cas9 protein  (New England Biolabs)


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    Name:
    Cas9 Nuclease S pyogenes
    Description:
    Cas9 Nuclease S pyogenes 2 000 pmol
    Catalog Number:
    M0386M
    Price:
    540
    Category:
    Other Endonucleases
    Size:
    2 000 pmol
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    Structured Review

    New England Biolabs cas9 protein
    Cas9 Nuclease S pyogenes
    Cas9 Nuclease S pyogenes 2 000 pmol
    https://www.bioz.com/result/cas9 protein/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cas9 protein - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "Biochemically diverse CRISPR-Cas9 orthologs"

    Article Title: Biochemically diverse CRISPR-Cas9 orthologs

    Journal: bioRxiv

    doi: 10.1101/2020.04.29.066654

    Target DNA cleavage patterns produced by Cas9 orthologs. Cleavage sites and resultant dsDNA ends are depicted as heatmaps that show the proportion of cleaved ends recovered by DNA sequencing at each position of a target DNA. Intensity of the blue color indicates the proportion of mapped cleavage ends. (A) Control digests using restriction enzymes recover 5’-overhangs, 3’-overhangs, and blunt ends. TS indicates top strand; BS indicates bottom strand. (B) SpyCas9 and SauCas9 cleaved DNA ends. Heatmaps represent mapped cleavage ends as the averages at each position in 5 different dsDNA targets. Position of the DNA bases and PAM sequences is depicted above the heatmaps. NTS indicates non-target strand; TS indicates target strand. (C) Blunt and staggered-end cleavage. Examples of blunt, one base 5’-overhang staggered cleavage, and multiple base 5’-overhang cleavage are depicted as heatmaps that show the proportion of cleaved ends as the averages at each position in 5 different dsDNA targets. Position of the DNA bases and PAM sequences is depicted above the heatmaps. NTS indicates non-target strand; TS indicates target strand.
    Figure Legend Snippet: Target DNA cleavage patterns produced by Cas9 orthologs. Cleavage sites and resultant dsDNA ends are depicted as heatmaps that show the proportion of cleaved ends recovered by DNA sequencing at each position of a target DNA. Intensity of the blue color indicates the proportion of mapped cleavage ends. (A) Control digests using restriction enzymes recover 5’-overhangs, 3’-overhangs, and blunt ends. TS indicates top strand; BS indicates bottom strand. (B) SpyCas9 and SauCas9 cleaved DNA ends. Heatmaps represent mapped cleavage ends as the averages at each position in 5 different dsDNA targets. Position of the DNA bases and PAM sequences is depicted above the heatmaps. NTS indicates non-target strand; TS indicates target strand. (C) Blunt and staggered-end cleavage. Examples of blunt, one base 5’-overhang staggered cleavage, and multiple base 5’-overhang cleavage are depicted as heatmaps that show the proportion of cleaved ends as the averages at each position in 5 different dsDNA targets. Position of the DNA bases and PAM sequences is depicted above the heatmaps. NTS indicates non-target strand; TS indicates target strand.

    Techniques Used: Produced, DNA Sequencing

    Activity of Cas9 orthologs at varying temperatures. The cleavage activity of Cas9 orthologs was measured using in vitro DNA cleavage assays using fluorophore-labeled dsDNA substrates. Cleaved fragments were quantitated and are represented in a heat map (A) showing overall activity at temperatures ranging from 10°C to 68°C. (B) Cas9 orthologs with activity at elevated temperatures. In vitro DNA cleavage activity for a subset of Cas9 orthologs with > 50% activity at 53°C is summarized in a heat map and plotted as proportion of DNA substrate cleaved at varied temperature. Points represent the mean +/- SEM of at least 3 independent experiments. (C) Cas9 orthologs with reduced activity at room temperature. In vitro DNA cleavage activity for a subset of Cas9 orthologs with
    Figure Legend Snippet: Activity of Cas9 orthologs at varying temperatures. The cleavage activity of Cas9 orthologs was measured using in vitro DNA cleavage assays using fluorophore-labeled dsDNA substrates. Cleaved fragments were quantitated and are represented in a heat map (A) showing overall activity at temperatures ranging from 10°C to 68°C. (B) Cas9 orthologs with activity at elevated temperatures. In vitro DNA cleavage activity for a subset of Cas9 orthologs with > 50% activity at 53°C is summarized in a heat map and plotted as proportion of DNA substrate cleaved at varied temperature. Points represent the mean +/- SEM of at least 3 independent experiments. (C) Cas9 orthologs with reduced activity at room temperature. In vitro DNA cleavage activity for a subset of Cas9 orthologs with

    Techniques Used: Activity Assay, In Vitro, Labeling

    Cas9 PAM Interacting (PI) domain similarity. Cas9 PI domains clustered by their pairwise sequence similarity. Lines connect sequences with P-value ≤ 1e-11. Line shading corresponds to P-values according to the scale in the top-right corner (light and long lines connect distantly related sequences). Major clusters are shown in bold. Cluster 1 was so named to emphasize that it contains first experimentally characterized Cas9. Clusters 2 to 10 were named beginning from the one with the most members. Sequences having known structures are marked red, their PDB code is shown in parentheses.
    Figure Legend Snippet: Cas9 PAM Interacting (PI) domain similarity. Cas9 PI domains clustered by their pairwise sequence similarity. Lines connect sequences with P-value ≤ 1e-11. Line shading corresponds to P-values according to the scale in the top-right corner (light and long lines connect distantly related sequences). Major clusters are shown in bold. Cluster 1 was so named to emphasize that it contains first experimentally characterized Cas9. Clusters 2 to 10 were named beginning from the one with the most members. Sequences having known structures are marked red, their PDB code is shown in parentheses.

    Techniques Used: Sequencing

    Cas9 diversity and characterization approach. (A) Phylogenetic representation of the diversity provided by Cas9 orthologs. Type II-A, B, and C systems are color-coded, red, blue, and green, respectively. Distinct phylogenetic clades are numbered I-X. Those selected for study are indicated with a black dot. Cas9s whose structure has been determined are also designated. (B) Biochemical approach used to directly capture target cleavage and assess PAM recognition. Experiments were assembled using Cas9 protein produced by IVT.
    Figure Legend Snippet: Cas9 diversity and characterization approach. (A) Phylogenetic representation of the diversity provided by Cas9 orthologs. Type II-A, B, and C systems are color-coded, red, blue, and green, respectively. Distinct phylogenetic clades are numbered I-X. Those selected for study are indicated with a black dot. Cas9s whose structure has been determined are also designated. (B) Biochemical approach used to directly capture target cleavage and assess PAM recognition. Experiments were assembled using Cas9 protein produced by IVT.

    Techniques Used: Produced

    Cas9 tracrRNA sequence and secondary structure similarity. Circles are scaled based on the number of sequences belonging to each covariance model (CM) and colored according to the designated cluster. The width of the connecting lines indicates the percentage of similarity or relatedness among CMs. Representative tracrRNAs from each cluster are indicated with the associated color. CMs not assigned to a cluster are in grey.
    Figure Legend Snippet: Cas9 tracrRNA sequence and secondary structure similarity. Circles are scaled based on the number of sequences belonging to each covariance model (CM) and colored according to the designated cluster. The width of the connecting lines indicates the percentage of similarity or relatedness among CMs. Representative tracrRNAs from each cluster are indicated with the associated color. CMs not assigned to a cluster are in grey.

    Techniques Used: Sequencing

    2) Product Images from "Morphogenesis is transcriptionally coupled to neurogenesis during peripheral olfactory organ development"

    Article Title: Morphogenesis is transcriptionally coupled to neurogenesis during peripheral olfactory organ development

    Journal: bioRxiv

    doi: 10.1101/725705

    Neurog1 directly controls cxcr4b expression via an upstream Cis-regulatory module (CRM). ( A ) Schematic representation of the cxcr4b locus indicating the position of exons of the cxcr4b gene (orange) and E-box clusters, which are color-coded depending on the nature of the E-box sequences. Also presented is the position of the genomic sequences found in the TgFOS(cxcr4b:eGFP) transgene. ( B ) qPCR analysis of the effect of Neurog1-Ty1mRNA mis-expression on the relative mRNA levels of the known Neurog1 target gene deltaA and cxcr4b . A significant increase in expression is detected for both genes. Shown are mean ± s.e.m, p values are calculated using a two-tailed Student’s t-test, *p=0.01, ***p=0.0001. ( C ) Chromatin immunoprecipitation (ChIP) using an antibody against Ty1 and chromatin prepared from 15 hpf embryos mis-expressing Neurog1-Ty1 mRNA (Grey). Control (Black) represents ChIP with IgG alone. Shown are mean ± s.e.m, p values are calculated using a two-tailed Student’s t-test, n.s. not significant, *p=0.01. ( D ) Single confocal sections of TgFOS(cxcr4b:eGFP) embryos at 24 hpf showing eGFP expression in the olfactory cups, and either HuC/D expression or nuclear labelling (TOPRO). Embryos were injected with an sgRNA pair flanking the E-box containing CRM at the cxcr4b locus plus or minus Cas9 as a control. Insets show HuC/D expression in both conditions.
    Figure Legend Snippet: Neurog1 directly controls cxcr4b expression via an upstream Cis-regulatory module (CRM). ( A ) Schematic representation of the cxcr4b locus indicating the position of exons of the cxcr4b gene (orange) and E-box clusters, which are color-coded depending on the nature of the E-box sequences. Also presented is the position of the genomic sequences found in the TgFOS(cxcr4b:eGFP) transgene. ( B ) qPCR analysis of the effect of Neurog1-Ty1mRNA mis-expression on the relative mRNA levels of the known Neurog1 target gene deltaA and cxcr4b . A significant increase in expression is detected for both genes. Shown are mean ± s.e.m, p values are calculated using a two-tailed Student’s t-test, *p=0.01, ***p=0.0001. ( C ) Chromatin immunoprecipitation (ChIP) using an antibody against Ty1 and chromatin prepared from 15 hpf embryos mis-expressing Neurog1-Ty1 mRNA (Grey). Control (Black) represents ChIP with IgG alone. Shown are mean ± s.e.m, p values are calculated using a two-tailed Student’s t-test, n.s. not significant, *p=0.01. ( D ) Single confocal sections of TgFOS(cxcr4b:eGFP) embryos at 24 hpf showing eGFP expression in the olfactory cups, and either HuC/D expression or nuclear labelling (TOPRO). Embryos were injected with an sgRNA pair flanking the E-box containing CRM at the cxcr4b locus plus or minus Cas9 as a control. Insets show HuC/D expression in both conditions.

    Techniques Used: Expressing, Genomic Sequencing, Real-time Polymerase Chain Reaction, Two Tailed Test, Chromatin Immunoprecipitation, Injection

    3) Product Images from "CRISPR-Cas9 Causes Chromosomal Instability and Rearrangements in Cancer Cell Lines, Detectable by Cytogenetic Methods"

    Article Title: CRISPR-Cas9 Causes Chromosomal Instability and Rearrangements in Cancer Cell Lines, Detectable by Cytogenetic Methods

    Journal: The CRISPR Journal

    doi: 10.1089/crispr.2019.0006

    CRISPR target loci and Sanger traces showing expected mutations. (A) Mutation of single nucleotide polymorphism (SNP) rs1800734 in the MLH1 promoter in COLO320 cells. The top panel shows the genomic location, target sequence (orange rectangle), and screening polymerase chain reaction (PCR) amplicon (green rectangle). The lower panel shows aligned sequence traces of the parental and two CRISPR-Cas9 mutated clones. The box shows the position of the heterozygous (A/G) SNP location and an AA and GG homozygous trace. (B) Reversion of mutation in POLE exon 9 in HCC2998 cells. The top panel shows the genomic location, and two target sequences due to the double-nicking strategy (orange rectangles) and screening PCR amplicon (green rectangle). The lower panel shows aligned sequence traces of the parental and a wild-type revertant clone. The box shows the position of the heterozygous (C to G) mutation location and a homozygous C (wild type) trace. (C) Knockout of NFE2L2 exon 4 in SW1463 cells. The top panel shows the genomic location, target sequence (orange rectangle), and screening PCR amplicon (green rectangle). The lower panel shows aligned sequence traces of the parental and clones with heterozygous deletions obtained from the reverse sequencing primer (right to left). The box shows the position of the guide and PAM sequence, within which the clean trace becomes disrupted due to a deletion.
    Figure Legend Snippet: CRISPR target loci and Sanger traces showing expected mutations. (A) Mutation of single nucleotide polymorphism (SNP) rs1800734 in the MLH1 promoter in COLO320 cells. The top panel shows the genomic location, target sequence (orange rectangle), and screening polymerase chain reaction (PCR) amplicon (green rectangle). The lower panel shows aligned sequence traces of the parental and two CRISPR-Cas9 mutated clones. The box shows the position of the heterozygous (A/G) SNP location and an AA and GG homozygous trace. (B) Reversion of mutation in POLE exon 9 in HCC2998 cells. The top panel shows the genomic location, and two target sequences due to the double-nicking strategy (orange rectangles) and screening PCR amplicon (green rectangle). The lower panel shows aligned sequence traces of the parental and a wild-type revertant clone. The box shows the position of the heterozygous (C to G) mutation location and a homozygous C (wild type) trace. (C) Knockout of NFE2L2 exon 4 in SW1463 cells. The top panel shows the genomic location, target sequence (orange rectangle), and screening PCR amplicon (green rectangle). The lower panel shows aligned sequence traces of the parental and clones with heterozygous deletions obtained from the reverse sequencing primer (right to left). The box shows the position of the guide and PAM sequence, within which the clean trace becomes disrupted due to a deletion.

