The mutagenesis of CAST was performed using primer pairs (table S4). The target amino acid position was coded by NNK (sense strand), where N = A, G, C, or T and K = G or T. The whole-plasmid PCR method was performed using Proofast Super-Fidelity DNA polymerase with the plasmid DNA isolated from the second round of FACS enrichment as the template DNA using the following PCR thermocycling profile: 95°C for 3 min, 30 cycles of 95°C for 15 s, 55°C for 15 s, 72°C for 5 min, and 72°C for 10 min. The PCR products were digested with DpnI to remove the parent plasmid and then were cleaned using a DNA purification kit (Shanghai Life iLab Biotech, China). The resulting DNA mixture was transformed into E. coli JM107* cells for screening.

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