Analyzing the process of mutation accumulation in MEK1 evolution experiment using amplicon sequencing

Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. we employed REPLACE to drive the continuous intracellular evolution of the cancer-related protein MEK1 with the aim of conferring resistance to Cobimetinib. To investigate the accumulation of mutations during this evolutionary process, we conducted amplicon sequencing on experimental materials collected at different stages. The results revealed intricate relationships among different mutations, highlighting the complex nature of the evolutionary landscape. MEK1 cDNA from H. sapiens was cloned into the repRNA-v4 plasmid, and the resulting replicative RNAs were electroporated into BHK-21 cells expressing nsP4 (stable cell line containing ML700 plasmid). Medium was replaced with fresh medium containing 5 μg/ml puromycin 24 h post-electroporation (i.e., Day 1). After 4 d of selection (i.e., Day 5), 2 μM of molnupiravir was added to the medium. On Day 7, cobimetinib was added to the medium. The cells were continued to be cultured and selected in medium with molnupiravir and cobimetinib (2-5 μM) for 7 days. To analyze the enrichment process of the screened mutations, amplicon sequencing was performed on MEK1 RNA extracted from cells at Day 1, and Day 14.