    Techniques Used: CRISPR, Mutagenesis, Sequencing, Polymerase Chain Reaction, Amplification, Clone Assay, Knock-Out

    CRIPSR-Cas9 mutation of cell lines: Experimental strategy. Schematic showing (A) CRISPR-Cas9 mutation design, (B) cell transfection and selection, and (C) mutation screening by Sanger sequencing and cytogenetic clone analysis
    Figure Legend Snippet: CRIPSR-Cas9 mutation of cell lines: Experimental strategy. Schematic showing (A) CRISPR-Cas9 mutation design, (B) cell transfection and selection, and (C) mutation screening by Sanger sequencing and cytogenetic clone analysis

    Techniques Used: Mutagenesis, CRISPR, Transfection, Selection, Sequencing

    4) Product Images from "Citrullination regulates wound responses and tissue regeneration in zebrafish"

    Article Title: Citrullination regulates wound responses and tissue regeneration in zebrafish

    Journal: bioRxiv

    doi: 10.1101/2019.12.27.889378

    Characterization of zebrafish Padi2. (A) Citrullination activity of bacterially expressed zebrafish Padi2 201a and 202 splice variants in total lysates with and without calcium. Absorbance of light was measured and expressed as mean (± SEM) relative light units (RLU), normalized for protein level. Data represent 3 independent replicates. (B) Citrullination activity of Padi2 201a and individual point mutations in calcium binding and catalytic amino acids. Fold change of enzymatic activity is shown relative to wild-type Padi2 201a. Data represent 2 independent replicates and wild-type values are also represented in A. (C) Schematic of padi2 gene with exon 7 gRNA sequence highlighted for CRISPR/Cas9 mutagenesis. gRNA sequence in blue, PAM site in red. (D) Sequence alignment of wild-type and padi2 −/− 20 bp mutation in exon 7. MwoI restriction site for genotyping highlighted in pink, predicted early stop codon highlighted in red. (E) RT-qPCR of padi2 exon5/6 on individual larvae from a padi2 +/- incross. Data are from three pooled independent replicates with the means and SEM reported and a one-sample t test performed. (F) Representative western blot for zebrafish Padi2 and Actin from pooled larvae (representative of 4 experiments). (G) Citrullination activity of pooled zebrafish lysates expressed as relative light units (RLU). Data are from 3 independent replicates with the means and SEM reported and an ANOVA performed. (H) Citrullination activity of pooled embryo lysates during development. Fold change of enzymatic activity is shown as a ratio of calcium-treated to no calcium for each condition. Data are from 3 independent replicates. (I) Representative western blot for zebrafish Padi2 and Actin from pooled zebrafish through stages of development (representative of 3 and 2 experiments).
    Figure Legend Snippet: Characterization of zebrafish Padi2. (A) Citrullination activity of bacterially expressed zebrafish Padi2 201a and 202 splice variants in total lysates with and without calcium. Absorbance of light was measured and expressed as mean (± SEM) relative light units (RLU), normalized for protein level. Data represent 3 independent replicates. (B) Citrullination activity of Padi2 201a and individual point mutations in calcium binding and catalytic amino acids. Fold change of enzymatic activity is shown relative to wild-type Padi2 201a. Data represent 2 independent replicates and wild-type values are also represented in A. (C) Schematic of padi2 gene with exon 7 gRNA sequence highlighted for CRISPR/Cas9 mutagenesis. gRNA sequence in blue, PAM site in red. (D) Sequence alignment of wild-type and padi2 −/− 20 bp mutation in exon 7. MwoI restriction site for genotyping highlighted in pink, predicted early stop codon highlighted in red. (E) RT-qPCR of padi2 exon5/6 on individual larvae from a padi2 +/- incross. Data are from three pooled independent replicates with the means and SEM reported and a one-sample t test performed. (F) Representative western blot for zebrafish Padi2 and Actin from pooled larvae (representative of 4 experiments). (G) Citrullination activity of pooled zebrafish lysates expressed as relative light units (RLU). Data are from 3 independent replicates with the means and SEM reported and an ANOVA performed. (H) Citrullination activity of pooled embryo lysates during development. Fold change of enzymatic activity is shown as a ratio of calcium-treated to no calcium for each condition. Data are from 3 independent replicates. (I) Representative western blot for zebrafish Padi2 and Actin from pooled zebrafish through stages of development (representative of 3 and 2 experiments).

    Techniques Used: Activity Assay, Binding Assay, Sequencing, CRISPR, Mutagenesis, Quantitative RT-PCR, Western Blot

    5) Product Images from "Genomic Access to Monarch Migration Using TALEN and CRISPR/Cas9-Mediated Targeted Mutagenesis"

    Article Title: Genomic Access to Monarch Migration Using TALEN and CRISPR/Cas9-Mediated Targeted Mutagenesis

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.116.027029

    Heritable CRISPR/Cas9-mediated targeted mutagenesis of monarch butterfly clock . (A) Schematic of part of the monarch clock genomic locus containing the CRISPR/Cas9 target site. The purple region in exon 2 is expanded to provide the sequence targeted for genome editing by a single guide RNA (sgRNA, purple letters). The protospacer adjacent motif (PAM site, 5′-NGG-3′) 3′ of the target sequence is highlighted in gray. Arrows on top and bottom represent the positions of the primers used to amplify the 839 bp ( clock F2, clock R1; Table S1 ) and 756 bp ( clock F2, clock R2; Table S1 ) targeted regions for analysis of mutagenic lesions in C and E, respectively. (B) Diagram of the in vitro cleavage assay used to detect mutagenic lesions showing a PCR amplicon subjected to the sgRNA and the Cas9 protein. Blue and red lines correspond to a wild-type (WT) genomic fragment cleaved by Cas9. Fragments with mutations induced by NHEJ at the site targeted by the sgRNA (red boxes) are uncleaved (black line). (C) Detection of mutagenic lesions at the clock locus in somatic cells of founder G 0 butterflies. For each founder, a PCR fragment was subjected to a Cas9-based in vitro cleavage assay. Blue and red arrows, WT fragments. Black arrow, amplicons carrying mutations (mut) at the site targeted by the sgRNA. Estimation of the frequency of NHEJ-mediated indels is provided under each founder. Black stars, founders selected for crosses to determine germline targeting rates. (D) CRISPR/Cas9-induced mutations in somatic and germline cells of founders 17 and 18. The sgRNA binding site is underlined on the wild-type sequence. Red dashes and red letters, deletion and insertions, respectively. (E) Genotyping of G 1 butterflies from founders 17 and 18 backcrossed to WT using the Cas9 in vitro cleavage assay. Heterozygote butterflies carrying the mutated allele are robustly discriminated by the presence of an additional uncleaved PCR fragment. CRISPR, clustered regularly interspaced short palindromic repeats; NHEJ, nonhomologous end-joining; PCR, polymerase chain reaction.
    Figure Legend Snippet: Heritable CRISPR/Cas9-mediated targeted mutagenesis of monarch butterfly clock . (A) Schematic of part of the monarch clock genomic locus containing the CRISPR/Cas9 target site. The purple region in exon 2 is expanded to provide the sequence targeted for genome editing by a single guide RNA (sgRNA, purple letters). The protospacer adjacent motif (PAM site, 5′-NGG-3′) 3′ of the target sequence is highlighted in gray. Arrows on top and bottom represent the positions of the primers used to amplify the 839 bp ( clock F2, clock R1; Table S1 ) and 756 bp ( clock F2, clock R2; Table S1 ) targeted regions for analysis of mutagenic lesions in C and E, respectively. (B) Diagram of the in vitro cleavage assay used to detect mutagenic lesions showing a PCR amplicon subjected to the sgRNA and the Cas9 protein. Blue and red lines correspond to a wild-type (WT) genomic fragment cleaved by Cas9. Fragments with mutations induced by NHEJ at the site targeted by the sgRNA (red boxes) are uncleaved (black line). (C) Detection of mutagenic lesions at the clock locus in somatic cells of founder G 0 butterflies. For each founder, a PCR fragment was subjected to a Cas9-based in vitro cleavage assay. Blue and red arrows, WT fragments. Black arrow, amplicons carrying mutations (mut) at the site targeted by the sgRNA. Estimation of the frequency of NHEJ-mediated indels is provided under each founder. Black stars, founders selected for crosses to determine germline targeting rates. (D) CRISPR/Cas9-induced mutations in somatic and germline cells of founders 17 and 18. The sgRNA binding site is underlined on the wild-type sequence. Red dashes and red letters, deletion and insertions, respectively. (E) Genotyping of G 1 butterflies from founders 17 and 18 backcrossed to WT using the Cas9 in vitro cleavage assay. Heterozygote butterflies carrying the mutated allele are robustly discriminated by the presence of an additional uncleaved PCR fragment. CRISPR, clustered regularly interspaced short palindromic repeats; NHEJ, nonhomologous end-joining; PCR, polymerase chain reaction.

    Techniques Used: CRISPR, Mutagenesis, Sequencing, In Vitro, Cleavage Assay, Polymerase Chain Reaction, Amplification, Non-Homologous End Joining, Binding Assay

    Highly efficient CRISPR/Cas9-mediated targeted mutations and deletions of monarch butterfly cry2 using dual sgRNAs. (A) Schematic of part of the monarch cry2 genomic locus containing the two CRISPR/Cas9 target sites. The orange and green regions in exons 2 and 3 are expanded to provide the sequences targeted for genome editing by each single guide RNA (sgRNA). Protospacer adjacent motifs (PAM site, 5′-NGG-3′) are highlighted in gray. Arrows on top represent the positions of the primers used to amplify the 1403 bp targeted region for analysis of mutagenic lesions in B ( cry2 F2, cry2 R2 in Table S1 ). (B) Detection of mutagenic lesions at the cry2 loci targeted by sgRNA1 (left) and sgRNA2 (right) in somatic cells of founder G 0 butterflies (9 out of 13 are shown). For each founder, a PCR fragment was subjected to a Cas9-based in vitro cleavage assay with each of the sgRNAs. Black arrowhead, amplicons carrying mutations (mut). Estimation of the frequency of NHEJ-mediated indels is provided under each founder. Red arrowhead, amplicons carrying genomic deletions (del) at the sites targeted by the sgRNAs. Blue and gray arrows, cleaved wild-type (WT) PCR fragments. Black stars, founders selected for crosses to determine germline targeting rates. (C) Left, CRISPR/Cas9-induced mutations in somatic and germline cells with sgRNA1. Guide RNA binding site is underlined on the wild-type sequence. Red dashes and red letters, deletion and insertions, respectively. Right, Dual sgRNA-mediated genomic deletion in somatic cells. Positions of sgRNA1 and sgRNA2 are underlined on the sequence and represented by colored boxes on a schematic representation. CRISPR, clustered regularly interspaced short palindromic repeats; NHEJ, nonhomologous end-joining; PCR, polymerase chain reaction.
    Figure Legend Snippet: Highly efficient CRISPR/Cas9-mediated targeted mutations and deletions of monarch butterfly cry2 using dual sgRNAs. (A) Schematic of part of the monarch cry2 genomic locus containing the two CRISPR/Cas9 target sites. The orange and green regions in exons 2 and 3 are expanded to provide the sequences targeted for genome editing by each single guide RNA (sgRNA). Protospacer adjacent motifs (PAM site, 5′-NGG-3′) are highlighted in gray. Arrows on top represent the positions of the primers used to amplify the 1403 bp targeted region for analysis of mutagenic lesions in B ( cry2 F2, cry2 R2 in Table S1 ). (B) Detection of mutagenic lesions at the cry2 loci targeted by sgRNA1 (left) and sgRNA2 (right) in somatic cells of founder G 0 butterflies (9 out of 13 are shown). For each founder, a PCR fragment was subjected to a Cas9-based in vitro cleavage assay with each of the sgRNAs. Black arrowhead, amplicons carrying mutations (mut). Estimation of the frequency of NHEJ-mediated indels is provided under each founder. Red arrowhead, amplicons carrying genomic deletions (del) at the sites targeted by the sgRNAs. Blue and gray arrows, cleaved wild-type (WT) PCR fragments. Black stars, founders selected for crosses to determine germline targeting rates. (C) Left, CRISPR/Cas9-induced mutations in somatic and germline cells with sgRNA1. Guide RNA binding site is underlined on the wild-type sequence. Red dashes and red letters, deletion and insertions, respectively. Right, Dual sgRNA-mediated genomic deletion in somatic cells. Positions of sgRNA1 and sgRNA2 are underlined on the sequence and represented by colored boxes on a schematic representation. CRISPR, clustered regularly interspaced short palindromic repeats; NHEJ, nonhomologous end-joining; PCR, polymerase chain reaction.

    Techniques Used: CRISPR, Polymerase Chain Reaction, In Vitro, Cleavage Assay, Non-Homologous End Joining, RNA Binding Assay, Sequencing

    6) Product Images from "Lamb1a regulates atrial growth by limiting excessive, contractility-dependent second heart field addition during zebrafish heart development"

    Article Title: Lamb1a regulates atrial growth by limiting excessive, contractility-dependent second heart field addition during zebrafish heart development

    Journal: bioRxiv

    doi: 10.1101/2021.03.10.434727

    Laminins perform multiple roles during zebrafish heart morphogenesis A-D: mRNA in situ hybridization analysis of myl7 expression in control embryos injected with lamc1 -targeting gRNAs only (A,C) or with lamc1 -targeting gRNAs together with Cas9 protein ( Lamc1 F0 , B-D) at 55hpf and 72hpf. E-H: Quantitative analysis of looping ratio (E, G) and myl7 area (F,H) in gRNA-injected controls (55hpf: n=44; 72hpf: n=44) and lamc1 F0 crispants (55hpf: n=47; 72hpf: n=44). lamc1 crispants exhibit reduced heart looping at 55hpf and 72hpf, and an increased area of myl7 expression at 72hpf. Horizontal bars indicate median with interquartile range, comparative statistics performed using Kruskal-Wallis test. I-L: mRNA in situ hybridization analysis of myl7 expression in sibling (I,K) and lamb1a Δ25 mutants (J,L) at 55hpf and 72hpf. M-P: Quantitative analysis of looping ratio (M,O) and myl7 area (N,P) in sibling (55hpf: n=70; 72hpf: n=56) and lamb1a Δ25 mutant embryos (55hpf: n=25; 72hpf: n=34). lamb1a Δ25 mutants exhibit a mild reduction in heart looping at 55hpf, and an increased area of myl7 expression at 55hpf and 72hpf. Scale bars: 50μm. Comparative statistics performed using Mann Whitney test, **** = p
    Figure Legend Snippet: Laminins perform multiple roles during zebrafish heart morphogenesis A-D: mRNA in situ hybridization analysis of myl7 expression in control embryos injected with lamc1 -targeting gRNAs only (A,C) or with lamc1 -targeting gRNAs together with Cas9 protein ( Lamc1 F0 , B-D) at 55hpf and 72hpf. E-H: Quantitative analysis of looping ratio (E, G) and myl7 area (F,H) in gRNA-injected controls (55hpf: n=44; 72hpf: n=44) and lamc1 F0 crispants (55hpf: n=47; 72hpf: n=44). lamc1 crispants exhibit reduced heart looping at 55hpf and 72hpf, and an increased area of myl7 expression at 72hpf. Horizontal bars indicate median with interquartile range, comparative statistics performed using Kruskal-Wallis test. I-L: mRNA in situ hybridization analysis of myl7 expression in sibling (I,K) and lamb1a Δ25 mutants (J,L) at 55hpf and 72hpf. M-P: Quantitative analysis of looping ratio (M,O) and myl7 area (N,P) in sibling (55hpf: n=70; 72hpf: n=56) and lamb1a Δ25 mutant embryos (55hpf: n=25; 72hpf: n=34). lamb1a Δ25 mutants exhibit a mild reduction in heart looping at 55hpf, and an increased area of myl7 expression at 55hpf and 72hpf. Scale bars: 50μm. Comparative statistics performed using Mann Whitney test, **** = p

    Techniques Used: In Situ Hybridization, Expressing, Injection, Mutagenesis, MANN-WHITNEY

    7) Product Images from "Disruption of PD-1 Enhanced the Anti-tumor Activity of Chimeric Antigen Receptor T Cells Against Hepatocellular Carcinoma"

    Article Title: Disruption of PD-1 Enhanced the Anti-tumor Activity of Chimeric Antigen Receptor T Cells Against Hepatocellular Carcinoma

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.01118

    CRISPR/Cas9 efficiently disrupted the gene expressing PD-1 in GPC3-CAR T cells. (A) Indels observed by clonal sequence analysis of PCR amplicons from the CRISPR-edited region in the gene expressing PD-1. Blue base or dot in the clonal sequences indicated insertion or deletion base, respectively. The number prefixed a “+” or “–” character in the bracket before a clonal sequence indicated the number of insertions or deletions in the corresponding clonal sequence, respectively. The number prefixed with a “×” character in the bracket before a clonal sequence indicated the number of the corresponding indels profile in the sixty clonal amplicons. Arrows indicated the putative cleavage sites. (B,C) The chromatograms from the Sanger sequencing of the PCR amplicon spanning the PD-1 CRISPR gRNAs [PD-1-gRNA-1 (B) and PD-1-gRNA-2 (C)] target sites within the exon 1 of the gene expressing PD-1. (D) Detection of the CRISPR-mediated disruption of PD-1 by a mismatch-selective T7EN1 nuclease assay on the DNA (spanning the gRNAs target sites) amplified from the genomic DNA of the cells shown.
    Figure Legend Snippet: CRISPR/Cas9 efficiently disrupted the gene expressing PD-1 in GPC3-CAR T cells. (A) Indels observed by clonal sequence analysis of PCR amplicons from the CRISPR-edited region in the gene expressing PD-1. Blue base or dot in the clonal sequences indicated insertion or deletion base, respectively. The number prefixed a “+” or “–” character in the bracket before a clonal sequence indicated the number of insertions or deletions in the corresponding clonal sequence, respectively. The number prefixed with a “×” character in the bracket before a clonal sequence indicated the number of the corresponding indels profile in the sixty clonal amplicons. Arrows indicated the putative cleavage sites. (B,C) The chromatograms from the Sanger sequencing of the PCR amplicon spanning the PD-1 CRISPR gRNAs [PD-1-gRNA-1 (B) and PD-1-gRNA-2 (C)] target sites within the exon 1 of the gene expressing PD-1. (D) Detection of the CRISPR-mediated disruption of PD-1 by a mismatch-selective T7EN1 nuclease assay on the DNA (spanning the gRNAs target sites) amplified from the genomic DNA of the cells shown.

