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pu6  (Addgene inc)


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    Structured Review

    Addgene inc pu6
    Schematic for <t>CRISPR-Cas9-mediated</t> knockin of exogenous DNA fragment into human AAVS1 locus in HEK293 cells Top: the human AAVS1 allele. gRNA sequences are designated (g1 and g2) and located upstream of exon 2. Center: donor plasmid is represented schematically. Dashed lines show homology arms (∼800 bp on each side of donor) between genomic locus and plasmid DNA. A splice acceptor sequence is inserted upstream of RFP gene to allow proper RNA splicing using the endogenous transcriptional promoter. <t>T2A</t> sequences are inserted both upstream and between RFP/AncUOX to allow the release of the individual proteins after translation. SV40 poly(A) signal sequence is designated downstream of the donor DNA. Bottom: engineered allele. Post Cas9 cuts at the site corresponding to the gRNA sequences, donor DNA is integrated into the breakpoint through homology directed repair (HDR).
    Pu6, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pu6+bbs1+mcherry/pU6-(BbsI)_CBh-Cas9-T2A-mcherry-P2A-Ad4E4orf6+(Plasmid+%2364222)/pmc08463316-121-3-10
    Average 93 stars, based on 7 article reviews
    pu6 - by Bioz Stars, 2026-09
    93/100 stars

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    1) Product Images from "CRISPR-Cas9-mediated reactivation of the uricase pseudogene in human cells prevents acute hyperuricemia"

    Article Title: CRISPR-Cas9-mediated reactivation of the uricase pseudogene in human cells prevents acute hyperuricemia

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2021.08.002

    Schematic for CRISPR-Cas9-mediated knockin of exogenous DNA fragment into human AAVS1 locus in HEK293 cells Top: the human AAVS1 allele. gRNA sequences are designated (g1 and g2) and located upstream of exon 2. Center: donor plasmid is represented schematically. Dashed lines show homology arms (∼800 bp on each side of donor) between genomic locus and plasmid DNA. A splice acceptor sequence is inserted upstream of RFP gene to allow proper RNA splicing using the endogenous transcriptional promoter. T2A sequences are inserted both upstream and between RFP/AncUOX to allow the release of the individual proteins after translation. SV40 poly(A) signal sequence is designated downstream of the donor DNA. Bottom: engineered allele. Post Cas9 cuts at the site corresponding to the gRNA sequences, donor DNA is integrated into the breakpoint through homology directed repair (HDR).
    Figure Legend Snippet: Schematic for CRISPR-Cas9-mediated knockin of exogenous DNA fragment into human AAVS1 locus in HEK293 cells Top: the human AAVS1 allele. gRNA sequences are designated (g1 and g2) and located upstream of exon 2. Center: donor plasmid is represented schematically. Dashed lines show homology arms (∼800 bp on each side of donor) between genomic locus and plasmid DNA. A splice acceptor sequence is inserted upstream of RFP gene to allow proper RNA splicing using the endogenous transcriptional promoter. T2A sequences are inserted both upstream and between RFP/AncUOX to allow the release of the individual proteins after translation. SV40 poly(A) signal sequence is designated downstream of the donor DNA. Bottom: engineered allele. Post Cas9 cuts at the site corresponding to the gRNA sequences, donor DNA is integrated into the breakpoint through homology directed repair (HDR).

