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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/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

    Related Articles

    Amplification:

    Article Title: Establishment of an efficient CRISPR-dCas9-SunTag transcriptional activation system in Aspergillus nidulans for enhancing emodin production
    Article Snippet: This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record.. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article.. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

    Article Title: Systematic epigenome editing captures the context-dependent instructive function of chromatin modifications
    Article Snippet: .. The gRNA plasmid, carrying an enhanced gRNA scaffold, was amplified from Addgene plasmid 60955 and cloned into a piggyBac recipient vector, which are also designed for constitutively expression of a puromycin resistance gene and TagBFP. .. All gRNA species used to target the epigenetic editing system were designed using the GPP Web Portal (Broad Institute). gRNA forward and reverse strands carrying appropriate overhangs (final concentration of 10 μM) were annealed in buffer containing 10 mM Tris, pH 7.5–8.0, 60 mM NaCl and 1 mM EDTA at 95 °C for 3 min and allowed to cool down at room temperature for >30 min. Annealed gRNA was ligated with T4 DNA ligase (NEB, M0202S) for 1 h at 37 °C into the piggyBac recipient vector previously digested with BlpI (NEB, R0585S) and BstXI (NEB, R0113S) restriction enzymes.

    Sequencing:

    Article Title: Establishment of an efficient CRISPR-dCas9-SunTag transcriptional activation system in Aspergillus nidulans for enhancing emodin production
    Article Snippet: This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record.. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article.. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

    Article Title: An unbiased survey of distal element-gene regulatory interactions with direct-capture targeted Perturb-seq
    Article Snippet: .. We constructed the sgOpti-CS vector by replacing the gRNA scaffold of the sgOpti plasmid (Addgene #85681) with the capture sequence containing scaffold from the pBA904 plasmid (Addgene #122238). .. We digested the sgOpti plasmid with NsiI and EcoRI (New England Biolabs) and used the same enzymes to digest a gBlock (IDT) containing the scaffold with the capture sequence.

    Functional Assay:

    Article Title: Establishment of an efficient CRISPR-dCas9-SunTag transcriptional activation system in Aspergillus nidulans for enhancing emodin production
    Article Snippet: This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record.. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article.. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

    Expressing:

    Article Title: Establishment of an efficient CRISPR-dCas9-SunTag transcriptional activation system in Aspergillus nidulans for enhancing emodin production
    Article Snippet: This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record.. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article.. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

    Article Title: ZBTB48 is a priming factor regulating B-cell-specific CIITA expression.
    Article Snippet: The sgRNAs were cloned into lentiCRISPRv2-puro vector and confirmed by Sanger sequencing (1st BASE). .. The lentiCRISPRv2-puro vector containing the Cas9 expression cassette and the gRNA scaffold was a gift from Brett Stringer (Addgene plasmid #98290; http://n2t.net/ addgene:98290; RRID:Addgene_98290). .. For transduction, 700 μl of lentiviral supernatant, generated using HEK293T cells and supplemented with 10 mM HEPES and 8 μg/mL polybrene (Sigma-Aldrich), was combined with 100 μl of cell suspension containing 5 × 105 SUDHL4 cells in 12-well plates and spininoculated by centrifugation at 2000 rpm for 1.5 h at 32 °C.

    Article Title: ZBTB48 is a priming factor regulating B-cell-specific CIITA expression
    Article Snippet: The sgRNAs were cloned into lentiCRISPRv2-puro vector and confirmed by Sanger sequencing (1st BASE). .. The lentiCRISPRv2-puro vector containing the Cas9 expression cassette and the gRNA scaffold was a gift from Brett Stringer (Addgene plasmid #98290; http://n2t.net/addgene:98290 ; RRID: Addgene_98290 ). .. For transduction, 700 μl of lentiviral supernatant, generated using HEK293T cells and supplemented with 10 mM HEPES and 8 μg/mL polybrene (Sigma-Aldrich), was combined with 100 μl of cell suspension containing 5 × 10 SUDHL4 cells in 12-well plates and spin-inoculated by centrifugation at 2000 rpm for 1.5 h at 32 °C.

