RNA G-quadruplex promote codon repeat-associated ribosomal frameshifting in human gene
A dualfluorescence reporter system was constructed by inserting an 84bp HDAC1 fragment nucleotides 4 88 between zsGreenP2A and mCherry in the pLVXpuroGCaMP6s backbone, with mCherry shifted to the 1 reading frame and its start codon removed CMV promoterdriven. This design enabled mCherryzsGreen fluorescence ratio to directly quantify frameshift efficiency. Lentivirus was produced in HEK293T cells via cotransfection of pMD2.G , psPAX2 , and lentiCRISPRv2 library using PEI, with viral supernatants harvested at 24, 48, and 72 h. Reporter HEK293T cells were transduced, selected with puromycin, and subjected to FACS to isolate populations with altered fluorescence ratios BD Aria SORP. Genomic DNA from sorted cells was analyzed by sgRNA sequencing to identify genes modulating frameshifting activity, with triplicate biological replicates ensuring robustness.