pen-stack 6.9.2__tar.gz → 6.10.1__tar.gz

{pen_stack-6.9.2 → pen_stack-6.10.1}/CHANGELOG.md

@@ -3,6 +3,76 @@
 All notable changes to PEN-STACK are documented here. This file follows
 [Keep a Changelog](https://keepachangelog.com/) and the program's phase structure.
 
+## [6.10.1] - 2026-06-20 - WS-OFFTARGET rigor pass + retroactive honesty audit (v6.7–v6.9)
+
+**PATCH — complete the Stage E plan and disclose every remaining deviation.** Closes the v6.10.0 gaps and adds a
+standing [DEVIATIONS_AND_DISCLOSURES.md](docs/DEVIATIONS_AND_DISCLOSURES.md) ledger; also retro-audited v6.7/v6.8/v6.9
+(plan vs shipped vs code) and fixed/disclosed the findings. No item is reported complete if it is a silent
+substitution/partial/deferral.
+
+### Added / changed (Stage E completion)
+- **Off-Target-Bench expanded to FOUR unbiased assays.** Added **CHANGE-seq** (Lazzarotto 2020, `10.1038/s41587-020-0555-7`)
+  + **SITE-seq** (Cameron 2017, `10.1038/nmeth.4284`) on **independent broad guide panels** (20 + 11 guides). The real
+  **CRISOT-Score** (MD-physics, **assay-agnostic → leakage-clean**) beats the homology baseline on **all four**:
+  AUPRC 0.646/0.520/0.541/0.521 vs 0.467/0.266/0.249/0.233; per-guide bootstrap CI excludes 0 on each.
+- **Chromatin wired to the real Stage B track + relabelled honestly.** `locus_accessibility(chrom, bin, ct)` reads
+  `phase_1/features/chromatin_{ct}.parquet` (ATAC/DNase) when present and **abstains** otherwise; the docs now say
+  "chromatin-accessibility modifier" (not "chromatin-aware engine") — the bare wheel / deployed atlas do not ship the
+  raw track, so the modifier is inactive there (disclosed).
+- **Off-target task added to the Genome-Writing Challenge** (`benchmarks/genome_writing_challenge/harness.py`) —
+  non-circular (label = wet-lab Active call), data-gated on the fixture; the reference solver nominates correctly.
+- **Disclosed (not eliminated):** CRISOT is used instead of the plan-named CCLMoff/CRISMER (CRISMER ships no license;
+  CCLMoff is GPU + trained on these assays → leakage; CRISOT is the leakage-clean, license-clean, CPU choice). A
+  genomic-coordinate **locus split** is not possible (harmonized data is coordinate-free) → held-out-guide + cross-assay.
+
+### Fixed (retroactive honesty audit)
+- **v6.9:** dropped the inaccurate "OOD-gated" claim in the MHC-II axis status (it is **coverage-gated**, abstains
+  when uncached — no distributional OOD gate); corrected the stale `AXIS_STATUS["mhc2_writer"].reason` + the
+  `immune_mhc2.py` module docstring (they still described the dropped v6.9.0 P1-anchor proxy as "the method" — the
+  production axis is the real NetMHCIIpan-4.0). Disclosed: the plan-named MHC-II tool ensemble + IEDB/ImmunoSeq/FVIII
+  ADA datasets were not used (single-tool NetMHCIIpan-4.0; the recovery bench is a 4-protein sanity check, not an
+  IEDB held-out leaderboard); the 6.9.1→6.9.2 MHC-II metric change (peptide-fraction → residue-coverage).
+- **v6.8:** disclosed that v6.8.0's attB was a poly-G/C **schematic** (fixed with the real Bxb1 attB in v6.9.2);
+  inter-curator agreement is N/A (single contributor); "≥1 external submission" is unmet (forward-looking).
+- **v6.7:** corrected the "chromosome-Mondrian" served-interval wording (per-chromosome Mondrian qhats are computed,
+  but the **global** qhat is served — correct, since a query has no chromosome at serve time); disclosed the prereg
+  consolidation + the deferred HEK293T-OOD demo / scope-manifest entry.
+
+See the full ledger in [docs/DEVIATIONS_AND_DISCLOSURES.md](docs/DEVIATIONS_AND_DISCLOSURES.md).
+
+## [6.10.0] - 2026-06-20 - WS-OFFTARGET (PEN-OFFTGT: cross-writer-family off-target nomination)
+
+**Series III, Stage E.** Off-target moves from a single-family bridge pseudosite scan to a **cross-writer-family,
+chromatin-aware NOMINATION engine** grounded in unbiased genome-wide assays — completing the safety triad
+(site B + writer C + off-target E). Nomination is scrupulously framed as **not a clearance**: every candidate
+ships with the empirical assay that would confirm it.
+
+### Added
+- **Off-Target-Bench** (`benchmarks/offtarget/`) — a real, leakage-controlled nomination benchmark over canonical
+  Cas9 guides (EMX1/VEGFA1-3/FANCF/HEK293) with **experimentally validated off-targets** from GUIDE-seq
+  (Tsai 2015, `10.1038/nbt.3117`) and CIRCLE-seq (Tsai 2017, `10.1038/nmeth.4278`). Held-out-guide split, per-assay
+  provenance, SHA256SUMS. **Gate E-G2 PASSES on real data + real tool:** the licensed **CRISOT-Score** predictor
+  (Chen et al., Nat Commun 2023, `10.1038/s41467-023-42695-4`; XGBoost RNA-DNA fingerprint) BEATS the sequence-
+  homology baseline — GUIDE-seq AUPRC **0.646 vs 0.467** (gap +0.179, CI [0.015, 0.340]); CIRCLE-seq **0.520 vs
+  0.266** (gap +0.253, CI [0.140, 0.361]); per-guide bootstrap CI excludes 0 on both assays.
+- `pen_stack/wgenome/offtarget_data.py` — validated assay/predictor provenance, a GROUNDED mismatch→active-fraction
+  risk calibration (real-data: GUIDE-seq 0-1mm→100% active, 2mm→76%, 3mm→23%, 4mm→3.3%), the bench fixture loader.
+- `pen_stack/wgenome/offtarget_predict.py` — `nominate_offtargets(writer_family, ...)`: **nuclease** (mismatch-
+  calibrated risk band + the real cached CRISOT score + a documented chromatin modifier, Lazzarotto 2020);
+  **serine integrase** (cryptic **pseudo-attB** scan on the real documented Bxb1 attB core GCGGTCTC/GT);
+  **bridge** (delegates to the existing Perry-DMS pseudosite engine). Abstains without inputs; never fabricates sites.
+- `pen_stack/wgenome/offtarget_assay.py` — validation-assay recommender (GUIDE/CHANGE/CIRCLE-seq for nucleases;
+  Cryptic-seq/HIDE-seq for integrases; **honest gap** for bridge recombinases — NO published genome-wide unbiased
+  off-target assay or predictor exists, verified).
+- **Surfaces:** REST `POST /offtarget` + `GET /offtarget/assay`, MCP `offtarget_scan`, manifest `nominate_offtargets`
+  (fabricates=False), an `offtarget_nomination` scope card, and a web **Off-Target** page.
+
+### Honest limits
+- Nomination is NOT a clearance; genome-wide candidate ENUMERATION needs the on-VM Cas-OFFinder/genome scan (this
+  engine SCORES + RANKS + risk-bands supplied candidates). The CRISOT predictor is CC-BY-NC — it runs only on the
+  VM and its weights are NEVER redistributed; only derived scores are cached (CI-safe). Bridge/integrase off-target
+  is data-thin/unmodeled and is flagged extrapolative; IntQuery (Tome Biosciences) is a paper-only reference.
+
 ## [6.9.2] - 2026-06-19 - WS-IMMUNE2 real-tool rigor pass (no proxies / no heuristics across the immune + writer axes)
 
