ti e widefield epifluorescence microscope  (Nikon)

Journal: bioRxiv

Article Title: Improved cloning-free one-step CRISPR-Cas12a-assisted tagging of mammalian genes using PCR generated reagents (PCR tagging)

doi: 10.64898/2025.12.02.690677

Figure Lengend Snippet: ( a ) Schematic of PCR tagging strategy: The tagging cassette contains the tag-2A-marker fusion which is followed by a polyA adenylation site for proper transcript termination. The cassette also contains a functional gRNA gene. The protospacer sequence of the gRNA is provided by one of the PCR primers, thus allowing the use of generic PCR templates with gene-specific primers . Upon transfection the gRNA gene is expressed and targets Cas12a (cotransfected on a plasmid) to induce a DNA DSB at the target location, followed by integration of the PCR cassette via homology-directed repair (HDR) or alternative pathways. ( b ) Representative microscopy image from HEK293T-N cells showing HNRNPA1 tagging. mNeonGreen fluorescence and HOECHST staining are shown. Images acquired using Nikon Ti-E widefield epifluorescence microscope with a 60x objective. Scale bar, 50 µm. ( c ) Schematic of sequencing read and integration outcomes. ( d ) Experimental workflow of Tn5-Anchor-Seq: 1) Isolated genomic DNA (gDNA) was fragmented using Tn5 transposase, which was loaded with indexed adapters comprised of a Tn5 mosaic end (ME), an 11-bp index (bc), and a nanopore sequencing primer binding site (NBS). 2) DNA fragments containing the anchor sequence were PCR-amplified with biotinylated primers specific to anchor sequence and adapter-specific primers directed at the NBS. 3) Biotinylated PCR products were enriched via a biotin-streptavidin capture step and nanopore barcodes and adapters were introduced by a nested PCR. 4) Final anchor-specific amplicons were sequenced on a Nanopore MinION platform. ( e ) Schematics illustrating genomic integration scenarios following PCR tagging: i. HDR-mediated integration; ii. NHEJ-mediated deletions and insertions; iii. Off-target integration; iv. Concatemeric integration. Corresponding IGV snapshots show representative sequencing reads for the indicated regions. ( f ) The frequencies of genomic integrations at PSMD10 locus.

Article Snippet: Images were acquired on a Nikon Ti-E widefield epifluorescence microscope with a 60x ApoTIRF oil-immersion objective (1.49-NA(numerical aperture); Nikon), an LED light engine (SpectraX, Lumencor), a 2048 x 2048 pixel (6.5 μm) sCMOS camera (Flash4, Hamamatsu) and an autofocus system (Perfect Focus System, Nikon) with either bright field or 469/35-nm excitation filter (Semrock) and 525/50-nm emission filter (Chroma).

Techniques: Marker, Functional Assay, Sequencing, Transfection, Plasmid Preparation, Microscopy, Fluorescence, Staining, Isolation, Nanopore Sequencing, Binding Assay, Amplification, Nested PCR

Journal: bioRxiv

Article Title: Improved cloning-free one-step CRISPR-Cas12a-assisted tagging of mammalian genes using PCR generated reagents (PCR tagging)

doi: 10.64898/2025.12.02.690677

Figure Lengend Snippet: ( a ) Schematic of seamless and standard PCR tagging strategies. ( b ) Experimental workflow of seamless PCR tagging and validation: HEK293T-N cells were transfected with two PCR cassettes and Cas12a helper plasmid, followed by neomycin selection and single-cell sorting by flow cytometry. The resulting clonal cell populations were subjected to junction PCR and amplicon sequencing for validation. ( c ) Representative fluorescent microscopy image from HEK293T-N cells showing HNRNPA1 and SLC16A1 tagging. mScarlet-i fluorescence and HOECHST staining are shown. Images acquired using Nikon Ti-E widefield epifluorescence microscope with a 60x objective. Scale bar, 10 µm. ( d ) Schematic showing the insertion outcomes following seamless PCR tagging at the HNRNPA1 locus and the junction PCR strategy. Red arrows indicate primers binding to the 3’ end of the ORF and the downstream of 3’-UTR. A representative gel image of junction PCR results is shown.

Article Snippet: Images were acquired on a Nikon Ti-E widefield epifluorescence microscope with a 60x ApoTIRF oil-immersion objective (1.49-NA(numerical aperture); Nikon), an LED light engine (SpectraX, Lumencor), a 2048 x 2048 pixel (6.5 μm) sCMOS camera (Flash4, Hamamatsu) and an autofocus system (Perfect Focus System, Nikon) with either bright field or 469/35-nm excitation filter (Semrock) and 525/50-nm emission filter (Chroma).

Techniques: Biomarker Discovery, Transfection, Plasmid Preparation, Selection, FACS, Flow Cytometry, Amplification, Sequencing, Microscopy, Fluorescence, Staining, Binding Assay