Sequence analysis of transgene 5′ junctions suggests several mechanisms of their formation. (A) Categorization of insertion 5′ junctions yielded by RNA templates with TriCasA, SL-28, and gRz 5′ modules. 5′ junctions are assessed as either full-length, indicating that the full amount of the nonhomology region (in the case of annealing) or total (in the case of other categories) transgene cDNA length is detected, or truncated. Within “full-length” and “truncated,” 5′ junctions are classified according to the categories in C. (B) Quantification of transgene 5′ end-joining positions for insertions yielded by different 5′ module RNA templates. Colored lines above templates indicate 5′ module regions, with white corresponding to the L8 leader, red corresponding to each Rz, and blue corresponding to SL-28. The remaining transgene sequence is an identical 98 nt 3′ module. Black vertical dashed line indicates the end of the region of rDNA homology. Red vertical dashed line indicates the end of 5′ module. (C) Depiction of different mechanisms of 5′ junction formation deduced from WGS analysis of cells with transgene insertions yielded by cotransfection of ZoAl R2p mRNA and RNA templates with different 5′ modules. (D) Quantification of number of nucleotides of inferred microhomology between transgene cDNA and rDNA at 5′ junctions. Nucleotides of microhomology (gray bars) are compared to the expected frequency of microhomology by chance (black lines, see Materials and Methods). (E) rDNA-transgene join position frequency for insertions with no detected microhomology yielded by different 5′ module RNA templates. Join positions are determined relative to the position of first-strand nicking, defined as 0. Black, full-length insertion events. Gray, truncated insertion events.