    Article Title: The interplay of DNA repair context with target sequence predictably biases Cas9-generated mutations
    Article Snippet: After each PCR, amplicons were purified using Monarch PCR & DNA Cleanup Kit (NEB). .. A lentiviral gRNA expression vector lacking the scaffold, pKLV2—U6(BbsI)—PGKpuro—2 A—mCherry—W, was generated by removing the improved gRNA scaffold from pKLV2—U6—gRNA5(BbsI)—PGKpuro—mCherry—W (Addgene 67977; see Allen 2018, with minor changes ). .. The amplicons were cloned into the vector using Gibson Assembly mix reactions (NEBuilder HiFi DNA Assembly Cloning Kit) according to manufacturer’s specifications in two or three separate reactions.

    Article Title: Systematic epigenome editing captures the context-dependent instructive function of chromatin modifications
    Article Snippet: .. The gRNA plasmid, carrying an enhanced gRNA scaffold, was amplified from Addgene plasmid 60955 and cloned into a piggyBac recipient vector, which are also designed for constitutively expression of a puromycin resistance gene and TagBFP. .. All gRNA species used to target the epigenetic editing system were designed using the GPP Web Portal (Broad Institute). gRNA forward and reverse strands carrying appropriate overhangs (final concentration of 10 μM) were annealed in buffer containing 10 mM Tris, pH 7.5–8.0, 60 mM NaCl and 1 mM EDTA at 95 °C for 3 min and allowed to cool down at room temperature for >30 min. Annealed gRNA was ligated with T4 DNA ligase (NEB, M0202S) for 1 h at 37 °C into the piggyBac recipient vector previously digested with BlpI (NEB, R0585S) and BstXI (NEB, R0113S) restriction enzymes.

    Plasmid Preparation:

    Article Title: ZBTB48 is a priming factor regulating B-cell-specific CIITA expression.
    Article Snippet: The sgRNAs were cloned into lentiCRISPRv2-puro vector and confirmed by Sanger sequencing (1st BASE). .. The lentiCRISPRv2-puro vector containing the Cas9 expression cassette and the gRNA scaffold was a gift from Brett Stringer (Addgene plasmid #98290; http://n2t.net/ addgene:98290; RRID:Addgene_98290). .. For transduction, 700 μl of lentiviral supernatant, generated using HEK293T cells and supplemented with 10 mM HEPES and 8 μg/mL polybrene (Sigma-Aldrich), was combined with 100 μl of cell suspension containing 5 × 105 SUDHL4 cells in 12-well plates and spininoculated by centrifugation at 2000 rpm for 1.5 h at 32 °C.

    Article Title: ZBTB48 is a priming factor regulating B-cell-specific CIITA expression
    Article Snippet: The sgRNAs were cloned into lentiCRISPRv2-puro vector and confirmed by Sanger sequencing (1st BASE). .. The lentiCRISPRv2-puro vector containing the Cas9 expression cassette and the gRNA scaffold was a gift from Brett Stringer (Addgene plasmid #98290; http://n2t.net/addgene:98290 ; RRID: Addgene_98290 ). .. For transduction, 700 μl of lentiviral supernatant, generated using HEK293T cells and supplemented with 10 mM HEPES and 8 μg/mL polybrene (Sigma-Aldrich), was combined with 100 μl of cell suspension containing 5 × 10 SUDHL4 cells in 12-well plates and spin-inoculated by centrifugation at 2000 rpm for 1.5 h at 32 °C.

    Article Title: Systematic epigenome editing captures the context-dependent instructive function of chromatin modifications
    Article Snippet: .. The gRNA plasmid, carrying an enhanced gRNA scaffold, was amplified from Addgene plasmid 60955 and cloned into a piggyBac recipient vector, which are also designed for constitutively expression of a puromycin resistance gene and TagBFP. .. All gRNA species used to target the epigenetic editing system were designed using the GPP Web Portal (Broad Institute). gRNA forward and reverse strands carrying appropriate overhangs (final concentration of 10 μM) were annealed in buffer containing 10 mM Tris, pH 7.5–8.0, 60 mM NaCl and 1 mM EDTA at 95 °C for 3 min and allowed to cool down at room temperature for >30 min. Annealed gRNA was ligated with T4 DNA ligase (NEB, M0202S) for 1 h at 37 °C into the piggyBac recipient vector previously digested with BlpI (NEB, R0585S) and BstXI (NEB, R0113S) restriction enzymes.

