Characterization of gRNA-dependent and gRNA-independent off-target binding sites of PspCas13b and RfxCas13d in mammalian cells
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Updated: Feb 24, 2026
Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens
Published on: June 6, 2020
Huanhuan Feng1,2, Zhixue Li1, Hongdao Zhang1
1State Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Key Laboratory of Molecular Andrology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.
Abstract:
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 gRNA-independent 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. Further analysis demonstrated that some off-target binding events led to changes in gene expression at the messenger RNA and/or protein level. Collectively, 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.
Insights
CRISPR-Cas13 RNA editing systems show promise but have off-target binding. This study mapped Cas13 binding sites, revealing both guide RNA-dependent and independent interactions, crucial for improving therapeutic RNA editing tools.
Area of Science:
- Molecular Biology
- RNA Therapeutics
- Gene Editing Technologies
Background:
- CRISPR-Cas13 systems offer RNA-guided transcriptome editing for therapeutic potential.
- Understanding in vivo target specificity and recognition rules for Cas13 is critical but challenging.
Purpose of the Study:
- To map transcriptome-wide binding sites of catalytically inactive PspCas13b (dPspCas13b) and RfxCas13d (dRfxCas13d) using various single guide RNAs (gRNAs).
- To investigate the characteristics of gRNA-dependent and gRNA-independent off-target binding for these Cas13 systems.
Main Methods:
- Employed the uSpyCLIP method for sensitive and specific identification of RNA-binding protein (RBP) binding sites.
- Analyzed binding sites of dPspCas13b and dRfxCas13d in HEK293T cells across the transcriptome.
Main Results:
- Identified both gRNA-dependent and gRNA-independent off-target binding sites for both dCas13 complexes.
- Discovered distinct RNA structural and sequence signatures for gRNA-independent binding (dPspCas13b: structural features; dRfxCas13d: sequence motifs).
- Found that specific gRNA regions (DR-distal and middle) are crucial for gRNA-dependent binding specificity and that some off-target binding impacts gene expression.
Conclusions:
- Provided insights into gRNA-dependent and gRNA-independent off-target binding characteristics of PspCas13b and RfxCas13d.
- Findings offer guidance for optimizing Cas13 and gRNA design in future RNA editing applications.
- Highlights the importance of considering both sequence and structural elements in RNA-target recognition by Cas13 systems.
