Fig. 4: Programmable in-cell genomic integration with type V-K CAST.

A, B Schematic representation of the dual plasmid system (left) and all-in-one plasmid system (right) expressing CAST and either an untargeted or targeted single guide RNA. The donor plasmid (pDonor) contains LE and RE, flanking either a reporter gene, a therapeutic transcript, or a selection marker. B Integration junction PCR products in the forward (Fwd) LE direction using the dual plasmid system indicate programmable integration to the human genome. Mock: transfection control (no bands expected). Lanes labeled 8, 12, and 15 correspond to LINE-1a 3’ targets 8, 12, and 15. C Junction PCR of Fwd integration events targeting two different AAVS1 sites (T1 and T2) confirm integration of the donor in the human genome. D Sanger sequencing trace of T1 target junction PCR products. Reference sequence (topmost sequence) with annotated Target and LE represents the expected integration junction sequence 63 bp away from the PAM. A 3-bp deletion is observed. E, F On-target integration efficiency determined from NGS for the expected LE integration junction product for MG64-1 with and without ClpX (E, n = 6 biological replicates for the target condition (T1) and n = 3 biological replicates for the non-targeting control (Null), with one standard deviation from the mean), and for MG64-1 and ShCAST (F, n = 4 biological replicates for both target condition (T1) and non-targeting control (Null), with one standard deviation from the mean). G Schematic of the on and off-target integration assay. Integration reads containing the target-to-LE junction will align upstream from the integration site (blue arrows), while reads containing the RE-to-target junction will align downstream from the integration site (pink arrows), leaving a target site duplication (TDS). H Convergence of AAVS1-T1 on-target reads, indicated by LE and RE reads aligning to the human genome with overlap due to the TDS confirms complete integration of the donor to the target site (T1). Experiments were done in duplicate. I Venn diagram of independent and shared off targets from two biological replicates of AAVS1-T1 sgRNA-containing samples. Source data are provided as a Source Data file.