    Article Title: An unbiased survey of distal element-gene regulatory interactions with direct-capture targeted Perturb-seq
    Article Snippet: .. We constructed the sgOpti-CS vector by replacing the gRNA scaffold of the sgOpti plasmid (Addgene #85681) with the capture sequence containing scaffold from the pBA904 plasmid (Addgene #122238). .. We digested the sgOpti plasmid with NsiI and EcoRI (New England Biolabs) and used the same enzymes to digest a gBlock (IDT) containing the scaffold with the capture sequence.

    Generated:

    Article Title: The interplay of DNA repair context with target sequence predictably biases Cas9-generated mutations
    Article Snippet: After each PCR, amplicons were purified using Monarch PCR & DNA Cleanup Kit (NEB). .. A lentiviral gRNA expression vector lacking the scaffold, pKLV2—U6(BbsI)—PGKpuro—2 A—mCherry—W, was generated by removing the improved gRNA scaffold from pKLV2—U6—gRNA5(BbsI)—PGKpuro—mCherry—W (Addgene 67977; see Allen 2018, with minor changes ). .. The amplicons were cloned into the vector using Gibson Assembly mix reactions (NEBuilder HiFi DNA Assembly Cloning Kit) according to manufacturer’s specifications in two or three separate reactions.

    other:

    Article Title: Systematic epigenome editing captures the context-dependent instructive function of chromatin modifications.
    Article Snippet: All gRNA species used to target the epigenetic editing system were designed using the GPP Web Portal (Broad Institute). gRNA forward and reverse strands carrying appropriate overhangs (final concentration of 10 μM) were annealed in buffer containing 10 mM Tris, pH 7.5–8.0, 60 mM NaCl and 1 mM EDTA at 95 °C for 3 min and allowed to cool down at room temperature for >30 min. Annealed gRNA was ligated with T4 DNA ligase (NEB, M0202S) for 1 h at 37 °C into the piggyBac recipient vector previously digested with BlpI (NEB, R0585S) and BstXI (NEB, R0113S) restriction enzymes.

    Clone Assay:

    Article Title: Rational engineering of minimally immunogenic nucleases for gene therapy
    Article Snippet: .. Guides were cloned into gRNA scaffold-containing constructs for SaCas9 (Addgene #70709) and AsCas12a (Addgene #pY020) respectively. ..

    Article Title: Systematic epigenome editing captures the context-dependent instructive function of chromatin modifications
    Article Snippet: .. The gRNA plasmid, carrying an enhanced gRNA scaffold, was amplified from Addgene plasmid 60955 and cloned into a piggyBac recipient vector, which are also designed for constitutively expression of a puromycin resistance gene and TagBFP. .. All gRNA species used to target the epigenetic editing system were designed using the GPP Web Portal (Broad Institute). gRNA forward and reverse strands carrying appropriate overhangs (final concentration of 10 μM) were annealed in buffer containing 10 mM Tris, pH 7.5–8.0, 60 mM NaCl and 1 mM EDTA at 95 °C for 3 min and allowed to cool down at room temperature for >30 min. Annealed gRNA was ligated with T4 DNA ligase (NEB, M0202S) for 1 h at 37 °C into the piggyBac recipient vector previously digested with BlpI (NEB, R0585S) and BstXI (NEB, R0113S) restriction enzymes.

    Construct:

    Article Title: Rational engineering of minimally immunogenic nucleases for gene therapy
    Article Snippet: .. Guides were cloned into gRNA scaffold-containing constructs for SaCas9 (Addgene #70709) and AsCas12a (Addgene #pY020) respectively. ..

    Article Title: An unbiased survey of distal element-gene regulatory interactions with direct-capture targeted Perturb-seq
    Article Snippet: .. We constructed the sgOpti-CS vector by replacing the gRNA scaffold of the sgOpti plasmid (Addgene #85681) with the capture sequence containing scaffold from the pBA904 plasmid (Addgene #122238). .. We digested the sgOpti plasmid with NsiI and EcoRI (New England Biolabs) and used the same enzymes to digest a gBlock (IDT) containing the scaffold with the capture sequence.



