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Addgene inc cas9 open reading frame
Deletion of the Fpr1-3 gene cluster by two sgRNAs spanning 95 kb. ( a ) Schematic overview of the strategy to delete a 95 kb DNA fragment on chromosome 17. The exons Fpr1 and Fpr3 are labeled in blue and purple respectively, and the target sites are indicated by arrowheads. PAM sequences are in red following the target sequence highlighted in blue or purple. After deletion of the DNA fragment, the resulting genomic sequence is composed of the 5′ part of Fpr1 (blue) and the 3′ portion of Fpr3 (purple). The locations of PCR primers (F, Forward; R, reverse) are indicated by arrows. ( b ) (Top) Genotyping of the founders. PCR analysis of F0 mice injected with <t>Cas9</t> protein and sgRNAs listed in ( a ). Arrowheads indicate the founders with deletion of the 95 kb genomic DNA fragment. (Below) Sequencing data of the PCR products from two founders showing the joint Fpr1/Fpr3 genomic sequence. M, DNA marker. ( c ) Genotyping analysis of F1 progenies from two founders showing germ line transmission of the 95 kb deletion. M, DNA marker.
Cas9 Open Reading Frame, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos"

Article Title: Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos

Journal: Scientific Reports

doi: 10.1038/srep17517

Deletion of the Fpr1-3 gene cluster by two sgRNAs spanning 95 kb. ( a ) Schematic overview of the strategy to delete a 95 kb DNA fragment on chromosome 17. The exons Fpr1 and Fpr3 are labeled in blue and purple respectively, and the target sites are indicated by arrowheads. PAM sequences are in red following the target sequence highlighted in blue or purple. After deletion of the DNA fragment, the resulting genomic sequence is composed of the 5′ part of Fpr1 (blue) and the 3′ portion of Fpr3 (purple). The locations of PCR primers (F, Forward; R, reverse) are indicated by arrows. ( b ) (Top) Genotyping of the founders. PCR analysis of F0 mice injected with Cas9 protein and sgRNAs listed in ( a ). Arrowheads indicate the founders with deletion of the 95 kb genomic DNA fragment. (Below) Sequencing data of the PCR products from two founders showing the joint Fpr1/Fpr3 genomic sequence. M, DNA marker. ( c ) Genotyping analysis of F1 progenies from two founders showing germ line transmission of the 95 kb deletion. M, DNA marker.
Figure Legend Snippet: Deletion of the Fpr1-3 gene cluster by two sgRNAs spanning 95 kb. ( a ) Schematic overview of the strategy to delete a 95 kb DNA fragment on chromosome 17. The exons Fpr1 and Fpr3 are labeled in blue and purple respectively, and the target sites are indicated by arrowheads. PAM sequences are in red following the target sequence highlighted in blue or purple. After deletion of the DNA fragment, the resulting genomic sequence is composed of the 5′ part of Fpr1 (blue) and the 3′ portion of Fpr3 (purple). The locations of PCR primers (F, Forward; R, reverse) are indicated by arrows. ( b ) (Top) Genotyping of the founders. PCR analysis of F0 mice injected with Cas9 protein and sgRNAs listed in ( a ). Arrowheads indicate the founders with deletion of the 95 kb genomic DNA fragment. (Below) Sequencing data of the PCR products from two founders showing the joint Fpr1/Fpr3 genomic sequence. M, DNA marker. ( c ) Genotyping analysis of F1 progenies from two founders showing germ line transmission of the 95 kb deletion. M, DNA marker.

Techniques Used: Labeling, Sequencing, Polymerase Chain Reaction, Mouse Assay, Injection, Marker, Transmission Assay

Comparison of the knock-in efficiency between injection of Cas9 protein and Cas9 mRNA. ( a ) Schematic overview of the strategy to introduce specific mutations in the Sirt3 locus. PAM sequence is labeled in red, designed mutations are blue. ( b ) The sequence of F0 pups generated from Cas9 mRNA (left) or protein (right) injection are listed. Founders with the desired mutation are labeled with a red asterisk. PAM sequence is red, designed mutations are blue, random mutations are green.
Figure Legend Snippet: Comparison of the knock-in efficiency between injection of Cas9 protein and Cas9 mRNA. ( a ) Schematic overview of the strategy to introduce specific mutations in the Sirt3 locus. PAM sequence is labeled in red, designed mutations are blue. ( b ) The sequence of F0 pups generated from Cas9 mRNA (left) or protein (right) injection are listed. Founders with the desired mutation are labeled with a red asterisk. PAM sequence is red, designed mutations are blue, random mutations are green.

