Transcriptomic Analysis of csn2 Gene Mutant Strains of Streptococcus mutans CRISPR-Cas9 System

HE Xiao-ya, ZHANG An-qi, GONG Tao, LI Yu-qing

Abstract

To explore the differences in transcriptional levels between mutant strains of csn2 gene of CRISPR-Cas9 system of Streptococcus mutans (S. mutans) and wild-type strains.  Methods  The S. mutans UA159, csn2-gene-deleted strains (Δcsn2) and csn2-gene-covering strains (Δcsn2/pDL278-csn2) of S. mutans were cultivated. Total RNA was extracted, and high-throughput sequencing technology was used for transcriptome sequencing. Based on the GO analysis and the KEGG analysis of the differentially expressed genes, the biological processes involved were thoroughly examined. The qRT-PCR method was used to verify the transcriptome sequencing results.  Results  The transcriptome results showed that, compared with UA159, there were 176 genes in Δcsn2 whose gene expression changed more than one fold (P<0.05), of which 72 were up-regulated and 104 were down-regulated. The GO enrichment analysis and the KEGG enrichment analysis revealed that both the up-regulated and down-regulated differentially expressed genes (DEG) were involved in amino acid transport and metabolism. In addition, the biological processes that up-regulated DEGs participated in were mainly related to carbohydrate metabolism, energy production and conversion, and transcription; down-regulated DEGs were mainly related to lipid metabolism, DNA replication, recombination and repair, signal transduction mechanisms, nucleotide transport and metabolism. The functions of some DEGs were still unclear. Results of qRT-PCR verified that the expressions of leuA, leuC and leuD (genes related to the formation of branched-chain amino acids) were significantly down-regulated in Δcsn2 when compared with UA159 and Δcsn2/pDL278-csn2.  Conclusion  Through transcriptome sequencing and qRT-PCR verification, it was found that the expression of genes related to branched-chain amino acid synthesis and cell membrane permeability in Δcsn2 changed significantly.

 

Keywords: Streptococcus mutans, CRISPR-Cas, csn2, Transcriptome

 

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References


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