 **PATCH — a top-to-bottom audit replacing every remaining proxy/heuristic in the immune & writer-design stack with

{pen_stack-6.9.2 → pen_stack-6.10.1}/CITATION.cff

@@ -1,7 +1,7 @@
 cff-version: 1.2.0
 message: "If you use PEN-STACK, please cite it as below."
 title: "PEN-STACK: open infrastructure for genome writing"
-version: 6.9.2
+version: 6.10.1
 date-released: 2026-06-20
 authors:
   - family-names: "Mahaboob Ali"

{pen_stack-6.9.2 → pen_stack-6.10.1}/PKG-INFO

@@ -1,6 +1,6 @@
 Metadata-Version: 2.4
 Name: pen-stack
-Version: 6.9.2
+Version: 6.10.1
 Summary: Open infrastructure for genome writing: the Writable Genome atlas, the Writer Atlas, and the Write Planner.
 Author-email: Anees Ahmed Mahaboob Ali <ahmedaneesm@gmail.com>
 License: MIT
@@ -90,7 +90,7 @@ every design against rule-grounded mechanism, reports calibrated confidence, cit
 [![codecov](https://codecov.io/gh/ahmedanees-m/pen-stack/branch/main/graph/badge.svg)](https://codecov.io/gh/ahmedanees-m/pen-stack)
 [![License: MIT](https://img.shields.io/badge/License-MIT-informational.svg)](LICENSE)
 [![Python 3.11+](https://img.shields.io/badge/python-3.11%2B-blue.svg)](https://www.python.org/)
-[![Version](https://img.shields.io/badge/version-6.9.2-blue.svg)](CHANGELOG.md)
+[![Version](https://img.shields.io/badge/version-6.10.1-blue.svg)](CHANGELOG.md)
 [![Status](https://img.shields.io/badge/status-1.0%20First%20Stable-success.svg)](docs/STABILITY.md)
 [![Tests](https://img.shields.io/badge/tests-378%20passing-success.svg)](tests/)
 [![Lint: ruff](https://img.shields.io/badge/lint-ruff-purple.svg)](https://github.com/astral-sh/ruff)
@@ -148,6 +148,31 @@ demonstrated (v5.12) and the benchmark went public (v5.13).
 > unknown funnel remains — made legible (scope flags, known-unknowns, honest baselines, no fabrication), not
 > hidden.
 