Techniques Used: Knock-In, Injection, Introduce, Sequencing, Labeling, Generated, Mutagenesis

Site-specific insertion of IRES-Cre ERT2 -pA into the mouse Nfatc1 locus . ( a ) Schematic of the strategy for insertion of the CreERT2 cassette into the mouse Nfatc1 locus. Cas9/sgRNA targeted the 3′UTR of the Nfatc1 gene in Exon 9. The HDR donor sequence consists of IRES-CreERT2-polyA (2.7 kb) flanked by two homologous arms 650 bp (left-arm) and 600 bp (right-arm) in length. Positions of genotyping primers are indicated by arrows. ( b ) Genotyping of Nfatc1-CreERT2 mice. Upper, T7E1 assay to check the indels created by CRISPR/Cas. Middle, identification of founder (F0) and F1 Nfatc1-CreERT2 mice by primer pairs: F1 + R1 and F2 + R2. Arrowheads: desired bands of site-specific inserted pups. M, DNA marker. ( c ) Lineage tracing of skin Nfatc1 stem cells in Nfatc1-CreERT2 +/− :Rosa26-LacZ +/− mouse via X-gal staining. Lineage tracing began at the age of 4 weeks by intraperitoneal injection of tamoxifen. Mice dorsal skin tissue was stained 2 days (middle) or 4 weeks (right) after induction. Scale bar, 100 μm.
Figure Legend Snippet: Site-specific insertion of IRES-Cre ERT2 -pA into the mouse Nfatc1 locus . ( a ) Schematic of the strategy for insertion of the CreERT2 cassette into the mouse Nfatc1 locus. Cas9/sgRNA targeted the 3′UTR of the Nfatc1 gene in Exon 9. The HDR donor sequence consists of IRES-CreERT2-polyA (2.7 kb) flanked by two homologous arms 650 bp (left-arm) and 600 bp (right-arm) in length. Positions of genotyping primers are indicated by arrows. ( b ) Genotyping of Nfatc1-CreERT2 mice. Upper, T7E1 assay to check the indels created by CRISPR/Cas. Middle, identification of founder (F0) and F1 Nfatc1-CreERT2 mice by primer pairs: F1 + R1 and F2 + R2. Arrowheads: desired bands of site-specific inserted pups. M, DNA marker. ( c ) Lineage tracing of skin Nfatc1 stem cells in Nfatc1-CreERT2 +/− :Rosa26-LacZ +/− mouse via X-gal staining. Lineage tracing began at the age of 4 weeks by intraperitoneal injection of tamoxifen. Mice dorsal skin tissue was stained 2 days (middle) or 4 weeks (right) after induction. Scale bar, 100 μm.

Techniques Used: Sequencing, Mouse Assay, CRISPR, Marker, Staining, Injection

2) Product Images from "CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus"

Article Title: CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus

Journal: Microbial Cell Factories

doi: 10.1186/s12934-017-0835-1

Deletion of ScURA3 in IMX1187 and IMX585 using RNA III polymerase dependent ( SNR52p ) gRNA expression. a Representation of the native and deleted ScURA3 . The plasmid pUDR107 carried a gRNA under the control of the SNR52p . Primers used for validation of the deletion are indicated. b Validation of transformants of the S. pastorianus IMX1187 strain with pUDR107 in presence or not of a 120 bp repair DNA. The PCR reactions were performed with the primers 9314 and 4728. All lanes (1–20) showed a PCR product of 1698 bp corresponding to the wildtype allele. The lane labelled with L designated the position of the DNA ladder [Gene ruler DNA ladder Mix (ThermoFischer Scientific #SM0332)]. c Sanger sequencing results of purified PCR fragments of ten transformants derived from the transformation of IMX1187 with pUD107 (gRNA URA3 ). The gRNA spacer used to direct Cas9 is indicated in bold and the PAM sequence is underlined. d Validation of transformants of the S. cerevisiae IMX585 strain with pUDR107 in presence or not of a 120 bp repair DNA. The PCR reactions were performed with the primers 4727 and 4728. The lanes (1–10) corresponding to transformants obtained with repair DNA showed a PCR product of 1440 bp corresponding to the deleted allele. The control lane labelled CEN.PK113-7D showed the wild type fragment at 2244 bp. The lane labelled with L designated the position of the DNA ladder
Figure Legend Snippet: Deletion of ScURA3 in IMX1187 and IMX585 using RNA III polymerase dependent ( SNR52p ) gRNA expression. a Representation of the native and deleted ScURA3 . The plasmid pUDR107 carried a gRNA under the control of the SNR52p . Primers used for validation of the deletion are indicated. b Validation of transformants of the S. pastorianus IMX1187 strain with pUDR107 in presence or not of a 120 bp repair DNA. The PCR reactions were performed with the primers 9314 and 4728. All lanes (1–20) showed a PCR product of 1698 bp corresponding to the wildtype allele. The lane labelled with L designated the position of the DNA ladder [Gene ruler DNA ladder Mix (ThermoFischer Scientific #SM0332)]. c Sanger sequencing results of purified PCR fragments of ten transformants derived from the transformation of IMX1187 with pUD107 (gRNA URA3 ). The gRNA spacer used to direct Cas9 is indicated in bold and the PAM sequence is underlined. d Validation of transformants of the S. cerevisiae IMX585 strain with pUDR107 in presence or not of a 120 bp repair DNA. The PCR reactions were performed with the primers 4727 and 4728. The lanes (1–10) corresponding to transformants obtained with repair DNA showed a PCR product of 1440 bp corresponding to the deleted allele. The control lane labelled CEN.PK113-7D showed the wild type fragment at 2244 bp. The lane labelled with L designated the position of the DNA ladder

Techniques Used: Expressing, Plasmid Preparation, Polymerase Chain Reaction, Sequencing, Purification, Derivative Assay, Transformation Assay

Related Articles

Clone Assay:

Article Title: Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos
Article Snippet: .. Recombinant Cas9 expression and purification The Cas9 open reading frame was amplified from pX330 (Addgene #42230) and cloned into pET28a in frame with an N-terminal His-6 tag for in vitro expression. .. Cas9 protein was expressed in Transetta (DE3) (Transgen Biotech), induced at OD 0.8 with 0.2 mM IPTG followed by at least 16 hours of expression at 18 °C.