    Techniques Used: CRISPR, Expressing, Sequencing, Polymerase Chain Reaction, Amplification, Nuclease Assay

    8) Product Images from "CRISPR Repair Reveals Causative Mutation in a Preclinical Model of Retinitis Pigmentosa"

    Article Title: CRISPR Repair Reveals Causative Mutation in a Preclinical Model of Retinitis Pigmentosa

    Journal: Molecular Therapy

    doi: 10.1038/mt.2016.107

    CRISPR/Cas9-mediated repair of Y347X was achieved in a mosaic fashion in 2 of 11 Founders (F 0 ) . ( a ) sgRNA targets exon 7 of the Pde6b locus (yellow box, top). Donor template (blue box, middle) encoding the wild-type allele corrects Y347X through homology-directed
    Figure Legend Snippet: CRISPR/Cas9-mediated repair of Y347X was achieved in a mosaic fashion in 2 of 11 Founders (F 0 ) . ( a ) sgRNA targets exon 7 of the Pde6b locus (yellow box, top). Donor template (blue box, middle) encoding the wild-type allele corrects Y347X through homology-directed

    Techniques Used: CRISPR

    9) Product Images from "In Vitro CRISPR/Cas9 System for Efficient Targeted DNA Editing"

    Article Title: In Vitro CRISPR/Cas9 System for Efficient Targeted DNA Editing

    Journal: mBio

    doi: 10.1128/mBio.01714-15

    Targeted insertion of bla gene in plasmid pYH285 by ICE system. (A) Schematic representation of the target site in pYH285 plasmid. The target region is shown as the top panel, in which the Cas9 cleavage site is indicated by a red triangle and the PAM sequence is highlighted in red. (B) DNA sequencing confirmation of bla insertion in a recombinant plasmid.
    Figure Legend Snippet: Targeted insertion of bla gene in plasmid pYH285 by ICE system. (A) Schematic representation of the target site in pYH285 plasmid. The target region is shown as the top panel, in which the Cas9 cleavage site is indicated by a red triangle and the PAM sequence is highlighted in red. (B) DNA sequencing confirmation of bla insertion in a recombinant plasmid.

    Techniques Used: Plasmid Preparation, Sequencing, DNA Sequencing, Recombinant

    Flowchart of targeted gene deletion of biosynthetic gene cluster using ICE system. The target gene (in pink) flanking by protospacers is cleaved 3 bp upstream of PAM by the Cas9-sgRNA complex. After phenol-chloroform extraction and ethanol precipitation, the linearized DNA fragments mixture is subsequently end blunted, self-ligated, and then introduced into E. coli . The desired colonies will be screened out from the pool of recirculation plasmids by PCR and subsequently confirmed by restriction mapping.
    Figure Legend Snippet: Flowchart of targeted gene deletion of biosynthetic gene cluster using ICE system. The target gene (in pink) flanking by protospacers is cleaved 3 bp upstream of PAM by the Cas9-sgRNA complex. After phenol-chloroform extraction and ethanol precipitation, the linearized DNA fragments mixture is subsequently end blunted, self-ligated, and then introduced into E. coli . The desired colonies will be screened out from the pool of recirculation plasmids by PCR and subsequently confirmed by restriction mapping.

    Techniques Used: Ethanol Precipitation, Polymerase Chain Reaction

    Recombination of a Cas9-created DNA fragment with and without end repair. (A) Schematic representation of four selected protospacers in the pUC18 plasmid. The transcribed sgRNAs matched the specific protospacers are shown in orange. PAM sequences are highlighted in pink, and “NGG” is underlined. The vertical blue lines between nucleotides indicate the Cas9-mediated DSB sites matched with specific protospacers. The 3′→5′ exonuclease trimming is shown with horizontal arrows, and the sequential trimming occurred in the noncomplementary strand is shown in gradient gray. ori , origin of replication of plasmid pUC18; bla , ampicillin resistance gene; lacZ , β-galactosidase gene. (B to E) End-joint sequencing results from cleavage of Cas9-sgRNAS3 (B), Cas9-sgRNAS1/Cas9-sgRNAS4 (C), Cas9-sgRNAS2/Cas9-sgRNAS4 (D), and Cas9-sgRNAS2/Cas9-sgRNAS3 (E) without end repair. For each combination of Cas9-sgRNA complex, the desired sequence is shown at the top, with the PAM sequence highlighted in pink and the joint interface indicated with a vertical blue line. Deletions are shown as a pink dashed line. The net change in length caused by exonuclease trimming is shown to the right of each sequence (−, deletion). (F to I) End-joint sequencing results from cleavage of Cas9-sgRNAS3 (F), Cas9-sgRNAS1/Cas9-sgRNAS4 (G), Cas9-sgRNAS2/Cas9-sgRNAS4 (H), and Cas9-sgRNAS2/Cas9-sgRNAS3 (I) with an additional end-repairing procedure that is parallel to the left column, respectively.
    Figure Legend Snippet: Recombination of a Cas9-created DNA fragment with and without end repair. (A) Schematic representation of four selected protospacers in the pUC18 plasmid. The transcribed sgRNAs matched the specific protospacers are shown in orange. PAM sequences are highlighted in pink, and “NGG” is underlined. The vertical blue lines between nucleotides indicate the Cas9-mediated DSB sites matched with specific protospacers. The 3′→5′ exonuclease trimming is shown with horizontal arrows, and the sequential trimming occurred in the noncomplementary strand is shown in gradient gray. ori , origin of replication of plasmid pUC18; bla , ampicillin resistance gene; lacZ , β-galactosidase gene. (B to E) End-joint sequencing results from cleavage of Cas9-sgRNAS3 (B), Cas9-sgRNAS1/Cas9-sgRNAS4 (C), Cas9-sgRNAS2/Cas9-sgRNAS4 (D), and Cas9-sgRNAS2/Cas9-sgRNAS3 (E) without end repair. For each combination of Cas9-sgRNA complex, the desired sequence is shown at the top, with the PAM sequence highlighted in pink and the joint interface indicated with a vertical blue line. Deletions are shown as a pink dashed line. The net change in length caused by exonuclease trimming is shown to the right of each sequence (−, deletion). (F to I) End-joint sequencing results from cleavage of Cas9-sgRNAS3 (F), Cas9-sgRNAS1/Cas9-sgRNAS4 (G), Cas9-sgRNAS2/Cas9-sgRNAS4 (H), and Cas9-sgRNAS2/Cas9-sgRNAS3 (I) with an additional end-repairing procedure that is parallel to the left column, respectively.

    Techniques Used: Plasmid Preparation, Sequencing

    10) Product Images from "Biochemically diverse CRISPR-Cas9 orthologs"

    Article Title: Biochemically diverse CRISPR-Cas9 orthologs

    Journal: bioRxiv

    doi: 10.1101/2020.04.29.066654

    Target DNA cleavage patterns produced by Cas9 orthologs. Cleavage sites and resultant dsDNA ends are depicted as heatmaps that show the proportion of cleaved ends recovered by DNA sequencing at each position of a target DNA. Intensity of the blue color indicates the proportion of mapped cleavage ends. (A) Control digests using restriction enzymes recover 5’-overhangs, 3’-overhangs, and blunt ends. TS indicates top strand; BS indicates bottom strand. (B) SpyCas9 and SauCas9 cleaved DNA ends. Heatmaps represent mapped cleavage ends as the averages at each position in 5 different dsDNA targets. Position of the DNA bases and PAM sequences is depicted above the heatmaps. NTS indicates non-target strand; TS indicates target strand. (C) Blunt and staggered-end cleavage. Examples of blunt, one base 5’-overhang staggered cleavage, and multiple base 5’-overhang cleavage are depicted as heatmaps that show the proportion of cleaved ends as the averages at each position in 5 different dsDNA targets. Position of the DNA bases and PAM sequences is depicted above the heatmaps. NTS indicates non-target strand; TS indicates target strand.
    Figure Legend Snippet: Target DNA cleavage patterns produced by Cas9 orthologs. Cleavage sites and resultant dsDNA ends are depicted as heatmaps that show the proportion of cleaved ends recovered by DNA sequencing at each position of a target DNA. Intensity of the blue color indicates the proportion of mapped cleavage ends. (A) Control digests using restriction enzymes recover 5’-overhangs, 3’-overhangs, and blunt ends. TS indicates top strand; BS indicates bottom strand. (B) SpyCas9 and SauCas9 cleaved DNA ends. Heatmaps represent mapped cleavage ends as the averages at each position in 5 different dsDNA targets. Position of the DNA bases and PAM sequences is depicted above the heatmaps. NTS indicates non-target strand; TS indicates target strand. (C) Blunt and staggered-end cleavage. Examples of blunt, one base 5’-overhang staggered cleavage, and multiple base 5’-overhang cleavage are depicted as heatmaps that show the proportion of cleaved ends as the averages at each position in 5 different dsDNA targets. Position of the DNA bases and PAM sequences is depicted above the heatmaps. NTS indicates non-target strand; TS indicates target strand.

    Techniques Used: Produced, DNA Sequencing

    Activity of Cas9 orthologs at varying temperatures. The cleavage activity of Cas9 orthologs was measured using in vitro DNA cleavage assays using fluorophore-labeled dsDNA substrates. Cleaved fragments were quantitated and are represented in a heat map (A) showing overall activity at temperatures ranging from 10°C to 68°C. (B) Cas9 orthologs with activity at elevated temperatures. In vitro DNA cleavage activity for a subset of Cas9 orthologs with > 50% activity at 53°C is summarized in a heat map and plotted as proportion of DNA substrate cleaved at varied temperature. Points represent the mean +/- SEM of at least 3 independent experiments. (C) Cas9 orthologs with reduced activity at room temperature. In vitro DNA cleavage activity for a subset of Cas9 orthologs with
    Figure Legend Snippet: Activity of Cas9 orthologs at varying temperatures. The cleavage activity of Cas9 orthologs was measured using in vitro DNA cleavage assays using fluorophore-labeled dsDNA substrates. Cleaved fragments were quantitated and are represented in a heat map (A) showing overall activity at temperatures ranging from 10°C to 68°C. (B) Cas9 orthologs with activity at elevated temperatures. In vitro DNA cleavage activity for a subset of Cas9 orthologs with > 50% activity at 53°C is summarized in a heat map and plotted as proportion of DNA substrate cleaved at varied temperature. Points represent the mean +/- SEM of at least 3 independent experiments. (C) Cas9 orthologs with reduced activity at room temperature. In vitro DNA cleavage activity for a subset of Cas9 orthologs with

    Techniques Used: Activity Assay, In Vitro, Labeling

    Cas9 PAM Interacting (PI) domain similarity. Cas9 PI domains clustered by their pairwise sequence similarity. Lines connect sequences with P-value ≤ 1e-11. Line shading corresponds to P-values according to the scale in the top-right corner (light and long lines connect distantly related sequences). Major clusters are shown in bold. Cluster 1 was so named to emphasize that it contains first experimentally characterized Cas9. Clusters 2 to 10 were named beginning from the one with the most members. Sequences having known structures are marked red, their PDB code is shown in parentheses.
    Figure Legend Snippet: Cas9 PAM Interacting (PI) domain similarity. Cas9 PI domains clustered by their pairwise sequence similarity. Lines connect sequences with P-value ≤ 1e-11. Line shading corresponds to P-values according to the scale in the top-right corner (light and long lines connect distantly related sequences). Major clusters are shown in bold. Cluster 1 was so named to emphasize that it contains first experimentally characterized Cas9. Clusters 2 to 10 were named beginning from the one with the most members. Sequences having known structures are marked red, their PDB code is shown in parentheses.

    Techniques Used: Sequencing

    Cas9 diversity and characterization approach. (A) Phylogenetic representation of the diversity provided by Cas9 orthologs. Type II-A, B, and C systems are color-coded, red, blue, and green, respectively. Distinct phylogenetic clades are numbered I-X. Those selected for study are indicated with a black dot. Cas9s whose structure has been determined are also designated. (B) Biochemical approach used to directly capture target cleavage and assess PAM recognition. Experiments were assembled using Cas9 protein produced by IVT.
    Figure Legend Snippet: Cas9 diversity and characterization approach. (A) Phylogenetic representation of the diversity provided by Cas9 orthologs. Type II-A, B, and C systems are color-coded, red, blue, and green, respectively. Distinct phylogenetic clades are numbered I-X. Those selected for study are indicated with a black dot. Cas9s whose structure has been determined are also designated. (B) Biochemical approach used to directly capture target cleavage and assess PAM recognition. Experiments were assembled using Cas9 protein produced by IVT.

    Techniques Used: Produced

    Cas9 tracrRNA sequence and secondary structure similarity. Circles are scaled based on the number of sequences belonging to each covariance model (CM) and colored according to the designated cluster. The width of the connecting lines indicates the percentage of similarity or relatedness among CMs. Representative tracrRNAs from each cluster are indicated with the associated color. CMs not assigned to a cluster are in grey.
    Figure Legend Snippet: Cas9 tracrRNA sequence and secondary structure similarity. Circles are scaled based on the number of sequences belonging to each covariance model (CM) and colored according to the designated cluster. The width of the connecting lines indicates the percentage of similarity or relatedness among CMs. Representative tracrRNAs from each cluster are indicated with the associated color. CMs not assigned to a cluster are in grey.

