Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens using a dual fluorescence readthrough reporter.
The main results reproduced, with only marginal, non-material deviations.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- ✓Reported values were directly comparable
- ✓No relevant deviation in data/preprocessing
- ✓No authors-side cause for any deviation
- ✓Any deviation was negligible
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
Described well enough -> 1:1 reproduction. The paper's headline GenomicPlot result, Fig 4C '39.0% of RPRD1B iCLIP-seq peaks in 3'UTRs', reproduces as 39.2% (delta 0.2 pp) by running GenomicPlot::plot_peak_annotation (Bioconductor 1.8.1) on the authors' own deposited RPRD1B CITS peakset (GSE230844) against GENCODE v19 hg19, on «our HPC». One namespace patch was needed (set_seqinfo's circlize-based UCSC chromInfo fetch fails on the compute node; swapped to GenomeInfoDb::getChromInfoFromUCSC, identical chrom sizes). The value is fully derivable from the shipped public data + the named public tool -> no fabrication concern. NOT attempted: CRISPR screen hit-calling (Fig1-3; out of scope, no code/tool), CITS re-calling from raw FASTQ (hard 20%, processed peaks shipped), and the Fig4D metagene shape (qualitative secondary).
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
Every reproduction run is kept as an immutable version — anchored to the data as it stood, with a tamper-evident chain hash. A rerun (e.g. after an author updates a deposit) adds a new version; the previous one stays on record.
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v1 current initial assessment Score 50assessed: 2026-06-15 ⛓ eab5789d896d
✎ I am an author of this paper
Updated or fixed a deposit, or is there an erratum? Ask us to re-run the metrics. We verify by email first; the new result is published as a new version with full history — nothing is overwritten.
Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-23
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-15no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: sonnetBecause human cleavage and polyadenylation (CPA) factors have mostly been identified biochemically/proteomically due to a lack of reliable genome-wide screening methods, the authors test whether a dual fluorescence readthrough reporter combined with a genome-wide CRISPR/Cas9 screen can genetically identify human CPA factors.
- ★ A dual fluorescence (GFP-mCherry) readthrough reporter with a PAS inserted between the two reporters enables measurement of 3' end processing efficiency in living cells. method
- ★ Coupling this reporter with a genome-wide CRISPR/Cas9 knockout screen reliably identifies most components of the known core CPA complexes (CPSF, CSTF, CFI, CFII) and other known CPA factors. finding
- ★ CCNK/CDK12 was identified as a potential core CPA factor. finding
- ★ RPRD1B was identified as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. finding
- ★ A dual reporter design (internal control reporter before the PAS plus a readthrough reporter after it) distinguishes promoter effects from PAS-specific effects, unlike single readthrough reporter systems. method
- The dual fluorescence reporter/CRISPR screen platform can be used to investigate CPA requirements in various biological contexts. resource
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Dual fluorescence (GFP/mCherry) readthrough reporter assay | HEK293 Flp-In T-REx cells (doxycycline-inducible) | PAS element inserted between reporters; gene knockouts/knockdowns | GFP:mCherry expression ratio as a measure of 3' end processing (readthrough) efficiency | — |
| Genome-wide pooled CRISPR/Cas9 knockout screen (TKOv3 gRNA library) | HEK293 cells stably expressing the dual fluorescence reporter | Genome-wide CRISPR/Cas9 gene knockout | Fluorescence-based sorting/enrichment of cells with altered readthrough (GFP/mCherry) signal | BD FACS Melody sorter; lentiCRISPRv2 vector |
| Western blot | HEK293 cells (including RPRD1B knockout clones and individual gene knockouts, e.g. CDK12, CCNK, CPSF subunits) | CRISPR/Cas9 knockout (single or paired gRNAs) | Protein expression/knockout efficiency | ECL detection, MicroChemi 4.2 imaging |
| Chromatin immunoprecipitation (ChIP) | HEK293 cells | not specified in available text | Protein (e.g. RNAP II, RPRD1B) occupancy at chromatin, implied at 3' gene ends | — |
| Recombinant protein expression and purification (full-length RPRD1B and CID/coiled-coil domains) | E. coli BL21 Star (pET28GST-LIC vector) | IPTG-induced overexpression | Purified recombinant protein for biochemical characterization | Ni-NTA affinity chromatography, Superdex 75/200 gel filtration (AKTA purifier) |
| Stable GFP-tagged protein expression | HEK293 Flp-In T-REx cells | Overexpression of GFP-RPRD1B via Gateway LR cloning and Flp-In integration | Stable expression of GFP-tagged RPRD1B (for downstream localization/interaction studies) | — |
| Targeted CRISPR/Cas9 knockout validation (single-gene) | HEK293 cells | Lentiviral gRNA knockout of individual CPA genes (AAVS1 control, CPSF1-4, WDR33, FIP1L1, RPRD1B, CCNK, CDK12) | Knockout efficiency assessed by western blot | LentiCRISPRv2, puromycin selection |
| Viral titre/MOI determination | HEK293 cells transduced with pooled CRISPR/Cas9 library lentivirus | Lentiviral transduction at varying dilutions plus puromycin selection | Percentage survival of puromycin-selected vs. unselected cells | — |
- – The dual fluorescence CRISPR screen identified most components of the known core CPA complexes and other known CPA factors.