Article Title: Development of a CRISPR/Cas9 System for Methylococcus capsulatusIn Vivo Gene Editing
Article Snippet: .. The Cas9 open reading frame was amplified, using primers TT16 and CAH537 , from Addgene plasmid no. 42876 ( ) and cloned into pCAH01SpR via Gibson assembly. .. The Cas9D10A nickase variant was generated by site-directed mutagenesis with primers TT143 and TT144 ( ) using the QuikChange primer design program and protocol (Agilent).

Amplification:

Article Title: Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos
Article Snippet: .. Recombinant Cas9 expression and purification The Cas9 open reading frame was amplified from pX330 (Addgene #42230) and cloned into pET28a in frame with an N-terminal His-6 tag for in vitro expression. .. Cas9 protein was expressed in Transetta (DE3) (Transgen Biotech), induced at OD 0.8 with 0.2 mM IPTG followed by at least 16 hours of expression at 18 °C.

Article Title: Development of a CRISPR/Cas9 System for Methylococcus capsulatusIn Vivo Gene Editing
Article Snippet: .. The Cas9 open reading frame was amplified, using primers TT16 and CAH537 , from Addgene plasmid no. 42876 ( ) and cloned into pCAH01SpR via Gibson assembly. .. The Cas9D10A nickase variant was generated by site-directed mutagenesis with primers TT143 and TT144 ( ) using the QuikChange primer design program and protocol (Agilent).

Article Title: CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus
Article Snippet: .. The Streptococcus pyogenes cas9 open reading frame (cas9 D147Y P411T [ ]) was amplified from the plasmid pCT (Addgene plasmid #60621) ( https://www.addgene.org/ ) using the primers 9390 and 9391. .. The AaTEF1 promoter flanked upstream by short homology flank (SHF) B was amplified from the plasmid pUD528 using the primers 3841 and 9394.

In Vitro:

Article Title: Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos
Article Snippet: .. Recombinant Cas9 expression and purification The Cas9 open reading frame was amplified from pX330 (Addgene #42230) and cloned into pET28a in frame with an N-terminal His-6 tag for in vitro expression. .. Cas9 protein was expressed in Transetta (DE3) (Transgen Biotech), induced at OD 0.8 with 0.2 mM IPTG followed by at least 16 hours of expression at 18 °C.

Purification:

Article Title: Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos
Article Snippet: .. Recombinant Cas9 expression and purification The Cas9 open reading frame was amplified from pX330 (Addgene #42230) and cloned into pET28a in frame with an N-terminal His-6 tag for in vitro expression. .. Cas9 protein was expressed in Transetta (DE3) (Transgen Biotech), induced at OD 0.8 with 0.2 mM IPTG followed by at least 16 hours of expression at 18 °C.

Expressing:

Article Title: Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos
Article Snippet: .. Recombinant Cas9 expression and purification The Cas9 open reading frame was amplified from pX330 (Addgene #42230) and cloned into pET28a in frame with an N-terminal His-6 tag for in vitro expression. .. Cas9 protein was expressed in Transetta (DE3) (Transgen Biotech), induced at OD 0.8 with 0.2 mM IPTG followed by at least 16 hours of expression at 18 °C.

Recombinant:

Article Title: Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos
Article Snippet: .. Recombinant Cas9 expression and purification The Cas9 open reading frame was amplified from pX330 (Addgene #42230) and cloned into pET28a in frame with an N-terminal His-6 tag for in vitro expression. .. Cas9 protein was expressed in Transetta (DE3) (Transgen Biotech), induced at OD 0.8 with 0.2 mM IPTG followed by at least 16 hours of expression at 18 °C.

Plasmid Preparation:

Article Title: Generation of Highly Enriched V2a Interneurons from Mouse Embryonic Stem Cells
Article Snippet: .. The p3s-Cas9HC vector contains the Cas9 open reading frame (Addgene plasmid #43945) ( ). .. 1x107 RW4 ESCs were resuspended in electroporation buffer (20 mM HEPES pH 7.5, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4 , and 6 mM dextrose) with 8 μg Chx10-Puro selection vector, 1 μg Cas9 vector, and 1 μg gChx10 vector.

