Extended Data Fig. 7: Additional evaluation of SpCas9^H840A-R2Tg^Δ1-183 and SpCas9^H840A-R2Tocc^Δ1-169 STITCHR systems at the endogenous AAVS1 and NOLC1 loci and reporter targets.

a) Schematic of SpCas9^H840A fused to N- and C-terminal truncations of R2Tg at different amino acid positions. Not all tested constructs are shown. b) Gluc payload insertion by different SpCas9^H840A-R2Tg^Δ1-183 fusions, according to the schematic in (a), into the endogenous AAVS1 locus quantified by NGS. N-term and C-term denote either N-terminal or C-terminal fusions of the full length R2Tg protein. Denoted residue positions indicate the starting amino acid position of N-terminal R2Tg truncations that are fused to the C-terminal of SpCas9^H840A. c) Gluc integration at the endogenous AAVS1 target by SpCas9^H840A-R2Tg^Δ1-183, SpCas9^H840A-R2Tg^{Δ1-183,F876A/A877L/D878A/D879A/L880A/V881A/L882A} (RTmut), and SpCas9^H840A-R2Tg^{Δ1-183,Δ(875-885)}, and SpCas9^H840A alone. Editing rates were quantified by NGS (left) and ddPCR (right). d) TPRT activity in HEK293FT cells with SpCas9^H840A alone or fused to R2Tg, R2Tg^{Δ1-183,F876A/A877L/D878A/D879A/L880A/V881A/L882A} (RTmut), or R2Tg^{Δ1-183,Δ875-885} into the NOLC1 genomic target with dual guides. EGFP payload contains the full 5′ and 3′ UTRs for R2Tg. e) Gluc payload insertion into a 28S plasmid reporter in HEK293FT cells by selected nLTR retrotransposons fused to SpCas9^H840A, with either targeting or non-targeting guides, quantified by Gluc production normalized to a control Cluc. Data is shown as ratio of targeting signal to non-targeting signal. f) Gluc payload insertion into the endogenous AAVS1 locus in HEK293FT cells by selected nLTR retrotransposons fused to SpCas9^H840A, with either targeting or non-targeting guides, profiled by next generation sequencing. Editing outcomes are quantified as perfect insertions, insertions with indels, and indels at the unmodified WT target site. g) Gluc payload insertion into the endogenous AAVS1 locus in HEK293FT cells by selected nLTR retrotransposons fused to so SpCas9^H840A and an AAVS1-targeting or non-targeting sgRNA control, quantified by ddPCR h) Validation of the AAVS1 3-primer NGS assay using mixes of genomic DNA from unedited or heterozygously inserted cells at AAVS1, 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) R2Tocc retrotransposition of a synthetic RNA payload into top- and bottom-strand labeled 28S DNA. The top strand is FAM labeled (red); the bottom strand is Cy5 labeled (green). Schematics on the side of the gel indicate the expected size of each band, including the TPRT product and cleaved target fragments. j) Indels or substitutions found in the sequencing reads of AAVS1 in vitro TPRT shown in Fig. 5b, analyzed by CRISPResso. Above, a reference sequence consisting of correct insertion of the Gluc payload into AAVS1 DNA. Below, a schematic of the different insertion outcomes found by sequencing, the raw number of reads and % of total reads which these correspond to. Error bars represent mean +/− (b,g) s.d. or (d, e, f, h) s.d. n = 3 where n represents 3 biological replicates.