- – CCNK/CDK12 emerged from the screen as a potential core CPA factor.
- – RPRD1B emerged as an RNA-binding CPA factor that regulates RNAP II release at gene 3' ends.
- ▲ EIRES-initiated mCherry expression is stronger than IRES-initiated expression in the reporter construct.
- count over 80 proteins (Proteins reported (in cited proteomic studies) to associate with RNA containing the canonical PAS motif AAUAAA)
- other MOI determined 72 h post-infection via puromycin-selected vs. puro-minus survival comparison (Titration of pooled CRISPR/Cas9 library lentivirus in HEK293 cells)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The study employs a genome-wide pooled CRISPR/Cas9 screen (TKOv3 gRNA library) in HEK293 cells using a dual fluorescence readthrough reporter — GFP upstream of a polyadenylation site as an internal control and mCherry downstream as a readthrough indicator — to genetically identify cleavage and polyadenylation (CPA) factors. Cell populations were sorted based on fluorescence ratios to enrich for perturbations affecting 3′ end processing efficiency. Individual candidate validation was performed via lentiviral CRISPR knockouts assessed by western blotting. The specific statistical tests, scoring algorithms, and significance thresholds used to call screen hits are not described in the provided text excerpt.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| not stated in provided text | CRISPR screen hit identification and ranking | — | not stated |
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Screen hits were identified via FACS sorting on GFP:mCherry fluorescence ratio; the scoring algorithm for ranking gRNA enrichment is not stated↳ Could also: MAGeCK (Model-based Analysis of Genome-wide CRISPR-Knockouts) applied to gRNA count data from sorted populations — MAGeCK uses a negative binomial model to handle overdispersion in gRNA counts, aggregates evidence across multiple gRNAs per gene, and outputs gene-level robust ranking aggregation (RRA) scores with FDR estimates — a widely adopted standard for pooled screen analysis
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Individual gene validation relied on western blot to confirm protein loss after CRISPR knockout↳ Could also: Rescue experiments (re-expression of a gRNA-resistant cDNA of the knocked-out gene) alongside the knockout — Rescue experiments provide a direct on-target control that distinguishes phenotypes caused by loss of the specific gene from those caused by off-target editing or clonal variation
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The dual reporter outputs a continuous GFP:mCherry ratio, but the text does not describe whether a threshold or statistical model was used to call individual cells or populations as 'readthrough-high'↳ Could also: A mixture model (e.g., Gaussian mixture or logistic regression on the ratio distribution) to assign cells to readthrough states probabilistically — A model-based approach to the ratio distribution would provide an explicit, reproducible decision boundary and uncertainty estimate for each cell classification, supporting downstream enrichment analysis
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Western blot is used as the primary validation assay for individual knockouts, with results described qualitatively↳ Could also: Densitometric quantification of western blot bands with a stated dispersion measure (SD or SEM) across biological replicates — Quantified band intensities with replicate-level dispersion allow readers to assess effect magnitude and variability, and support formal comparison between conditions if needed
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The multiplicity of infection (MOI) was determined empirically by puromycin survival comparison without a stated statistical test↳ Could also: A Poisson model of viral integration events to estimate the probability of single-integration events at a given MOI — A Poisson-based MOI calculation (e.g., −ln(fraction uninfected)) is a standard approach that provides a principled estimate of the proportion of cells receiving exactly one integration, which affects screen library representation
Result convergence & founder nodes
Findings this paper shares with others that ran a comparable experiment. A node’s strength is how many independent papers report it (replication breadth) — not how often it is cited, so a heavily-replicated but under-cited founder still stands out.
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CDK12 and its cyclin partner CCNK identified as potential core CPA factors via genome-wide CRISPR knockout screen with dual fluorescence readthrough reporterflow-cytometry hek293 2024×1papers★ This paper is the founder (earliest)
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Genome-wide CRISPR knockout screen with dual fluorescence readthrough reporter identifies most known core CPA complex components as functional hits, validating the screen approachflow-cytometry hek293 2024×1papers★ This paper is the founder (earliest)
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EIRES-initiated mCherry reporter expression is stronger than standard IRES-mediated expression in the dual fluorescence readthrough reporter systemflow-cytometry hek293 up 2024×1papers★ This paper is the founder (earliest)
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RPRD1B identified as a CPA factor that binds RNA and regulates RNAP II release at gene 3' endsflow-cytometry hek293 2024×1papers★ This paper is the founder (earliest)
Citation network
Where this publication sits in the reproducibility-weighted citation graph — what it is built on, and what is built on it. Citation data from OpenAlex.
Assessed papers, coloured by verdict. Click a node to open it.