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    grna scaffold template - by Bioz Stars, 2026-09
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    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

    Z7 enhances DNA binding and cleavage activity of AtCas9 at low magnesium condition. (a) In vitro cleavage assay of WT and Z7 proteins with different concentrations of Mg 2+ (0.5, 1, 2, 5 mM). Three target loci ( Spacer-21a , CTNNB1-10g , and VEGFA-11g ) were tested. (b) Temperature-dependent cleavage efficiency of WT and Z7. (c) Thermal shift analysis of melting temperatures of the AtCas9 proteins. (d-e) EMSA analysis of binding affinities between Cas9 variants and sgRNA (d), or Cas9 RNP complexes with DNA substrates (e), under the indicated magnesium concentrations.

    Journal: Cell Insight

    Article Title: Loop engineering of AtCas9 for effective and broad genome editing

    doi: 10.1016/j.cellin.2025.100286

    Figure Lengend Snippet: Z7 enhances DNA binding and cleavage activity of AtCas9 at low magnesium condition. (a) In vitro cleavage assay of WT and Z7 proteins with different concentrations of Mg 2+ (0.5, 1, 2, 5 mM). Three target loci ( Spacer-21a , CTNNB1-10g , and VEGFA-11g ) were tested. (b) Temperature-dependent cleavage efficiency of WT and Z7. (c) Thermal shift analysis of melting temperatures of the AtCas9 proteins. (d-e) EMSA analysis of binding affinities between Cas9 variants and sgRNA (d), or Cas9 RNP complexes with DNA substrates (e), under the indicated magnesium concentrations.

    Article Snippet: A U6 promoter-driven AtCas9 sgRNA mammalian expression plasmid (designated Gcl203) was created by replacing the sgRNA scaffold in the gRNA_cloning Vector (Addgene plasmid 41824) with the AtCas9 sgRNA optimized scaffold.

    Techniques: Binding Assay, Activity Assay, In Vitro, Cleavage Assay

    Structure-guided protein engineering improves the editing efficiency of AtCas9 . (a) Schematic representation of sgRNA: DNA target recognition by AtCas9 variants. Residue substitutions in the E535 and E5116 variants are highlighted. (b) Adenine base editing efficiencies of AtCas9 mutants at C-myc-41g and CTNNB1-10g target sites. (c) Solvent-accessible surface area (SASA) analysis of interactions between different AtCas9 variants and nucleic acids, reflecting changes in protein–nucleic acid interface accessibility. (d) (left) A-to-G editing efficiencies of wildtype and AtCas9 E5116 mutant. (right) Median fold change of editing efficiency. Data were normalized to WT AtCas9-ABE. For (b) and (d), data are mean ± SD of n = 3 biologically independent experiments. Statistical significance was determined by unpaired t -test (∗ p ​< ​0.033, ∗∗∗ p ​< ​0.001). Each dot represents one biological experiment.

    Journal: Cell Insight

    Article Title: Loop engineering of AtCas9 for effective and broad genome editing

    doi: 10.1016/j.cellin.2025.100286

    Figure Lengend Snippet: Structure-guided protein engineering improves the editing efficiency of AtCas9 . (a) Schematic representation of sgRNA: DNA target recognition by AtCas9 variants. Residue substitutions in the E535 and E5116 variants are highlighted. (b) Adenine base editing efficiencies of AtCas9 mutants at C-myc-41g and CTNNB1-10g target sites. (c) Solvent-accessible surface area (SASA) analysis of interactions between different AtCas9 variants and nucleic acids, reflecting changes in protein–nucleic acid interface accessibility. (d) (left) A-to-G editing efficiencies of wildtype and AtCas9 E5116 mutant. (right) Median fold change of editing efficiency. Data were normalized to WT AtCas9-ABE. For (b) and (d), data are mean ± SD of n = 3 biologically independent experiments. Statistical significance was determined by unpaired t -test (∗ p ​< ​0.033, ∗∗∗ p ​< ​0.001). Each dot represents one biological experiment.

    Article Snippet: A U6 promoter-driven AtCas9 sgRNA mammalian expression plasmid (designated Gcl203) was created by replacing the sgRNA scaffold in the gRNA_cloning Vector (Addgene plasmid 41824) with the AtCas9 sgRNA optimized scaffold.

    Techniques: Drug discovery, Residue, Solvent, Mutagenesis