Fig. 1: Development of a targeted integration system based on Cas9 and PB transposase.

a FiCAT technology deployed in the reporter cell line. FiCAT: Cas9 (purple) is combined with an engineered PB transposase domain (in red). Reporter cell line was generated by insertion of a C-terminal fragment of GFP preceded by a splice acceptor and gRNAs target sites in HEK293T cell line. PB transposon was generated by introducing the complementary N-terminal fragment of GFP followed by a splice donor under the control of a CAG promoter between ITRs. Gray triangles: PB ITRs; SA splice acceptor, SD splice donor, Target: targeted insertion site containing AAVS1-3 and TRAC-1 target sequences. b On-target and overall efficiency of cas9 catalytic variants fused to hyPB in different topologies, targeting AAVS1-3 site in reporter cell line. Nuclease cas9_PB fusion shows better results in targeted and overall insertion as opposed to dead cas9 (dcas9) or nickase cas9 (ncas9) fusions. Efficiency is represented indicating the percentage of insertion reported by GFP (on-target) and RFP (overall) positive cells by flow cytometry. Targeted insertion (light purple) and off-target insertion (yellow) Mean ±  SD of n = 2 technical replicates plotted. c On-target integration efficiency of PB variants fused to Cterm-cas9. PB mutants involved in target DNA binding (R372A, K375A, R376A, E377A, E380A) or enhanced excision (M194V, D450N) (details in Supplementary Table 2) using gRNA targeting the AAVS1 site (light pink), TRAC site (dark purple), or a non-targeting gRNA control (dark pink) in the reporter cell line. Mean ± SD of n = 4 independent experiments plotted. d Junction PCR between 3′ ITR and TRAC locus is shown (down panel) in + strand (1, 3) and − strand (2, 4) payload insertion, comparing FiCAT R372A_K375A_D450N (1, 2) and episomal (3, 4) from flow cytometry enriched populations. Representative image of n = 3. e Colocalization of double stranded breaks (DSB) and targeted DNA binding effects on PB-mediated targeted insertion. ZnF targeting the reporter cell line target site was fused to PB variants and cotransfected with cas9 and either TRAC or non-targeting gRNA to induce DSB. Mean ± SD of n = 2 technical replicates plotted. Technical replicates graphs are a representative image of n = 3 biological replicates. Source data are provided as a Source Data file.