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grna scaffold sequence  (Addgene inc)


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    Addgene inc grna scaffold sequence
    Results of CRISPR–Cas9 plasmids for targeted gene disruption in A. fijiensis . ( a ) <t>The</t> <t>sgRNA</t> scaffold sequence was amplified from plasmid pX330 using primers <t>gRNA-scaffold-F</t> and gRNA-scaffold-R. The predicted endogenous U6 promoter was amplified from the A. fijiensis genome using adaptor primers U6-1-F and U6-1-R containing 5′ overlapping sequences of the sgRNA scaffold. the 20 bp pyrG -targeting sgRNA sequence was seamlessly inserted between the U6 promoter and sgRNA scaffold using primers U6- pyrG -F and U6-1- pyrG -R. The plasmid backbone containing the Cas9 open reading frame and the AMA1 autonomous replication element was amplified from plasmid FM-6 using primers pAMA-1-F and pAMA1-R. The U6- pyrG -sgRNA expression cassette was amplified from pPu6- pyrG -sgRNA using primers U6-1-F and gRNA-scaffold-R, and subsequently inserted into the Cas9-containing backbone by homologous recombination to generate the final plasmid AFM-Δ pyrG . For clarity, DNA elements in the schematic are not drawn to scale. ( b ) Targeted disruption of the pyrG gene mediated by plasmid-based CRISPR-Cas9 editing in A. fijiensis . Diagnostic PCR analysis of genomic DNA from independent transformants in pyrG locus. The DNA molecular weight marker used was a 100–5000 bp DNA Marker III (Biosharp BL103A, Anhui, China). PCR amplification was performed using primers flanking the targeted integration region to distinguish wild-type and disrupted alleles.
    Grna Scaffold Sequence, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 2984 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/u6+grna+scaffold/pX330-U6-Chimeric_BB-CBh-hSpCas9+(Plasmid+%2342230)/pmc13027702-112-13-19
    Average 96 stars, based on 2984 article reviews
    grna scaffold sequence - by Bioz Stars, 2026-09
    96/100 stars

    Images

    1) Product Images from "A High-Efficiency CRISPR–Cas9 Ribonucleoprotein Genome Editing System in Aspergillus fijiensis Enabled by Microhomology-Mediated End Joining"

    Article Title: A High-Efficiency CRISPR–Cas9 Ribonucleoprotein Genome Editing System in Aspergillus fijiensis Enabled by Microhomology-Mediated End Joining

    Journal: Journal of Fungi

    doi: 10.3390/jof12030165

    Results of CRISPR–Cas9 plasmids for targeted gene disruption in A. fijiensis . ( a ) The sgRNA scaffold sequence was amplified from plasmid pX330 using primers gRNA-scaffold-F and gRNA-scaffold-R. The predicted endogenous U6 promoter was amplified from the A. fijiensis genome using adaptor primers U6-1-F and U6-1-R containing 5′ overlapping sequences of the sgRNA scaffold. the 20 bp pyrG -targeting sgRNA sequence was seamlessly inserted between the U6 promoter and sgRNA scaffold using primers U6- pyrG -F and U6-1- pyrG -R. The plasmid backbone containing the Cas9 open reading frame and the AMA1 autonomous replication element was amplified from plasmid FM-6 using primers pAMA-1-F and pAMA1-R. The U6- pyrG -sgRNA expression cassette was amplified from pPu6- pyrG -sgRNA using primers U6-1-F and gRNA-scaffold-R, and subsequently inserted into the Cas9-containing backbone by homologous recombination to generate the final plasmid AFM-Δ pyrG . For clarity, DNA elements in the schematic are not drawn to scale. ( b ) Targeted disruption of the pyrG gene mediated by plasmid-based CRISPR-Cas9 editing in A. fijiensis . Diagnostic PCR analysis of genomic DNA from independent transformants in pyrG locus. The DNA molecular weight marker used was a 100–5000 bp DNA Marker III (Biosharp BL103A, Anhui, China). PCR amplification was performed using primers flanking the targeted integration region to distinguish wild-type and disrupted alleles.
    Figure Legend Snippet: Results of CRISPR–Cas9 plasmids for targeted gene disruption in A. fijiensis . ( a ) The sgRNA scaffold sequence was amplified from plasmid pX330 using primers gRNA-scaffold-F and gRNA-scaffold-R. The predicted endogenous U6 promoter was amplified from the A. fijiensis genome using adaptor primers U6-1-F and U6-1-R containing 5′ overlapping sequences of the sgRNA scaffold. the 20 bp pyrG -targeting sgRNA sequence was seamlessly inserted between the U6 promoter and sgRNA scaffold using primers U6- pyrG -F and U6-1- pyrG -R. The plasmid backbone containing the Cas9 open reading frame and the AMA1 autonomous replication element was amplified from plasmid FM-6 using primers pAMA-1-F and pAMA1-R. The U6- pyrG -sgRNA expression cassette was amplified from pPu6- pyrG -sgRNA using primers U6-1-F and gRNA-scaffold-R, and subsequently inserted into the Cas9-containing backbone by homologous recombination to generate the final plasmid AFM-Δ pyrG . For clarity, DNA elements in the schematic are not drawn to scale. ( b ) Targeted disruption of the pyrG gene mediated by plasmid-based CRISPR-Cas9 editing in A. fijiensis . Diagnostic PCR analysis of genomic DNA from independent transformants in pyrG locus. The DNA molecular weight marker used was a 100–5000 bp DNA Marker III (Biosharp BL103A, Anhui, China). PCR amplification was performed using primers flanking the targeted integration region to distinguish wild-type and disrupted alleles.

