RNA sequencing of BHK-21 cells carrying alphaviral RNA replication system

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. RNA-seq of 3 different cells was performed to analyze the effect of introducing different versions of RNA replication systems (i.e., repRNA-v3 and repRNA-v4) into BHK-21 cells on endogenous gene expression. Analysis of the transcriptome profiling data indicated that repRNA-v4 stimulated lower interferon signaling and may have lower cytopathicity. In this study, repRNA-v3 was electroporated into BHK-21 cells and repRNA-v4 was electroporated into BHK-21 cells expressing nsP4 in trans. The cells were cultured for ~3 weeks with the presence of 10 μg/ml of puromycin. 300,000 of BHK-21 cells expressing nsP4 in trans, 300,000 of repRNA-v3 cells and 300,000 of repRNA-v4 cells were collected for RNA isolation and library preparation. RNA-seq was performed on a DNBSEQ-T7 platform. Cleaned paired-end 150 bp reads were aligned to the Mesocricetus auratus genome (GCF_017639785.1_BCM_Maur_2.0_genomic.fna) and the alphaviral RNA replication system genome using STAR 2.7.8a. Genome annotation file (GCF_017639785.1_BCM_Maur_2.0_genomic.gtf) was downloaded from NCBI and edited to add the information of the alphaviral RNA replication system. Transcript abundances were estimated using featureCounts, then normalized and statistically compared using DESeq2 (i.e., the transcriptome difference between the repRNA-v4 cells and the BHK-21 cells expressing nsP4 in trans, the transcriptome difference between the repRNA-v3 cells and the repRNA-v4 cells).