Analyzing the process of mutation accumulation in KRAS 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. Using REPLACE, we attempted continuous intracellular evolution of the negative dominant mutant KRAS (S17N). To analyze the process of mutation accumulation, we performed amplicon sequencing on experimental materials at different stages and under different treatment conditions. The results indicated that the mutations generated by this system were primarily induced by Monanunavir, and the addition of Monanunavir significantly accelerated the rate of evolution. Overall design: KRAS (S17N) 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. The cells were continued to be cultured and selected in medium with molnupiravir and puromycin for 10 days. To analyze the enrichment process of the screened mutations, amplicon sequencing was performed on KRAS (S17N) DNA, in vitro transcribed RNA, and RNA extracted from cells at Day 1, Day 5, Day 9, Day 12, and Day 14. Additionally, to compare the effect of molnupiravir treatment, amplicon sequencing was performed on RNA from cells at Day 9 that did not undergo molnupiravir (Day 5-Day 9) treatment.