Skip to main content
. 2022 Oct 10;41(4):488–499. doi: 10.1038/s41587-022-01494-w

Fig. 2. Development of efficient recombinases for landing pads.

Fig. 2

a, Schematic of plasmid recombination assay. Cells are co-transfected with three plasmids, and, upon recombination, mCherry gains a promoter and is expressed. b, Plasmid recombination assay of predicted LSRs and att sites in HEK293FT cells, shown as corrected mCherry MFI. Error = s.d. (n = 3). P value was determined by one-tailed t-test. c, Example mCherry distributions for all three plasmids (LSR + attB + attP) compared to the attP-only negative control. d, Plasmid recombination assay between pairs of LSR + attP and attB in K562 cells (n = 1). e, Schematic of genomic landing pad assay. An EF-1α promoter, attB and LSR are integrated via lentivirus. Upon attP donor transfection and successful integration into the landing pad, mCherry is expressed, and the LSR and GFP are displaced and knocked out. f, Donor integration into polyclonal genomic landing pad (LP) K562 cell lines, measured after 5 days (n = 2 independently transduced and then electroporated replicates). g, Donor integration into clonal LP cells. Asterisks show significance for comparison with Bxb1 (P = 0.0012, one-way ANOVA, n = 3 clonal cell lines for Pa01 and n = 4 clonal cell lines for others at 1,000-ng dose, error = s.e.m.). h, Pa01 clonal LP line electroporated twice in rapid succession. i, Plasmid recombination assay for a new batch of LSRs selected for higher quality (Methods) in HEK293FT cells, shown as corrected mCherry MFI. Error = s.d. (n = 3 transfections). Controls are labeled in bold, and the previous batch is in italics. The dotted line indicates the positive control Bxb1. P value was determined by one-tailed t-test. j, Representative mCherry distributions for all three plasmids (LSR + attB + attP) compared to the attP-only negative control.