+## What is new in v6.10 — PEN-OFFTGT (cross-writer-family off-target nomination)
+
+Off-target prediction was a single-family bridge pseudosite scan that abstained for nucleases and integrases.
+v6.10 makes it a **cross-writer-family NOMINATION engine** with a real-data-calibrated risk band and a documented
+chromatin-accessibility modifier — completing the safety triad (site + writer + off-target). It is scrupulously
+honest that **nomination is not a clearance**: every candidate ships with the empirical assay that would confirm it.
+
+- **A real, validated benchmark** (`benchmarks/offtarget/`) over **experimentally validated off-targets** from
+  **four** unbiased assays — GUIDE-seq (Tsai 2015) + CIRCLE-seq (Tsai 2017) on canonical guides, and CHANGE-seq
+  (Lazzarotto 2020) + SITE-seq (Cameron 2017) on **independent broad guide panels**. The licensed **CRISOT** predictor
+  (Chen et al. *Nat Commun* 2023; MD-physics, assay-agnostic → leakage-clean) **beats the sequence-homology baseline
+  on all four** — AUPRC 0.65/0.52/0.54/0.52 vs 0.47/0.27/0.25/0.23; per-guide bootstrap CI excludes 0 on each.
+  CRISOT is CC-BY-NC and runs only on the VM — only derived scores are cached (like the licensed MHC tools).
+- **Grounded, calibrated risk** — the risk band IS the real-data fraction of candidates at *k* mismatches that were
+  experimentally validated-active (GUIDE-seq: 0–1 mm → 100%, 2 mm → 76%, 3 mm → 23%, 4 mm → 3.3%), not a guessed curve.
+- **Cross-family** — nucleases (mismatch-calibrated risk + the real CRISOT score + a chromatin-accessibility modifier
+  that reads the Stage B ATAC/DNase track when present, else abstains), serine integrases (cryptic **pseudo-attB**
+  scan on the real documented Bxb1 attB core), bridge recombinases (the existing Perry-DMS engine). Bridge/integrase
+  off-target is **honestly flagged data-thin** — there is no published genome-wide unbiased assay or predictor for
+  bridge recombinases (verified).
+- **Surfaces** — REST `POST /offtarget`, MCP `offtarget_scan`, manifest `nominate_offtargets`, an Off-Target web
+  page, and an off-target task in the Genome-Writing Challenge. Abstains without inputs; never fabricates sites.
+  *(Plan deviations — e.g. the CRISOT-for-CCLMoff/CRISMER substitution — are disclosed in
+  [docs/DEVIATIONS_AND_DISCLOSURES.md](docs/DEVIATIONS_AND_DISCLOSURES.md).)*
+
 ## What is new in v6.9 — PEN-IMMUNE (MHC-II/CD4 + ADA + the writer enzyme as a distinct antigen)
 
 The immune profile did **CD8/MHC-I only** (capsid epitope load via MHCflurry) — but the **dominant** immunogenicity

{pen_stack-6.9.2 → pen_stack-6.10.1}/README.md

@@ -15,7 +15,7 @@ every design against rule-grounded mechanism, reports calibrated confidence, cit
 [![codecov](https://codecov.io/gh/ahmedanees-m/pen-stack/branch/main/graph/badge.svg)](https://codecov.io/gh/ahmedanees-m/pen-stack)
 [![License: MIT](https://img.shields.io/badge/License-MIT-informational.svg)](LICENSE)
 [![Python 3.11+](https://img.shields.io/badge/python-3.11%2B-blue.svg)](https://www.python.org/)
-[![Version](https://img.shields.io/badge/version-6.9.2-blue.svg)](CHANGELOG.md)
+[![Version](https://img.shields.io/badge/version-6.10.1-blue.svg)](CHANGELOG.md)
 [![Status](https://img.shields.io/badge/status-1.0%20First%20Stable-success.svg)](docs/STABILITY.md)
 [![Tests](https://img.shields.io/badge/tests-378%20passing-success.svg)](tests/)
 [![Lint: ruff](https://img.shields.io/badge/lint-ruff-purple.svg)](https://github.com/astral-sh/ruff)
@@ -73,6 +73,31 @@ demonstrated (v5.12) and the benchmark went public (v5.13).
 > unknown funnel remains — made legible (scope flags, known-unknowns, honest baselines, no fabrication), not
 > hidden.
 
