Cas9 nuclease

IL-17C HEK-Blue reporter cells (Invivogen) were transfected in 12-well plates with single guide RNA targeting either IL17RA or IL17RB and Cas9 nuclease (Synthego) using Lipofectamine CRISPRMAX Cas9 Transfection Reagent (Thermo Fisher). Five days post-transfection, cells were seeded into 10-cm dishes under limiting dilution. After 2–3 weeks, well-separated single colonies were transferred (via cloning discs) into 24-well plates, then expanded and screened for loss of receptor expression and responsiveness to IL-25.

CRIPSR/RNP transfection was performed as previously described.^{51 (link)} RNP electroporation was performed with naive CD8⁺ T cells (for LCMV Cl13 infection), or total CD8⁺ T cells (for tumor experiments) isolated from spleens of donor P14 mice using EasySep immunomagnetic negative selection kits from STEMCELL. Briefly, Cas9 (Alt-R S.p. Cas9 Nuclease, IDT) and sgRNAs (Synthego) were combined and incubated at RT for 10 min. For each target, two sgRNAs was used to increase knockout efficiency. Electroporation was performed using the 4D-NucleofectorTM 4 Core Unit and P3 primary cell 4D-NucleofectorTM5 X kit S with program DN100. Following the electroporation, cells were kept in an incubator for 10 min at 37°C. For LCMV Cl13 infection studies, 2,500 cells were immediately adoptively transferred to naive C57BL/6 recipient mice via i.v. injection followed by LCMV Cl13 infection. For tumor studies, the cells were activated with anti-CD3 and anti-CD28 for 3 days and 1x10⁶ cells were adoptively transferred to separate groups of tumor-bearing mice. For co-transfer tumor experiments, cells were mixed at a 1:1 ratio (1x10⁶) before adoptive transfer.