    Techniques Used: Sequencing

    11) Product Images from "Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly"

    Article Title: Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly

    Journal: Genome Medicine

    doi: 10.1186/s13073-021-00850-w

    Targeted CRISPR/Cas9 disruption of copb1 exon 8 generates a range of insertion-deletion changes in vivo. Xenopus tropicalis copb1 has 21 exons, including an untranslated first exon ( a ). CRISPR/Cas9 directed indel formation disrupting X. tropicalis exon 8 using 2 sgRNAs (red: sgRNA3 and sgRNA4 ( b )) results in homozygous frameshift and splice changes akin to those identified within the patient subpopulation. Genotyping analysis of X. tropicalis crispants details the range of indels across three groups of 10 pooled tadpoles (NF41) injected with sgRNA3 following PCR amplification and Sanger sequencing of subclones ( c ). The 41-bp deletion observed in gDNA subclones extends into the intronic region and is proposed to induce exon 8 skipping in crispant X. tropicalis tadpoles. Total RNA was obtained from 4 groups (un-injected control (1), copb1 sgRNA 3 crispant set 1 (2) and copb1 sgRNA 3 crispant set 2 (3), injected control (4)) of 10 pooled individuals as outlined in Guille 1999 [ 51 ] and cDNA synthesised using Primer Design’s Reverse Transcription Premix 2. Amplification across the copb1 region of interest revealed a band at 382 bp (exons: 7, 8 and 9) and an additional band in crispants at 262 bp (exons: 7 and 9, Additional file 1 : Fig. S1D)
    Figure Legend Snippet: Targeted CRISPR/Cas9 disruption of copb1 exon 8 generates a range of insertion-deletion changes in vivo. Xenopus tropicalis copb1 has 21 exons, including an untranslated first exon ( a ). CRISPR/Cas9 directed indel formation disrupting X. tropicalis exon 8 using 2 sgRNAs (red: sgRNA3 and sgRNA4 ( b )) results in homozygous frameshift and splice changes akin to those identified within the patient subpopulation. Genotyping analysis of X. tropicalis crispants details the range of indels across three groups of 10 pooled tadpoles (NF41) injected with sgRNA3 following PCR amplification and Sanger sequencing of subclones ( c ). The 41-bp deletion observed in gDNA subclones extends into the intronic region and is proposed to induce exon 8 skipping in crispant X. tropicalis tadpoles. Total RNA was obtained from 4 groups (un-injected control (1), copb1 sgRNA 3 crispant set 1 (2) and copb1 sgRNA 3 crispant set 2 (3), injected control (4)) of 10 pooled individuals as outlined in Guille 1999 [ 51 ] and cDNA synthesised using Primer Design’s Reverse Transcription Premix 2. Amplification across the copb1 region of interest revealed a band at 382 bp (exons: 7, 8 and 9) and an additional band in crispants at 262 bp (exons: 7 and 9, Additional file 1 : Fig. S1D)

    Techniques Used: CRISPR, In Vivo, Injection, Polymerase Chain Reaction, Amplification, Sequencing

    12) Product Images from "Nicotinic Acetylcholine Receptor Subtype Alpha-9 Mediates Triple-Negative Breast Cancers Based on a Spontaneous Pulmonary Metastasis Mouse Model"

    Article Title: Nicotinic Acetylcholine Receptor Subtype Alpha-9 Mediates Triple-Negative Breast Cancers Based on a Spontaneous Pulmonary Metastasis Mouse Model

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2017.00336

    Genome editing assay based on α9-nAChR gene sequencing. (A) Schematic representation of the α9-nAChR DNA locus and two protospacer sequences (blue underline) for editing. The arrowhead indicates the expected Cas9 cleavage site. The protospacer adjacent motif (PAM, red underline) is the motif required for Cas9 nuclease activity. Sanger sequencing of the α9-nAChR DNA region was performed in 2LM cells. Scrambled SC sgRNA and α9-nAChR gene editing were accomplished by lentiviral delivery with a multiplicity of infection (MOI) = 5. Post transfection, DNA from virus-infected cells was purified and Sanger-sequenced for the α9-nAChR exon2 DNA locus, and 2LM cells expressing SC sgRNA were used as the control. (B,C) had wild-type sequences, while cells expressing (D) α9-nAChR sgRNA_1 and (E) α9-nAChR sgRNA_2 showed a mixture of sequences around the expected Cas9 cleavage point. TIDE decomposition algorithm analysis of the α9-nAChR gene-edited sequence (indel –insertion and deletion) in 2LM cells showed high editing efficiency at the expected cleavage point by (F) α9-nAChR sgRNA_1 and (G) α9-nAChR sgRNA_2 delivered via lentivirus. The commonly sequenced variants for CRISPR-targeted α9-nAChR transgenes in 2LM cells are shown. The pie charts show the indel percentages of the α9-nAChR gene edited by (H) α9-nAChR sgRNA_1 and (I) α9-nAChR sgRNA_2. The gene editing efficiency of the two sgRNAs are presented in green (left panel), while –1 and +1 indels are presented in orange and gray, respectively. The panels illustrate the aberrant sequence signal in the scrambled (black), (J) α9-nAChR_sgRNA1, and (K) α9-nAChR sgRNA_2 (green) cell pools and the expected cleavage site (vertical dotted line). Several α9-nAChR sgRNA_1- and α9-nAChR sgRNA_2-null MDA-MB-231 cell clones were isolated and expanded in culture. The selected cell lysates from each α9-nAChR sgRNA_1 null clone were assessed for (L) α9-nAChR protein expression by western blotting and (M) α9-nAChR gene editing by the RGEN-RFLP assay.
    Figure Legend Snippet: Genome editing assay based on α9-nAChR gene sequencing. (A) Schematic representation of the α9-nAChR DNA locus and two protospacer sequences (blue underline) for editing. The arrowhead indicates the expected Cas9 cleavage site. The protospacer adjacent motif (PAM, red underline) is the motif required for Cas9 nuclease activity. Sanger sequencing of the α9-nAChR DNA region was performed in 2LM cells. Scrambled SC sgRNA and α9-nAChR gene editing were accomplished by lentiviral delivery with a multiplicity of infection (MOI) = 5. Post transfection, DNA from virus-infected cells was purified and Sanger-sequenced for the α9-nAChR exon2 DNA locus, and 2LM cells expressing SC sgRNA were used as the control. (B,C) had wild-type sequences, while cells expressing (D) α9-nAChR sgRNA_1 and (E) α9-nAChR sgRNA_2 showed a mixture of sequences around the expected Cas9 cleavage point. TIDE decomposition algorithm analysis of the α9-nAChR gene-edited sequence (indel –insertion and deletion) in 2LM cells showed high editing efficiency at the expected cleavage point by (F) α9-nAChR sgRNA_1 and (G) α9-nAChR sgRNA_2 delivered via lentivirus. The commonly sequenced variants for CRISPR-targeted α9-nAChR transgenes in 2LM cells are shown. The pie charts show the indel percentages of the α9-nAChR gene edited by (H) α9-nAChR sgRNA_1 and (I) α9-nAChR sgRNA_2. The gene editing efficiency of the two sgRNAs are presented in green (left panel), while –1 and +1 indels are presented in orange and gray, respectively. The panels illustrate the aberrant sequence signal in the scrambled (black), (J) α9-nAChR_sgRNA1, and (K) α9-nAChR sgRNA_2 (green) cell pools and the expected cleavage site (vertical dotted line). Several α9-nAChR sgRNA_1- and α9-nAChR sgRNA_2-null MDA-MB-231 cell clones were isolated and expanded in culture. The selected cell lysates from each α9-nAChR sgRNA_1 null clone were assessed for (L) α9-nAChR protein expression by western blotting and (M) α9-nAChR gene editing by the RGEN-RFLP assay.

    Techniques Used: Sequencing, Activity Assay, Infection, Transfection, Purification, Expressing, CRISPR, Multiple Displacement Amplification, Clone Assay, Isolation, Western Blot, RFLP Assay

    13) Product Images from "High-Efficiency CRISPR/Cas9 Mutagenesis of the white Gene in the Milkweed Bug Oncopeltus fasciatus"

    Article Title: High-Efficiency CRISPR/Cas9 Mutagenesis of the white Gene in the Milkweed Bug Oncopeltus fasciatus

    Journal: Genetics

    doi: 10.1534/genetics.120.303269

    High rates of somatic mutation were observed after Of-w CRISPR mutagenesis. Examples of first-instar nymph hatchlings at varying stages (A–D) or adults (E–I) after injection of embryos with buffer or gRNAs and Cas9, are shown. (A) Nymph post-melanization, control injection with buffer; (B) nymph mid-melanization, gRNA-B; large translucent patches can be seen in the abdomen; and (C) nymph premelanization, gRNA-B; mosaic translucent patches are obvious in the right half of the head and patches in the thorax, abdomen, and appendages. (D) Nymph pre-melanization, gRNA-A; translucent regions can be seen throughout the body; (E) adult compound eye, wild-type; (F) adult compound eye, gRNA-A, individual A-14; and (G) adult compound eye, gRNA-A, individual A-13. The black pigmentation seen in wild-type is absent from sections of the eye in these two individuals (F and G). (H) Wild-type pigmentation in adult body, ventral view. (I) Mosaic loss of pigmentation in lateral patch (dotted white outline) in adult body, gRNA-A, individual A-13, ventral view. CRISPR, clustered regularly interspaced short palindromic repeats; gRNA, guide RNA.
    Figure Legend Snippet: High rates of somatic mutation were observed after Of-w CRISPR mutagenesis. Examples of first-instar nymph hatchlings at varying stages (A–D) or adults (E–I) after injection of embryos with buffer or gRNAs and Cas9, are shown. (A) Nymph post-melanization, control injection with buffer; (B) nymph mid-melanization, gRNA-B; large translucent patches can be seen in the abdomen; and (C) nymph premelanization, gRNA-B; mosaic translucent patches are obvious in the right half of the head and patches in the thorax, abdomen, and appendages. (D) Nymph pre-melanization, gRNA-A; translucent regions can be seen throughout the body; (E) adult compound eye, wild-type; (F) adult compound eye, gRNA-A, individual A-14; and (G) adult compound eye, gRNA-A, individual A-13. The black pigmentation seen in wild-type is absent from sections of the eye in these two individuals (F and G). (H) Wild-type pigmentation in adult body, ventral view. (I) Mosaic loss of pigmentation in lateral patch (dotted white outline) in adult body, gRNA-A, individual A-13, ventral view. CRISPR, clustered regularly interspaced short palindromic repeats; gRNA, guide RNA.

    Techniques Used: Mutagenesis, CRISPR, Injection

    Somatic mosaicism and hatch rate varied after CRISPR/Cas9 embryonic injection with different guide RNAs. (A) Hatch rate of embryos injected with gRNAs (A–C) compared to those injected with buffer. (B) Frequency of mosaic pigmentation phenotype in G0s. Embryos injected with gRNAs A or B clearly showed much higher rates of mosaicism than embryos injected with gRNA-C. CRISPR, clustered regularly interspaced short palindromic repeats; gRNA, guide RNA; WT, wild-type.
    Figure Legend Snippet: Somatic mosaicism and hatch rate varied after CRISPR/Cas9 embryonic injection with different guide RNAs. (A) Hatch rate of embryos injected with gRNAs (A–C) compared to those injected with buffer. (B) Frequency of mosaic pigmentation phenotype in G0s. Embryos injected with gRNAs A or B clearly showed much higher rates of mosaicism than embryos injected with gRNA-C. CRISPR, clustered regularly interspaced short palindromic repeats; gRNA, guide RNA; WT, wild-type.

    Techniques Used: CRISPR, Injection

    Oncopeltus CRISPR/Cas9 mutagenesis workflow. (A) (i) Genomic structure of Of-w ; exon boundaries are based on transcriptome data and the alignment of a cDNA sequenced in this study to the genome; however, exon 4 was not found in the genome. The dsRNA target region (519 bp) used in this study is shown as an orange bar (exons only). (ii) The target location gRNAs (A–C) used in this study are in red. (iii) The primers used to PCR-amplify exon 2 for our two genotyping assays are shown as black arrows. The amplicon is 513 bp and includes some intronic regions surrounding the exon. Stars mark the predicted loci of Cas9 cleavage and thus likely mutation; the predicted Surveyor cleavage product sizes (in base pairs) for each gRNA are shown above the strands. (B) Gels showing representative results from the Surveyor digest and the heteroduplex mobility assays used to genotype G1s. (i) In the Surveyor assay, PCR products were digested with Surveyor nuclease, resulting in cleavage in samples derived from heterozygous individuals. All gRNA A individuals screened here (lanes 1–7) show the expected cleavage products (∼100 and 413 bp), as do all gRNA B individuals (lanes 8–9; ∼277 and 236 bp, asterisks), identifying all individuals shown here as heterozygotes. (ii) In the heteroduplex mobility assay, 5 μl of PCR product was electrophoresed on a 4% agarose gel for > 5 hr at 80 V, allowing visualization of heteroduplexes, which migrate more slowly than homoduplexes, in samples from heterozygotes (lanes 11–14); thus, samples from homozygous individuals (WT) should produce only the 513-bp band (lane 10). (C) Sequenced mutant alleles induced by (i) gRNA-A/Cas9 and (ii) gRNA-B/Cas9. Red and blue lettering indicate the PAM site and insertions, respectively. (i) The mutant allele from individual A6-17 has a 16-bp insertion (blue) that adds a premature stop codon (red box); the allele from individual A12-10 has a 15-bp insertion (blue) and 1-bp deletion that induces a frameshift. (ii) The allele from individual B4-4 has a 7-bp deletion, resulting in a frameshift; the allele from individual B5-16 is complex, showing substitutions (blue) replacing sequence (underlined) 5′ and 3′ of the PAM site (red), including mutation of the splicing donor site (black box). cDNA, complementary DNA; CRISPR, clustered regularly interspaced short palindromic repeats; dsRNA, double-stranded RNA; gRNA, guide RNA; PAM, protospacer-adjacent motif; WT, wild-type.
    Figure Legend Snippet: Oncopeltus CRISPR/Cas9 mutagenesis workflow. (A) (i) Genomic structure of Of-w ; exon boundaries are based on transcriptome data and the alignment of a cDNA sequenced in this study to the genome; however, exon 4 was not found in the genome. The dsRNA target region (519 bp) used in this study is shown as an orange bar (exons only). (ii) The target location gRNAs (A–C) used in this study are in red. (iii) The primers used to PCR-amplify exon 2 for our two genotyping assays are shown as black arrows. The amplicon is 513 bp and includes some intronic regions surrounding the exon. Stars mark the predicted loci of Cas9 cleavage and thus likely mutation; the predicted Surveyor cleavage product sizes (in base pairs) for each gRNA are shown above the strands. (B) Gels showing representative results from the Surveyor digest and the heteroduplex mobility assays used to genotype G1s. (i) In the Surveyor assay, PCR products were digested with Surveyor nuclease, resulting in cleavage in samples derived from heterozygous individuals. All gRNA A individuals screened here (lanes 1–7) show the expected cleavage products (∼100 and 413 bp), as do all gRNA B individuals (lanes 8–9; ∼277 and 236 bp, asterisks), identifying all individuals shown here as heterozygotes. (ii) In the heteroduplex mobility assay, 5 μl of PCR product was electrophoresed on a 4% agarose gel for > 5 hr at 80 V, allowing visualization of heteroduplexes, which migrate more slowly than homoduplexes, in samples from heterozygotes (lanes 11–14); thus, samples from homozygous individuals (WT) should produce only the 513-bp band (lane 10). (C) Sequenced mutant alleles induced by (i) gRNA-A/Cas9 and (ii) gRNA-B/Cas9. Red and blue lettering indicate the PAM site and insertions, respectively. (i) The mutant allele from individual A6-17 has a 16-bp insertion (blue) that adds a premature stop codon (red box); the allele from individual A12-10 has a 15-bp insertion (blue) and 1-bp deletion that induces a frameshift. (ii) The allele from individual B4-4 has a 7-bp deletion, resulting in a frameshift; the allele from individual B5-16 is complex, showing substitutions (blue) replacing sequence (underlined) 5′ and 3′ of the PAM site (red), including mutation of the splicing donor site (black box). cDNA, complementary DNA; CRISPR, clustered regularly interspaced short palindromic repeats; dsRNA, double-stranded RNA; gRNA, guide RNA; PAM, protospacer-adjacent motif; WT, wild-type.

