Abstract
BackgroundCRISPR/CAS9 (epi)genome editing revolutionized the field of gene and cell therapy. Our previous study demonstrated that a rapid and robust reactivation of the HIV latent reservoir by a catalytically-deficient Cas9 (dCas9)-synergistic activation mediator (SAM) via HIV long terminal repeat (LTR)-specific MS2-mediated single guide RNAs (msgRNAs) directly induces cellular suicide without additional immunotherapy. However, potential off-target effect remains a concern for any clinical application of Cas9 genome editing and dCas9 epigenome editing. After dCas9 treatment, potential off-target responses have been analyzed through different strategies such as mRNA sequence analysis, and functional screening. In this study, a comprehensive analysis of the host transcriptome including mRNA, lncRNA, and alternative splicing was performed using human cell lines expressing dCas9-SAM and HIV-targeting msgRNAs.ResultsThe 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 the LTR_L and LTR_Zero groups.ConclusionThis is an extremely comprehensive study demonstrating the rare off-target effects of the HIV-specific dCas9-SAM system in human cells. This finding is encouraging for the safe application of dCas9-SAM technology to induce target-specific reactivation of latent HIV for an effective “shock-and-kill” strategy.
Highlights
CRISPR/CAS9genome editing revolutionized the field of gene and cell therapy
The aim of this study is to further explore the deficient Cas9 (dCas9)-synergistic activation mediator (SAM)-related potential off-target effects by generating deep sequence coverage of the entire transcriptome, comprehensively analyzing mRNAs, Long noncoding RNA (lncRNA), alternative splicing, genetic mutations including single-nucleotide polymorphisms (SNPs) and indels in TZM-bI cells stably expressing dCas9-SAM and HIV-specific MS2-mediated single guide RNA (msgRNA)
Very similar expression profiles at the whole transcriptome level among the three conditions In previous studies, 16 msgRNAs targeting the U3 region of the HIV long terminal repeat (LTR) were screened for their efficiency in guiding dCas9-SAM to activate HIV promoter activity [31]
Summary
CRISPR/CAS9 (epi)genome editing revolutionized the field of gene and cell therapy. Our previous study demonstrated that a rapid and robust reactivation of the HIV latent reservoir by a catalytically-deficient Cas (dCas9)-synergistic activation mediator (SAM) via HIV long terminal repeat (LTR)-specific MS2-mediated single guide RNAs (msgRNAs) directly induces cellular suicide without additional immunotherapy. A comprehensive analysis of the host transcriptome including mRNA, lncRNA, and alternative splicing was performed using human cell lines expressing dCas9-SAM and HIV-targeting msgRNAs. Recently, CRISPR/Cas genome editing technology has been rapidly developed and attracted extensive attention in biomedical research, with preclinical examples and potential clinical trials in genetic diseases, cancer biology, and infectious diseases [1,2,3,4,5,6,7]. In addition to transcriptional activation, the dCas property is extensively repurposed for transcriptional repression and DNA (de)methylation [12, 33,34,35] These epigenome-editing approaches can alter the epigenetic code of the target region, and offer a durable manipulation of many genes important in infectious diseases, cancer, and chronic noninfectious diseases [12, 36]. Permanent silencing of target genes in all cell types may require a combination of several epigenetic effectors [12]
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