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

Journal: Cancer Research Communications

Article Title: Key Early Changes in Oral Squamous Cell Carcinogenesis Are Accelerated by Ectopic BMI1 Expression

doi: 10.1158/2767-9764.CRC-25-0580

Figure Lengend Snippet: BMI1 KO in the human OSCC line SCC-25 decreases the expression of HIF1α and glycolysis-associated protein GLUT1. A, A scheme of Crispr/Cas9 technology strategy indicating the three gRNAs designed in exons 6 and 9 of the human BMI1 gene. Treatment with gRNA B (in red) resulted in the highest percentage of editing. B, Discordance plots detailing the level of alignment per base between the parental (control) and the edited (B#5 or B#6) SCC-25 cell lines in the interference windows, based on Sanger sequencing of fragments amplified via PCR from genomic DNA of treated cells. On the plots, green and orange lines are close together before the cutsite (dashed line) and remain far apart after this site. These plots were generated using the online tool provided by Synthego, ICE. Immunoblotting of the protein levels of ( C ) BMI1 and GAPDH, ( D ) HIF1α and GAPDH, and ( E ) GLUT1 and GAPDH in parental, B#5, and B#6 SCC-25 cells (triplicates). Quantifications of ( F ) HIF1α and ( G ) GLUT1 immunoblottings (relative to GAPDH) are included. Statistical significance was determined relative to parental SCC-25 cells using Welch t test (*, 0.01 < P < 0.05).

Article Snippet: We seeded SCC-25 cells in 24-well tissue culture plates at a density of 50,000 cells/well, and after 24 hours, we treated cells with the three gRNA sequences (Supplementary Table S6), following the manufacturers’ instructions (Synthego).

Techniques: Expressing, CRISPR, Control, Sequencing, Amplification, Generated, Western Blot