Extended Data Fig. 5: Analysis of integration outcomes at the 28S target and NOLC1 locus with payload variations.

a) Gaussia luciferase exon 2 (Gluc) payload insertion by wild type and ___domain inactivated mutants of R2Tg into a 28S plasmid reporter, with editing outcomes profiled by NGS at the upstream (left) junction. Mutants tested are WT R2Tg and R2Tg^D1275A (RLE mutant) and outcomes are classified as perfect insertions, insertions with indels, or WT locus indels. b) Schematic of additional payload variant with internal homology arms against the 28S target. c) Gaussia luciferase exon 2 (Gluc) payload insertion by wild type R2Tg into a 28S plasmid reporter with payload variants shown in part B, with editing outcomes profiled by NGS at the upstream (left) junction. Outcomes are classified as perfect insertions, insertions with indels, or WT locus indels. d) Size analysis by gel electrophoresis of 5′ and 3′ insertion junctions at the 28S target reporter for payload designs from part (b) and (c) after R2Tg integration. Payload numbers correspond to those in B. e) Gluc exon 2 payload insertion by WT R2Tg, R2Tg^D1275A, or the RT ___domain deletion R2Tg^Δ(875-885) into a 28S plasmid reporter with payloads containing 28S or AAVS1 targeting homology arms, profiled by NGS. Statistics were calculated using unpaired t-test. f) Biochemical retrotransposition of different RNA payloads into the AAVS1 DNA target with the R2Tg protein, dNTPs, and MgCl₂, as indicated. Either no payload was used or the following two payloads were used: 1) payload with a 5′ UTR targeting AAVS1 and containing a Gluc insert, or 2) a payload with 5′ and 3′ UTRs targeting NOLC1 and containing an EGFP insert. Labels on the gel indicate the specific TPRT product, DNA target band, and R2Tg produced nicked fragments. g) Validation of AAVS1 NGS method. Synthetic eblocks containing editing and unedited DNA sequences were mixed at defined ratios (x-axis) and measured by NGS (y-axis). Agreement between the known editing percentage (x-axis) and measured editing percentage was calculated by linear regression and is shown inset. h) Validation of the NOLC1 3-primer NGS assay using mixes of genomic DNA from unedited or heterozygously inserted cells at NOLC1, as measured by ddPCR. Shown is the known pre-mixed ratio of edited and unedited gDNA (x-axis) vs the measured editing rate by NGS (y-axis). Inset, coefficient of determination between values on x- and y-axes. i) Schematic of payload engineering for R2Tg reprogramming to the NOLC1 locus. j) EGFP payload insertion at human endogenous NOLC1 locus by natural reprogrammed wild-type R2Tg as well as R2Tg^D1275A and R2Tg^RTmut. Insertion is quantified by ddPCR. Statistics calculated with unpaired t-test. k) Payload insertion by SpCas9^H840A-R2Tg^Δ1-183 or SpCas9^H840A-R2Tg^{Δ1-183,D1275A} into the endogenous NOLC1 locus, mediated by dual guides or non-targeting guides and quantified by ddPCR. Inset shows payload design and locus schematic with homology arms colored and top guide in red and bottom guide in blue. Statistics calculated with unpaired t-test. l) Secondary structure analysis of the 5′ UTR of R2Tg, including the full length, 15 nt truncated variant, and the 15 nt truncated variant with the 50 nt 28S homology sequence upstream. m) Validation of the 3-primer NGS assay for analysis of AAVS1 integration via the left insertion junction. Standards consist of edited and WT amplicons that are mixed in the listed ratios (x-axis) and the measured editing is determined by the 3-primer NGS assay (y-axis). n) Gluc integration at the endogenous AAVS1 locus via the SpCas9^H840A-R2Tg^Δ1-183 fusion using payloads with the full length or 15-nt truncated 5′ UTR, an upstream 28S 50 nt sequence, and internal AAVS1 homology arms. Integration is quantified by next-generation sequencing (left) and ddPCR (right). Error bars represent mean +/− s.e.m. n = 3 where n represents three biological replicates.