Comprehensive Off-target Analysis of dCas9-SAM-mediated HIV Reactivation via Long Noncoding RNA and mRNA Profiling

CRISPR/CAS9 (epi)genome editing brought revolutionary changes to the field of gene and cell therapy. However, a major concern is the potential off-target effects. A comprehensive analysis of the host transcriptome including mRNA, lncRNA, and alternative splicing was done using human cell line expressing the dead Cas9-mediated synergistic activation mediator (dCas9-SAM) plus HIV long terminal repeat (LTR)-specific MS2-mediated single guide RNA (msgRNA). The control scrambled msgRNA (LTR-Zero), and two LTR-specific msgRNAs (LTR_L and LTR_O) groups show very similar expression profiles of the whole transcriptome. Among 839 identified lncRNAs, none exhibited significantly different expression in LTR_L vs. LTR-Zero group. In LTR_O group, only TERC and scaRNA2 lncRNAs were significantly decreased. Among 142,791 mRNAs, four genes were differentially expressed in LTR_L vs. LTR-Zero group. There were 21 genes significantly downregulated in LTR_O vs. either LTR-Zero or LTR_L group and one third of them are histone related. The distributions of different types of alternative splicing were very similar either within or between groups. There were no apparent changes in all the lncRNA and mRNA transcripts between LTR_L and LTR-Zero groups. This is the most comprehensive study of its kind to date demonstrating the rare off-target effects of the HIV-specific dCas9-SAM system in human cells.