Article Title: Development of a CRISPR/Cas9 System for Methylococcus capsulatusIn Vivo Gene Editing
Article Snippet: .. The Cas9 open reading frame was amplified, using primers TT16 and CAH537 , from Addgene plasmid no. 42876 ( ) and cloned into pCAH01SpR via Gibson assembly. .. The Cas9D10A nickase variant was generated by site-directed mutagenesis with primers TT143 and TT144 ( ) using the QuikChange primer design program and protocol (Agilent).

Article Title: CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus
Article Snippet: .. The Streptococcus pyogenes cas9 open reading frame (cas9 D147Y P411T [ ]) was amplified from the plasmid pCT (Addgene plasmid #60621) ( https://www.addgene.org/ ) using the primers 9390 and 9391. .. The AaTEF1 promoter flanked upstream by short homology flank (SHF) B was amplified from the plasmid pUD528 using the primers 3841 and 9394.

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    Addgene inc hdqp1 cas9 expressing cells
    GREP1 regulates GDF15. a) Cytokine profiling in HEK293T cells with transient ectopic GREP1 or GFP overexpression, ZR-75-1 cells with stable GREP1 knockout, or <t>HDQP1</t> cells with stable GREP1 knockout. The change in signal abundance was calculated for each control/GREP1 pair. To rank cytokines, the average of the absolute values for the individual signal changes was plotted. b) GDF15 abundance by ELISA in ZR-75-1 and CAMA-1 cells overexpressing a GREP1 or GFP cDNA plasmid. c) Spearman’s rho for GREP1 expression correlation with GDF15, EMILIN2, or FN1 in the indicated TCGA datasets. d) Spearman’s p value for the GREP1 correlation coefficient for GREP1 correlation with GDF15, EMILIN2, or FN1 in the indicated TCGA datasets. e-g) Recombinant GDF15 partially rescues GREP1 knockout. CAMA-1, ZR-75-1 or T47D <t>Cas9</t> cells were infected with the indicated sgRNAs. 24 hours after infection, cells were treated with vehicle control or increasing concentration of recombinant human GDF15 as shown. Relative abundance was measured 7 days after infection. All error bars represent standard deviation.
    Hdqp1 Cas9 Expressing Cells, supplied by Addgene inc, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hdqp1 cas9 expressing cells/product/Addgene inc
    Average 84 stars, based on 1 article reviews
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    91
    Addgene inc cas9 cds
    Strategy for episome-based <t>Cas9</t> genome editing. ( A ) Key features of the Cas9- and the sgRNA-episome. Black arrow: PARP (procyclic acidic repetitive protein) promoter; HA: HA-epitope; NLS: SV40 nuclear localization signal; hSpCas9 : human codon-optimized Cas9 ; PUR R : puromycin N-acetyl transferase <t>CDS</t> conferring resistance to puromycin; G418 R : neomycin phosphotransferase conferring resistance to G418; HH: hammerhead ribozyme; crRNA: CRISPR-RNA; tracrRNA: trans activating RNA; HDV: hepatitis delta virus ribozyme; repair: sequence providing the DNA repair template carrying the desired mutations. ( B ) Sequence of the genomic target on Chr 11, 140,068-140,155. SCD6 ORF: black, uppercase; SCD6 3′ UTR: black, lower case; protospacer: purple highlight; PAM: green. ( C ) Strategy for the marker-free tagging of the SCD6 ORF. The purple triangle indicates the protospacer location targeted by the crRNA (not drawn to scale). The repair template encompasses the eGFP ORF flanked by 220 bp of the 3′-end of the SCD6 ORF and 220 bp of its 3′-UTR.
    Cas9 Cds, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cas9 cds/product/Addgene inc
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    Price from $9.99 to $1999.99
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    88
    Addgene inc cas9 open reading frame
    Deletion of the Fpr1-3 gene cluster by two sgRNAs spanning 95 kb. ( a ) Schematic overview of the strategy to delete a 95 kb DNA fragment on chromosome 17. The exons Fpr1 and Fpr3 are labeled in blue and purple respectively, and the target sites are indicated by arrowheads. PAM sequences are in red following the target sequence highlighted in blue or purple. After deletion of the DNA fragment, the resulting genomic sequence is composed of the 5′ part of Fpr1 (blue) and the 3′ portion of Fpr3 (purple). The locations of PCR primers (F, Forward; R, reverse) are indicated by arrows. ( b ) (Top) Genotyping of the founders. PCR analysis of F0 mice injected with <t>Cas9</t> protein and sgRNAs listed in ( a ). Arrowheads indicate the founders with deletion of the 95 kb genomic DNA fragment. (Below) Sequencing data of the PCR products from two founders showing the joint Fpr1/Fpr3 genomic sequence. M, DNA marker. ( c ) Genotyping analysis of F1 progenies from two founders showing germ line transmission of the 95 kb deletion. M, DNA marker.
    Cas9 Open Reading Frame, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cas9 open reading frame/product/Addgene inc
    Average 88 stars, based on 1 article reviews
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    GREP1 regulates GDF15. a) Cytokine profiling in HEK293T cells with transient ectopic GREP1 or GFP overexpression, ZR-75-1 cells with stable GREP1 knockout, or HDQP1 cells with stable GREP1 knockout. The change in signal abundance was calculated for each control/GREP1 pair. To rank cytokines, the average of the absolute values for the individual signal changes was plotted. b) GDF15 abundance by ELISA in ZR-75-1 and CAMA-1 cells overexpressing a GREP1 or GFP cDNA plasmid. c) Spearman’s rho for GREP1 expression correlation with GDF15, EMILIN2, or FN1 in the indicated TCGA datasets. d) Spearman’s p value for the GREP1 correlation coefficient for GREP1 correlation with GDF15, EMILIN2, or FN1 in the indicated TCGA datasets. e-g) Recombinant GDF15 partially rescues GREP1 knockout. CAMA-1, ZR-75-1 or T47D Cas9 cells were infected with the indicated sgRNAs. 24 hours after infection, cells were treated with vehicle control or increasing concentration of recombinant human GDF15 as shown. Relative abundance was measured 7 days after infection. All error bars represent standard deviation.

