Fig. 1: Adding a U7 or U1 promoter and SmOPT U7 hairpin to the antisense gRNA improves RNA editing.

a 100 nt antisense guide RNAs targeting adenosines in the RAB7A 3’UTR, FANCC 3’UTR, SMAD4 coding sequence, or SOD1 start codon (TIS) were expressed using the hU6, hU7, or mU7 promoters, either alone or possessing an additional SmOPT sequence and human or mouse U7 hairpin on the 3’end. Plasmids also contained the CMV promoter to overexpress ADAR2 (gray) or GFP (endogenous ADAR, yellow). RNA was measured from HEK-293T cells 2 days post transfection. Mean values ± SD are shown; n = 3 biological replicates. P values were calculated by Mixed-effects analysis using Dunnett’s multiple comparisons test (two-sided). b Sequences containing two or three copies of the hnRNP A1 binding motif (UAGGGW, underlined) were added onto the 5′ end of antisense guide RNAs expressed using a human U1 promoter and possessing a SmOPT mU7 hairpin. RNA was measured from HEK-293T cells with endogenous ADAR levels 2 days after plasmid transfection. Mean values ± SD are shown; n = 5 biological replicates. P values were calculated by Mixed-effects analysis using Dunnett’s multiple comparisons test (two-sided). c Landing Pad system for testing guide RNAs at a single copy per cell. A Landing Pad construct allowing doxycycline-inducible expression of FusionRed, BxB1 integrase, and a blasticidin-resistance marker was integrated into a single AAVS1 safe harbor genomic locus in HEK-293T cells. When the resulting Landing Pad cell line was transfected with plasmids expressing a guide RNA and selectable marker, B × B1 integrase recombines the attB2 and attP2 sites to incorporate exactly one guide RNA construct into a single site in the genome. d Antisense guide RNAs possessing various 5′ hnRNP A1 domains were constructed using the hU6 promoter with no 3’hairpin or the hU1 promoter with a SmOPT mU7 hairpin as described above (Linear) or containing additional circularizing ribozyme sequences flanking the guide RNA (Circular). Constructs were transfected into HEK-293T Landing Pad cells; RNA from successfully integrated single-copy gRNA constructs was measured 13 days after the initial plasmid transfection. For all panels, negative controls were measured from samples where the gRNA targeted a different gene. Mean values ± SD are shown; n = 3 biological replicates. Source data are provided as a Source Data file.