Comprehensive off-target analysis of dCas9-SAM-mediated HIV reactivation via long noncoding RNA and mRNA profiling

Abstract

Background: 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 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.

Results: 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 the LTR_L and LTR_Zero groups.

Conclusion: This 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.

Keywords: CRISPR; Genome editing; HIV; Latency; Off-target; RNA sequencing; Shock and kill; Transcriptome.

Fig. 1

No difference in the entire RNA transcripts among the three experimental conditions. a Diagram showing the HIV proviral activation by the dCas9-SAM system with msgRNAs targeting LTR_L or LTR_O. b Box plot and density plot for the distribution of transcript expression levels measured by FPKM (averaged within replicates) of the three conditions. The plotted region of the box plot represents the maximum, upper quartile, median, lower quartile, and minimum, respectively, from top to bottom. c Hierarchical clustering of samples based on Pearson correlation coefficient of transcript expression levels for all the pairwise comparisons of the samples

Fig. 2

No difference in the lncRNAs among the three experimental conditions. a Predicted lncRNAs based on four coding potential filtering methods. CPC, Coding-Potential Calculator; PFAM, Protein FAMily analysis; PhyloCSF, Phylogenetic Codon Substitution Frequency; CNCI, Coding-Non-Coding Index. b Expression level distribution of the 839 lncRNAs in the six samples (FPKM values are z-score normalized)

Fig. 3

Hierarchical clustering of the six samples based on the Jaccard index for SNPs (a) and indels (b)

Fig. 4

Summary statistics of the 12 types of alternative splicing in the six samples. The number of events for each type is log₁₀ transformed

Fig. 5

The sashimi plot showing exon skipping in TOPORS that exhibits significant differential regulation between the LTR_O group and the control group. The top left panel shows the FPKM of reads that supports the corresponding exons and exon junctions in the two LTR_O samples and two control samples, respectively. The top right panel shows the posterior distribution of Ψ (the fraction of inclusive isoform), with the red line denoting the estimated Ψ and grey lines the 95% confidence interval of Ψ. The bottom panel shows the two transcripts due to exon skipping in the bottom transcript