Analysis of the accumulation of mutations in self-replicating RNAs using barcoded EGFP library

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. To investigate the process of mutation accumulation in REPLACE system, we constructed a repRNA-v4 plasmid library containing 64 barcodes. Using this library, we analyzed the differences in mutation accumulation for different RNAs upon entry into cells, before and after molnupiravir treatment, and before and after FACS sorting. The results demonstrated that these barcoded RNAs undergo similar processes of mutation accumulation, providing evidence that mutations are commonly accumulated across different RNAs. To investigate the process of mutation accumulation in the REPLACE system, a repRNA-v4 plasmid library containing 64 barcodes was constructed. The library was generated by amplifying barcoded sequences from the repRNA-v4 plasmid using primers containing degenerate bases (F primer: gaagcaatccgcgaaaaatgcccggtcgacc, R primer: cattggggcgtactagtatcgatgccgctcctaggcttcScWaScWcSgWgatttaggcaccgggcttgcgggt). The original fragment was then replaced with the barcoded PCR products through enzymatic digestion using SalI and AvrII enzymes (NEB). The resulting ligation products were transformed into competent Escherichia coli cells (Trelief 5α) for plasmid amplification. Next, the plasmids were subjected to in vitro transcription, and 10 μg of RNA was electroporated into 4 million BHK-21 cells (with nsP4 expressing in trans). Twenty-four hours post-electroporation (referred to as T1), the medium was replaced with fresh medium containing 5 μg/ml puromycin. After four days of selection (referred to as T5), 10 μM of molnupiravir was added to the medium for a 2-day treatment. Subsequently, the cells were allowed to grow for four days (referred to as T3) and then subjected to flow cytometry sorting. The sorted cells were cultured for approximately one week (referred to as T4) and subsequently treated with molnupiravir for another 2 days (referred to as T5). Cells at each time point (T1 to T5) were collected, and total RNA was extracted using Trizol reagent. The extracted RNA was subjected to reverse transcription, followed by amplification using primers specific to genomic RNA targeting the EGFP-PuroR-barcode region. The purified PCR products underwent adapter ligation, fragment selection, primer and polymerase binding, and were then sequenced using a PacBio Sequel IIe instrument to obtain Circular Consensus Sequencing (CCS) reads.