Fig. 4: Deployment of FiCAT in additional cell lines models and in vivo in mice liver.

a C2C12 cells were transduced with RFP transposon alone (episomal), or in combination with FiCAT R372A_K375A_D450N and gRNA targeting Lama2 gene (spacer 271.1), RFP positive cells were monitored for 2 weeks after transduction. Mean ± SD of n = 2 technical replicates plotted. Representative image of n = 3. b Karyoplot showing detected insertions in the c2c12 genome. c Junction PCR between 3′ ITR and Lama2 locus is shown (down panel) in + strand (1, 3) and − strand (2, 4) payload insertion comparing FiCAT (3, 4) and episomal (1, 2) treated enriched populations. Representative image of n = 3. d Coverage at the on-target junction (Lama2 site). e RFP transposon alone (episomal) or together with hyPB or FiCAT R372A_K375A_D450N mRNA delivered with in vivo JetPEI reagent were used to target Rosa26 safe harbor in mouse genome. Relative copy number of RFP transgene in liver was measured by semiquantitative qPCR and normalized to relative double copies of Tfrc gene (diploid genomes). Mean ± SD of n = 2 animals/condition. f Liver integration of minicircle luciferase transposon. Minicircle luciferase transposon, sgRNA targeting Rosa26 locus and FiCAT R372A_K375A_D450N mRNA were delivered by hydrodynamic injection and luciferase signal was monitored. A representative experiment of n = 3 is shown. g Junction PCR between transposon 3′ ITR and Rosa26 locus in liver genomic DNA. Mice were injected hydrodynamically with FiCAT R372A_K375A_D450N plasmid DNA or mRNA, gRNA targeting Rosa26 locus and minicircle transposon GFP payload and sacrificed 5 weeks after injection (detailed in vivo delivery methods and payloads in Supplementary Fig. 12a). PCR was performed amplifying genomic + strand integration. n = 2–3 animals/condition, numbers correspond to different individuals. 66% of treated mice with FiCAT mRNA or pDNA shows targeted insertion. Size of the band detected in FiCAT corresponds to the expected size of the amplified insertion. A higher size band is detected in the episomal sample considered background. Source data are provided as a Source Data file.