA portion is shown in red. X axis indicates the relative indel frequency (%). Only (cr)RNA - Cpf1 treated (dark blue) and combination with dCas9 (blue) or nCas9 (pink) treated samples were indicated, respectively. dCas9 and nCas9 indicates deactivated Cas9 (D10A, H840A) and nickase Cas9 (D10A), respectively. Indel ratio (%) is calculated by targeted amplicon sequencing from DNMT1 site in HEK293FT cells (indel frequency (%) = mutant DNA read number / total DNA read number) and normalized to wild-type (cr)RNA (crRNA 1 for DNMT1 ). Data are shown as means ± s.e.m. from three independent experiments. P -values are calculated using a two-tailed Student’s t-test (ns: not significant, P*:

    Article Snippet: Chimeric DNA-RNA guides (bioneer) were batch synthesized according to the target sequences in each target gene (Table S1) .

    Techniques: Amplification, Sequencing, Mutagenesis, Two Tailed Test

    Intracellular genome editing using Cpf1 and chimeric DNA-RNA guides. (A) Endogenous locus ( DNMT1 ) editing in HEK293FT cells using chimeric DNA-RNA guides with serial 4-nt DNA substitutions from the 3′-end of the (cr)RNA. The RNA portion of the Cpf1 (cr)RNA is shown in blue, and the DNA portion is shown in red (‘D’ indicates a DNA and the number of substituted DNA nucleotides is indicated). (B) Endogenous locus ( DNMT1 ) editing in HEK293FT cells using chimeric DNA-RNA guides with DNA substitutions in the seed region of the (cr)RNA. (C) Endogenous locus ( DNMT1 ) editing in HEK293FT cells using chimeric DNA-RNA guides with serial 4-nt DNA substitutions from the 5′-end of the (cr)RNA. (D) Representative genome editing pattern of AsCpf1 using chimeric DNA-RNA guides. Sequencing data is obtained from DNMT1 targeted amplicon sequencing. Deleted base is shown by dashed line. PAM sequence (TTTN) and target sequence for AsCpf1 is shown in cyon and red color, respectively. All the relative indel ratio was calculated from targeted amplicon sequencing (indel frequency (%) = mutant DNA read number / total DNA read number) and normalized to wild-type (cr)RNA (crRNA 1 for DNMT1 ). Data are shown as means ± s.e.m. from three independent experiments. P -values are calculated using a two-tailed Student’s t-test (ns: not significant, P*:

    Journal: bioRxiv

    Article Title: Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

    doi: 10.1101/2020.02.04.933614

    Figure Lengend Snippet: Intracellular genome editing using Cpf1 and chimeric DNA-RNA guides. (A) Endogenous locus ( DNMT1 ) editing in HEK293FT cells using chimeric DNA-RNA guides with serial 4-nt DNA substitutions from the 3′-end of the (cr)RNA. The RNA portion of the Cpf1 (cr)RNA is shown in blue, and the DNA portion is shown in red (‘D’ indicates a DNA and the number of substituted DNA nucleotides is indicated). (B) Endogenous locus ( DNMT1 ) editing in HEK293FT cells using chimeric DNA-RNA guides with DNA substitutions in the seed region of the (cr)RNA. (C) Endogenous locus ( DNMT1 ) editing in HEK293FT cells using chimeric DNA-RNA guides with serial 4-nt DNA substitutions from the 5′-end of the (cr)RNA. (D) Representative genome editing pattern of AsCpf1 using chimeric DNA-RNA guides. Sequencing data is obtained from DNMT1 targeted amplicon sequencing. Deleted base is shown by dashed line. PAM sequence (TTTN) and target sequence for AsCpf1 is shown in cyon and red color, respectively. All the relative indel ratio was calculated from targeted amplicon sequencing (indel frequency (%) = mutant DNA read number / total DNA read number) and normalized to wild-type (cr)RNA (crRNA 1 for DNMT1 ). Data are shown as means ± s.e.m. from three independent experiments. P -values are calculated using a two-tailed Student’s t-test (ns: not significant, P*:

    Article Snippet: Chimeric DNA-RNA guides (bioneer) were batch synthesized according to the target sequences in each target gene (Table S1) .

