Tracing the lineage of replicative RNAs in the process of KRAS (S17N) evolution

A dominant-negative gene therapy approach has been proposed and tested on proto-oncogene KRAS, wherein the oncogenic activity (and cell proliferation) of KRAS can be suppressed by introducing a dominant-negative KRAS allele (S17N). We employed REPLACE to conduct continuous evolution on KRAS (S17N) and examined its potential pathways for conferring resistance in this gene therapy methodology.To explore the accumulation of mutations in various RNAs during the KRAS (S17N) evolution experiment, we established a barcoded library and conducted lineage tracing of replicative RNAs carrying KRAS (S17N) throughout the evolution process. To investigate the process of mutation accumulation in different RNAs in the KRAS (S17N) evolution experiment, we generated a library of tens of thousands of barcodes by incorporating a 20-bp random sequence (20 Ns) downstream of the stop codon in KRAS (S17N)-P2A-PuroR gene. The library, after undergoing transformation, in vitro transcription, and electroporation, was delivered to the BHK-21 host cells. Medium was replaced with fresh medium containing 10 μg/mL puromycin 24 h post-electroporation (i.e., Day 1). After 4 d of selection (i.e., Day 5), approximately 2 million cells were transferred to a new 10-cm dish and subjected to RNA mutagenesis using molnupiravir (2 μM). When the plate reached approximately 90% confluence, the cells were consistently subcultured at a 1:5 ratio and the medium was daily replenished with 10 μg/mL puromycin and 2 μM molnupiravir treatment. The remaining cells were partially cryopreserved as backup and partially utilized for RNA extraction and mutation analysis. Cells at day 1, day 5, day 7, and day 14 were collected for total RNA isolation and reverse transcription. The KRAS (S17N)-T2A-PuroR-barcode region was amplified from both the DNA library plasmid and the aforementioned cDNA libraries using indexed primers. The purified PCR products were mixed and subjected to adapter ligation, fragment selection, primer and polymerase binding, followed by sequencing using a PacBio Sequel IIe instrument for Circular Consensus Sequencing (CCS) reads acquisition.