CD45-deficient cell lines were generated using the pX330 vector (38) and guide RNAs targeting CD45 (59). The pX330 vector was introduced using electroporation and cultured for ~4 days. Cells were stained for surface CD45, and cells with low CD45 expression were sorted into 96-well plates using a BD FACSAria. Cells were expanded and analyzed for CD45 expression using flow cytometry and immunoblot to confirm CD45-deficient clones. Mutations to PTPRC, which encodes CD45, were confirmed by sequencing. The target site was amplified by polymerase chain reaction using genomic DNA isolated from cell lines and cloned into a Topo vector. Cell lines that were reconstituted with CskAS were confirmed by immunoblot using anti-Csk and anti-Myc tag antibodies.

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