Co-editing generates PSMB6 T35A mutant cells with reduced proteasome caspase-like activity. (A,B) Threonine 35 at the active site of the proteasome PSMB6 subunit is mutated to alanine using sgRNA (marked in green) and ssODN shown. (C,D). Caspase-like and chymotrypsin-like activities of control HEK293 TAF1ts repaired cells, and the T35A PSMB6 mutants were measured using the fluorescent substrates Z-LLE-AMC and Suc-LLVY-AMC as described in Methods. N = 4, * p < 0.05, ** p < 0.01,*** p < 0.001. The structures for the 20S proteasome and the PSMB6 subunit were derived from RSCB PDB ID 5LE5 [26,27].