    Techniques Used: CRISPR, Mutagenesis, Polymerase Chain Reaction, Amplification, Derivative Assay, Agarose Gel Electrophoresis, Sequencing

    14) Product Images from "A catalogue of biochemically diverse CRISPR-Cas9 orthologs"

    Article Title: A catalogue of biochemically diverse CRISPR-Cas9 orthologs

    Journal: Nature Communications

    doi: 10.1038/s41467-020-19344-1

    Cas9 tracrRNA sequence and secondary structure similarity. Circles are scaled based on the number of sequences belonging to each covariance model (CM) and colored according to the designated cluster. The width of the connecting lines indicates the percentage of similarity or relatedness among CMs. Representative tracrRNAs from each cluster are indicated with the associated color. CMs not assigned to a cluster are in gray.
    Figure Legend Snippet: Cas9 tracrRNA sequence and secondary structure similarity. Circles are scaled based on the number of sequences belonging to each covariance model (CM) and colored according to the designated cluster. The width of the connecting lines indicates the percentage of similarity or relatedness among CMs. Representative tracrRNAs from each cluster are indicated with the associated color. CMs not assigned to a cluster are in gray.

    Techniques Used: Sequencing

    Activity of Cas9 orthologs at varying temperatures. The cleavage activity of Cas9 orthologs was measured using in vitro DNA cleavage assays using fluorophore-labeled double-stranded DNA (dsDNA) substrates. Cleaved fragments were quantitated and are represented in a heatmap a showing overall activity at temperatures ranging from 10 °C to 68 °C. The intensity of the blue color indicates the proportion of substrate cleaved. Source data are provided in the Source Data file. b Cas9 orthologs with activity at elevated temperatures. In vitro DNA cleavage activity for a subset of Cas9 orthologs with > 50% activity at 53 °C is summarized in a heatmap and plotted as the proportion of DNA substrate cleaved at varied temperatures. The intensity of the blue color in heatmaps indicates the proportion of substrate cleaved. Points represent the mean ± SEM of at least three independent experiments. Green shading highlights the temperature range above 53 °C. c Cas9 orthologs with reduced activity at room temperature. In vitro DNA cleavage activity for a subset of Cas9 orthologs with
    Figure Legend Snippet: Activity of Cas9 orthologs at varying temperatures. The cleavage activity of Cas9 orthologs was measured using in vitro DNA cleavage assays using fluorophore-labeled double-stranded DNA (dsDNA) substrates. Cleaved fragments were quantitated and are represented in a heatmap a showing overall activity at temperatures ranging from 10 °C to 68 °C. The intensity of the blue color indicates the proportion of substrate cleaved. Source data are provided in the Source Data file. b Cas9 orthologs with activity at elevated temperatures. In vitro DNA cleavage activity for a subset of Cas9 orthologs with > 50% activity at 53 °C is summarized in a heatmap and plotted as the proportion of DNA substrate cleaved at varied temperatures. The intensity of the blue color in heatmaps indicates the proportion of substrate cleaved. Points represent the mean ± SEM of at least three independent experiments. Green shading highlights the temperature range above 53 °C. c Cas9 orthologs with reduced activity at room temperature. In vitro DNA cleavage activity for a subset of Cas9 orthologs with

    Techniques Used: Activity Assay, In Vitro, Labeling

    Cas9 protospacer adjacent motif (PAM) interacting (PI) domain similarity. Cas9 PI domains clustered by their pairwise sequence similarity. Sequences were clustered using CLANS (BLAST option). Lines connect sequences with P value ≤ 1e − 11. Line shading corresponds to P values according to the scale in the top-right corner (light and long lines connect distantly related sequences). For details on how P values are calculated, please see the “Methods” section. Major clusters are shown in bold. Cluster 1 was so named to emphasize that it contains the first experimentally characterized Cas9, Spy. Clusters 2–10 were named beginning from the one with the most members. Different clusters are indicated, and PAM sequences recognized by members of each cluster are highlighted with the associated color. The Cas9 which belongs to the same clade is outlined by a black dashed line. Sequences having known structures are marked red; their PDB code is shown in parentheses.
    Figure Legend Snippet: Cas9 protospacer adjacent motif (PAM) interacting (PI) domain similarity. Cas9 PI domains clustered by their pairwise sequence similarity. Sequences were clustered using CLANS (BLAST option). Lines connect sequences with P value ≤ 1e − 11. Line shading corresponds to P values according to the scale in the top-right corner (light and long lines connect distantly related sequences). For details on how P values are calculated, please see the “Methods” section. Major clusters are shown in bold. Cluster 1 was so named to emphasize that it contains the first experimentally characterized Cas9, Spy. Clusters 2–10 were named beginning from the one with the most members. Different clusters are indicated, and PAM sequences recognized by members of each cluster are highlighted with the associated color. The Cas9 which belongs to the same clade is outlined by a black dashed line. Sequences having known structures are marked red; their PDB code is shown in parentheses.

    Techniques Used: Sequencing

    Cas9 diversity and characterization approach. a Phylogenetic representation of the diversity provided by Cas9 orthologs. Type II-A, B, and C systems are color-coded, red, blue, and green, respectively. Distinct phylogenetic clades are numbered I–X. Those selected for the study are indicated with a black dot. Cas9s whose structure has been determined are also designated. b Biochemical approach used to directly capture target cleavage and assess protospacer adjacent motif (PAM) recognition. Experiments were assembled using Cas9 protein produced by IVT.
    Figure Legend Snippet: Cas9 diversity and characterization approach. a Phylogenetic representation of the diversity provided by Cas9 orthologs. Type II-A, B, and C systems are color-coded, red, blue, and green, respectively. Distinct phylogenetic clades are numbered I–X. Those selected for the study are indicated with a black dot. Cas9s whose structure has been determined are also designated. b Biochemical approach used to directly capture target cleavage and assess protospacer adjacent motif (PAM) recognition. Experiments were assembled using Cas9 protein produced by IVT.

    Techniques Used: Produced

    Target DNA cleavage patterns produced by Cas9 orthologs. Cleavage sites and resultant double-stranded DNA (dsDNA) ends are depicted as heatmaps that show the proportion of cleaved ends recovered by DNA sequencing at each position of a target DNA. The intensity of the blue color indicates the proportion of mapped cleavage ends. a Control digests using restriction enzymes showed that blunt ends, 5′-overhangs and 3′-overhangs might be recovered with our approach. TS indicates the top strand; BS indicates the bottom strand. b Spy Cas9 and Sau Cas9 cleaved DNA ends. Heatmaps represent mapped cleavage ends as the averages at each position in five different dsDNA targets. The position of the DNA bases and protospacer adjacent motif (PAM) sequences is depicted above the heatmaps. NTS indicates a non-target strand; TS indicates the target strand. c Blunt and staggered-end cleavage. Examples of blunt, one base 5′-overhang staggered cleavage, and multiple base 5′-overhang cleavage are depicted as heatmaps that show the proportion of cleaved ends as the averages at each position in five different dsDNA targets. The position of the DNA bases and PAM sequences is depicted above the heatmaps. NTS indicates a non-target strand; TS indicates the target strand. Source data are provided in Supplementary Data 5.
    Figure Legend Snippet: Target DNA cleavage patterns produced by Cas9 orthologs. Cleavage sites and resultant double-stranded DNA (dsDNA) ends are depicted as heatmaps that show the proportion of cleaved ends recovered by DNA sequencing at each position of a target DNA. The intensity of the blue color indicates the proportion of mapped cleavage ends. a Control digests using restriction enzymes showed that blunt ends, 5′-overhangs and 3′-overhangs might be recovered with our approach. TS indicates the top strand; BS indicates the bottom strand. b Spy Cas9 and Sau Cas9 cleaved DNA ends. Heatmaps represent mapped cleavage ends as the averages at each position in five different dsDNA targets. The position of the DNA bases and protospacer adjacent motif (PAM) sequences is depicted above the heatmaps. NTS indicates a non-target strand; TS indicates the target strand. c Blunt and staggered-end cleavage. Examples of blunt, one base 5′-overhang staggered cleavage, and multiple base 5′-overhang cleavage are depicted as heatmaps that show the proportion of cleaved ends as the averages at each position in five different dsDNA targets. The position of the DNA bases and PAM sequences is depicted above the heatmaps. NTS indicates a non-target strand; TS indicates the target strand. Source data are provided in Supplementary Data 5.

    Techniques Used: Produced, DNA Sequencing

    15) Product Images from "Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity"

    Article Title: Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03927-0

    BNA NC incorporation broadly improves specificity in vitro. Heat maps showing DNA cleavage specificity scores across > 10 12 off-target sequences for either unmodified (top) or BNA NC -modified crRNAs (bottom) targeting a WAS or b EMX1. Specificity scores of 1.0 (dark blue) correspond to 100% enrichment for, while scores of −1.0 (dark red) correspond to 100% enrichment against a specific base-pair at a specific position. Black boxes denote the intended target nucleotides. Bar graph showing the quantitative difference in specificity score at each position in the 20 base-pair target site and 2 base-pair PAM (N of NGG excluded), between the unmodified and BNA NC -modified crRNA for c WAS or d EMX1 target sequences. A score of zero indicates no change in specificity. Difference in specificity was calculated as specificity score BNA NC −specificity score RNA . Experiments were performed with 200 nM pre-selection library and 1000 nM Cas9 RNP complex
    Figure Legend Snippet: BNA NC incorporation broadly improves specificity in vitro. Heat maps showing DNA cleavage specificity scores across > 10 12 off-target sequences for either unmodified (top) or BNA NC -modified crRNAs (bottom) targeting a WAS or b EMX1. Specificity scores of 1.0 (dark blue) correspond to 100% enrichment for, while scores of −1.0 (dark red) correspond to 100% enrichment against a specific base-pair at a specific position. Black boxes denote the intended target nucleotides. Bar graph showing the quantitative difference in specificity score at each position in the 20 base-pair target site and 2 base-pair PAM (N of NGG excluded), between the unmodified and BNA NC -modified crRNA for c WAS or d EMX1 target sequences. A score of zero indicates no change in specificity. Difference in specificity was calculated as specificity score BNA NC −specificity score RNA . Experiments were performed with 200 nM pre-selection library and 1000 nM Cas9 RNP complex

    Techniques Used: In Vitro, Modification, Selection

    BNA NC incorporation influences conformational transitions. a Schematic diagram for smFRET experiments showing a Cas9 RNP complex consisting of Cy5-labeled crRNA, tracrRNA, and Cas9, bound to a Cy3-labeled dsDNA immobilized on a quartz surface. b Histograms showing FRET efficiency after equilibration for the WAS DNA (upper) or WAS-OT4 DNA (lower) target sequences using WAS-RNA (dark blue) or WAS-BNA-3 (light blue) crRNAs; black curves represent Gaussian fits. c Time trace for Cas9 on WAS-OT4 DNA using WAS-BNA-3 crRNA indicating repetitive transitions between the open and zipped conformations. Dwell time in each conformation is indicated as ∆ τ . d Comparison of Cas9 dwell times between WAS-RNA and WAS-BNA-3 crRNA using the WAS-OT4 DNA template; mean ± SD shown
    Figure Legend Snippet: BNA NC incorporation influences conformational transitions. a Schematic diagram for smFRET experiments showing a Cas9 RNP complex consisting of Cy5-labeled crRNA, tracrRNA, and Cas9, bound to a Cy3-labeled dsDNA immobilized on a quartz surface. b Histograms showing FRET efficiency after equilibration for the WAS DNA (upper) or WAS-OT4 DNA (lower) target sequences using WAS-RNA (dark blue) or WAS-BNA-3 (light blue) crRNAs; black curves represent Gaussian fits. c Time trace for Cas9 on WAS-OT4 DNA using WAS-BNA-3 crRNA indicating repetitive transitions between the open and zipped conformations. Dwell time in each conformation is indicated as ∆ τ . d Comparison of Cas9 dwell times between WAS-RNA and WAS-BNA-3 crRNA using the WAS-OT4 DNA template; mean ± SD shown

    Techniques Used: Labeling

    BNA NC incorporation increases Cas9 specificity in cells. Gel showing relative cellular cleavage efficiencies of the unmodified, and 9 BNA NC -modified crRNAs targeting a WAS or b EMX1 on-target (top) or off-target (bottom) sequences, as determined by T7 endonuclease I digestion. Mock transfections lacking guide RNA were used as controls. Modification frequencies were determined using densitometry (ImageJ) and are indicated below each lane. Lanes in which no cleavage products were observed are marked as undetected (UD)
    Figure Legend Snippet: BNA NC incorporation increases Cas9 specificity in cells. Gel showing relative cellular cleavage efficiencies of the unmodified, and 9 BNA NC -modified crRNAs targeting a WAS or b EMX1 on-target (top) or off-target (bottom) sequences, as determined by T7 endonuclease I digestion. Mock transfections lacking guide RNA were used as controls. Modification frequencies were determined using densitometry (ImageJ) and are indicated below each lane. Lanes in which no cleavage products were observed are marked as undetected (UD)

