Figure 1.

PD-1-targeting CRISPR-Cas9 is compatible with a TIL-based ACT workflow

(A) Overview of the TIL-based ACT workflow from surgical resection to patient treatment, including the addition of PD-1 CRISPR-Cas9. (B) Base-pair level depiction of PDCD1-targeting gRNA binding to the target site (black bars). Expected cut site is marked with scissors, and the protospacer adjacent motif (PAM) for Cas9 binding is in boldface. Chromosomal location, strand direction, and sense are annotated. (C) Electroporation was used to deliver non- (mock) or PDCD1- (edited) targeting RNPs to pre-REP TILs. Cell recovery 1 h post-electroporation is shown. Statistical significance was calculated via paired t test (p = 0.0095). (D) Mock and edited pre-REP TILs were expanded with the 14-day REP protocol. TILs were counted, and fold expansion was calculated at days 7, 10, and 14. (E) Comparison of final fold expansions (day 14) for melanoma and non-melanoma REPs. (F) Calculated CD4/CD8 ratio in day 14 REP samples. Statistical significance calculated via paired t test (p = 0.0302). See also Figure S1. (B–F) Each point represents the average of two replicates per sample. Mock samples are shown as black dots on a white bar and edited samples as black triangles on a shaded bar. Bars signify median of 10 samples. (A–B) Created with BioRender.com.