    Techniques: Sequencing, Amplification, Mutagenesis, Two Tailed Test

    Off-target cleavage assay of Cpf1 using chimeric DNA-RNA guides. (A) Target and off-target DNMT1 DNA cleavage experiments to confirm the target specificity of AsCpf1 using a chimeric DNA-RNA guide (serial 4-nt DNA substitutions from the 3’-end; on-target (dark brown), off-target 1 (blue), off-target 2 (orange), off-target 3 (pink)). The RNA portion of the Cpf1 (cr)RNA is shown in blue, and the DNA portion is shown in red (‘D’ indicates a DNA and the number of substituted DNA nucleotides is indicated). (B) Target and off-target DNMT1 DNA cleavage experiments to confirm the target specificity of AsCpf1 using a chimeric DNA-RNA guide (DNA substitutions in the seed region of (cr)RNA; on-target (dark brown), off-target 1 (blue), off-target 2 (orange), off-target 3 (pink)). (C) Relative cleavage efficiency of target (dark brown) and off-target (off1: blue, off2: orange) of FANCF DNA using chimeric DNA-RNA guided (3’-end 4-bp, 8-bp DNA substitutions) AsCpf1. (D) Relative cleavage efficiency of target (dark brown) and off-target (off1: blue, off2: orange) of GRIN2B DNA using chimeric DNA-RNA guided (3’-end 4-bp, 8-bp DNA substitutions) AsCpf1. (E) Relative cleavage efficiency of target (dark brown) and off-target (off1: blue) of EMX1 DNA using chimeric DNA-RNA guided (3’-end 4-bp, 8-bp DNA substitutions) AsCpf1. All the cleavage efficiency was calculated from agarose gel separated band intensity (cleaved fragment intensity (%) / total fragment intensity (%)) and normalized to wild-type (cr)RNA (crRNA 1 for DNMT1 , crRNA 81 for FANCF , crRNA 83 for GRIN2B , crRNA 85 for EMX1 ). Data are shown as means ± s.e.m. from three independent experiments. P -values are calculated using a two-tailed Student’s t-test (ns: not significant, P*:

    Journal: bioRxiv

    Article Title: Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

    doi: 10.1101/2020.02.04.933614

    Figure Lengend Snippet: Off-target cleavage assay of Cpf1 using chimeric DNA-RNA guides. (A) Target and off-target DNMT1 DNA cleavage experiments to confirm the target specificity of AsCpf1 using a chimeric DNA-RNA guide (serial 4-nt DNA substitutions from the 3’-end; on-target (dark brown), off-target 1 (blue), off-target 2 (orange), off-target 3 (pink)). The RNA portion of the Cpf1 (cr)RNA is shown in blue, and the DNA portion is shown in red (‘D’ indicates a DNA and the number of substituted DNA nucleotides is indicated). (B) Target and off-target DNMT1 DNA cleavage experiments to confirm the target specificity of AsCpf1 using a chimeric DNA-RNA guide (DNA substitutions in the seed region of (cr)RNA; on-target (dark brown), off-target 1 (blue), off-target 2 (orange), off-target 3 (pink)). (C) Relative cleavage efficiency of target (dark brown) and off-target (off1: blue, off2: orange) of FANCF DNA using chimeric DNA-RNA guided (3’-end 4-bp, 8-bp DNA substitutions) AsCpf1. (D) Relative cleavage efficiency of target (dark brown) and off-target (off1: blue, off2: orange) of GRIN2B DNA using chimeric DNA-RNA guided (3’-end 4-bp, 8-bp DNA substitutions) AsCpf1. (E) Relative cleavage efficiency of target (dark brown) and off-target (off1: blue) of EMX1 DNA using chimeric DNA-RNA guided (3’-end 4-bp, 8-bp DNA substitutions) AsCpf1. All the cleavage efficiency was calculated from agarose gel separated band intensity (cleaved fragment intensity (%) / total fragment intensity (%)) and normalized to wild-type (cr)RNA (crRNA 1 for DNMT1 , crRNA 81 for FANCF , crRNA 83 for GRIN2B , crRNA 85 for EMX1 ). Data are shown as means ± s.e.m. from three independent experiments. P -values are calculated using a two-tailed Student’s t-test (ns: not significant, P*:

    Article Snippet: Chimeric DNA-RNA guides (bioneer) were batch synthesized according to the target sequences in each target gene (Table S1) .