    Journal: bioRxiv

    Article Title: Non-canonical open reading frames encode functional proteins essential for cancer cell survival

    doi: 10.1101/2020.03.10.981001

    Figure Lengend Snippet: GREP1 regulates GDF15. a) Cytokine profiling in HEK293T cells with transient ectopic GREP1 or GFP overexpression, ZR-75-1 cells with stable GREP1 knockout, or HDQP1 cells with stable GREP1 knockout. The change in signal abundance was calculated for each control/GREP1 pair. To rank cytokines, the average of the absolute values for the individual signal changes was plotted. b) GDF15 abundance by ELISA in ZR-75-1 and CAMA-1 cells overexpressing a GREP1 or GFP cDNA plasmid. c) Spearman’s rho for GREP1 expression correlation with GDF15, EMILIN2, or FN1 in the indicated TCGA datasets. d) Spearman’s p value for the GREP1 correlation coefficient for GREP1 correlation with GDF15, EMILIN2, or FN1 in the indicated TCGA datasets. e-g) Recombinant GDF15 partially rescues GREP1 knockout. CAMA-1, ZR-75-1 or T47D Cas9 cells were infected with the indicated sgRNAs. 24 hours after infection, cells were treated with vehicle control or increasing concentration of recombinant human GDF15 as shown. Relative abundance was measured 7 days after infection. All error bars represent standard deviation.

    Article Snippet: For stable knockout cell lines, ZR-75-1 Cas9 and HDQP1 Cas9 expressing cells were infected with lentivirus for the indicated sgRNAs which had been cloned into the LentiGuide-Puro plasmid (plasmid #52963, Addgene) with 4ug/mL of polybrene.

    Techniques: Over Expression, Knock-Out, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Expressing, Recombinant, Infection, Concentration Assay, Standard Deviation

    Strategy for episome-based Cas9 genome editing. ( A ) Key features of the Cas9- and the sgRNA-episome. Black arrow: PARP (procyclic acidic repetitive protein) promoter; HA: HA-epitope; NLS: SV40 nuclear localization signal; hSpCas9 : human codon-optimized Cas9 ; PUR R : puromycin N-acetyl transferase CDS conferring resistance to puromycin; G418 R : neomycin phosphotransferase conferring resistance to G418; HH: hammerhead ribozyme; crRNA: CRISPR-RNA; tracrRNA: trans activating RNA; HDV: hepatitis delta virus ribozyme; repair: sequence providing the DNA repair template carrying the desired mutations. ( B ) Sequence of the genomic target on Chr 11, 140,068-140,155. SCD6 ORF: black, uppercase; SCD6 3′ UTR: black, lower case; protospacer: purple highlight; PAM: green. ( C ) Strategy for the marker-free tagging of the SCD6 ORF. The purple triangle indicates the protospacer location targeted by the crRNA (not drawn to scale). The repair template encompasses the eGFP ORF flanked by 220 bp of the 3′-end of the SCD6 ORF and 220 bp of its 3′-UTR.

    Journal: Nucleic Acids Research

    Article Title: Exploiting CRISPR–Cas9 technology to investigate individual histone modifications

    doi: 10.1093/nar/gky517

    Figure Lengend Snippet: Strategy for episome-based Cas9 genome editing. ( A ) Key features of the Cas9- and the sgRNA-episome. Black arrow: PARP (procyclic acidic repetitive protein) promoter; HA: HA-epitope; NLS: SV40 nuclear localization signal; hSpCas9 : human codon-optimized Cas9 ; PUR R : puromycin N-acetyl transferase CDS conferring resistance to puromycin; G418 R : neomycin phosphotransferase conferring resistance to G418; HH: hammerhead ribozyme; crRNA: CRISPR-RNA; tracrRNA: trans activating RNA; HDV: hepatitis delta virus ribozyme; repair: sequence providing the DNA repair template carrying the desired mutations. ( B ) Sequence of the genomic target on Chr 11, 140,068-140,155. SCD6 ORF: black, uppercase; SCD6 3′ UTR: black, lower case; protospacer: purple highlight; PAM: green. ( C ) Strategy for the marker-free tagging of the SCD6 ORF. The purple triangle indicates the protospacer location targeted by the crRNA (not drawn to scale). The repair template encompasses the eGFP ORF flanked by 220 bp of the 3′-end of the SCD6 ORF and 220 bp of its 3′-UTR.