    Techniques Used: CRISPR, Disruption, Sequencing, Amplification, Plasmid Preparation, Expressing, Homologous Recombination, Diagnostic Assay, Molecular Weight, Marker



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    Results of CRISPR–Cas9 plasmids for targeted gene disruption in A. fijiensis . ( a ) <t>The</t> <t>sgRNA</t> scaffold sequence was amplified from plasmid pX330 using primers <t>gRNA-scaffold-F</t> and gRNA-scaffold-R. The predicted endogenous U6 promoter was amplified from the A. fijiensis genome using adaptor primers U6-1-F and U6-1-R containing 5′ overlapping sequences of the sgRNA scaffold. the 20 bp pyrG -targeting sgRNA sequence was seamlessly inserted between the U6 promoter and sgRNA scaffold using primers U6- pyrG -F and U6-1- pyrG -R. The plasmid backbone containing the Cas9 open reading frame and the AMA1 autonomous replication element was amplified from plasmid FM-6 using primers pAMA-1-F and pAMA1-R. The U6- pyrG -sgRNA expression cassette was amplified from pPu6- pyrG -sgRNA using primers U6-1-F and gRNA-scaffold-R, and subsequently inserted into the Cas9-containing backbone by homologous recombination to generate the final plasmid AFM-Δ pyrG . For clarity, DNA elements in the schematic are not drawn to scale. ( b ) Targeted disruption of the pyrG gene mediated by plasmid-based CRISPR-Cas9 editing in A. fijiensis . Diagnostic PCR analysis of genomic DNA from independent transformants in pyrG locus. The DNA molecular weight marker used was a 100–5000 bp DNA Marker III (Biosharp BL103A, Anhui, China). PCR amplification was performed using primers flanking the targeted integration region to distinguish wild-type and disrupted alleles.
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    Image Search Results


    Results of CRISPR–Cas9 plasmids for targeted gene disruption in A. fijiensis . ( a ) The sgRNA scaffold sequence was amplified from plasmid pX330 using primers gRNA-scaffold-F and gRNA-scaffold-R. The predicted endogenous U6 promoter was amplified from the A. fijiensis genome using adaptor primers U6-1-F and U6-1-R containing 5′ overlapping sequences of the sgRNA scaffold. the 20 bp pyrG -targeting sgRNA sequence was seamlessly inserted between the U6 promoter and sgRNA scaffold using primers U6- pyrG -F and U6-1- pyrG -R. The plasmid backbone containing the Cas9 open reading frame and the AMA1 autonomous replication element was amplified from plasmid FM-6 using primers pAMA-1-F and pAMA1-R. The U6- pyrG -sgRNA expression cassette was amplified from pPu6- pyrG -sgRNA using primers U6-1-F and gRNA-scaffold-R, and subsequently inserted into the Cas9-containing backbone by homologous recombination to generate the final plasmid AFM-Δ pyrG . For clarity, DNA elements in the schematic are not drawn to scale. ( b ) Targeted disruption of the pyrG gene mediated by plasmid-based CRISPR-Cas9 editing in A. fijiensis . Diagnostic PCR analysis of genomic DNA from independent transformants in pyrG locus. The DNA molecular weight marker used was a 100–5000 bp DNA Marker III (Biosharp BL103A, Anhui, China). PCR amplification was performed using primers flanking the targeted integration region to distinguish wild-type and disrupted alleles.