    Techniques: Cleavage Assay, Agarose Gel Electrophoresis, Two Tailed Test

    Comparison of target specificity of chimeric DNA-RNA guides. (A) Comparison of the DNMT1 target specificity (on/off cleavage ratio (%)) of AsCpf1 using chimeric DNA-RNA guides with serial 4-nt DNA substitutions from the 3’-end of the (cr)RNA (Substituted DNA nucleotides in (cr)RNA is shown in red color and RNA is shown in blue color, respectively). Target specificity was calculated from the results in ( Figure 3 ) by dividing on-target cleavage efficiency by off-target cleavage efficiency. Target specificity is shown in black (on/off 1), dark gray (on/off 2), and light gray (on/off 3). (B) Comparison of the DNMT1 target specificity of AsCpf1 using chimeric DNA-RNA guides with DNA substitutions in the seed region of the (cr)RNA. Target specificity is shown in black (on/off 1), dark gray (on/off 2) and light gray (on/off 3). (C-E) Comparison of FANCF, GRIN2B , and EMX1 target specificities of AsCpf1 using chimeric DNA-RNA guides with serial 4-nt DNA substitutions from the 3’-end of the (cr)RNA). Target specificity is shown in black (on/off 1) and gray (on/off2). The number of substituted DNA nucleotides in AsCpf1 (cr)RNA used for each gene targeting is indicated by red color.

    Journal: bioRxiv

    Article Title: Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

    doi: 10.1101/2020.02.04.933614

    Figure Lengend Snippet: Comparison of target specificity of chimeric DNA-RNA guides. (A) Comparison of the DNMT1 target specificity (on/off cleavage ratio (%)) of AsCpf1 using chimeric DNA-RNA guides with serial 4-nt DNA substitutions from the 3’-end of the (cr)RNA (Substituted DNA nucleotides in (cr)RNA is shown in red color and RNA is shown in blue color, respectively). Target specificity was calculated from the results in ( Figure 3 ) by dividing on-target cleavage efficiency by off-target cleavage efficiency. Target specificity is shown in black (on/off 1), dark gray (on/off 2), and light gray (on/off 3). (B) Comparison of the DNMT1 target specificity of AsCpf1 using chimeric DNA-RNA guides with DNA substitutions in the seed region of the (cr)RNA. Target specificity is shown in black (on/off 1), dark gray (on/off 2) and light gray (on/off 3). (C-E) Comparison of FANCF, GRIN2B , and EMX1 target specificities of AsCpf1 using chimeric DNA-RNA guides with serial 4-nt DNA substitutions from the 3’-end of the (cr)RNA). Target specificity is shown in black (on/off 1) and gray (on/off2). The number of substituted DNA nucleotides in AsCpf1 (cr)RNA used for each gene targeting is indicated by red color.

    Article Snippet: Chimeric DNA-RNA guides (bioneer) were batch synthesized according to the target sequences in each target gene (Table S1) .

    Techniques:

    Target DNA cleavage by CRISPR-Cas12a (Cpf1) using chimeric DNA-RNA guides. (A) Schematic representation of interactions of AsCpf1 (cr)RNA (colored in cyon) with target DNA (colored in purple) [ 6 ]. The AsCpf1 (cr)RNA was numbered from 5’-end to 3’-end. (B) The target DNA amplicon cleavage efficiency of AsCpf1 with partial DNA substitution of (cr)RNA was determined. The (cr)RNA was replaced with DNA from 3’-end with 4nt interval. The RNA portion of the (cr)RNA is shown in blue, and the DNA portion is shown in red, respectively (‘D’ indicates a DNA and the number of substituted DNA nucleotides is indicated). X axis indicates for efficiency of the target gene ( DNMT1 (orange), CCR5 (green)) cleavage by AsCpf1 using various chimeric DNA-RNA guides (DNA substitution of 4-nt from the 3′-end of the (cr)RNA). Y axis indicates for used chimeric (cr)RNAs in cleavage experiment. (C) Target gene ( DNMT1 (orange)) cleavage by AsCpf1 using chimeric DNA-RNA guides (Serial 4-nt DNA substitution from the 5′-end of the (cr)RNA). All the cleavage efficiency was calculated from agarose gel separated band intensity (cleaved fragment intensity (%) / total fragment intensity (%)) and normalized to wild-type (cr)RNA (crRNA 1 or crRNA 51). Data are shown as means ± s.e.m. from three independent experiments. P -values are calculated using a two-tailed Student’s t-test (ns: not significant, P*:

    Journal: bioRxiv

    Article Title: Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

    doi: 10.1101/2020.02.04.933614

    Figure Lengend Snippet: Target DNA cleavage by CRISPR-Cas12a (Cpf1) using chimeric DNA-RNA guides. (A) Schematic representation of interactions of AsCpf1 (cr)RNA (colored in cyon) with target DNA (colored in purple) [ 6 ]. The AsCpf1 (cr)RNA was numbered from 5’-end to 3’-end. (B) The target DNA amplicon cleavage efficiency of AsCpf1 with partial DNA substitution of (cr)RNA was determined. The (cr)RNA was replaced with DNA from 3’-end with 4nt interval. The RNA portion of the (cr)RNA is shown in blue, and the DNA portion is shown in red, respectively (‘D’ indicates a DNA and the number of substituted DNA nucleotides is indicated). X axis indicates for efficiency of the target gene ( DNMT1 (orange), CCR5 (green)) cleavage by AsCpf1 using various chimeric DNA-RNA guides (DNA substitution of 4-nt from the 3′-end of the (cr)RNA). Y axis indicates for used chimeric (cr)RNAs in cleavage experiment. (C) Target gene ( DNMT1 (orange)) cleavage by AsCpf1 using chimeric DNA-RNA guides (Serial 4-nt DNA substitution from the 5′-end of the (cr)RNA). All the cleavage efficiency was calculated from agarose gel separated band intensity (cleaved fragment intensity (%) / total fragment intensity (%)) and normalized to wild-type (cr)RNA (crRNA 1 or crRNA 51). Data are shown as means ± s.e.m. from three independent experiments. P -values are calculated using a two-tailed Student’s t-test (ns: not significant, P*:

    Article Snippet: Chimeric DNA-RNA guides (bioneer) were batch synthesized according to the target sequences in each target gene (Table S1) .

    Techniques: CRISPR, Amplification, Agarose Gel Electrophoresis, Two Tailed Test

    Intracellular genome editing of Cpf1 using 3′-end chemically modified chimeric DNA-RNA guides. (A) DNMT1 on/off-target amplicon cleavage by AsCpf1 using chimeric DNA-RNA guides with 3’-end 4-nt or 8-nt DNA substitution and 3’-end PS modification. The relative cleavage efficiency (%) of on-target (dark brown), off-target 1 (dark pink), off-target 2 (light purple) and off-target 3 (light pink)) is indicated by different colors. The RNA portion of the Cpf1 (cr)RNA is shown in blue, and the substituted DNA portion is shown in red. Asterisk indicates the 3’-end PS modification of (cr)RNA. All the cleavage efficiency was calculated from agarose gel separated band intensity (cleavage efficiency (%) = cleaved fragment intensity / total fragment intensity) and normalized to wild-type (cr)RNA (crRNA 1 for DNMT1 ). Data are shown as means ± s.e.m. from three independent experiments. P -values are calculated using a two-tailed Student’s t-test (ns: not significant, P*:

    Journal: bioRxiv

    Article Title: Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

    doi: 10.1101/2020.02.04.933614

    Figure Lengend Snippet: Intracellular genome editing of Cpf1 using 3′-end chemically modified chimeric DNA-RNA guides. (A) DNMT1 on/off-target amplicon cleavage by AsCpf1 using chimeric DNA-RNA guides with 3’-end 4-nt or 8-nt DNA substitution and 3’-end PS modification. The relative cleavage efficiency (%) of on-target (dark brown), off-target 1 (dark pink), off-target 2 (light purple) and off-target 3 (light pink)) is indicated by different colors. The RNA portion of the Cpf1 (cr)RNA is shown in blue, and the substituted DNA portion is shown in red. Asterisk indicates the 3’-end PS modification of (cr)RNA. All the cleavage efficiency was calculated from agarose gel separated band intensity (cleavage efficiency (%) = cleaved fragment intensity / total fragment intensity) and normalized to wild-type (cr)RNA (crRNA 1 for DNMT1 ). Data are shown as means ± s.e.m. from three independent experiments. P -values are calculated using a two-tailed Student’s t-test (ns: not significant, P*:

    Article Snippet: Chimeric DNA-RNA guides (bioneer) were batch synthesized according to the target sequences in each target gene (Table S1) .

