Journal: bioRxiv

Article Title: CRISPR-based engineering of RNA viruses

doi: 10.1101/2023.05.19.541219

Figure Lengend Snippet: ( A ) Diagram showing a pipeline for deleting 12 nucleotides from the GFP open reading frame (ORF) of the Sindbis-GFP (SINV-GFP) virus. See Fig. S2A for annotated genome map and Fig. S4A for additional experimental details. The deletion eliminates 4 codons encoding amino acids (L64, T65, Y66, G67) forming the chromophore of the fluorescent protein, which is expected to ablate fluorescence (“dead” GFP, dGFP). ( B ) RNA aliquots collected after the cleavage with the Csm complex (red) and ligation (blue) with T4 RNA ligase were reverse-transcribed and quantified with qPCR. Primers were designed to amplify cDNA across the target site. Relative quantities were calculated by normalizing to the uncut RNA control. ( C ) Melt curve analysis of the qPCR products generated with RNA after ligation in (B). Peaks indicate the melting temperature of the qPCR products. ( D ) BHK-21 cells were transfected with unedited or edited RNA of the SINV-GFP (two replicates each), seeded in 6-well plates, and imaged 24 hours later to capture the GFP signal (excitation 488 nm, emission 520 nm). ( E ) Melt curve analysis of the RT-qPCR products that were generated with RNA extracted from supernatants of BHK-21 cells 24 hours post-transfection. Primers were the same as in (B) and (C).

Article Snippet: Products of RNA cleavage with gfp -targeting SthCsm were sequenced with Oxford Nanopore using direct cDNA sequencing kit (SQK-DCS109).

Techniques: Virus, Fluorescence, Ligation, Reverse Transcription, Control, Generated, Transfection, Quantitative RT-PCR