    Article Snippet: To add an HA-tag and an SV40 NLS to hSpCas9 CDS, the Cas9 CDS was amplified from pX330 (Addgene plasmid #42230, ( )) using the oligo pair ocas_9/10 from pX330, and ligated into pRPa ( ) after both fragments were digested with HindIII and BamHI, yielding pCW20.

    Techniques: CRISPR, Sequencing, Marker

    Cas9 allows multiple genome edits. ( A ) Strategy used for the removal of the sgRNA-episome. ( B ) PCR-based analysis of cells sequentially transfected with the Cas9-episome and sgRNA-episome(ΔH3.V) or sgRNA-episome(ΔH4.V). Top panel: outline indicating primer binding sites (half arrows). Bottom panel: agarose gel revealing presence or absence of H3.V (left) or H4.V (right). Homozygous deletions of H4.V were only observed after the culture had been diluted a second time. ( C ) Sanger sequencing demonstrated that all editing events exactly matched the repair template. The deleted CDSs of H3.V and H4.V are represented on top and highlighted in orange. The repair templates are shown in the middle (5′-UTR in green, 5′-end of the H3.V CDS in orange and 3′-UTR in purple. The sequencing chromatograms are depicted at the bottom.

    Journal: Nucleic Acids Research

    Article Title: Exploiting CRISPR–Cas9 technology to investigate individual histone modifications

    doi: 10.1093/nar/gky517

    Figure Lengend Snippet: Cas9 allows multiple genome edits. ( A ) Strategy used for the removal of the sgRNA-episome. ( B ) PCR-based analysis of cells sequentially transfected with the Cas9-episome and sgRNA-episome(ΔH3.V) or sgRNA-episome(ΔH4.V). Top panel: outline indicating primer binding sites (half arrows). Bottom panel: agarose gel revealing presence or absence of H3.V (left) or H4.V (right). Homozygous deletions of H4.V were only observed after the culture had been diluted a second time. ( C ) Sanger sequencing demonstrated that all editing events exactly matched the repair template. The deleted CDSs of H3.V and H4.V are represented on top and highlighted in orange. The repair templates are shown in the middle (5′-UTR in green, 5′-end of the H3.V CDS in orange and 3′-UTR in purple. The sequencing chromatograms are depicted at the bottom.

    Article Snippet: To add an HA-tag and an SV40 NLS to hSpCas9 CDS, the Cas9 CDS was amplified from pX330 (Addgene plasmid #42230, ( )) using the oligo pair ocas_9/10 from pX330, and ligated into pRPa ( ) after both fragments were digested with HindIII and BamHI, yielding pCW20.

    Techniques: Polymerase Chain Reaction, Transfection, Binding Assay, Agarose Gel Electrophoresis, Sequencing

    Strategy for episome-based Cas9 genome editing. ( A ) Key features of the Cas9- and the sgRNA-episome. Black arrow: PARP (procyclic acidic repetitive protein) promoter; HA: HA-epitope; NLS: SV40 nuclear localization signal; hSpCas9 : human codon-optimized Cas9 ; PUR R : puromycin N-acetyl transferase CDS conferring resistance to puromycin; G418 R : neomycin phosphotransferase conferring resistance to G418; HH: hammerhead ribozyme; crRNA: CRISPR-RNA; tracrRNA: trans activating RNA; HDV: hepatitis delta virus ribozyme; repair: sequence providing the DNA repair template carrying the desired mutations. ( B ) Sequence of the genomic target on Chr 11, 140,068-140,155. SCD6 ORF: black, uppercase; SCD6 3′ UTR: black, lower case; protospacer: purple highlight; PAM: green. ( C ) Strategy for the marker-free tagging of the SCD6 ORF. The purple triangle indicates the protospacer location targeted by the crRNA (not drawn to scale). The repair template encompasses the eGFP ORF flanked by 220 bp of the 3′-end of the SCD6 ORF and 220 bp of its 3′-UTR.

    Journal: Nucleic Acids Research

    Article Title: Exploiting CRISPR–Cas9 technology to investigate individual histone modifications

    doi: 10.1093/nar/gky517

    Figure Lengend Snippet: Strategy for episome-based Cas9 genome editing. ( A ) Key features of the Cas9- and the sgRNA-episome. Black arrow: PARP (procyclic acidic repetitive protein) promoter; HA: HA-epitope; NLS: SV40 nuclear localization signal; hSpCas9 : human codon-optimized Cas9 ; PUR R : puromycin N-acetyl transferase CDS conferring resistance to puromycin; G418 R : neomycin phosphotransferase conferring resistance to G418; HH: hammerhead ribozyme; crRNA: CRISPR-RNA; tracrRNA: trans activating RNA; HDV: hepatitis delta virus ribozyme; repair: sequence providing the DNA repair template carrying the desired mutations. ( B ) Sequence of the genomic target on Chr 11, 140,068-140,155. SCD6 ORF: black, uppercase; SCD6 3′ UTR: black, lower case; protospacer: purple highlight; PAM: green. ( C ) Strategy for the marker-free tagging of the SCD6 ORF. The purple triangle indicates the protospacer location targeted by the crRNA (not drawn to scale). The repair template encompasses the eGFP ORF flanked by 220 bp of the 3′-end of the SCD6 ORF and 220 bp of its 3′-UTR.