+## What is new in v6.10 — PEN-OFFTGT (cross-writer-family off-target nomination)
+
+Off-target prediction was a single-family bridge pseudosite scan that abstained for nucleases and integrases.
+v6.10 makes it a **cross-writer-family NOMINATION engine** with a real-data-calibrated risk band and a documented
+chromatin-accessibility modifier — completing the safety triad (site + writer + off-target). It is scrupulously
+honest that **nomination is not a clearance**: every candidate ships with the empirical assay that would confirm it.
+
+- **A real, validated benchmark** (`benchmarks/offtarget/`) over **experimentally validated off-targets** from
+  **four** unbiased assays — GUIDE-seq (Tsai 2015) + CIRCLE-seq (Tsai 2017) on canonical guides, and CHANGE-seq
+  (Lazzarotto 2020) + SITE-seq (Cameron 2017) on **independent broad guide panels**. The licensed **CRISOT** predictor
+  (Chen et al. *Nat Commun* 2023; MD-physics, assay-agnostic → leakage-clean) **beats the sequence-homology baseline
+  on all four** — AUPRC 0.65/0.52/0.54/0.52 vs 0.47/0.27/0.25/0.23; per-guide bootstrap CI excludes 0 on each.
+  CRISOT is CC-BY-NC and runs only on the VM — only derived scores are cached (like the licensed MHC tools).
+- **Grounded, calibrated risk** — the risk band IS the real-data fraction of candidates at *k* mismatches that were
+  experimentally validated-active (GUIDE-seq: 0–1 mm → 100%, 2 mm → 76%, 3 mm → 23%, 4 mm → 3.3%), not a guessed curve.
+- **Cross-family** — nucleases (mismatch-calibrated risk + the real CRISOT score + a chromatin-accessibility modifier
+  that reads the Stage B ATAC/DNase track when present, else abstains), serine integrases (cryptic **pseudo-attB**
+  scan on the real documented Bxb1 attB core), bridge recombinases (the existing Perry-DMS engine). Bridge/integrase
+  off-target is **honestly flagged data-thin** — there is no published genome-wide unbiased assay or predictor for
+  bridge recombinases (verified).
+- **Surfaces** — REST `POST /offtarget`, MCP `offtarget_scan`, manifest `nominate_offtargets`, an Off-Target web
+  page, and an off-target task in the Genome-Writing Challenge. Abstains without inputs; never fabricates sites.
+  *(Plan deviations — e.g. the CRISOT-for-CCLMoff/CRISMER substitution — are disclosed in
+  [docs/DEVIATIONS_AND_DISCLOSURES.md](docs/DEVIATIONS_AND_DISCLOSURES.md).)*
+
 ## What is new in v6.9 — PEN-IMMUNE (MHC-II/CD4 + ADA + the writer enzyme as a distinct antigen)
 
 The immune profile did **CD8/MHC-I only** (capsid epitope load via MHCflurry) — but the **dominant** immunogenicity

pen_stack-6.10.1/benchmarks/offtarget/SHA256SUMS

@@ -0,0 +1,4 @@
+8752efa5b6ada36cdf07707999380150f749e819178008be04dcc6b8cd70c084  split.json
+e149d144c5b74499010b062cf9d3a7e2e290e71c8d3720d7b75811bf2ad7dbf3  offtarget_bench_fixture.csv
+e699f399c1c640968aea1d4fe6cc679f035db3a653a5766fed795ab5eec2cecb  offtarget_bench_metrics.json
+42556ae9915037d8a041259daafa93265a5c3eb9f6377245fdb425a4c3418448  offtarget_calibration.json

{pen_stack-6.9.2 → pen_stack-6.10.1}/configs/oracles/scope_cards.yaml

@@ -139,6 +139,23 @@ oracles:
     generalizes_to_unseen_loci: false
     license: "open (this work; MHCflurry 10.1016/j.cels.2020.06.010, HLA-I supertypes 10.1186/1471-2172-9-1)"
 
+  offtarget_nomination:         # v6.10 WS-OFFTARGET: cross-family off-target NOMINATION (not clearance)
+    family: genome
+    version: "crisot+homology-2026"
+    output_kind: baseline       # ranks/risk-bands CANDIDATE sites, not generative
+    valid_for: "RELATIVE off-target NOMINATION for a writer: rank candidate sites so validated off-targets
+      surface first, with a mismatch-CALIBRATED empirical risk band (real GUIDE-seq/CIRCLE-seq active fractions).
+      Nucleases use the real CRISOT-Score (beats a homology baseline on held-out guides, CI excludes 0); serine
+      integrases use a documented pseudo-attB cryptic scan; bridge recombinases delegate to the Perry-DMS engine"
+    not_valid_for: "a safety CLEARANCE (nomination != validation; every result ships with the empirical assay
+      that would confirm it); genome-wide candidate ENUMERATION (needs the on-VM Cas-OFFinder/genome scan; this
+      engine SCORES supplied candidates); a per-site cleavage/recombination PROBABILITY; bridge-recombinase
+      off-target magnitude (NO published genome-wide unbiased assay/predictor exists -> extrapolative/abstains);
+      structural variants/translocations beyond nominated sites"
+    generalizes_to_unseen_loci: false
+    license: "open (this work; GUIDE-seq 10.1038/nbt.3117, CIRCLE-seq 10.1038/nmeth.4278, CHANGE-seq
+      10.1038/s41587-020-0555-7, CRISOT 10.1038/s41467-023-42695-4 CC-BY-NC run on the VM only)"
+
   innate_sensing:               # v5.4 WS-INNATE: computed nucleic-acid innate-sensing motif load
     family: genome
     version: "cpg-oe+dsrna-2026"

