Extended Data Fig. 9: Evaluation of viral templates for PASTE and characterization of editing in non-dividing cells.

a) Schematic of PASTE performance in the presence of cell cycle inhibition. Cells are transfected with plasmids for insertion with PASTE or Cas9-induced HDR and treated with aphidicolin to arrest cell division. Efficiency of PASTE and HDR are read out with ddPCR or amplicon sequencing, respectively. b) Editing efficiency of single mutations by HDR at EMX1 locus with two Cas9 guides in the presence or absence of cell division read out with amplicon sequencing. Data are mean (n = 3) ± s.e.m. c) HDR mediated editing of the EMX1 locus is significantly diminished in non-dividing HEK293FT cells blocked by 5 µM aphidicolin treatment. Data are mean (n = 3) ± s.e.m. d) Integration efficiency of various sized GFP inserts up to 13.3 kb at the ACTB locus with PASTE in the presence or absence of cell division. Data are mean (n = 3) ± s.e.m. e) Effect of insert minicircle DNA amount on PASTE-mediated insertion at the ACTB locus in dividing and non-dividing HEK293FT cells blocked by 5 µM aphidicolin treatment. Data are mean (n = 3) ± s.e.m. f) PASTE efficiency of EGFP integration at the ACTB locus in K562 cells. Data are mean (n = 3) ± s.e.m. g) Insertion templates delivered via AAV transduction. Templates were co-delivered via AAV dosing at levels indicated. Data are mean (n = 3) ± s.e.m. h) PASTE integration of GFP at the ACTB locus with the GFP template delivered via AAV in HEK293FT cells. i) PASTE integration of GFP at the ACTB locus with the GFP template delivered via AAV at different doses in HEK293FT cells. Data are mean (n = 3) ± s.e.m. j) Integration efficiency of AdV delivery of integrase, guides, and cargo in HEK293FT and HepG2 cells. BxbINT and guide RNAs or cargo were delivered either via plasmid transfection (Pl), AdV transduction (AdV), or omitted (-). SpCas9-RT was only delivered as plasmid or omitted. Data are mean (n = 3) ± s.e.m. k) Delivery of PASTE system components with mRNA and synthetic guides, paired with either AdV or plasmid cargo. Data are mean (n = 3) ± s.e.m. l) Attachment site insertion efficiency at the LMNB1 locus using PASTE delivered as mRNA with synthetic atgRNA and nicking guides. Data are mean (n = 3) ± s.e.m. m) Integration efficiency at the LMNB1 locus using PASTE delivered as mRNA (Trilink versions), synthetic atgRNA and nicking guides, and adenoviral delivered EGFP cargo. All conditions contain full length PASTE mRNA and are optionally supplemented with additional Bxb1 mRNA as indicated. Data are mean (n = 2) ± s.e.m.