# DMD Minimum Perturbome Stage A Panel Draft

## Identity and state

- Protocol: `PROTOCOL:v1.2.0-measured-dmd-evidence:DMD-MIN-PERTURBOME:1.1-PANEL-DRAFT`
- Panel: `PANEL:v1.2.0-measured-dmd-evidence:DMD-MIN-PERTURBOME:STAGE-A:0.1-DRAFT`
- Assignment checksum: `e18d58935bf32500c833f1e0b530c73e90ee64ea0da04b0e346010f0410d0aec`
- Slots assigned: 24/24
- Candidate genes: 14
- Calibration controls: 10
- Registered studies: 0
- Experiments: 0
- Outcomes: 0

This is a provisional panel assignment, not an immutable registered experiment and not a DMD efficacy claim.

## Selection contract

The panel contains six muscle-context candidates, four high-uncertainty candidates, four mechanism-diversity candidates, four known myogenesis controls, four negative controls and two toxicity controls. Historical numeric ranks are not used. Within-stratum display order is not a target ranking.

Six candidate slots, two per candidate stratum, are reserved as future prediction-validation roles by a deterministic SHA-256 rule. Their perturbation identities are public, but they enter that track only after an eligible model exists and the required Prediction objects are immutable. Other pilot conditions may generate truth without a Prediction.

## Primary endpoint and estimand

- Endpoint: fusion index, defined as the percentage of analyzable nuclei within myosin-heavy-chain-positive syncytia containing at least two nuclei.
- Planning timepoint: day 5, with a day 4-7 blinded calibration window.
- Planning minimally important effect: 10 absolute percentage points.
- Planning negligible-effect margin: +/-5 absolute percentage points.
- Primary estimand: For each candidate, (DMD perturbation − DMD non-targeting control) − (matched-control perturbation − matched-control non-targeting control) on fusion index.

The numeric thresholds are provisional. They may be retained or replaced only after blinded assay calibration and before any candidate outcome is accessed.

## Replication and QC planning defaults

- Two independent reagents per gene.
- At least four donor or isogenic pairs, with six targeted where feasible.
- At least two differentiation batches per pair.
- At least three technical wells per reagent, context and batch; wells are not biological replicates.
- CRISPRi mRNA reduction of at least 70% for an engagement-qualified reagent.
- Candidate viability of at least 80% of non-targeting control.
- At least 500 analyzable nuclei per well and non-targeting-control inter-well CV no greater than 20%.
- Both qualified reagents must agree in direction; discordance is inconclusive.

## Primary-source anchors

- [PMID:33355126: Human myotube formation is determined by the MyoD-Myomixer/Myomaker axis](https://pubmed.ncbi.nlm.nih.gov/33355126/) - Human myogenesis and fusion-control rationale
- [PMID:26858401: Structure-function analysis of myomaker domains required for myoblast fusion](https://pubmed.ncbi.nlm.nih.gov/26858401/) - CRISPR MYMK loss-of-function fusion control
- [PMID:35642635: Impaired activity of the fusogenic micropeptide Myomixer causes myopathy](https://pubmed.ncbi.nlm.nih.gov/35642635/) - Human MYMX fusion-defect evidence
- [PMID:29246312: CRISPR deletion of CTG expansions in patient-derived myogenic cells](https://pubmed.ncbi.nlm.nih.gov/29246312/) - Fusion-index measurement definition in human myogenic cells
- [PMCID:PMC6926425: A solid-phase transfection platform for arrayed CRISPR screens](https://pmc.ncbi.nlm.nih.gov/articles/PMC6926425/) - POLR2A viability-control evidence
- [DOI:10.1038/s41419-025-07587-z: Genome-wide CRISPR screen using RPA3 as a cell-essential control](https://doi.org/10.1038/s41419-025-07587-z) - RPA3 viability-control evidence

## Registration blockers

- GATE-A1: freeze the 24 slot assignments and selection rules - PANEL_DRAFT_COMPLETE_SIGNOFF_PENDING
- GATE-A2: freeze one primary endpoint and numeric minimally important effect - PROVISIONAL_DEFAULT_DEFINED_BLINDED_CALIBRATION_PENDING
- GATE-A3: freeze negligible-effect margin - PROVISIONAL_DEFAULT_DEFINED_BLINDED_CALIBRATION_PENDING
- GATE-A4: freeze sample size or pilot variance rule - BLINDED_VARIANCE_RULE_DRAFTED_SIGNOFF_PENDING
- GATE-A5: freeze target-engagement, viability and imaging QC thresholds - PLANNING_THRESHOLDS_DEFINED_SYSTEM_CALIBRATION_PENDING
- GATE-A6: confirm biospecimen, institutional and laboratory approvals - BLOCKED_EXTERNAL_APPROVAL_RECORDS_MISSING
- GATE-A7: register an immutable Study before execution; freeze a Prediction only for conditions assigned to prediction validation - AWAITING_REGISTERED_TRUTH_GENERATION_STUDY

## Claim boundary

This is a provisional 24-slot Panel Draft, not a registered experiment. Fourteen candidate genes and ten calibration controls are assigned, but reagent sequences, the final cell system, blinded calibration, approvals, an immutable truth-generation Study and measured outcomes remain absent. Prediction objects belong only to a later validation track. No assignment is a treatment, efficacy or validated DMD-response claim.