    Journal: Journal of Fungi

    Article Title: A High-Efficiency CRISPR–Cas9 Ribonucleoprotein Genome Editing System in Aspergillus fijiensis Enabled by Microhomology-Mediated End Joining

    doi: 10.3390/jof12030165

    Figure Lengend Snippet: Results of CRISPR–Cas9 plasmids for targeted gene disruption in A. fijiensis . ( a ) The sgRNA scaffold sequence was amplified from plasmid pX330 using primers gRNA-scaffold-F and gRNA-scaffold-R. The predicted endogenous U6 promoter was amplified from the A. fijiensis genome using adaptor primers U6-1-F and U6-1-R containing 5′ overlapping sequences of the sgRNA scaffold. the 20 bp pyrG -targeting sgRNA sequence was seamlessly inserted between the U6 promoter and sgRNA scaffold using primers U6- pyrG -F and U6-1- pyrG -R. The plasmid backbone containing the Cas9 open reading frame and the AMA1 autonomous replication element was amplified from plasmid FM-6 using primers pAMA-1-F and pAMA1-R. The U6- pyrG -sgRNA expression cassette was amplified from pPu6- pyrG -sgRNA using primers U6-1-F and gRNA-scaffold-R, and subsequently inserted into the Cas9-containing backbone by homologous recombination to generate the final plasmid AFM-Δ pyrG . For clarity, DNA elements in the schematic are not drawn to scale. ( b ) Targeted disruption of the pyrG gene mediated by plasmid-based CRISPR-Cas9 editing in A. fijiensis . Diagnostic PCR analysis of genomic DNA from independent transformants in pyrG locus. The DNA molecular weight marker used was a 100–5000 bp DNA Marker III (Biosharp BL103A, Anhui, China). PCR amplification was performed using primers flanking the targeted integration region to distinguish wild-type and disrupted alleles.

    Article Snippet: The sgRNA expression plasmid pPu6- pyrG -sgRNA was first generated by amplifying the gRNA scaffold sequence from pX330 plasmid (Addgene, Watertown, MA, USA, #42230) using primers gRNA-scaffold-F and gRNA-scaffold-R ( ).

    Techniques: CRISPR, Disruption, Sequencing, Amplification, Plasmid Preparation, Expressing, Homologous Recombination, Diagnostic Assay, Molecular Weight, Marker