    Article Snippet: To add an HA-tag and an SV40 NLS to hSpCas9 CDS, the Cas9 CDS was amplified from pX330 (Addgene plasmid #42230, ( )) using the oligo pair ocas_9/10 from pX330, and ligated into pRPa ( ) after both fragments were digested with HindIII and BamHI, yielding pCW20.

    Techniques: CRISPR, Sequencing, Marker

    Cas9 allows multiple genome edits. ( A ) Strategy used for the removal of the sgRNA-episome. ( B ) PCR-based analysis of cells sequentially transfected with the Cas9-episome and sgRNA-episome(ΔH3.V) or sgRNA-episome(ΔH4.V). Top panel: outline indicating primer binding sites (half arrows). Bottom panel: agarose gel revealing presence or absence of H3.V (left) or H4.V (right). Homozygous deletions of H4.V were only observed after the culture had been diluted a second time. ( C ) Sanger sequencing demonstrated that all editing events exactly matched the repair template. The deleted CDSs of H3.V and H4.V are represented on top and highlighted in orange. The repair templates are shown in the middle (5′-UTR in green, 5′-end of the H3.V CDS in orange and 3′-UTR in purple. The sequencing chromatograms are depicted at the bottom.

    Journal: Nucleic Acids Research

    Article Title: Exploiting CRISPR–Cas9 technology to investigate individual histone modifications

    doi: 10.1093/nar/gky517

    Figure Lengend Snippet: Cas9 allows multiple genome edits. ( A ) Strategy used for the removal of the sgRNA-episome. ( B ) PCR-based analysis of cells sequentially transfected with the Cas9-episome and sgRNA-episome(ΔH3.V) or sgRNA-episome(ΔH4.V). Top panel: outline indicating primer binding sites (half arrows). Bottom panel: agarose gel revealing presence or absence of H3.V (left) or H4.V (right). Homozygous deletions of H4.V were only observed after the culture had been diluted a second time. ( C ) Sanger sequencing demonstrated that all editing events exactly matched the repair template. The deleted CDSs of H3.V and H4.V are represented on top and highlighted in orange. The repair templates are shown in the middle (5′-UTR in green, 5′-end of the H3.V CDS in orange and 3′-UTR in purple. The sequencing chromatograms are depicted at the bottom.

    Article Snippet: To add an HA-tag and an SV40 NLS to hSpCas9 CDS, the Cas9 CDS was amplified from pX330 (Addgene plasmid #42230, ( )) using the oligo pair ocas_9/10 from pX330, and ligated into pRPa ( ) after both fragments were digested with HindIII and BamHI, yielding pCW20.

    Techniques: Polymerase Chain Reaction, Transfection, Binding Assay, Agarose Gel Electrophoresis, Sequencing

    Deletion of the Fpr1-3 gene cluster by two sgRNAs spanning 95 kb. ( a ) Schematic overview of the strategy to delete a 95 kb DNA fragment on chromosome 17. The exons Fpr1 and Fpr3 are labeled in blue and purple respectively, and the target sites are indicated by arrowheads. PAM sequences are in red following the target sequence highlighted in blue or purple. After deletion of the DNA fragment, the resulting genomic sequence is composed of the 5′ part of Fpr1 (blue) and the 3′ portion of Fpr3 (purple). The locations of PCR primers (F, Forward; R, reverse) are indicated by arrows. ( b ) (Top) Genotyping of the founders. PCR analysis of F0 mice injected with Cas9 protein and sgRNAs listed in ( a ). Arrowheads indicate the founders with deletion of the 95 kb genomic DNA fragment. (Below) Sequencing data of the PCR products from two founders showing the joint Fpr1/Fpr3 genomic sequence. M, DNA marker. ( c ) Genotyping analysis of F1 progenies from two founders showing germ line transmission of the 95 kb deletion. M, DNA marker.

    Journal: Scientific Reports

    Article Title: Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos

    doi: 10.1038/srep17517

    Figure Lengend Snippet: Deletion of the Fpr1-3 gene cluster by two sgRNAs spanning 95 kb. ( a ) Schematic overview of the strategy to delete a 95 kb DNA fragment on chromosome 17. The exons Fpr1 and Fpr3 are labeled in blue and purple respectively, and the target sites are indicated by arrowheads. PAM sequences are in red following the target sequence highlighted in blue or purple. After deletion of the DNA fragment, the resulting genomic sequence is composed of the 5′ part of Fpr1 (blue) and the 3′ portion of Fpr3 (purple). The locations of PCR primers (F, Forward; R, reverse) are indicated by arrows. ( b ) (Top) Genotyping of the founders. PCR analysis of F0 mice injected with Cas9 protein and sgRNAs listed in ( a ). Arrowheads indicate the founders with deletion of the 95 kb genomic DNA fragment. (Below) Sequencing data of the PCR products from two founders showing the joint Fpr1/Fpr3 genomic sequence. M, DNA marker. ( c ) Genotyping analysis of F1 progenies from two founders showing germ line transmission of the 95 kb deletion. M, DNA marker.