    Techniques Used: CRISPR, Knock-In, Plasmid Preparation, Sequencing

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    Schematic for <t>CRISPR-Cas9-mediated</t> knockin of exogenous DNA fragment into human AAVS1 locus in HEK293 cells Top: the human AAVS1 allele. gRNA sequences are designated (g1 and g2) and located upstream of exon 2. Center: donor plasmid is represented schematically. Dashed lines show homology arms (∼800 bp on each side of donor) between genomic locus and plasmid DNA. A splice acceptor sequence is inserted upstream of RFP gene to allow proper RNA splicing using the endogenous transcriptional promoter. <t>T2A</t> sequences are inserted both upstream and between RFP/AncUOX to allow the release of the individual proteins after translation. SV40 poly(A) signal sequence is designated downstream of the donor DNA. Bottom: engineered allele. Post Cas9 cuts at the site corresponding to the gRNA sequences, donor DNA is integrated into the breakpoint through homology directed repair (HDR).
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    Schematic for <t>CRISPR-Cas9-mediated</t> knockin of exogenous DNA fragment into human AAVS1 locus in HEK293 cells Top: the human AAVS1 allele. gRNA sequences are designated (g1 and g2) and located upstream of exon 2. Center: donor plasmid is represented schematically. Dashed lines show homology arms (∼800 bp on each side of donor) between genomic locus and plasmid DNA. A splice acceptor sequence is inserted upstream of RFP gene to allow proper RNA splicing using the endogenous transcriptional promoter. <t>T2A</t> sequences are inserted both upstream and between RFP/AncUOX to allow the release of the individual proteins after translation. SV40 poly(A) signal sequence is designated downstream of the donor DNA. Bottom: engineered allele. Post Cas9 cuts at the site corresponding to the gRNA sequences, donor DNA is integrated into the breakpoint through homology directed repair (HDR).
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    Schematic for <t>CRISPR-Cas9-mediated</t> knockin of exogenous DNA fragment into human AAVS1 locus in HEK293 cells Top: the human AAVS1 allele. gRNA sequences are designated (g1 and g2) and located upstream of exon 2. Center: donor plasmid is represented schematically. Dashed lines show homology arms (∼800 bp on each side of donor) between genomic locus and plasmid DNA. A splice acceptor sequence is inserted upstream of RFP gene to allow proper RNA splicing using the endogenous transcriptional promoter. <t>T2A</t> sequences are inserted both upstream and between RFP/AncUOX to allow the release of the individual proteins after translation. SV40 poly(A) signal sequence is designated downstream of the donor DNA. Bottom: engineered allele. Post Cas9 cuts at the site corresponding to the gRNA sequences, donor DNA is integrated into the breakpoint through homology directed repair (HDR).
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    Schematic for CRISPR-Cas9-mediated knockin of exogenous DNA fragment into human AAVS1 locus in HEK293 cells Top: the human AAVS1 allele. gRNA sequences are designated (g1 and g2) and located upstream of exon 2. Center: donor plasmid is represented schematically. Dashed lines show homology arms (∼800 bp on each side of donor) between genomic locus and plasmid DNA. A splice acceptor sequence is inserted upstream of RFP gene to allow proper RNA splicing using the endogenous transcriptional promoter. T2A sequences are inserted both upstream and between RFP/AncUOX to allow the release of the individual proteins after translation. SV40 poly(A) signal sequence is designated downstream of the donor DNA. Bottom: engineered allele. Post Cas9 cuts at the site corresponding to the gRNA sequences, donor DNA is integrated into the breakpoint through homology directed repair (HDR).

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: CRISPR-Cas9-mediated reactivation of the uricase pseudogene in human cells prevents acute hyperuricemia

    doi: 10.1016/j.omtn.2021.08.002

    Figure Lengend Snippet: Schematic for CRISPR-Cas9-mediated knockin of exogenous DNA fragment into human AAVS1 locus in HEK293 cells Top: the human AAVS1 allele. gRNA sequences are designated (g1 and g2) and located upstream of exon 2. Center: donor plasmid is represented schematically. Dashed lines show homology arms (∼800 bp on each side of donor) between genomic locus and plasmid DNA. A splice acceptor sequence is inserted upstream of RFP gene to allow proper RNA splicing using the endogenous transcriptional promoter. T2A sequences are inserted both upstream and between RFP/AncUOX to allow the release of the individual proteins after translation. SV40 poly(A) signal sequence is designated downstream of the donor DNA. Bottom: engineered allele. Post Cas9 cuts at the site corresponding to the gRNA sequences, donor DNA is integrated into the breakpoint through homology directed repair (HDR).

    Article Snippet: To construct the pU6-(Bbs1)_CBh-Cas9-T2A-GFP-p2A-Ad4E4orf6 plasmid (CRISPR plasmid), we digested px458 (Addgene, #48138) and pU6-(BbsI)_CBh-Cas9-T2A-mCherry-P2A-Ad4E4orf6 (Addgene, #64222) with FseI/BsrGI (New England Biolabs).

    Techniques: CRISPR, Knock-In, Plasmid Preparation, Sequencing