pen_stack-6.10.1/docs/DEVIATIONS_AND_DISCLOSURES.md

@@ -0,0 +1,57 @@
+# PEN-STACK — Deviations & Disclosures Ledger
+
+A standing, honest record of every place a shipped release **deviated** from its execution plan — a dataset/model
+that was substituted, a deliverable that was narrowed or deferred, or wording that overclaimed — together with the
+correction or the justification. Maintained from v6.10.1 onward (the rule: *no item is reported complete if it is a
+silent substitution/partial/deferral; it is either fixed or disclosed here*). Audited retroactively for v6.7–v6.9.
+
+Legend: **FIXED** = corrected in code/docs · **DISCLOSED** = a justified, permanent deviation · **N/A** = not
+applicable (e.g. single contributor).
+
+---
+
+## v6.10 / v6.10.1 — PEN-OFFTGT (Stage E, off-target nomination)
+
+| Item | Status | Detail |
+|---|---|---|
+| Nuclease predictor named in plan (CCLMoff / CRISMER) vs shipped (CRISOT) | **DISCLOSED** | We use **CRISOT-Score** (Chen 2023, CC-BY-NC, CPU), not the plan's CCLMoff/CRISMER. Justified: **CRISMER ships no license** (cannot be redistributed/wrapped); **CCLMoff** is a GPU RNA-language-model stack AND was trained on these very assays (evaluating it on them would be **leakage**), whereas **CRISOT-Score is MD-physics, assay-agnostic → a leakage-clean held-out evaluation**. CRISOT is the more rigorous and license-appropriate choice. A genuine substitution, disclosed. |
+| Assay coverage (plan listed GUIDE/CIRCLE/CHANGE/SITE/SURRO/TTISS/Digenome-seq) | **FIXED (v6.10.1)** | v6.10.0 used GUIDE-seq + CIRCLE-seq only. v6.10.1 adds **CHANGE-seq + SITE-seq** (independent broad guide panels) — CRISOT beats homology on **all four** (per-guide bootstrap CI excludes 0). SURRO-seq is targeted/biased (kept as an orthogonal reference, not genome-wide truth); TTISS/Digenome-seq remain **DISCLOSED** as not-yet-folded-in. |
+| "Chromatin-aware via the Stage B feature store" | **FIXED (v6.10.1)** | v6.10.0 had only a caller-supplied accessibility hook and the docs said "chromatin-aware" (overclaim). v6.10.1 adds a **real** `locus_accessibility(chrom, bin, ct)` that reads the Stage B `chromatin_{ct}.parquet` ATAC/DNase track when present, **abstains** when absent (bare wheel / CI / current deployed atlas do not ship the raw track), and accepts a caller-supplied scalar otherwise. Docs relabelled to "chromatin-accessibility modifier". |
+| Held-out **guide** AND **locus** splits | **DISCLOSED** | Held-out-**guide** is implemented; a genomic-coordinate **locus** split is **not possible** — the harmonized assay data ships sequences, not coordinates. Instead we add **cross-assay generalization** (the assay-agnostic CRISOT-Score evaluated on GUIDE/CIRCLE canonical guides + CHANGE/SITE independent panels). This is the leakage-clean substitute and is stated in `benchmarks/offtarget/split.json`. |
+| Learned **integrase** off-target scorer (plan: "learned on HIDE/Cryptic-seq") | **DISCLOSED** | Shipped as a documented **pseudo-attB cryptic scan** on the real Bxb1 attB core, not a learned model — Cryptic-seq/HIDE-seq are recent preprints and IntQuery has **no public weights**, so a real learned integrase predictor is not groundable. Flagged extrapolative; IntQuery cited as paper-only. |
+| Bench "joins the Challenge" | **FIXED (v6.10.1)** | An `offtarget` nomination task is added to the Genome-Writing Challenge (`benchmarks/genome_writing_challenge/harness.py`) — non-circular (label = wet-lab Active call), data-gated on the fixture. |
+| "≥1 external use" (success criterion) | **DISCLOSED** | Forward-looking; not yet met (no external submission). The surfaces (REST/MCP/manifest/web) exist to enable it. |
+| HIDE-seq / Cryptic-seq integrase **data** | **DISCLOSED** | Validated + cited, **not used as data** (single-company Bxb1-centric preprint; bridge off-target is unmodeled). Used only to ground the assay recommender. |
+
+## v6.9 — PEN-IMMUNE (Stage G) — *audited retroactively, corrected in v6.10.1*
+
+| Item | Status | Detail |
+|---|---|---|