    Techniques Used: Modification, Transfection

    BNA NC incorporation reduces off-target cleavage in vitro. a Chemical structures of RNA, LNA (2′,4′-BNA), and BNA NC (2′,4′-BNA NC [NMe]) nucleotides. b WAS and c EMX1 on-target and off-target sequences used for in vitro and cellular cleavage assays. Mismatches are indicated by red lowercase lettering. Heat map showing in vitro cleavage specificity for the unmodified crRNA and 9 BNA NC -modified crRNAs toward either d WAS or e EMX1 on-target and off-target sequences (as listed in Fig. 1b, c); mean shown ( n = 2). crRNA and BNA NC -modified sequences are shown to the left of the corresponding heat map. BNA NC modifications are indicated in black. Targets that were highly cleaved in vitro are indicated in red, while targets that were not cleaved are indicated in blue. Gel showing relative cleavage efficiencies of the unmodified and most specific BNA NC -modified crRNAs on a 1-kb DNA fragment containing either the f WAS or g EMX1 on-target and off-target sequences. The two bottom bands are cleavage products, while the top band is full-length substrate. The molar ratio of Cas9 RNP complex to target DNA was 30:1 for these experiments. Quantification of cleavage percentages was determined using densitometry (ImageJ), and are shown below each lane. Lanes in which no cleavage products were observed are marked as undetected (UD). Values used to generate heatmaps are presented in Supplementary Table 1
    Figure Legend Snippet: BNA NC incorporation reduces off-target cleavage in vitro. a Chemical structures of RNA, LNA (2′,4′-BNA), and BNA NC (2′,4′-BNA NC [NMe]) nucleotides. b WAS and c EMX1 on-target and off-target sequences used for in vitro and cellular cleavage assays. Mismatches are indicated by red lowercase lettering. Heat map showing in vitro cleavage specificity for the unmodified crRNA and 9 BNA NC -modified crRNAs toward either d WAS or e EMX1 on-target and off-target sequences (as listed in Fig. 1b, c); mean shown ( n = 2). crRNA and BNA NC -modified sequences are shown to the left of the corresponding heat map. BNA NC modifications are indicated in black. Targets that were highly cleaved in vitro are indicated in red, while targets that were not cleaved are indicated in blue. Gel showing relative cleavage efficiencies of the unmodified and most specific BNA NC -modified crRNAs on a 1-kb DNA fragment containing either the f WAS or g EMX1 on-target and off-target sequences. The two bottom bands are cleavage products, while the top band is full-length substrate. The molar ratio of Cas9 RNP complex to target DNA was 30:1 for these experiments. Quantification of cleavage percentages was determined using densitometry (ImageJ), and are shown below each lane. Lanes in which no cleavage products were observed are marked as undetected (UD). Values used to generate heatmaps are presented in Supplementary Table 1

    Techniques Used: In Vitro, Modification

    16) Product Images from "Simultaneous lineage tracing and cell-type identification using CRISPR/Cas9-induced genetic scars"

    Article Title: Simultaneous lineage tracing and cell-type identification using CRISPR/Cas9-induced genetic scars

    Journal: Nature biotechnology

    doi: 10.1038/nbt.4124

    Using the CRISPR/Cas9 system for massively parallel single cell lineage tracing. (a) Cas9 creates insertions or deletions in an RFP transgene. These genetic scars can be used as lineage barcodes. Using the fish line zebrabow M , which has 16-32 integrations of the RFP transgene, enables us to record complex lineage trees with a single sgRNA. Simultaneous transcriptome profiling by scRNA-seq allows unbiased cell type identification. (b) Sketch of the experimental protocol. Injection of Cas9 and sgRNA for RFP into the zygote marks cells with genetic scars at an early developmental stage. Scars can be read out together with the transcriptome by scRNA-seq at a later stage. (c) Approach for simultaneous detection of scars and transcriptome from single cells. Cells are captured by droplet microfluidics, followed by lysis, reverse transcription, and amplification. After amplification, the material is split and processed into a whole transcriptome library and a targeted RFP library for scar detection. (d ) t-SNE representation of scRNA-seq data and identified cell types for dissociated zebrafish larvae (5 dpf, n=7 animals). Cell types were grouped into 8 categories as indicated by the color code. (e) Probability distribution of scars, measured in bulk experiments on the DNA level. Pie chart shows fractions of different types of scars (deletion, insertion, single nucleotide polymorphism (SNP), complex scars). (f) Length distributions for deletions and insertions for the data shown in (e). (g) Scarring dynamics as measured on the DNA and RNA level, with exponential fit.
    Figure Legend Snippet: Using the CRISPR/Cas9 system for massively parallel single cell lineage tracing. (a) Cas9 creates insertions or deletions in an RFP transgene. These genetic scars can be used as lineage barcodes. Using the fish line zebrabow M , which has 16-32 integrations of the RFP transgene, enables us to record complex lineage trees with a single sgRNA. Simultaneous transcriptome profiling by scRNA-seq allows unbiased cell type identification. (b) Sketch of the experimental protocol. Injection of Cas9 and sgRNA for RFP into the zygote marks cells with genetic scars at an early developmental stage. Scars can be read out together with the transcriptome by scRNA-seq at a later stage. (c) Approach for simultaneous detection of scars and transcriptome from single cells. Cells are captured by droplet microfluidics, followed by lysis, reverse transcription, and amplification. After amplification, the material is split and processed into a whole transcriptome library and a targeted RFP library for scar detection. (d ) t-SNE representation of scRNA-seq data and identified cell types for dissociated zebrafish larvae (5 dpf, n=7 animals). Cell types were grouped into 8 categories as indicated by the color code. (e) Probability distribution of scars, measured in bulk experiments on the DNA level. Pie chart shows fractions of different types of scars (deletion, insertion, single nucleotide polymorphism (SNP), complex scars). (f) Length distributions for deletions and insertions for the data shown in (e). (g) Scarring dynamics as measured on the DNA and RNA level, with exponential fit.

    Techniques Used: CRISPR, Fluorescence In Situ Hybridization, Injection, Lysis, Amplification

    17) Product Images from "Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity"

    Article Title: Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03927-0

    BNA NC incorporation broadly improves specificity in vitro. Heat maps showing DNA cleavage specificity scores across > 10 12 off-target sequences for either unmodified (top) or BNA NC -modified crRNAs (bottom) targeting a WAS or b EMX1. Specificity scores of 1.0 (dark blue) correspond to 100% enrichment for, while scores of −1.0 (dark red) correspond to 100% enrichment against a specific base-pair at a specific position. Black boxes denote the intended target nucleotides. Bar graph showing the quantitative difference in specificity score at each position in the 20 base-pair target site and 2 base-pair PAM (N of NGG excluded), between the unmodified and BNA NC -modified crRNA for c WAS or d EMX1 target sequences. A score of zero indicates no change in specificity. Difference in specificity was calculated as specificity score BNA NC −specificity score RNA . Experiments were performed with 200 nM pre-selection library and 1000 nM Cas9 RNP complex
    Figure Legend Snippet: BNA NC incorporation broadly improves specificity in vitro. Heat maps showing DNA cleavage specificity scores across > 10 12 off-target sequences for either unmodified (top) or BNA NC -modified crRNAs (bottom) targeting a WAS or b EMX1. Specificity scores of 1.0 (dark blue) correspond to 100% enrichment for, while scores of −1.0 (dark red) correspond to 100% enrichment against a specific base-pair at a specific position. Black boxes denote the intended target nucleotides. Bar graph showing the quantitative difference in specificity score at each position in the 20 base-pair target site and 2 base-pair PAM (N of NGG excluded), between the unmodified and BNA NC -modified crRNA for c WAS or d EMX1 target sequences. A score of zero indicates no change in specificity. Difference in specificity was calculated as specificity score BNA NC −specificity score RNA . Experiments were performed with 200 nM pre-selection library and 1000 nM Cas9 RNP complex

    Techniques Used: In Vitro, Modification, Selection

    BNA NC incorporation influences conformational transitions. a Schematic diagram for smFRET experiments showing a Cas9 RNP complex consisting of Cy5-labeled crRNA, tracrRNA, and Cas9, bound to a Cy3-labeled dsDNA immobilized on a quartz surface. b Histograms showing FRET efficiency after equilibration for the WAS DNA (upper) or WAS-OT4 DNA (lower) target sequences using WAS-RNA (dark blue) or WAS-BNA-3 (light blue) crRNAs; black curves represent Gaussian fits. c Time trace for Cas9 on WAS-OT4 DNA using WAS-BNA-3 crRNA indicating repetitive transitions between the open and zipped conformations. Dwell time in each conformation is indicated as ∆ τ . d Comparison of Cas9 dwell times between WAS-RNA and WAS-BNA-3 crRNA using the WAS-OT4 DNA template; mean ± SD shown
    Figure Legend Snippet: BNA NC incorporation influences conformational transitions. a Schematic diagram for smFRET experiments showing a Cas9 RNP complex consisting of Cy5-labeled crRNA, tracrRNA, and Cas9, bound to a Cy3-labeled dsDNA immobilized on a quartz surface. b Histograms showing FRET efficiency after equilibration for the WAS DNA (upper) or WAS-OT4 DNA (lower) target sequences using WAS-RNA (dark blue) or WAS-BNA-3 (light blue) crRNAs; black curves represent Gaussian fits. c Time trace for Cas9 on WAS-OT4 DNA using WAS-BNA-3 crRNA indicating repetitive transitions between the open and zipped conformations. Dwell time in each conformation is indicated as ∆ τ . d Comparison of Cas9 dwell times between WAS-RNA and WAS-BNA-3 crRNA using the WAS-OT4 DNA template; mean ± SD shown

    Techniques Used: Labeling

    BNA NC incorporation increases Cas9 specificity in cells. Gel showing relative cellular cleavage efficiencies of the unmodified, and 9 BNA NC -modified crRNAs targeting a WAS or b EMX1 on-target (top) or off-target (bottom) sequences, as determined by T7 endonuclease I digestion. Mock transfections lacking guide RNA were used as controls. Modification frequencies were determined using densitometry (ImageJ) and are indicated below each lane. Lanes in which no cleavage products were observed are marked as undetected (UD)
    Figure Legend Snippet: BNA NC incorporation increases Cas9 specificity in cells. Gel showing relative cellular cleavage efficiencies of the unmodified, and 9 BNA NC -modified crRNAs targeting a WAS or b EMX1 on-target (top) or off-target (bottom) sequences, as determined by T7 endonuclease I digestion. Mock transfections lacking guide RNA were used as controls. Modification frequencies were determined using densitometry (ImageJ) and are indicated below each lane. Lanes in which no cleavage products were observed are marked as undetected (UD)

    Techniques Used: Modification, Transfection

    BNA NC incorporation reduces off-target cleavage in vitro. a Chemical structures of RNA, LNA (2′,4′-BNA), and BNA NC (2′,4′-BNA NC [NMe]) nucleotides. b WAS and c EMX1 on-target and off-target sequences used for in vitro and cellular cleavage assays. Mismatches are indicated by red lowercase lettering. Heat map showing in vitro cleavage specificity for the unmodified crRNA and 9 BNA NC -modified crRNAs toward either d WAS or e EMX1 on-target and off-target sequences (as listed in Fig. 1b, c); mean shown ( n = 2). crRNA and BNA NC -modified sequences are shown to the left of the corresponding heat map. BNA NC modifications are indicated in black. Targets that were highly cleaved in vitro are indicated in red, while targets that were not cleaved are indicated in blue. Gel showing relative cleavage efficiencies of the unmodified and most specific BNA NC -modified crRNAs on a 1-kb DNA fragment containing either the f WAS or g EMX1 on-target and off-target sequences. The two bottom bands are cleavage products, while the top band is full-length substrate. The molar ratio of Cas9 RNP complex to target DNA was 30:1 for these experiments. Quantification of cleavage percentages was determined using densitometry (ImageJ), and are shown below each lane. Lanes in which no cleavage products were observed are marked as undetected (UD). Values used to generate heatmaps are presented in Supplementary Table 1
    Figure Legend Snippet: BNA NC incorporation reduces off-target cleavage in vitro. a Chemical structures of RNA, LNA (2′,4′-BNA), and BNA NC (2′,4′-BNA NC [NMe]) nucleotides. b WAS and c EMX1 on-target and off-target sequences used for in vitro and cellular cleavage assays. Mismatches are indicated by red lowercase lettering. Heat map showing in vitro cleavage specificity for the unmodified crRNA and 9 BNA NC -modified crRNAs toward either d WAS or e EMX1 on-target and off-target sequences (as listed in Fig. 1b, c); mean shown ( n = 2). crRNA and BNA NC -modified sequences are shown to the left of the corresponding heat map. BNA NC modifications are indicated in black. Targets that were highly cleaved in vitro are indicated in red, while targets that were not cleaved are indicated in blue. Gel showing relative cleavage efficiencies of the unmodified and most specific BNA NC -modified crRNAs on a 1-kb DNA fragment containing either the f WAS or g EMX1 on-target and off-target sequences. The two bottom bands are cleavage products, while the top band is full-length substrate. The molar ratio of Cas9 RNP complex to target DNA was 30:1 for these experiments. Quantification of cleavage percentages was determined using densitometry (ImageJ), and are shown below each lane. Lanes in which no cleavage products were observed are marked as undetected (UD). Values used to generate heatmaps are presented in Supplementary Table 1

    Techniques Used: In Vitro, Modification

    18) Product Images from "CRISPR/Cas9 Genome Editing of Epidermal Growth Factor Receptor Sufficiently Abolished Oncogenicity in Anaplastic Thyroid Cancer"

    Article Title: CRISPR/Cas9 Genome Editing of Epidermal Growth Factor Receptor Sufficiently Abolished Oncogenicity in Anaplastic Thyroid Cancer

    Journal: Disease Markers

    doi: 10.1155/2018/3835783

    Optimization of viral transduction conditions for human SW579 cells: (a) the purified and concentrated PLJM1-GFP lentivirus was measured as virus copy number by Q-PCR analysis. SW579 cells were seeded in a 6 cm dish and infected with 1000-, 3000-, 9000-, 15,000-, 30,000-, and 90,000-fold concentrations of virus to SW579 cell number for three days. The GFP-positive (infected) cell population was assessed using flow cytometry. (b) The image shows the linear curve comparison of virus input and the GFP-positive cell population. (c) The image shows the western blot analysis of EGFR protein expression on SC sgRNA control SW579 cells; EGFR levels were significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The downstream signaling proteins such as phosphor-AKT and phosphor-ERK were also analyzed in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The tumor-suppressive proteins such as P53 and P21 were found to be induced in EGFR gene-edited cells. GAPDH served as internal control. (d) TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in SW579 cells. The pie charts show the percentages of indels in the EGFR gene edited by EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and other mutations are presented in purple and pink, respectively. (e) The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (f) The EGFR gene in SW579 cells was analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific EGFR sgRNA_2 and Cas9 addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (g) The CRISPR Design website was used to predict off-target candidate genes for both the EGFR sgRNA_1 and EGFR sgRNA_2 viruses. To note, no potential off-target candidate gene was predicted for EGFR sgRNA_1 (mismatch
    Figure Legend Snippet: Optimization of viral transduction conditions for human SW579 cells: (a) the purified and concentrated PLJM1-GFP lentivirus was measured as virus copy number by Q-PCR analysis. SW579 cells were seeded in a 6 cm dish and infected with 1000-, 3000-, 9000-, 15,000-, 30,000-, and 90,000-fold concentrations of virus to SW579 cell number for three days. The GFP-positive (infected) cell population was assessed using flow cytometry. (b) The image shows the linear curve comparison of virus input and the GFP-positive cell population. (c) The image shows the western blot analysis of EGFR protein expression on SC sgRNA control SW579 cells; EGFR levels were significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The downstream signaling proteins such as phosphor-AKT and phosphor-ERK were also analyzed in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The tumor-suppressive proteins such as P53 and P21 were found to be induced in EGFR gene-edited cells. GAPDH served as internal control. (d) TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in SW579 cells. The pie charts show the percentages of indels in the EGFR gene edited by EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and other mutations are presented in purple and pink, respectively. (e) The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (f) The EGFR gene in SW579 cells was analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific EGFR sgRNA_2 and Cas9 addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (g) The CRISPR Design website was used to predict off-target candidate genes for both the EGFR sgRNA_1 and EGFR sgRNA_2 viruses. To note, no potential off-target candidate gene was predicted for EGFR sgRNA_1 (mismatch