    a Schematic view of the LV-reporter vector and the parental AAV vector carrying various repressor effectors. LV harbors a dual reporter system consisting of destabilized GFP (dGFP) and Nano-Luciferase (NLuc). Viral long terminal repeats – LTRs; SV40 polyadenylation signal- SV40pA are highlighted.pSV40 drives the expression of the puromycin marker. The vector was transduced at the MOI = 0.2 to enable the selection of the cells carried 1 copy/cell. Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE). The inverted terminal repeats (ITRs) of AAV are highlighted. The EFS-NC promoter that drives the expression of the dCas9-effector (black and red boxes, respectively) is highlighted. Human U6 promoter driving expression of gRNA is highlighted. b Schematic representation of Heterochromatin Protein 1 alpha (HP1a) and Heterochromatin Protein 1 beta (HP1b) The Chromodomain (CD) and Chromoshadow Domain (CSD) are highlighted. The Hinge region separating the CD and CSD domains is highlighted . c Schematic representation of Methyl-Binding Proteins (MBDs). Methyl-Binding Protein 1 (MBD1); Methyl-Binding Protein 2 (MBD2); Methyl-Binding Protein 3 (MBD3); and Methyl-CpG Binding Protein 2 (MeCP2) are highlighted. Methyl- Binding Domain (MBD) highlighted here, is responsible for the protein-DNA binding Transcription –Repression Domain (TRD) highlighted here is responsible for protein-protein interactions directly involved in gene silencing ( d ) Schematic representation of DNA methyltransferases (DNMTs). De novo methyl-transferase A and B (DNMT3A and B, respectively) are highlighted here. The PWWP domain, named for a conserved Pro-Trp-Trp-Pro motif is highlighted. The Methyl-transferase catalytic domains (MTase) of DNMT3A and DNMT3B are highlighted. e Schematic representation of Nuclear inhibitor of Protein phosphatase 1 (NIPP1) protein. Embryonic Ectoderm Development domain (EED) binding to Polycomb Repressive Complex 2 (PRC2) is highlighted. The Krüppel associated box (KRAB) consists of the repressive boxA and boxB domains which is schematically represented. f Schematic representation of CMV promoter targeted by AAV-KRAB-MeCP2(TRD) system. CjCas9 –matching gRNA1 and gRNA2 are highlighted. SaCas9 – matching gRNA1 and gRNA2 are highlighted. PAM of CjCas9 and SaCas9 are highlighted.

    Journal: Nature Communications

    Article Title: The therapeutic implications of all-in-one AAV-delivered epigenome-editing platform in neurodegenerative disorders

    doi: 10.1038/s41467-024-50515-6

    Figure Lengend Snippet: a Schematic view of the LV-reporter vector and the parental AAV vector carrying various repressor effectors. LV harbors a dual reporter system consisting of destabilized GFP (dGFP) and Nano-Luciferase (NLuc). Viral long terminal repeats – LTRs; SV40 polyadenylation signal- SV40pA are highlighted.pSV40 drives the expression of the puromycin marker. The vector was transduced at the MOI = 0.2 to enable the selection of the cells carried 1 copy/cell. Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE). The inverted terminal repeats (ITRs) of AAV are highlighted. The EFS-NC promoter that drives the expression of the dCas9-effector (black and red boxes, respectively) is highlighted. Human U6 promoter driving expression of gRNA is highlighted. b Schematic representation of Heterochromatin Protein 1 alpha (HP1a) and Heterochromatin Protein 1 beta (HP1b) The Chromodomain (CD) and Chromoshadow Domain (CSD) are highlighted. The Hinge region separating the CD and CSD domains is highlighted . c Schematic representation of Methyl-Binding Proteins (MBDs). Methyl-Binding Protein 1 (MBD1); Methyl-Binding Protein 2 (MBD2); Methyl-Binding Protein 3 (MBD3); and Methyl-CpG Binding Protein 2 (MeCP2) are highlighted. Methyl- Binding Domain (MBD) highlighted here, is responsible for the protein-DNA binding Transcription –Repression Domain (TRD) highlighted here is responsible for protein-protein interactions directly involved in gene silencing ( d ) Schematic representation of DNA methyltransferases (DNMTs). De novo methyl-transferase A and B (DNMT3A and B, respectively) are highlighted here. The PWWP domain, named for a conserved Pro-Trp-Trp-Pro motif is highlighted. The Methyl-transferase catalytic domains (MTase) of DNMT3A and DNMT3B are highlighted. e Schematic representation of Nuclear inhibitor of Protein phosphatase 1 (NIPP1) protein. Embryonic Ectoderm Development domain (EED) binding to Polycomb Repressive Complex 2 (PRC2) is highlighted. The Krüppel associated box (KRAB) consists of the repressive boxA and boxB domains which is schematically represented. f Schematic representation of CMV promoter targeted by AAV-KRAB-MeCP2(TRD) system. CjCas9 –matching gRNA1 and gRNA2 are highlighted. SaCas9 – matching gRNA1 and gRNA2 are highlighted. PAM of CjCas9 and SaCas9 are highlighted.

    Article Snippet: Then, we created a CjCas9- U6- promoter-gRNA scaffold using the corresponding fragment synthesized via the GenScript synthesis service.

    Techniques: Plasmid Preparation, Luciferase, Expressing, Marker, Selection, Virus, Binding Assay, Protein-Protein interactions