t7e1 digestion  (New England Biolabs)

Journal: bioRxiv

Article Title: Optimised genome editing for precise DNA insertion and substitution using Prime Editors in zebrafish

doi: 10.1101/2025.04.23.650248

Figure Lengend Snippet: a, Schematic illustration of the functional domains in the Ror2 protein and alignment of partial amino acid sequences within the tyrosine kinase domain. Sequences from multiple species, including those related to Robinow syndrome (RS) in humans W720X and the corresponding zebrafish W722X mutant, are aligned. The conserved tyrosine residue is highlighted. b, Schematic illustration of guide RNA (gRNA) designs for prime editing insertion in ror2 . c, Schematic illustration of prime editing insertion by Cas9-nuclease-based Prime Editor (PEn). An additional DNA fragment, reverse-transcribed at the target cleavage site, containing the programmed insertion, is integrated into the genome via homology-directed repair or non-homologous end joining. d, Agarose gel images of genomic PCR products from embryos injected with Prime Editor mRNA and pegRNA/springRNA. PCR products of the ror2 target region (top) and those after digestion with T7 endonuclease I (T7E1, bottom). e, Sequence alignment of the edits in the ror2 target site obtained from embryos injected with PEn/springRNA. Prime editing insertion (TGA) is outlined, and the gRNA target sequence is underlined. f, Quantitative comparison of editing outcomes using different combinations of Prime Editor and gRNA. The proportion of sequence reads with each type of edit in amplicon sequencing is presented.

Article Snippet: The PCR and T7E1-digested products were assessed on a 3% agarose gel alongside a 100 bp DNA ladder (New England Biolabs).

Techniques: Functional Assay, Mutagenesis, Residue, Reverse Transcription, Non-Homologous End Joining, Agarose Gel Electrophoresis, Injection, Sequencing, Comparison, Amplification

Journal: Journal of Translational Medicine

Article Title: SIRPB1 regulates inflammatory factor expression in the glioma microenvironment via SYK: functional and bioinformatics insights

doi: 10.1186/s12967-024-05149-z

Figure Lengend Snippet: SIRPB1 Knockout and Macrophage Polarization. A T7E1 assay results. WT wild-type, NC negative control, PC positive control. B SIRPB1 and FLAG-cas9 expression in THP-1 lines. C Sanger sequencing of SIRPB1 WT and SIRPB1 KO . D Protein expression post-M1/M2 treatments. E mRNA levels of M1/M2 markers (* P < 0.05, ** P < 0.01, *** P < 0.001, Dunnett’s test). F Flow cytometry of CD11b, CD86, CD206 in THP-1 lines

Article Snippet: Post-7-d culture, T7E1 digestion confirmed gene editing using a Genome-Editing Mutation Detection Kit (Beyotime).

Techniques: Knock-Out, Negative Control, Positive Control, Expressing, Sequencing, Flow Cytometry