    Techniques Used: Transduction, Purification, Polymerase Chain Reaction, Infection, Flow Cytometry, Cytometry, Western Blot, Expressing, Sequencing, RFLP Assay, CRISPR

    EGFR gene targeting in MDA-MB-231 cells using the CRISPR/CAS9 system: (a) schematic representation of the human EGFR DNA locus and two protospacer sequences (blue underline) for editing. The arrowhead indicates the expected Cas9 cleavage site. The protospacer adjacent motif (PAM, red underline) is the motif required for Cas9 nuclease activity. Scrambled (SC) sgRNA and EGFR sgRNA were delivered to MDA-MB-231 cells by lentivirus. After transduction, DNA from virus-infected cells was purified and subjected to Sanger sequencing of EGFR exon 3 and exon 9. (b, c) Wild-type EGFR sequences in MDA-MB-231 cells. (d) EGFR sgRNA_1 and (e) EGFR sgRNA_2 producing a mixture of sequences around the expected Cas9 cleavage point in a pool of gene-edited cells after lentivirus transduction. TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in MDA-MB-231 cells. The pie charts show the percentages of indels in the EGFR gene edited by (f) EGFR sgRNA_1 and (g) EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and −2 indels are presented in purple and blue, respectively. The original TIDE algorithm analysis is shown for (h) EGFR sgRNA_1 and (i) EGFR sgRNA_2 virus transfected on MDA-MB-231 cells, compared to SC MDA-MB-231 cells. The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (j) The EGFR gene in MDA-MB-231 cells analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific sgRNA and Cas9 addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (k) Western blot analysis of EGFR protein expression. (c) Image showing parental and SC sgRNA control MDA-MB-231 cells; EGFR expression was significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced MDA-MB-231 cells.
    Figure Legend Snippet: EGFR gene targeting in MDA-MB-231 cells using the CRISPR/CAS9 system: (a) schematic representation of the human EGFR DNA locus and two protospacer sequences (blue underline) for editing. The arrowhead indicates the expected Cas9 cleavage site. The protospacer adjacent motif (PAM, red underline) is the motif required for Cas9 nuclease activity. Scrambled (SC) sgRNA and EGFR sgRNA were delivered to MDA-MB-231 cells by lentivirus. After transduction, DNA from virus-infected cells was purified and subjected to Sanger sequencing of EGFR exon 3 and exon 9. (b, c) Wild-type EGFR sequences in MDA-MB-231 cells. (d) EGFR sgRNA_1 and (e) EGFR sgRNA_2 producing a mixture of sequences around the expected Cas9 cleavage point in a pool of gene-edited cells after lentivirus transduction. TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in MDA-MB-231 cells. The pie charts show the percentages of indels in the EGFR gene edited by (f) EGFR sgRNA_1 and (g) EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and −2 indels are presented in purple and blue, respectively. The original TIDE algorithm analysis is shown for (h) EGFR sgRNA_1 and (i) EGFR sgRNA_2 virus transfected on MDA-MB-231 cells, compared to SC MDA-MB-231 cells. The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (j) The EGFR gene in MDA-MB-231 cells analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific sgRNA and Cas9 addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (k) Western blot analysis of EGFR protein expression. (c) Image showing parental and SC sgRNA control MDA-MB-231 cells; EGFR expression was significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced MDA-MB-231 cells.

    Techniques Used: Multiple Displacement Amplification, CRISPR, Activity Assay, Transduction, Infection, Purification, Sequencing, Transfection, RFLP Assay, Western Blot, Expressing

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    In Vitro:

    Article Title: CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool
    Article Snippet: .. In vitro cleavage assay with Cas9 protein DNA cleavage assay was carried out based on [ ] with commercially available Cas9 enzyme (NEB). .. PCR amplified genomic fragments for each sgRNA-1 off-target site (OT#1_F 5’-AGAGGCAAGTAAAGGTCAAGTAGG-3’ , OT#1_R 5’-TCACATTGCAATGATGAGCACTTT-3’ ; OT#2_F 5’-CCAGCTCATGTTGAAAAGACACAT-3’ , OT#2_R 5’-CCCCCACAGATGAAATGAAAAGAC-3’ ; OT#3_F 5’-TACCCAAAAATTGTAAGCCAGCAG-3’ , OT#3_R 5’-AGATCTGATCCGGTTTCAAAGTGA-3’ ) were cloned into pGEM-T easy vector (Promega).

    Cleavage Assay:

    Article Title: CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool
    Article Snippet: .. In vitro cleavage assay with Cas9 protein DNA cleavage assay was carried out based on [ ] with commercially available Cas9 enzyme (NEB). .. PCR amplified genomic fragments for each sgRNA-1 off-target site (OT#1_F 5’-AGAGGCAAGTAAAGGTCAAGTAGG-3’ , OT#1_R 5’-TCACATTGCAATGATGAGCACTTT-3’ ; OT#2_F 5’-CCAGCTCATGTTGAAAAGACACAT-3’ , OT#2_R 5’-CCCCCACAGATGAAATGAAAAGAC-3’ ; OT#3_F 5’-TACCCAAAAATTGTAAGCCAGCAG-3’ , OT#3_R 5’-AGATCTGATCCGGTTTCAAAGTGA-3’ ) were cloned into pGEM-T easy vector (Promega).

    DNA Cleavage Assay:

    Article Title: CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool
    Article Snippet: .. In vitro cleavage assay with Cas9 protein DNA cleavage assay was carried out based on [ ] with commercially available Cas9 enzyme (NEB). .. PCR amplified genomic fragments for each sgRNA-1 off-target site (OT#1_F 5’-AGAGGCAAGTAAAGGTCAAGTAGG-3’ , OT#1_R 5’-TCACATTGCAATGATGAGCACTTT-3’ ; OT#2_F 5’-CCAGCTCATGTTGAAAAGACACAT-3’ , OT#2_R 5’-CCCCCACAGATGAAATGAAAAGAC-3’ ; OT#3_F 5’-TACCCAAAAATTGTAAGCCAGCAG-3’ , OT#3_R 5’-AGATCTGATCCGGTTTCAAAGTGA-3’ ) were cloned into pGEM-T easy vector (Promega).

    Plasmid Preparation:

    Article Title: Multiplex mutagenesis of four clustered CrRLK1L with CRISPR/Cas9 exposes their growth regulatory roles in response to metal ions
    Article Snippet: To this end, PCR primers were designed with following overhangs: E (forward primer) and P (reverse primer) for gRNA2 and P (forward primer) and F (reverse primer) for gRNA3 (primers no. 17–20 Supplemental Table ). .. For the Golden Gate reaction, 150 ng of the destination vector (pGGZ003) and 250 ng of each entry vector containing the promoter, Cas9, terminator, gRNA1, and BASTA resistance were mixed with 250 ng each of the gRNA2 and gRNA3 PCR products, 0.2 µL BSA protein (New England Biolabs, B9000S), 2 µL ligase buffer, 1.2 µL T4 DNA ligase (ThermoFisher, #EL0011), 1 µL BsaI (NEB, #R0535S) and distilled water (to 20 µL). .. To eliminate not fully-ligated intermediate products, we added 0.85 µL ATP (25 mM) and 1 µL Plasmid-Safe ATP-dependent DNase (Epicentre, E3101K) and incubated for 60 min at 37 °C and for 30 min at 70 °C.

    Polymerase Chain Reaction:

    Article Title: Multiplex mutagenesis of four clustered CrRLK1L with CRISPR/Cas9 exposes their growth regulatory roles in response to metal ions
    Article Snippet: To this end, PCR primers were designed with following overhangs: E (forward primer) and P (reverse primer) for gRNA2 and P (forward primer) and F (reverse primer) for gRNA3 (primers no. 17–20 Supplemental Table ). .. For the Golden Gate reaction, 150 ng of the destination vector (pGGZ003) and 250 ng of each entry vector containing the promoter, Cas9, terminator, gRNA1, and BASTA resistance were mixed with 250 ng each of the gRNA2 and gRNA3 PCR products, 0.2 µL BSA protein (New England Biolabs, B9000S), 2 µL ligase buffer, 1.2 µL T4 DNA ligase (ThermoFisher, #EL0011), 1 µL BsaI (NEB, #R0535S) and distilled water (to 20 µL). .. To eliminate not fully-ligated intermediate products, we added 0.85 µL ATP (25 mM) and 1 µL Plasmid-Safe ATP-dependent DNase (Epicentre, E3101K) and incubated for 60 min at 37 °C and for 30 min at 70 °C.

    Real-time Polymerase Chain Reaction:

    Article Title: Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease
    Article Snippet: .. For qPCR detection of template depletion, Cas9 cleavage assays were conducted using 5, 2.5, 1.25 or 0.625 nM Cas9 (New England Biolabs), 15 nM tracrXNA (Dharmacon and Eurogentec), 15 nM crXNA molecules (IDT and Eurogentec), 1× Cas9 reaction buffer (New England Biolabs), 0.75 nM eGFP and AAVS1 target DNA. ..

    Injection:

    Article Title: The primary role of zebrafish nanog is in extra-embryonic tissue
    Article Snippet: M nanog ; GESTALT donor embryos were generated from crosses of homozygous nanog −/− female fish to homozygous Tg(ubb:DsRed-barcodev7,myl7:EGFP) male fish ( ). .. These donor embryos were injected with Cas9 protein (NEB) and sgRNAs targeting the v7 barcode (sequence in ) ( ). ..

    Sequencing:

    Article Title: The primary role of zebrafish nanog is in extra-embryonic tissue
    Article Snippet: M nanog ; GESTALT donor embryos were generated from crosses of homozygous nanog −/− female fish to homozygous Tg(ubb:DsRed-barcodev7,myl7:EGFP) male fish ( ). .. These donor embryos were injected with Cas9 protein (NEB) and sgRNAs targeting the v7 barcode (sequence in ) ( ). ..

    other:

    Article Title: CRISPR-typing PCR (ctPCR), a new Cas9-based DNA detection method
    Article Snippet: Different amounts of HPV16 and HPV18 L1 gene plasmids were respectively cut with the Cas9 nuclease in complex with pairs of sgRNAs targeting to the HPV16 and HPV18 L1 genes.

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    New England Biolabs cas9 protein
    Optimization of viral transduction conditions for human SW579 cells: (a) the purified and concentrated PLJM1-GFP lentivirus was measured as virus copy number by Q-PCR analysis. SW579 cells were seeded in a 6 cm dish and infected with 1000-, 3000-, 9000-, 15,000-, 30,000-, and 90,000-fold concentrations of virus to SW579 cell number for three days. The GFP-positive (infected) cell population was assessed using flow cytometry. (b) The image shows the linear curve comparison of virus input and the GFP-positive cell population. (c) The image shows the western blot analysis of EGFR protein expression on SC sgRNA control SW579 cells; EGFR levels were significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The downstream signaling proteins such as phosphor-AKT and phosphor-ERK were also analyzed in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The tumor-suppressive proteins such as P53 and P21 were found to be induced in EGFR gene-edited cells. GAPDH served as internal control. (d) TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in SW579 cells. The pie charts show the percentages of indels in the EGFR gene edited by EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and other mutations are presented in purple and pink, respectively. (e) The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (f) The EGFR gene in SW579 cells was analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific EGFR sgRNA_2 and <t>Cas9</t> addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (g) The CRISPR Design website was used to predict off-target candidate genes for both the EGFR sgRNA_1 and EGFR sgRNA_2 viruses. To note, no potential off-target candidate gene was predicted for EGFR sgRNA_1 (mismatch
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    Optimization of viral transduction conditions for human SW579 cells: (a) the purified and concentrated PLJM1-GFP lentivirus was measured as virus copy number by Q-PCR analysis. SW579 cells were seeded in a 6 cm dish and infected with 1000-, 3000-, 9000-, 15,000-, 30,000-, and 90,000-fold concentrations of virus to SW579 cell number for three days. The GFP-positive (infected) cell population was assessed using flow cytometry. (b) The image shows the linear curve comparison of virus input and the GFP-positive cell population. (c) The image shows the western blot analysis of EGFR protein expression on SC sgRNA control SW579 cells; EGFR levels were significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The downstream signaling proteins such as phosphor-AKT and phosphor-ERK were also analyzed in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The tumor-suppressive proteins such as P53 and P21 were found to be induced in EGFR gene-edited cells. GAPDH served as internal control. (d) TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in SW579 cells. The pie charts show the percentages of indels in the EGFR gene edited by EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and other mutations are presented in purple and pink, respectively. (e) The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (f) The EGFR gene in SW579 cells was analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific EGFR sgRNA_2 and Cas9 addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (g) The CRISPR Design website was used to predict off-target candidate genes for both the EGFR sgRNA_1 and EGFR sgRNA_2 viruses. To note, no potential off-target candidate gene was predicted for EGFR sgRNA_1 (mismatch

    Journal: Disease Markers

    Article Title: CRISPR/Cas9 Genome Editing of Epidermal Growth Factor Receptor Sufficiently Abolished Oncogenicity in Anaplastic Thyroid Cancer

    doi: 10.1155/2018/3835783

    Figure Lengend Snippet: Optimization of viral transduction conditions for human SW579 cells: (a) the purified and concentrated PLJM1-GFP lentivirus was measured as virus copy number by Q-PCR analysis. SW579 cells were seeded in a 6 cm dish and infected with 1000-, 3000-, 9000-, 15,000-, 30,000-, and 90,000-fold concentrations of virus to SW579 cell number for three days. The GFP-positive (infected) cell population was assessed using flow cytometry. (b) The image shows the linear curve comparison of virus input and the GFP-positive cell population. (c) The image shows the western blot analysis of EGFR protein expression on SC sgRNA control SW579 cells; EGFR levels were significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The downstream signaling proteins such as phosphor-AKT and phosphor-ERK were also analyzed in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The tumor-suppressive proteins such as P53 and P21 were found to be induced in EGFR gene-edited cells. GAPDH served as internal control. (d) TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in SW579 cells. The pie charts show the percentages of indels in the EGFR gene edited by EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and other mutations are presented in purple and pink, respectively. (e) The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (f) The EGFR gene in SW579 cells was analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific EGFR sgRNA_2 and Cas9 addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (g) The CRISPR Design website was used to predict off-target candidate genes for both the EGFR sgRNA_1 and EGFR sgRNA_2 viruses. To note, no potential off-target candidate gene was predicted for EGFR sgRNA_1 (mismatch

    Article Snippet: Next, proteinase K (2 μ g) was added, and the reaction mixture was incubated for 15 minutes at 58°C to remove the Cas9 protein.

