Journal: Nucleic Acids Research

Article Title: Engineering of BZ transposase and transposon donor vector for enhanced efficiency and safety in gene delivery applications

doi: 10.1093/nar/gkaf935

Figure Lengend Snippet: BZ transposase engineering based on the conservation analysis. ( A ) Sequence alignment of the BZ wt and SB100X transposase. The alignment was generated by CLUSTALW embedded in Bioedit software and then used to highlight secondary structures of the BZ wt transposase (predicted by AlphaFold 3) with ESPript3.026. The three key residues (D, D, and E) of the catalytic DDE triad are marked by a star. ( B ) The transposition activity and gMFI of BZ transposase mutants in CHO cells were measured using flow cytometry analysis. CHO cells were co-transfected with a donor plasmid (GFP expression cassette with flanked BZ TIRs) and a helper plasmid of BZ transposase variant. ( C ) The transposition activity and gMFI for the combined mutants in CHO cells were measured using flow cytometry analysis. ( D ) The transposition activity of mutant Q71R\H110R and BZ wt in CHO and PBMCs measured by flow cytometry analysis at Days 5, 9, and 13 after transfection. ( E ) The transposition activity of Q71R\H110R and BZ wt in HeLa cells measured using binary transposition assay. Data are shown as the mean ± SEM in D, F and G. P values (* P < 0.05; ** P < 0.01) were calculated by one-way ANOVA with Dunnett’s multiple comparisons test (three independent replicates).

Article Snippet: The alignment between BZ wt and SB100X was generated using CLUSTALW embedded in BioEdit software and subsequently employed to highlight secondary structures of the BZ wt transposase with ESPript3.0 [ ].

Techniques: Sequencing, Generated, Software, Activity Assay, Flow Cytometry, Transfection, Plasmid Preparation, Expressing, Variant Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Engineering of BZ transposase and transposon donor vector for enhanced efficiency and safety in gene delivery applications

doi: 10.1093/nar/gkaf935

Figure Lengend Snippet: BZ transposase key motif and residues engineering and ZIP fusing. ( A ) KTPLLN motif identification. A KTPLLN motif within the linker region of BZ transposase, which bridges the DNA-binding and catalytic domains, was identified by aligning the Tc1 transposases. The red arrows indicate the first and last amino acids of the linker KTPLLN. ( B ) Targeted saturation mutagenesis of the K120 and N125 residues in the BZ transposase’s KTPLLN motif and transposition activity was measured by the binary transposition assay. ( C ) Partial sequence alignment of Mos1 , SB100X , and BZ wt transposases. The alignment was generated using CLUSTALW and then was used to highlight secondary structures of the Mos1 transposase (based on PDB ID:4U7B) with ESPript 3.026. Superposition of the BZ prediction model (purple) with the SB TCC (green, PDB ID: 5CR4) or the Mos1 STC (pink, PDB ID:5HOO) with structures shows that F188 in BZ aligns with R186 in Mos1 . The target DNA is depicted in dark gray (left) and K249 in BZ and K248 in SB . ( D ) Targeted saturation mutagenesis of the F188 and K249 residues and transposition activity measured using a binary transposition assay. ( E ) Schematic of Q71R\H110R variant fused with different leucine zipper (ZIP) motifs, which were derived from CEBPα, CREB3, CREPT, and TEF transcription factors, and their fusions were named as Q71R\H110R CEBPα , Q71R\H110R CREPT , Q71R\H110R TEF , and Q71R\H110R CREB3 . ( F ) The transposition activity of BZ transposase and ZIP fusions in HeLa cells was measured using binary transposition assay. ( G ) Transposition activities of the combined mutants were measured using a binary transposition assay. Data are shown as the mean ± SEM in panels (B), (D), (F), and (G). P values (* P < 0.05; ** P < 0.001) were calculated using one-way ANOVA with Dunnett’s multiple comparisons test (three independent replicates).

Article Snippet: The alignment between BZ wt and SB100X was generated using CLUSTALW embedded in BioEdit software and subsequently employed to highlight secondary structures of the BZ wt transposase with ESPript3.0 [ ].

Techniques: Binding Assay, Mutagenesis, Activity Assay, Sequencing, Generated, Variant Assay, Derivative Assay