Fig. 3: FiCAT benchmarking and optimization.

a Benchmarking of FiCAT R372A_K375A_D450N to Cas9 induced HDR (300 and 800 bp homology arms were used for left and right arms, respectively). Data were normalized to HDR activity at 4000 bp cargo. Cargo size indicates number of base pairs that compose the inserted payload. Transfection was performed on ½ GFP reporter cell line targeting AAVS1 site. Mean ± SD of n = 3 independent experiments plotted. b FiCAT R372A_K375A_D450N comparison to homology independent targeted integration (HITI) mediated by Cas9 fused to a catalytic dead mutant of hyPB (D268A, D346A, R372A, K375A, D450N). A payload GFP transposon under CMV promoter regulation that includes AAVS1-3 gRNA target sites adjacent to both ITRs was used. For the 9500 bp payload, the CDS of FVIII gene was cloned upstream of the split GFP cassette. FiCAT was performed by using TRAC-1 gRNA; while assisted HITI was done using the AAVS1-3 gRNA both targeting K-562. Mean ± SD of n = 2 technical replicates plotted. c Programmable insertion activity of FiCAT R372A_K375A_D450N using four different nuclease proteins. SpCas9 is used as control for programmable insertion with gRNA-TRAC-1 only (black). Each nuclease was used with three independent gRNAs (1–3) for targeted insertion in ½ GFP reporter cell line. In all cases a scramble gRNA was used for non-targeted activity measurement (gray). Mean ± SD of two technical replicates of a representative experiment out of 3 is shown. Upper panel denotes the relative position of the gRNA’s targets. d Programmable insertion activity of FiCAT R372A_K375A_D450N inserting a minicircle version of the reporter transposon or the full-length plasmid version of the transposon in the reporter cell line using AAVS-3 gRNA (left panel). The efficiency of programmable insertion for FiCAT was also tested using different fusion of Cas9 to one or two units of mutated hyPB (right panel). A representative experiment of an n = 3 is shown. Mean ± SD of two technical replicates plotted. FiCAT construct denotes the fusion of SpCas9 with hyPB mutant (R372A, K375A, D450N). Technical replicates graphs are a representative image of n = 3 biological replicates. Source data are provided as a Source Data file.