    Techniques: Modification, Amplification, Agarose Gel Electrophoresis, Two Tailed Test

    Genome-editing activity of the TALENs and CRISPR/Cas9 systems targeting HIV LTR. (A) LTR-driven GFP expression after TF with CRISPR/Cas9 or TALENs expressing plasmid DNA. Jurkat cells were infected with an LTIG lentiviral vector. Five days after infection, the GFP positive cells were sorted (Jurkat/LTIG) and co-transfected with T5 gRNA-expressing and hCas9-expressing DNA or LTR TALEN-L and -R expressing DNA. The level of GFP expression was analyzed by flow cytometry 5 days after TF. (B) Sequence analysis of TALENs targeting sites. The DNA sequences of the TAR and adjacent regions of LTR are indicated. Nineteen sequences were obtained from Jurkat/LTIG cells, which were transfected twice with TAR TALEN-LR. The WT reference sequence is shown at the top. The target sequences of TAR TALENs and T5 gRNA are indicated in orange and green, respectively. The putative cleavage site of T5 CRISPR is indicated with an orange arrowhead. (C) Genome-editing activity of TAR TALENs and T5 CRISPR in c19 cells, latently transduced with an LTIG lentiviral vector. The level of GFP expression after 48 hours of TNF-α stimulation is shown. The error bars in B and D show standard deviations (n = 3).

    Journal: PLoS ONE

    Article Title: A High Excision Potential of TALENs for Integrated DNA of HIV-Based Lentiviral Vector

    doi: 10.1371/journal.pone.0120047

    Figure Lengend Snippet: Genome-editing activity of the TALENs and CRISPR/Cas9 systems targeting HIV LTR. (A) LTR-driven GFP expression after TF with CRISPR/Cas9 or TALENs expressing plasmid DNA. Jurkat cells were infected with an LTIG lentiviral vector. Five days after infection, the GFP positive cells were sorted (Jurkat/LTIG) and co-transfected with T5 gRNA-expressing and hCas9-expressing DNA or LTR TALEN-L and -R expressing DNA. The level of GFP expression was analyzed by flow cytometry 5 days after TF. (B) Sequence analysis of TALENs targeting sites. The DNA sequences of the TAR and adjacent regions of LTR are indicated. Nineteen sequences were obtained from Jurkat/LTIG cells, which were transfected twice with TAR TALEN-LR. The WT reference sequence is shown at the top. The target sequences of TAR TALENs and T5 gRNA are indicated in orange and green, respectively. The putative cleavage site of T5 CRISPR is indicated with an orange arrowhead. (C) Genome-editing activity of TAR TALENs and T5 CRISPR in c19 cells, latently transduced with an LTIG lentiviral vector. The level of GFP expression after 48 hours of TNF-α stimulation is shown. The error bars in B and D show standard deviations (n = 3).

    Article Snippet: For TF of the CRISPR/Cas9 system, 1 μg of humanized Cas9 expression DNA and 1 μg of gRNA expression DNA (Addgene) were used.

    Techniques: Activity Assay, TALENs, CRISPR, Expressing, Plasmid Preparation, Infection, Transfection, Flow Cytometry, Cytometry, Sequencing, Transduction

    Characterization of the RNA nanoparticle/doxorubicin physical conjugates (a) Construct of Endo28-3WJ-sph1 RNA nanoparticle with predicted chelating sites for doxorubicin indicated by red stars. (b) Native PAGE showing the assembly of Endo28-3WJ-sph1 nanoparticles. Lane 1 is 100bp DNA ladder (Thermo Scientific). (c) Fluorescence spectra of doxorubicin solution (1.4μM) with increasing concentration of RNA nanoparticles. Insert: A hill plot for the nanoparticle titration (K d = 140nM, 0.1 equivalent of the RNA nanoparticles). (d) The percentage of doxorubicin released from the nanoparticle doxorubicin conjugates at 37°C. The doxorubicin concentration was quantified by its fluorescence intensity in release medium.