+| "OOD-gated" in the MHC-II axis status string | **FIXED (v6.10.1)** | No distributional OOD gate is computed for an in-panel sequence; the axis is **coverage-gated** (abstains when the antigen is uncached). Wording corrected in `immune_mhc2.py`. |
+| Stale `AXIS_STATUS["mhc2_writer"].reason` + module docstring described the dropped v6.9.0 P1-anchor proxy as "the method" | **FIXED (v6.10.1)** | Updated to state the production method is the real **NetMHCIIpan-4.0** (v6.9.1/6.9.2); the P1-anchor density is offline-triage only. |
+| Plan-named MHC-II tools (MixMHC2pred, Graph-pMHC, HLAIIPred, FIONA, iTope) + ADA datasets (IEDB held-out, ImmunoSeq-217, FVIII/ATHN/MLOF inhibitor sets) | **DISCLOSED** | The MHC-II axis is **NetMHCIIpan-4.0 only** (a single real tool, not the named ensemble); the Immuno-Bench panel is the 4 bundled origin-labelled proteins, **not** an IEDB/ADA held-out leaderboard. The named datasets/tools were **not used**. The single-tool result is real; the broader ensemble/benchmark is deferred. |
+| Immuno-Bench "recovery" circularity | **DISCLOSED** | `recovery()` scores 3 foreign + 1 self protein; because `ada_risk = density × foreignness(origin)` and `foreignness(self)=0`, the self control is **zeroed by construction** — "foreign > self" cannot fail. It is a sanity check, not the IEDB/ADA held-out leaderboard the plan named. The ADA-incidence absence (stays 🟡) was already disclosed; this circularity note is new. |
+| MHC-II metric changed (peptide-fraction → residue-coverage) between 6.9.1 and 6.9.2 | **DISCLOSED** | The 4× difference between the §6b (0.153…) and §6c/shipped (0.636…) numbers is a **metric-definition change** (now residue coverage), not a contradiction. No live surface emits the old numbers. |
+
+## v6.8 — PEN-WRITER (Stage C) — *audited retroactively*
+
+| Item | Status | Detail |
+|---|---|---|
+| attB was a poly-G/C **schematic** at v6.8.0, surfaced as a design candidate | **FIXED (v6.9.2)** | v6.8.0 emitted `("G"*18)+core+("C"*18)` and only tested the central dinucleotide, never disclosing the attB sequence was a placeholder. The **real documented Bxb1 minimal attB** (FlyBase FBto0000359; Ghosh 2003) replaced it in v6.9.2. Disclosed here as a v6.8.0 limitation. |
+| Inter-curator agreement (pre-registered C-WS1 acceptance) | **N/A** | Single contributor (`ahmedanees-m`) — inter-curator agreement is structurally inapplicable; the curation is single-author with per-row DOI + verbatim quote. |
+| "≥1 external submission" (success criterion) | **DISCLOSED** | Not met; forward-looking. |
+| Writer predictor gate (beats baseline on held-out **family**) | **OK (already disclosed)** | It wins on held-out **locus** only, **not** family; KB ranking retained. Stated verbatim in code/docs — no fix needed. |
+
+## v6.7 — PEN-EXPRESS (Stage H expression) — *audited retroactively*
+
+| Item | Status | Detail |
+|---|---|---|
+| TPE-Bench headline: held-out-**cell-type** (plan) vs held-out-**chromosome** (shipped) | **OK (already disclosed)** | The live track is labelled `chrom_holdout` and the cell-type track is labelled `DATA-GATED` everywhere — an honestly-disclosed scope narrowing, not covert. |
+| "chromosome-Mondrian" served interval | **FIXED (v6.10.1)** | Per-chromosome Mondrian qhats are computed, but the **served** band uses the global qhat (a query has no chromosome at serve time — which is correct). Wording corrected in `position_effect.py` to avoid implying per-query Mondrian serving. |
+| Prereg consolidation (`ws_expr_{d,m,u,cal}` → single `ws_expr2.yaml`) | **DISCLOSED** | The four planned preregs were consolidated into one; the `cal` (wet-lab) prereg was dropped with the user-scoped wet-lab omission. |
+| HEK293T-OOD acceptance demo + scope-manifest entry | **DISCLOSED** | Deferred with the data-gated cross-cell-type transfer track (needs a second cell-type epigenome). The OOD detector itself ships and is wired; titer/patient state are registered known-unknowns stack-wide. |
+
+---
+
+*This ledger is updated on every release. If you find a deviation not listed here, it is a bug in our honesty, not
+an intended omission — please flag it.*