    Article Snippet: Recombinant Cas9 expression and purification The Cas9 open reading frame was amplified from pX330 (Addgene #42230) and cloned into pET28a in frame with an N-terminal His-6 tag for in vitro expression.

    Techniques: Labeling, Sequencing, Polymerase Chain Reaction, Mouse Assay, Injection, Marker, Transmission Assay

    Comparison of the knock-in efficiency between injection of Cas9 protein and Cas9 mRNA. ( a ) Schematic overview of the strategy to introduce specific mutations in the Sirt3 locus. PAM sequence is labeled in red, designed mutations are blue. ( b ) The sequence of F0 pups generated from Cas9 mRNA (left) or protein (right) injection are listed. Founders with the desired mutation are labeled with a red asterisk. PAM sequence is red, designed mutations are blue, random mutations are green.

    Journal: Scientific Reports

    Article Title: Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos

    doi: 10.1038/srep17517

    Figure Lengend Snippet: Comparison of the knock-in efficiency between injection of Cas9 protein and Cas9 mRNA. ( a ) Schematic overview of the strategy to introduce specific mutations in the Sirt3 locus. PAM sequence is labeled in red, designed mutations are blue. ( b ) The sequence of F0 pups generated from Cas9 mRNA (left) or protein (right) injection are listed. Founders with the desired mutation are labeled with a red asterisk. PAM sequence is red, designed mutations are blue, random mutations are green.

    Article Snippet: Recombinant Cas9 expression and purification The Cas9 open reading frame was amplified from pX330 (Addgene #42230) and cloned into pET28a in frame with an N-terminal His-6 tag for in vitro expression.

    Techniques: Knock-In, Injection, Introduce, Sequencing, Labeling, Generated, Mutagenesis

    Site-specific insertion of IRES-Cre ERT2 -pA into the mouse Nfatc1 locus . ( a ) Schematic of the strategy for insertion of the CreERT2 cassette into the mouse Nfatc1 locus. Cas9/sgRNA targeted the 3′UTR of the Nfatc1 gene in Exon 9. The HDR donor sequence consists of IRES-CreERT2-polyA (2.7 kb) flanked by two homologous arms 650 bp (left-arm) and 600 bp (right-arm) in length. Positions of genotyping primers are indicated by arrows. ( b ) Genotyping of Nfatc1-CreERT2 mice. Upper, T7E1 assay to check the indels created by CRISPR/Cas. Middle, identification of founder (F0) and F1 Nfatc1-CreERT2 mice by primer pairs: F1 + R1 and F2 + R2. Arrowheads: desired bands of site-specific inserted pups. M, DNA marker. ( c ) Lineage tracing of skin Nfatc1 stem cells in Nfatc1-CreERT2 +/− :Rosa26-LacZ +/− mouse via X-gal staining. Lineage tracing began at the age of 4 weeks by intraperitoneal injection of tamoxifen. Mice dorsal skin tissue was stained 2 days (middle) or 4 weeks (right) after induction. Scale bar, 100 μm.

    Journal: Scientific Reports

    Article Title: Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos

    doi: 10.1038/srep17517

    Figure Lengend Snippet: Site-specific insertion of IRES-Cre ERT2 -pA into the mouse Nfatc1 locus . ( a ) Schematic of the strategy for insertion of the CreERT2 cassette into the mouse Nfatc1 locus. Cas9/sgRNA targeted the 3′UTR of the Nfatc1 gene in Exon 9. The HDR donor sequence consists of IRES-CreERT2-polyA (2.7 kb) flanked by two homologous arms 650 bp (left-arm) and 600 bp (right-arm) in length. Positions of genotyping primers are indicated by arrows. ( b ) Genotyping of Nfatc1-CreERT2 mice. Upper, T7E1 assay to check the indels created by CRISPR/Cas. Middle, identification of founder (F0) and F1 Nfatc1-CreERT2 mice by primer pairs: F1 + R1 and F2 + R2. Arrowheads: desired bands of site-specific inserted pups. M, DNA marker. ( c ) Lineage tracing of skin Nfatc1 stem cells in Nfatc1-CreERT2 +/− :Rosa26-LacZ +/− mouse via X-gal staining. Lineage tracing began at the age of 4 weeks by intraperitoneal injection of tamoxifen. Mice dorsal skin tissue was stained 2 days (middle) or 4 weeks (right) after induction. Scale bar, 100 μm.

    Article Snippet: Recombinant Cas9 expression and purification The Cas9 open reading frame was amplified from pX330 (Addgene #42230) and cloned into pET28a in frame with an N-terminal His-6 tag for in vitro expression.

    Techniques: Sequencing, Mouse Assay, CRISPR, Marker, Staining, Injection