Extended Data Fig. 7: Additional characterization of attP mutants for improved editing and multiplexing.

a) Integration efficiencies of wildtype and mutant attP sites with PASTE at the ACTB locus. b) attP single mutants are characterized for PASTE EGFP integration at the ACTB locus. c) Relative enrichment values (calculated as ratio of integrated reads to total reads) for the wildtype Bxb1 and top 5 mutants from the mutagenesis screen d) Comparison of integration efficiency between PASTEv3 and Twin-PE integration at the ACTB locus, with both single atgRNA (46 bp) or dual atgRNA with PASTE-Replace (38 bp). e) Comparison of integration efficiency and residual attB formation between PASTEv3 with PASTE-Replace and Twin-PE integration at the NOLC1 locus with dual atgRNAs containing either a 46 bp or 42 bp attB sequence. f) Comparison of integration efficiency and residual attB formation between PASTEv3 with PASTE-Replace and Twin-PE integration at the CCR5 locus with dual atgRNAs containing a 38 bp attB sequence. g) Comparison of residual attB formation between PASTEv3 with PASTE-Replace and Twin-PE integration at the ACTB locus. h) Characterization of integration of a 5 kb payload at the ACTB locus with all 16 possible dinucleotides for attB/attP pairs between the atgRNA and minicircle. i) Schematic of the pooled attB/attP dinucleotide orthogonality assay. Each attB dinucleotide sequence is co-transfected with a barcoded pool of all 16 attP dinucleotide sequences and BxbINT, and relative integration efficiencies are determined by next generation sequencing of barcodes. All 16 attB dinucleotides are profiled in an arrayed format with attP pools. j) Relative insertion preferences for all possible attB/attP dinucleotide pairs determined by the pooled orthogonality assay. k) Orthogonality of BxbINT dinucleotides as measured by a pooled reporter assay. Each web logo motif shows the relative integration of different attP sequences in a pool at a denoted attB sequence with the listed dinucleotide. l) Representative fluorescence images of multiplexed PASTE gene tagging of ACTB, LMNB1, and NOLC1. Data are mean (n = 3) ± s.e.m.