    Journal: Nanomedicine : nanotechnology, biology, and medicine

    Article Title: RNA Nanoparticles Harboring Annexin A2 Aptamer Can Target Ovarian Cancer for Tumor-Specific Doxorubicin Delivery

    doi: 10.1016/j.nano.2016.11.015

    Figure Lengend Snippet: Characterization of the RNA nanoparticle/doxorubicin physical conjugates (a) Construct of Endo28-3WJ-sph1 RNA nanoparticle with predicted chelating sites for doxorubicin indicated by red stars. (b) Native PAGE showing the assembly of Endo28-3WJ-sph1 nanoparticles. Lane 1 is 100bp DNA ladder (Thermo Scientific). (c) Fluorescence spectra of doxorubicin solution (1.4μM) with increasing concentration of RNA nanoparticles. Insert: A hill plot for the nanoparticle titration (K d = 140nM, 0.1 equivalent of the RNA nanoparticles). (d) The percentage of doxorubicin released from the nanoparticle doxorubicin conjugates at 37°C. The doxorubicin concentration was quantified by its fluorescence intensity in release medium.

    Article Snippet: Self-assembled DNA-Guided RNA Nanovector via Step-wise Dual Enzyme Polymerization (SDEP) for Carrier-free siRNA Delivery.

    Techniques: Construct, Clear Native PAGE, Fluorescence, Concentration Assay, Titration

    CRISPR-Cas9-mediated frameshift mutations of CXCR7 resulted in the onset of cellular senescence. ( a ) X-gal assay for SA-β-galactosidase (SA-gal) activity in C4-2B CXCR7 frameshift mutant cells. Positive SA-gal expression is indicated by dark blue coloration to the cells. Upper-left panel: C4-2B cells with wild-type (WT) CXCR7 expression untreated (UT) (negative control), Lower-left panel: WT C4-2B cells treated with 10 µM H 2 O 2 (positive control). Upper-middle, lower middle, and upper-right panels: representative images in CXCR7-CRISPR-mutated cells. Lower-right panel: Sanger sequencing chromatogram of the CXCR7-gRNA target site illustrating overlapping unique CXCR7 InDel mutations. ( b ) X-gal assay in LNCaP CXCR7 frameshift mutant cells and controls (UT or H 2 O 2 treated).

    Journal: Scientific Reports

    Article Title: Inhibition of androgen receptor promotes CXC-chemokine receptor 7-mediated prostate cancer cell survival

    doi: 10.1038/s41598-017-02918-3

    Figure Lengend Snippet: CRISPR-Cas9-mediated frameshift mutations of CXCR7 resulted in the onset of cellular senescence. ( a ) X-gal assay for SA-β-galactosidase (SA-gal) activity in C4-2B CXCR7 frameshift mutant cells. Positive SA-gal expression is indicated by dark blue coloration to the cells. Upper-left panel: C4-2B cells with wild-type (WT) CXCR7 expression untreated (UT) (negative control), Lower-left panel: WT C4-2B cells treated with 10 µM H 2 O 2 (positive control). Upper-middle, lower middle, and upper-right panels: representative images in CXCR7-CRISPR-mutated cells. Lower-right panel: Sanger sequencing chromatogram of the CXCR7-gRNA target site illustrating overlapping unique CXCR7 InDel mutations. ( b ) X-gal assay in LNCaP CXCR7 frameshift mutant cells and controls (UT or H 2 O 2 treated).

    Article Snippet: CRISPR-Cas9 mediated genomic DNA mutation CRISPR guide RNA (gRNA) sequence design and plasmid preparation were provided by Santa Cruz Biotechnologies (Dallas, TX), utilizing the Genome-Scale CRISPR Knock-Out (GeCKO) v2 library .

    Techniques: CRISPR, Activity Assay, Mutagenesis, Expressing, Negative Control, Positive Control, Sequencing

    CRISPR-Cas9-generated CXCR7 inframe mutation in LNCaP cells disrupts membrane protein interactions. ( a ) Left: PCR of CXCR7 genomic DNA in unmodified cells (WT) and CRISPR-edited LNCaP cells revealing a 394 nt deletion event (CX7-mutant). Right: Sanger sequencing chromatogram of the DNA sequence illustrating the contiguous sequence, Black arrow represents the expected 5′ CXCR7 sequence and the red arrow shows the 3′ CXCR7 sequence around the site of deletion event. ( b ) EGFR and ERK phosphorylation in WT and CX7-mutant cells with densitometry of phosphorylated protein relative to total. ( c ) Count of second generation spheroids formed per plate from 10,000 seeded WT and CX7-mutant cells (n = 2, mean ± SD; *p