    Techniques: Transduction, Purification, Polymerase Chain Reaction, Infection, Flow Cytometry, Cytometry, Western Blot, Expressing, Sequencing, RFLP Assay, CRISPR

    EGFR gene targeting in MDA-MB-231 cells using the CRISPR/CAS9 system: (a) schematic representation of the human EGFR DNA locus and two protospacer sequences (blue underline) for editing. The arrowhead indicates the expected Cas9 cleavage site. The protospacer adjacent motif (PAM, red underline) is the motif required for Cas9 nuclease activity. Scrambled (SC) sgRNA and EGFR sgRNA were delivered to MDA-MB-231 cells by lentivirus. After transduction, DNA from virus-infected cells was purified and subjected to Sanger sequencing of EGFR exon 3 and exon 9. (b, c) Wild-type EGFR sequences in MDA-MB-231 cells. (d) EGFR sgRNA_1 and (e) EGFR sgRNA_2 producing a mixture of sequences around the expected Cas9 cleavage point in a pool of gene-edited cells after lentivirus transduction. TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in MDA-MB-231 cells. The pie charts show the percentages of indels in the EGFR gene edited by (f) EGFR sgRNA_1 and (g) EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and −2 indels are presented in purple and blue, respectively. The original TIDE algorithm analysis is shown for (h) EGFR sgRNA_1 and (i) EGFR sgRNA_2 virus transfected on MDA-MB-231 cells, compared to SC MDA-MB-231 cells. The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (j) The EGFR gene in MDA-MB-231 cells analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific sgRNA and Cas9 addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (k) Western blot analysis of EGFR protein expression. (c) Image showing parental and SC sgRNA control MDA-MB-231 cells; EGFR expression was significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced MDA-MB-231 cells.

    Journal: Disease Markers

    Article Title: CRISPR/Cas9 Genome Editing of Epidermal Growth Factor Receptor Sufficiently Abolished Oncogenicity in Anaplastic Thyroid Cancer

    doi: 10.1155/2018/3835783

    Figure Lengend Snippet: EGFR gene targeting in MDA-MB-231 cells using the CRISPR/CAS9 system: (a) schematic representation of the human EGFR DNA locus and two protospacer sequences (blue underline) for editing. The arrowhead indicates the expected Cas9 cleavage site. The protospacer adjacent motif (PAM, red underline) is the motif required for Cas9 nuclease activity. Scrambled (SC) sgRNA and EGFR sgRNA were delivered to MDA-MB-231 cells by lentivirus. After transduction, DNA from virus-infected cells was purified and subjected to Sanger sequencing of EGFR exon 3 and exon 9. (b, c) Wild-type EGFR sequences in MDA-MB-231 cells. (d) EGFR sgRNA_1 and (e) EGFR sgRNA_2 producing a mixture of sequences around the expected Cas9 cleavage point in a pool of gene-edited cells after lentivirus transduction. TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in MDA-MB-231 cells. The pie charts show the percentages of indels in the EGFR gene edited by (f) EGFR sgRNA_1 and (g) EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and −2 indels are presented in purple and blue, respectively. The original TIDE algorithm analysis is shown for (h) EGFR sgRNA_1 and (i) EGFR sgRNA_2 virus transfected on MDA-MB-231 cells, compared to SC MDA-MB-231 cells. The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (j) The EGFR gene in MDA-MB-231 cells analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific sgRNA and Cas9 addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (k) Western blot analysis of EGFR protein expression. (c) Image showing parental and SC sgRNA control MDA-MB-231 cells; EGFR expression was significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced MDA-MB-231 cells.

    Article Snippet: Next, proteinase K (2 μ g) was added, and the reaction mixture was incubated for 15 minutes at 58°C to remove the Cas9 protein.

    Techniques: Multiple Displacement Amplification, CRISPR, Activity Assay, Transduction, Infection, Purification, Sequencing, Transfection, RFLP Assay, Western Blot, Expressing

    Long-term GESTALT fate mapping of transplanted cells lacking Nanog. (A) Diagram of cell transplantation at sphere stage from a M nanog ; GESTALT barcoded donor embryo into a wild-type host embryo. The donor embryo was injected with sgRNAs targeting CRISPR-Cas9 sites in the GESTALT barcode array. Host animals were grown to 90 days post-fertilization, when intestine, heart, eyes and brain were dissected ( n =20 animals across two independent trials). Genomic DNA was prepared and barcodes (corresponding to surviving descendants of M nanog ; GESTALT transplanted cells) were sequenced from each organ. (B) After sequence processing of libraries from each organ across all 20 adults, distinct barcodes corresponding to different clones of transplanted M nanog ; GESTALT cells were counted. Shown is a summary of all 428 clones of M nanog ; GESTALT cells found in host animal organs.

    Journal: Development (Cambridge, England)

    Article Title: The primary role of zebrafish nanog is in extra-embryonic tissue

    doi: 10.1242/dev.147793

    Figure Lengend Snippet: Long-term GESTALT fate mapping of transplanted cells lacking Nanog. (A) Diagram of cell transplantation at sphere stage from a M nanog ; GESTALT barcoded donor embryo into a wild-type host embryo. The donor embryo was injected with sgRNAs targeting CRISPR-Cas9 sites in the GESTALT barcode array. Host animals were grown to 90 days post-fertilization, when intestine, heart, eyes and brain were dissected ( n =20 animals across two independent trials). Genomic DNA was prepared and barcodes (corresponding to surviving descendants of M nanog ; GESTALT transplanted cells) were sequenced from each organ. (B) After sequence processing of libraries from each organ across all 20 adults, distinct barcodes corresponding to different clones of transplanted M nanog ; GESTALT cells were counted. Shown is a summary of all 428 clones of M nanog ; GESTALT cells found in host animal organs.

    Article Snippet: These donor embryos were injected with Cas9 protein (NEB) and sgRNAs targeting the v7 barcode (sequence in ) ( ).

    Techniques: Transplantation Assay, Injection, CRISPR, Sequencing, Clone Assay

    Experimental verification of sgRNA-1. (A) In vitro cleavage depending on sgRNA-1/Cas9 occurred on linearized plasmids containing eGFP but not eGFP var . Successful cleavage of eGFP plasmid (4128bp) resulted in a 2052bp and 2076bp fragment. The absence of expected fragments (627bp, 4025bp) demonstrated that eGFP var (4652bp) was not digested by sgRNA-1/Cas9. (B) A faint double band (2154bp, 2198bp, asterisk) indicated inefficient digestion of off-target 1 (OT#1) while OT#2 and OT#3 ( S1 Table ) were not cleaved. Note: contrast was enhanced for better visualization. (C) Silent mutations of sgRNA-1 target site in eGFP var . (D-E) Injections of sgRNA-1 and Cas9 mRNA into wimb -/+ embryos (D) resulted in strong inactivation of eGFP (E).

    Journal: PLoS ONE

    Article Title: CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool

    doi: 10.1371/journal.pone.0124633

    Figure Lengend Snippet: Experimental verification of sgRNA-1. (A) In vitro cleavage depending on sgRNA-1/Cas9 occurred on linearized plasmids containing eGFP but not eGFP var . Successful cleavage of eGFP plasmid (4128bp) resulted in a 2052bp and 2076bp fragment. The absence of expected fragments (627bp, 4025bp) demonstrated that eGFP var (4652bp) was not digested by sgRNA-1/Cas9. (B) A faint double band (2154bp, 2198bp, asterisk) indicated inefficient digestion of off-target 1 (OT#1) while OT#2 and OT#3 ( S1 Table ) were not cleaved. Note: contrast was enhanced for better visualization. (C) Silent mutations of sgRNA-1 target site in eGFP var . (D-E) Injections of sgRNA-1 and Cas9 mRNA into wimb -/+ embryos (D) resulted in strong inactivation of eGFP (E).

    Article Snippet: In vitro cleavage assay with Cas9 protein DNA cleavage assay was carried out based on [ ] with commercially available Cas9 enzyme (NEB).

    Techniques: In Vitro, Plasmid Preparation

    Single-molecule FRET analysis for sub-conformation of Cas9:gRNA:DNA. ( a ) Scheme for smFRET experiment. Cas9 with gRNAs that consist of Cy5 (acceptor)-labelled crRNA, tracrRNA, binds to Cy3 (donor)-labelled dsDNA. In the FRET measurement, a relatively low concentration of gRNAs (30 nM) compared with cleavage experiments was used to reduce the background fluorescence of Cy5-labelled crRNA. ( b ) A histogram of the FRET upon binding (we selected the molecules emitting Cy5 signal) to wild-type target DNA exhibits two peaks centred at 0.27 and 0.83, with Gaussian fits (black line). ( c ) A representative time trajectory representing the short-lived open conformation and zipped conformation exhibits two FRET states. The duration of each conformation is measured as the dwell time (Δ t ). The trajectory was imaged right after the injection of Cas9:gRNA into a single-molecule chamber with an integration time of 0.03 s. The points of binding and photo-bleaching were indicated by the black arrows. ( d ) Histograms of FRET upon binding with each PAM-distal mutant exhibit different conformational distribution (with Gaussian fits, black line). The ‘mis-crRNA' represents the sequence containing mismatched bases between target and crRNA in PAM-distal region (from +15 to +20) and the ‘bubble DNA' represents the sequence containing mismatched bases between nontarget and target strand in PAM-distal region (from +15 to +20). In b and d , diagrammatic representations are used for PAM (yellow), protospacer (black), crRNA (blue) and the approximate location of mismatched bases on target DNA (red cross).

    Journal: Nature Communications

    Article Title: Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease

    doi: 10.1038/ncomms13350

    Figure Lengend Snippet: Single-molecule FRET analysis for sub-conformation of Cas9:gRNA:DNA. ( a ) Scheme for smFRET experiment. Cas9 with gRNAs that consist of Cy5 (acceptor)-labelled crRNA, tracrRNA, binds to Cy3 (donor)-labelled dsDNA. In the FRET measurement, a relatively low concentration of gRNAs (30 nM) compared with cleavage experiments was used to reduce the background fluorescence of Cy5-labelled crRNA. ( b ) A histogram of the FRET upon binding (we selected the molecules emitting Cy5 signal) to wild-type target DNA exhibits two peaks centred at 0.27 and 0.83, with Gaussian fits (black line). ( c ) A representative time trajectory representing the short-lived open conformation and zipped conformation exhibits two FRET states. The duration of each conformation is measured as the dwell time (Δ t ). The trajectory was imaged right after the injection of Cas9:gRNA into a single-molecule chamber with an integration time of 0.03 s. The points of binding and photo-bleaching were indicated by the black arrows. ( d ) Histograms of FRET upon binding with each PAM-distal mutant exhibit different conformational distribution (with Gaussian fits, black line). The ‘mis-crRNA' represents the sequence containing mismatched bases between target and crRNA in PAM-distal region (from +15 to +20) and the ‘bubble DNA' represents the sequence containing mismatched bases between nontarget and target strand in PAM-distal region (from +15 to +20). In b and d , diagrammatic representations are used for PAM (yellow), protospacer (black), crRNA (blue) and the approximate location of mismatched bases on target DNA (red cross).

    Article Snippet: Cas9 and nucleic acids Recombinant Cas9 and dCas9 from S. pyogenes were overexpressed from Escherichia coli or purchased from New England Biolabs.

    Techniques: Concentration Assay, Fluorescence, Binding Assay, Injection, Mutagenesis, Sequencing

    Identification and quantification of inactive Cas9. ( a ) Three different procedures of our partial pre-incubation experiment. ( b , c ) Quantification of DNA cleavage efficiencies under different pre-incubating conditions: mixing process ( b ) or temperature during pre-incubation ( c ; mean±s.e.m., n ≥5). ( d ) CD spectra of Cas9 without tracrRNA at 25 °C (black) and 37 °C (green). Annealing was performed from 37 °C to 25 °C at a cooling rate of −1 °C min −1 (red dotted line). (e) Kinetic curves of DNA cleavage in the absence (grey) or presence (yellow: Cas9-crRNA, blue: Cas9 alone) of 37 °C pre-incubation. DNA incubation and fluorescence measurement were conducted at 37 °C for all plots in ( b , c , e ). Error bars in e represent s.d.

    Journal: Nature Communications

    Article Title: Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease

    doi: 10.1038/ncomms13350

    Figure Lengend Snippet: Identification and quantification of inactive Cas9. ( a ) Three different procedures of our partial pre-incubation experiment. ( b , c ) Quantification of DNA cleavage efficiencies under different pre-incubating conditions: mixing process ( b ) or temperature during pre-incubation ( c ; mean±s.e.m., n ≥5). ( d ) CD spectra of Cas9 without tracrRNA at 25 °C (black) and 37 °C (green). Annealing was performed from 37 °C to 25 °C at a cooling rate of −1 °C min −1 (red dotted line). (e) Kinetic curves of DNA cleavage in the absence (grey) or presence (yellow: Cas9-crRNA, blue: Cas9 alone) of 37 °C pre-incubation. DNA incubation and fluorescence measurement were conducted at 37 °C for all plots in ( b , c , e ). Error bars in e represent s.d.

    Article Snippet: Cas9 and nucleic acids Recombinant Cas9 and dCas9 from S. pyogenes were overexpressed from Escherichia coli or purchased from New England Biolabs.

    Techniques: Incubation, Fluorescence

    Cas9 cleavage activity with various mutated target DNA. ( a ) The relative target binding (grey bars) versus the fraction of cleaved product (red bars) for DNA sequences containing various positions of mismatched bases. (mean±s.e.m., n ≥5). ( b ) Histograms of FRET upon binding with each mismatched mutant with Gaussian fits (black line). The ratio of high-FRET states to all DNA-bound states calculated from the FRET histograms (white bars) versus the DNA cleavage efficiency (red bars) for different DNA sequences containing various positions of mismatched bases. The base positions are labelled by numbering from the 5′ end of PAM as shown on top panel.

    Journal: Nature Communications

    Article Title: Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease

    doi: 10.1038/ncomms13350

    Figure Lengend Snippet: Cas9 cleavage activity with various mutated target DNA. ( a ) The relative target binding (grey bars) versus the fraction of cleaved product (red bars) for DNA sequences containing various positions of mismatched bases. (mean±s.e.m., n ≥5). ( b ) Histograms of FRET upon binding with each mismatched mutant with Gaussian fits (black line). The ratio of high-FRET states to all DNA-bound states calculated from the FRET histograms (white bars) versus the DNA cleavage efficiency (red bars) for different DNA sequences containing various positions of mismatched bases. The base positions are labelled by numbering from the 5′ end of PAM as shown on top panel.

    Article Snippet: Cas9 and nucleic acids Recombinant Cas9 and dCas9 from S. pyogenes were overexpressed from Escherichia coli or purchased from New England Biolabs.

    Techniques: Activity Assay, Binding Assay, Mutagenesis

    Scheme of the conformational roles of both tracrRNA and crRNA during Cas9 nuclease activity. Model for the conformational regulation of gRNAs during target DNA binding and cleavage process by Cas9:gRNA complex.

    Journal: Nature Communications

    Article Title: Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease

    doi: 10.1038/ncomms13350

    Figure Lengend Snippet: Scheme of the conformational roles of both tracrRNA and crRNA during Cas9 nuclease activity. Model for the conformational regulation of gRNAs during target DNA binding and cleavage process by Cas9:gRNA complex.

    Article Snippet: Cas9 and nucleic acids Recombinant Cas9 and dCas9 from S. pyogenes were overexpressed from Escherichia coli or purchased from New England Biolabs.

    Techniques: Activity Assay, Binding Assay