Fig. 1: CRISPR-Cas9-mediated targeted integration of the α-PD-1 cassette into the GAPDH locus in HEK293T cells via IDLV delivery.

a Schematic overview of the donor plasmid, Cas9/sgRNA expression plasmid, and targeting strategy for α-PD-1 integration into GAPDH 3′-UTR. Positions of the PCR primers (black arrows) used for detection of integrated DNA fragments are indicated. Fine gray lines on donor plasmids indicate sections homologous to the GAPDH locus. Lightning shape, sgRNA target sequence, HR, homologous recombination-based strategy, HMEJ, homology-mediated end joining-based strategy, LHR/RHR, left/right arm of homology recombination, F1/R2, outer forward/reverse primer, F2/R1, inner forward/reverse primer. b The mismatch-sensitive endonuclease T7E1 assay results showed the different efficiencies of Cas9/sgRNA-1, 2, and 3 for targeting human HEK293T genome. HEK293T cells were transfected with Cas9/sgRNA-1, 2 or 3 expression plasmid, without donor plasmid. Genomic DNA was extracted for T7E1 assay at day 4 post transfection. c FACS analysis of HEK293T cells showed the knock-in efficiencies of the α-PD-1 mAb in HEK293T cells. IDLV with HR-donor alone, IDLV expressing Cas9/sgRNA alone, or the two IDLVs together, were allowed to infect HEK293T cells. Control without IDLV infection is shown on the top. d CD90⁺ cells were sorted for genomic PCR analysis. Two sets of primers specific for the 5′ or 3′ integration junctions were used. Primer pair F1/R1 and F2/R2 amplified the 5′-junction (1435 bp) and the 3′-junction (1008 bp) of the transgene integration respectively. Primers F1/R2 amplified two DNA fragments that represent the wild type (2176 bp) and modified gene (4929 bp), respectively. e Relative knock-in efficiencies of HR and HMEJ-based strategies in HEK293T cells. Cells were infected with IDLV carried HR-donor or HMEJ-donor along with IDLV expressing Cas9/sgRNA at different MOIs. CD90 expression was analyzed by FACS 5 days post infection. Data are representative of three independent experiments (means ± SEM), **P < 0.01, ns, no significant difference; two-tailed Student’s t test (e) was used.