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).

哺乳动物细胞中的定向进化（Directed evolution）可助力适配哺乳动物的生物分子工程改造，并为哺乳动物细胞赋予合成进化能力。我们构建了一套正交甲病毒RNA复制系统（orthogonal alphaviral RNA replication system），用于进化合成RNA器件，从而在活哺乳动物细胞中实现RNA聚合酶辅助的连续进化（REPLACE，RNA replicase-assisted continuous evolution）。为分析向BHK-21细胞中导入不同版本RNA复制系统（即repRNA-v3与repRNA-v4）对内源基因表达的影响，我们对3种不同细胞开展了RNA测序（RNA-seq）。转录组谱数据分析结果显示，repRNA-v4可激活较弱的干扰素信号通路，且可能具有更低的细胞致病性。本研究中，我们将repRNA-v3电转染至BHK-21细胞，同时将repRNA-v4电转染至反式表达nsP4的BHK-21细胞。将上述细胞置于含10 μg/ml嘌呤霉素的培养基中培养约3周。随后分别收集30万个反式表达nsP4的BHK-21细胞、30万个repRNA-v3细胞与30万个repRNA-v4细胞，用于RNA提取与文库制备。我们采用DNBSEQ-T7测序平台完成RNA测序。将质控后的150 bp双端读段通过STAR 2.7.8a比对至金仓鼠（Mesocricetus auratus）基因组（GCF_017639785.1_BCM_Maur_2.0_genomic.fna）与甲病毒RNA复制系统基因组。从美国国家生物技术信息中心（NCBI）下载基因组注释文件（GCF_017639785.1_BCM_Maur_2.0_genomic.gtf），并通过编辑补充甲病毒RNA复制系统的相关信息。使用featureCounts估算转录本丰度，随后通过DESeq2完成标准化处理与统计学比较：分别对比repRNA-v4细胞与反式表达nsP4的BHK-21细胞的转录组差异，以及repRNA-v3细胞与repRNA-v4细胞的转录组差异。