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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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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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https://www.bioz.com/result/grna sequence/product/Addgene inc
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grna sequence - by Bioz Stars, 2026-05
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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.
Guide Rna Scaffold Sequence, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/guide rna scaffold sequence/product/Addgene inc
Average 96 stars, based on 1 article reviews
guide rna scaffold sequence - by Bioz Stars, 2026-05
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guide rna sequences  (Broad Clinical Labs)
96
Broad Clinical Labs guide rna sequences
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.
Guide Rna Sequences, supplied by Broad Clinical Labs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/guide rna sequences/product/Broad Clinical Labs
Average 96 stars, based on 1 article reviews
guide rna sequences - by Bioz Stars, 2026-05
96/100 stars
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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