    Journal: Scientific Reports

    Article Title: Inhibition of androgen receptor promotes CXC-chemokine receptor 7-mediated prostate cancer cell survival

    doi: 10.1038/s41598-017-02918-3

    Figure Lengend Snippet: CRISPR-Cas9-generated CXCR7 inframe mutation in LNCaP cells disrupts membrane protein interactions. ( a ) Left: PCR of CXCR7 genomic DNA in unmodified cells (WT) and CRISPR-edited LNCaP cells revealing a 394 nt deletion event (CX7-mutant). Right: Sanger sequencing chromatogram of the DNA sequence illustrating the contiguous sequence, Black arrow represents the expected 5′ CXCR7 sequence and the red arrow shows the 3′ CXCR7 sequence around the site of deletion event. ( b ) EGFR and ERK phosphorylation in WT and CX7-mutant cells with densitometry of phosphorylated protein relative to total. ( c ) Count of second generation spheroids formed per plate from 10,000 seeded WT and CX7-mutant cells (n = 2, mean ± SD; *p

    Article Snippet: CRISPR-Cas9 mediated genomic DNA mutation CRISPR guide RNA (gRNA) sequence design and plasmid preparation were provided by Santa Cruz Biotechnologies (Dallas, TX), utilizing the Genome-Scale CRISPR Knock-Out (GeCKO) v2 library .

    Techniques: CRISPR, Generated, Mutagenesis, Polymerase Chain Reaction, Sequencing

    CRISPR/Cas9-mediated gene targeting in mouse iPSCs. ( a ) Schematic diagram showing the site of Arg1 gene modification using CRISPR/Cas9. The gRNA was designed to target a 20 nt region in intron 6 of Arg1 . ( b ) Cas9:gRNA off-target analysis. A table showing the output from CRISPR design tool. The on-site target is given at the top, followed by off-target sequences organized in decreasing Cas9-cleavage probability. ( c ) Detection of Cas9:gRNA-mediated on-target cleavage of Arg1 by the Surveyor nuclease assay. The cleavage products were shown as extra bands (149 bp + 197 bp) indicated by the arrows. Mutation frequency (indel) was calculated by measuring the band intensities. ( d ) Sequencing data from TOPO-cloned PCR amplicons of CRISPR/Cas9-modified genomic DNA showing a few examples of NHEJ-mediated indel mutations at the desired location. gRNA target site is in bold. Deletions are shown in dashes. Nucleotide substitution is shown in lowercase. The net change in length caused by each indel mutation is to the right of each sequence.

    Journal: Scientific Reports

    Article Title: Proof-of-Concept Gene Editing for the Murine Model of Inducible Arginase-1 Deficiency

    doi: 10.1038/s41598-017-02927-2

    Figure Lengend Snippet: CRISPR/Cas9-mediated gene targeting in mouse iPSCs. ( a ) Schematic diagram showing the site of Arg1 gene modification using CRISPR/Cas9. The gRNA was designed to target a 20 nt region in intron 6 of Arg1 . ( b ) Cas9:gRNA off-target analysis. A table showing the output from CRISPR design tool. The on-site target is given at the top, followed by off-target sequences organized in decreasing Cas9-cleavage probability. ( c ) Detection of Cas9:gRNA-mediated on-target cleavage of Arg1 by the Surveyor nuclease assay. The cleavage products were shown as extra bands (149 bp + 197 bp) indicated by the arrows. Mutation frequency (indel) was calculated by measuring the band intensities. ( d ) Sequencing data from TOPO-cloned PCR amplicons of CRISPR/Cas9-modified genomic DNA showing a few examples of NHEJ-mediated indel mutations at the desired location. gRNA target site is in bold. Deletions are shown in dashes. Nucleotide substitution is shown in lowercase. The net change in length caused by each indel mutation is to the right of each sequence.

    Article Snippet: Functional evaluation of targeted DNA cleavage by Cas9:gRNA Arg1 Δ iPSCs (3 × 106 ) were electroporated with 15 µg Cas9:gRNA plasmid DNA to induce double-stranded breaks (Bio-Rad Gene Pulser System: 250 V, 500 μF, 0.4 cm cuvettes).

    Techniques: CRISPR, Modification, Nuclease Assay, Mutagenesis, Sequencing, Clone Assay, Polymerase Chain Reaction, Non-Homologous End Joining