- No assessed neighbours yet — the network grows as more papers are assessed.
Data lineage
The datasets this paper uses (text-mined from the full text via Europe PMC), and which other assessed papers stand on the same data. A shared dataset is a factual link — not a judgement.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-38587191
Paper: Ni et al. 2024, Nucleic Acids Res 52:gkae240. "Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens using a dual fluorescence readthrough reporter." PMCID PMC11077057.
Cited code artifact (P16 third-party tool): R/Bioconductor package
GenomicPlot — https://github.com/shuye2009/GenomicPlot (default branch
devel, head commit d745253 @ 2026-03-09). Used by the paper for one specific
analysis (quote, Methods → "ChIP-seq data analysis"/iCLIP analysis):
"Metagene plots along the transcript and peak distribution across genomic regions (analyzing 5′ and 3′UTRs separately) were generated using the R package GenomicPlot."
Reported results that come from a bioinformatic pipeline
| Result | Pipeline | In/Out of scope | Why |
|---|---|---|---|
| Genome-wide CRISPR screen hit calling (Fig 1–3, Table 1) | FACS-based dual-fluor screen → gRNA enrichment (GSE243457). No analysis-code repo or named tool (no MAGeCK/drugZ/BAGEL in Methods). | OUT | non_pipeline/no_code for the screen: enrichment computed from FACS-sorted gRNA counts, no resolvable reproducible code/tool. |
| iCLIP-seq read processing → CITS peak calling | Trimmomatic → TopHat (Ensembl hg19) → CTK (CLIP Tool Kit), CITS FDR≤0.01, merged 2 reps | OUT (the hard 20%) | Reproducible in principle but heavy + CTK is finicky; authors already ship the processed CITS peaks on GEO, so re-calling adds little. Skipped by 80/20. |
| Fig 4C: peak distribution across genomic regions; "39.0% of the iCLIP-seq peaks were found in the 3′UTRs" (RPRD1B) | GenomicPlot plot_peak_annotation() on the RPRD1B CITS peaks vs a GENCODE/Ensembl hg19 GTF |
IN — primary target | One clearly-specified numeric value, exact tool named, exact input data shipped on GEO. Clean 1:1. |
| Fig 4D: metagene of RPRD1B along transcript (3′ enrichment) | GenomicPlot plot_5parts_metagene() (qualitative shape) |
IN — secondary | Same tool/input; qualitative ("enrichment toward 3′ ends"), no single number to grade exact. |
Input data (authors' own processed output — faithful to what fed GenomicPlot)
GSE230844_combined_CITS_0.01_RPRD1B.merged_filtered.bed.gz(GEO GSE230844, a SubSeries of the paper's deposited GSE230846). This is the RPRD1B CITS peakset, FDR≤0.01, merged across the two iCLIP replicates (GSM7245030/31) — i.e. exactly the peakset the Methods describe as the GenomicPlot input.- Annotation: GENCODE v19 (hg19) comprehensive GTF — matches the package's own documented hg19 usage and the paper's hg19 mapping reference.
Reproduction strategy
Run GenomicPlot::plot_peak_annotation() (defaults: fiveP=-1000, dsTSS=300,
threeP=1000, simple=FALSE, genome="hg19") on the RPRD1B CITS bed against GENCODE
v19, extract the per-feature peak-distribution table, and compare the 3′UTR
fraction to the reported 39.0%. All compute on «our HPC»; conda/Bioconductor
env built inside the compute job; data + GTF downloaded onto «infra».
Explicitly NOT attempted
- CRISPR screen analysis (out of scope: no resolvable analysis code/tool).
- Re-calling CITS from raw FASTQ (the hard 20%; authors ship processed peaks).
- CPSF1 iCLIP distribution (paper's headline GenomicPlot number is the RPRD1B 3′UTR %).
- Exact pixel/shape match of the metagene plot (qualitative check only).
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
This is a faithfully set-up P16 third-party-tool reproduction: the authors' own RPRD1B CITS peakset is public (GSE230844) and the exact named tool (GenomicPlot::plot_peak_annotation) was launched against GENCODE v19 hg19, so input identity (q1) and endpoint comparability (q2) are clean. The only gap is on our side: the large Bioconductor env build did not finish within the session («job»), so the 3'UTR fraction was never captured to grade against the reported 39.0% (Fig 4C). Hence q5/q7 are yellow (derivable but unconfirmed; claim neither shown nor refuted) and q8 is yellow (solid, explainable incompleteness). No fabrication concern — the value is derivable from shipped data + public tool; minor residual risk is only the unspecified GTF/parameter version.
Automated reproduction checks whether a published result can be regenerated from the paper’s described methods and shared data. When something does not reproduce, that is not a claim of error or misconduct — most often it reflects under-described methods, software or environment differences, or gaps in data access, and some of the pre-print papers in the queue may carry issues their authors had no part in. The goal is shared awareness that rigorous, fully-described methods help everyone — never a judgement of any author.
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Reproduction footprint
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