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

GSE291437 Homo sapiens (human) RNA-seq (gene expression) 66 samples Submitted 2026/01/27 Platform GPL24676
At a glance

RNA-seq (gene expression) · human · 66 samples

What it is
RNA-seq (gene expression)Gene activity measured by sequencing. The summary does not mention single-cell work, so this is most likely bulk: one average per sample, the usual starting point for comparing groups.
Organism
Homo sapiens (human)
Samples
66 samples: 21 conditions, mostly in triplicateGroups are read from the sample names, not from the paper. Check the list below.
Data you can get
A processed table (series matrix) and supplementary files from NCBI; the raw sequencing reads from ENA. Which of these hold counts depends on the study, so check the file list.
Paper
Characterization of gRNA-dependent and gRNA-independent off-target binding sites of PspCas13b and RfxCas13d in mammalian cellsNucleic acids research · 2026
Summary (written by the submitters)
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).
Read the full summary ↓
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.
Published in
Characterization of gRNA-dependent and gRNA-independent off-target binding sites of PspCas13b and RfxCas13d in mammalian cells
Feng H, Li Z, Zhang H et al. · Nucleic acids research 2026 · PMID 41728952 · doi:10.1093/nar/gkag112
This dataset
Open GSE291437 in finder → Find more human RNA-seq datasets →
Download · which file do I need?

Direct links to NCBI and ENA, no account and no request form. Pick by what you want to do: take the small processed table to look, the supplementary files to re-analyse, or the original raw data to start over.

1 · Just have a look

Processed table

The series matrix: one table of values per sample as the submitters processed it. The smallest download, and it opens in Excel or R. Start here to see what the data look like.

Series matrix ↓
2 · Re-analyse it

Supplementary files

The count tables and other files the authors uploaded. Take only the samples you need, or the whole study as GSE291437_RAW.tar (it can run to tens of gigabytes).

All supplementary files ↗ Whole study · RAW.tar ↓
3 · Start from the raw reads

Raw sequencing reads

The original FASTQ files, straight from the sequencer, held at ENA. Large, and you align them yourself, so only choose this if you need to re-process the study from scratch.

Raw reads on ENA ↗

What the files usually look like: For RNA-seq the counts are usually a table among the supplementary files (look for 'counts', 'expression', 'FPKM' or 'TPM' in the file name). The series matrix of a sequencing study is sometimes nearly empty.

Also filed as BioProject PRJNA1233596 and SRA study SRP568893. Searching any of these in the dataset finder brings you back here.

NCBI GEO page ↗ Paper (PMID 41728952) ↗
Study design
21 conditions, mostly in triplicate
Control ×6 dPspCas13b ×3 dPspCas13b-gNT ×3 dPspCas13b-EZH2g1 ×3 dPspCas13b-EZH2g2 ×3 dPspCas13b-TP53g1 ×3 dRfxCas13d ×3 dRfxCas13d-gNT ×3 +13 more

Supports a between-group comparison across 66 samples.

How we read this: 21 replicated groups read from 66 sample titles; they account for 66 of them. These groups are guessed from the sample names, not taken from the paper, so check them against the sample list below before relying on them.

Samples in this study

+ 26 more — browse all 66 samples with per-sample file links →

Datasets on a similar topic

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