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Fig. 5 | Genome Biology

Fig. 5

From: A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila

Fig. 5

Adaptation of the REPAIRv2 system to modify RNA in Drosophila Sg4 cell culture. a Schematic for the Drosophila-modified REPAIRv2 system (FREPAIRv2), to modify a mutant eCFP transcript. Mutant eCFP carries an early stop codon that normally encodes Tryptophan at residue 57 (W57*). By generating an A to C mismatch in the crRNA spacer that corresponds to the stop codon, the ADAR2DD domain will change the equivalent adenosine (A) to inosine (I). Inosine will be treated as guanosine by the translation machinery. b Schematic of FREPAIRv2 outcome. Originally, the mutant eCFP transcript harbors a stop codon at position 57, which will generate a short polypeptide with 56 amino acids. However, once modified by FREPAIRv2, codon 57 will be reverted to wild-type tryptophan and restore the production of a full-length polypeptide. c Western blotting monitoring eCFP productions relative to transfection time. d Fluorescence emitted by eCFP relative to transfection time. Nuclei were stained with nuclear green DCS1 (Abcam ab138905). Color was adjusted for color-blind-friendly purpose. eCFP and DsRed fluorescence were measured based on their natively emitted fluorescence. Scale bar = 50 μm. e Schematic of crRNAs that we used for FREPAIRv2. We considered two criteria for the crRNA design: (i) mismatch distance from the first nucleotide and (ii) spacer length. f Editing rate and off-target rate of FREPAIRv2 concerning mismatch distance when spacer length was kept at a constant 50 nucleotides. Error bars represent standard deviation. g Editing rate and off-target rates of FREPAIRv2 in relation to spacer lengths when the mismatch distance was kept at the constant position 26. Error bars represent standard deviation

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