pen_stack-6.10.1/docs/cards/offtarget_data.md

@@ -0,0 +1,69 @@
+# Data card — Off-Target-Bench (v6.10 PEN-OFFTGT)
+
+## Summary
+A real, leakage-controlled benchmark for cross-writer-family off-target **nomination**: given a guide and its
+candidate sites, rank the candidates so the experimentally validated off-targets surface first. Labels are the
+wet-lab assay calls (NON-circular — the label is the experiment, not a predictor).
+
+## Ground truth (independently verified 2026-06-19; 4 assays as of v6.10.1)
+| Assay | Setting | Guide panel | Citation | DOI |
+|---|---|---|---|---|
+| GUIDE-seq | cell-based, unbiased | canonical (8) | Tsai et al., *Nat Biotechnol* 2015 | `10.1038/nbt.3117` |
+| CIRCLE-seq | in vitro (cell-free), unbiased | canonical (8) | Tsai et al., *Nat Methods* 2017 | `10.1038/nmeth.4278` |
+| CHANGE-seq | in vitro, high-throughput | independent broad (20) | Lazzarotto et al., *Nat Biotechnol* 2020 | `10.1038/s41587-020-0555-7` |
+| SITE-seq | in vitro biochemical | independent broad (11) | Cameron et al., *Nat Methods* 2017 | `10.1038/nmeth.4284` |
+
+Canonical Cas9 guides: **EMX1, VEGFA site 1/2/3, FANCF, HEK293 site 2/3/4**. CHANGE/SITE-seq use **independent broad
+guide panels** (not the canonical 8) — a cross-assay generalization test. The harmonized candidate/label tables are
+sourced from the CRISOT data release (Zenodo `10.5281/zenodo.8420032`), which redistributes the public assay
+supplements; PEN-STACK cites the **original assay papers** as the ground-truth provenance.
+
+## Learned predictor (real tool, VM-only)
+**CRISOT-Score** — Chen et al., *Nat Commun* 2023, `10.1038/s41467-023-42695-4`; an XGBoost RNA-DNA interaction
+fingerprint. **License: CC-BY-NC** → it runs only on the VM (`crisot:tools` Docker, `xgboost`/`pandas`/`numpy`);
+its weights are NEVER redistributed. Only DERIVED scores are cached/committed (CI-safe), exactly like the licensed
+NetMHC tools.
+
+## Baseline (pre-registered)
+Sequence-homology nomination = ascending **mismatch count** (Hamming over the 20-nt protospacer).
+
+## Result (E-G2, full real data, on the VM)
+CRISOT-Score is the MD-physics, **assay-agnostic** scorer (not fit on these labels) → a leakage-clean held-out
+evaluation on every assay.
+
+| Assay | guide panel | CRISOT AUPRC | homology AUPRC | gap | 95% CI (held-out-guide bootstrap) | beats |
+|---|---|---|---|---|---|---|
+| GUIDE-seq | canonical (8) | 0.646 | 0.467 | +0.179 | [0.014, 0.329] | ✅ |
+| CIRCLE-seq | canonical (8) | 0.520 | 0.266 | +0.253 | [0.146, 0.370] | ✅ |
+| CHANGE-seq | independent (20) | 0.541 | 0.249 | +0.292 | [0.235, 0.348] | ✅ |
+| SITE-seq | independent (11) | 0.521 | 0.233 | +0.287 | [0.239, 0.335] | ✅ |
+
+The learned predictor beats the homology baseline on **all four** assays (per-guide bootstrap CI excludes 0),
+including two independent broad guide panels (cross-assay generalization).
+
+## Chromatin-accessibility modifier (honest scope)
+The nominator applies a documented chromatin modifier (open chromatin raises realized off-target activity;
+Lazzarotto 2020). It reads the **real Stage B accessibility track** (`phase_1/features/chromatin_{ct}.parquet`,
+ATAC/DNase) when a candidate's genomic locus + cell type are supplied AND the feature store is present, or accepts a
+caller-supplied scalar; it **abstains** otherwise. The bare wheel / CI / current deployed atlas do **not** ship the
+raw accessibility track, so the modifier is inactive there — this is a documented, honestly-bounded integration, not
+an autonomous "chromatin-aware" guarantee.
+
+## Risk calibration (grounded)
+The nomination risk band IS the empirical fraction of candidates at *k* mismatches that were validated-active
+(full real data): GUIDE-seq 0–1 mm → 1.00, 2 mm → 0.765, 3 mm → 0.231, 4 mm → 0.033, 5 mm → 0.0028, 6 mm → 0.00014.
+Mismatch counts outside the calibrated range abstain rather than extrapolate.
+
+## Files
+- `benchmarks/offtarget/offtarget_bench_fixture.csv` — real validated off-targets + cached CRISOT scores (CI-safe;
+  inactives downsampled with a fixed seed for a small committed file).
+- `benchmarks/offtarget/offtarget_bench_metrics.json` — the AUTHORITATIVE full-data metrics.
+- `benchmarks/offtarget/offtarget_calibration.json` — the full-data mismatch / CRISOT-decile calibration.
+- `benchmarks/offtarget/split.json`, `SHA256SUMS` — split definition + checksums.
+
+## Honest limits
+Nomination is **not** a clearance — every result ships with the empirical assay that would confirm it. Genome-wide
+candidate ENUMERATION needs the on-VM Cas-OFFinder/genome scan; this benchmark covers SCORING + RANKING of supplied
+candidates. Bridge/integrase off-target is data-thin: there is **no published genome-wide unbiased off-target assay
+or predictor for bridge recombinases** (verified), and the large-serine-integrase assays (Cryptic-seq/HIDE-seq) and
+predictor (IntQuery) are recent single-company preprints with no public weights.

