Journal: bioRxiv

Article Title: Novel mouse reporter models for the detection of genome editing events in vivo

doi: 10.64898/2026.04.29.721708

Figure Lengend Snippet: a) Schematic of TLR-2 reporter allele structure and outcomes following editing. The allele includes two open reading frames encoding different fluorescent proteins; an upstream mVenus (green) in the +1 frame and downstream TagRFP (red) in the +3 frame linked by a P2A sequence. The mVenus cassette is interrupted by a 108 bp polylinker sequence that includes several guide target sequences and a stop codon (blue square). Thus in its native configuration, neither fluorescent protein is expressed. Following CRISPR/Cas9 editing, repair via NHEJ will result in expression of TagRFP if the frame shift results in a -2 deletion, or multiple thereof. Alternatively, HDR can be detected with the inclusion of a plasmid donor designed to repair the mVenus gap. b) Mouse embryo reporter validation workflow. Fertilized zygotes from TLR-2 reporter mice (typically male homozygous to WT female) are either microinjected or electroporated with Cas9 RNP with or without a plasmid donor for HDR. The embryos are then cultured to the blastocyst stage where the outcome can be scored by fluorescent imaging, and followed up by PCR-Sanger sequencing to confirm the identify of specific edits.

Article Snippet: Sanger sequence traces were analyzed using the ICE (Inference of CRISPR Editing) deconvolution tool from Synthego. ( https://ice.synthego.com ; Synthego Performance Analysis, ICE Analysis.

Techniques: Sequencing, CRISPR, Expressing, Plasmid Preparation, Biomarker Discovery, Cell Culture, Imaging