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: 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

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