Fig. 3: R2Tg mediates transgene insertion in human cells.

A Schematic of the functional reporter system. R2Tg mRNA encodes the human codon optimized ORF, and the donor RNA contains a GFP reporter flanked by R2Tg’s UTRs. B Reporter integration (left) and GFP expression (right) when donors are cotransfected with wildtype R2Tg (WT), reverse transcriptase mutant (RTmut), or endonuclease mutant (ENmut) (repeated two additional times with similar results). “28S” products were diluted 5-fold relative to other products prior to gel electrophoresis. C Effects of progressively reducing the donor’s 5′ UTR from the 3′ direction (n = 3). Each condition was transfected with the same molar quantities of editor and donor RNAs (1:2 molar ratio). Filler RNA compensates for differences in total mass. D Effects of progressively reducing the donor’s 3′ UTR from the 5′ direction (“325” data in 3c as “0 nt 5′ UTR”) (n = 3). Each condition was transfected with the same molar quantities of editor and donor RNAs (1:2 molar ratio). Filler RNA compensates for differences in total mass. E Comparison of native donor and mini donor’s integration efficiencies (data from 3c,d) and theoretical insertion scar size (n = 3). F Effects of progressively reducing the donor’s 5′ homology from the 5′ direction (n = 3). G Effects of progressively reducing the donor’s 3′ homology from the 3′ direction (n = 3). H Effects of progressively increasing the cargo’s length with two different filler sequences (n = 3). Each condition was transfected with 400 ng of total RNA with the same molar quantities of editor and donor (1:2 molar ratio). Filler RNA compensates for differences in total mass. For bar graphs, each dot represents a technical replicate, the bars represent the mean, and the error bars represent the standard deviation. When relevant, p values are calculated with Student’s two-sided t test. Source data are provided as a Source Data file.