Characterization of gRNA-Dependent and gRNA-Independent Off-Target Binding Sites of PspCas13b and RfxCas13d in Mammalian Cells [RNA-Seq]

CRISPR-Cas13 systems, harnessed for RNA-guided transcriptome editing, hold significant promise for clinical and in vivo therapeutic applications. However, understanding their in vivo target specificity and recognition rules remains a challenge. In this study, we employed the uSpyCLIP method, which enhances sensitivity and specificity for identifying RNA-binding protein (RBP) binding sites, to map the transcriptome-wide binding sites of catalytically inactive PspCas13b (dPspCas13b) and RfxCas13d (dRfxCas13d) in HEK293T cells, using a variety of single guide RNAs (gRNAs). Surprisingly, we identified both gRNA-dependent and gRNA-independent off-target binding sites for both dCas13 complexes. These off-target sites exhibited distinct RNA structural and sequence signatures: dPspCas13b's gRNA-independent binding was associated with specific RNA structural features, while dRfxCas13d's was linked to unique sequence motifs. Analysis of gRNA-dependent off-target sites revealed the crucial role of the DR-distal and middle regions of the gRNA in determining binding specificity. mRNA sequencing and biochemical assays further indicated that while off-target binding does not directly affect mRNA levels, it can perturb the expression of certain endogenous proteins. Therefore, our findings provide important insights into the characteristics of gRNA-dependent and gRNA-independent off-target binding for PspCas13b and RfxCas13d, offering valuable guidance for optimizing Cas13 and gRNA design in future applications. Overall design: Lenti-X 293T cells transfected with empty vector, Lenti-X 293T cells transfected with dPspCas13b expression plasmid, Lenti-X 293T cells co-transfected with dPspCas13b expression plasmid and a non-targeting gRNA (gNT) constructs, Lenti-X 293T cells co-transfected with dPspCas13b expression plasmid and a targeting gRNA (EZH2-g1, EZH2-g2 or TP53-g1) constructs, Lenti-X 293T cells transfected with dRfxCas13d expression plasmid, Lenti-X 293T cells co-transfected with dRfxCas13d expression plasmid and a non-targeting gRNA (gNT) constructs, Lenti-X 293T cells co-transfected with dRfxCas13d expression plasmid and a targeting gRNA (EZH2-g1, EZH2-g2 or TP53-g1) constructs were prapared for RNA-seq.