Journal: Advanced Science

Article Title: Library‐Assisted Evolution in Eukaryotic Cells Yield Adenine Base Editors with Enhanced Editing Specificity

doi: 10.1002/advs.202309004

Figure Lengend Snippet: TadA library screening and efficiency plasmid testing. a) The E. Coli TadA8e (green and cyans) structure with spCas9 (orange) and DNA (blue and magenta). The mutant amino acids were red and lemon sticks. The protein data is from RCSB: ABE8e (6VPC). b) The library screening process. LTR is lentivirus parts. EF1α is ABE and EGFP's promotor. U6 is sgRNA's promotor. A BsmBI enzyme cutting site has been designed on TadA. c) The amino acids of Mut14, Mut 15, Mut25, and Mut32 compared to ABE8e. d) ABE8e and 31 mutants’ base editing results in HEK293T at nine genetic sites. Each mutant data is compared with ABE8e to show the A to G efficiency increasing ratio. The base editing frequencies are shown in Figure (Supporting Information). e) Average A‐to‐G editing frequencies of ABE8e, TadA8e‐Mut14, TadA8e‐Mut15, TadA8e‐Mut25, TadA8e‐Mut32, ABE9, and ABE9e. f. Adenine and cytosine editing efficiency of ABE8e, TadA8e‐Mut14, TadA8e‐Mut15, TadA8e‐Mut25, and TadA8e‐Mut32 at FANCF‐sg1 site. All data have three independent biological replicates (mean ± SD).

Article Snippet: The chemical‐modified synthetic sgRNA (2′‐O‐methyl 3′ phosphorothioate modifications in the first and last three nucleotides) was ordered from GenScript.

Techniques: Library Screening, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: An InDel Genomic Variant within a Bifunctional Super-Enhancer for LINC00636 and CD47 Regulation in Breast Cancer

doi: 10.1101/2025.11.05.684493

Figure Lengend Snippet: LINC00636 RNA expression is regulated by a bifunctional SE. (A) LINC00636 and CD47 RNA expression levels decrease upon BET inhibition in BCCL, and this reduction occurs at a greater extent in the cells where the SE is present. Cells were treated with 1 μM JQ1 or I-BET151 for 6 hours and mRNA levels were analyzed by qPCR. DMSO was used as vehicle control. Two-way ANOVA was performed, *p<0.05, **p<0.01, ***p<0.001. (B) CRISPRa of the SE locus increases LINC00636 and CD47 RNA expression levels, and this activation occurs at a greater extent in the cells where the SE is present. dCas9-VP64-expressing cells were transfected with scramble sgRNA or sgRNA targeting the E5 core element within the SE. mRNA levels were analyzed by qPCR 48 hours after transfection. Two-way ANOVA was performed, *p<0.05, **p<0.01, ***p<0.001. (C) LINC00636 overexpression does not affect CD47 RNA expression levels. Left image shows the CRISPRa approach used for LINC00636 overexpression targeting LINC00636 TSS. The plots show LINC00636 and CD47 RNA levels in dCas9-VP64-expressing MCF7 cells transfected with sgRNAs targeting two different sites on LINC00636 TSS. mRNA levels were analyzed by qPCR 48 hours after transfection. ANOVA was performed, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Synthetic modified sgRNAs (Synthego) for the target region or negative control were transfected at a final concentration of 50nM using TransIT-X2 Dynamic Delivery System (MIR 6004, Mirus) following manufacturer’s instructions.

Techniques: RNA Expression, Inhibition, Control, Activation Assay, Expressing, Transfection, Over Expression