pen_stack-6.10.1/docs/offtarget.md

@@ -0,0 +1,52 @@
+# Stage E — the off-target nomination engine (v6.10 PEN-OFFTGT)
+
+Stage E completes the **safety triad** — site (Stage B) + writer (Stage C) + **off-target** (Stage E) — by turning
+off-target from a single-family bridge pseudosite scan into a **cross-writer-family, chromatin-aware NOMINATION
+engine** with a real-data-calibrated risk band. The cardinal honesty invariant: **nomination is not a clearance.**
+A nominated off-target is a candidate, and every result ships with the empirical assay that would confirm it.
+
+## The engine (`pen_stack/wgenome/offtarget_predict.py`)
+
+`nominate_offtargets(writer_family, ...)` dispatches by writer family:
+
+- **Nuclease (Cas9):** given a guide + candidate sites (e.g. from a Cas-OFFinder scan), each candidate gets a
+  **mismatch-calibrated empirical risk** (the real active fraction at *k* mismatches — not a guessed curve), the
+  **real CRISOT-Score** when the (guide, site) is in the cached bench, and a documented **chromatin-accessibility
+  modifier** (open chromatin raises realized off-target activity; Lazzarotto 2020). The modifier reads the **real
+  Stage B accessibility track** (`phase_1/features/chromatin_{ct}.parquet`) when a candidate's genomic locus + cell
+  type are supplied and the store is present, accepts a caller-supplied scalar otherwise, and **abstains** when
+  neither is available (the bare wheel / current deployed atlas do not ship the raw track).
+- **Serine integrase (Bxb1):** a cryptic **pseudo-attB** scan that seeds on the *real documented* Bxb1 attB core
+  (`GCGGTCTC`, central GT; FlyBase FBto0000359, Ghosh 2003) and reports candidate cryptic sites by arm mismatches.
+- **Bridge recombinase:** delegates to the existing Perry-DMS pseudosite engine (`pen_stack.bridge.offtarget`).
+
+The engine **abstains without inputs** (no candidate sites → no fabricated sites) and is explicit that genome-wide
+candidate ENUMERATION needs the on-VM scan; this engine SCORES + RANKS + risk-bands supplied candidates.
+
+## The benchmark (E-G2, real data + real tool)
+
+`benchmarks/offtarget/` is a held-out-guide nomination benchmark over **four** unbiased assays — GUIDE-seq +
+CIRCLE-seq (canonical guides) and CHANGE-seq + SITE-seq (**independent broad guide panels**, a cross-assay test).
+The licensed **CRISOT-Score** predictor (CC-BY-NC, run on the VM; MD-physics and **assay-agnostic** → leakage-clean)
+beats the sequence-homology baseline on **all four** — AUPRC 0.65/0.52/0.54/0.52 vs 0.47/0.27/0.25/0.23, with the
+per-guide bootstrap CI on the gap excluding 0 on each. The Off-Target-Bench also contributes a nomination task to the
+**Genome-Writing Challenge**. Only derived CRISOT scores are cached/committed; the weights are never redistributed.
+A genomic-coordinate locus split is not possible (the harmonized data ships sequences, not coordinates) — held-out-
+guide + cross-assay is the leakage-clean evaluation. See `docs/cards/offtarget_data.md` and
+`docs/DEVIATIONS_AND_DISCLOSURES.md` for full provenance and disclosed plan deviations.
+
+## Validation-assay recommendation (`pen_stack/wgenome/offtarget_assay.py`)
+
+`recommend_assay(writer_family)` maps a writer to the empirical assay(s) that would confirm a nomination —
+GUIDE/CHANGE/CIRCLE-seq for nucleases, Cryptic-seq/HIDE-seq for serine integrases — and is **honest about the gap**
+for bridge recombinases: there is no published genome-wide unbiased off-target assay or predictor for them, so their
+nominations are flagged extrapolative and routed to targeted confirmation, never read as a clearance.
+
+## Surfaces
+REST `POST /offtarget` + `GET /offtarget/assay`, MCP `offtarget_scan`, manifest tool `nominate_offtargets`
+(`fabricates: false`), the `offtarget_nomination` scope card, and the **Off-Target** web page.
+
+## Honest limits
+Nomination ≠ validation. Bridge/integrase off-target is data-thin/unmodeled (verified) and flagged extrapolative.
+Chromatin-awareness depends on target-cell-type data. Translocations/structural variants beyond nominated sites are
+out of scope. The CRISOT learned predictor is CC-BY-NC and runs only on the VM; PEN-STACK ships only derived scores.

{pen_stack-6.9.2 → pen_stack-6.10.1}/pen_stack/__init__.py

@@ -1,2 +1,2 @@
 """PEN-STACK v3.0 - open infrastructure for genome writing."""
-__version__ = "6.9.2"
+__version__ = "6.10.1"

{pen_stack-6.9.2 → pen_stack-6.10.1}/pen_stack/agent/cite.py

@@ -39,6 +39,19 @@ def curated_dois() -> frozenset[str]:
     # v5.4 computed innate-sensing provenance (CpG-TLR9 / AAV CpG-depletion / RNA modification)
     from pen_stack.planner.innate_sensing import PROVENANCE_DOIS as _innate_dois
     dois.update(_innate_dois)
+    # v6.10 off-target nomination provenance (GUIDE/CIRCLE/CHANGE/SITE-seq assays + CRISOT predictor + LSI assays)
+    try:
+        from pen_stack.wgenome.offtarget_data import (
+            ASSAY_PROVENANCE,
+            INTEGRASE_ASSAY_PROVENANCE,
+            PREDICTOR_PROVENANCE,
+        )
+        for _src in (ASSAY_PROVENANCE, PREDICTOR_PROVENANCE, INTEGRASE_ASSAY_PROVENANCE):
+            for _rec in _src.values():
+                if _rec.get("doi"):
+                    dois.add(_rec["doi"])
+    except Exception:  # noqa: BLE001
+        pass
     # v5.5 anti-vector seroprevalence provenance (serosurveys)
     try:
         sp = yaml.safe_load(resource("configs/seroprevalence.yaml").read_text(encoding="utf-8"))

{pen_stack-6.9.2 → pen_stack-6.10.1}/pen_stack/agent/mcp_server.py

@@ -91,6 +91,18 @@ def immune_profile(design: dict) -> dict:
     return _ip(design)
 
 
+@mcp.tool()
+def offtarget_scan(writer_family: str, guide: str | None = None, candidate_sites: list | None = None,
+                   sequence: str | None = None, assay: str = "guideseq") -> dict:
+    """v6.10 PEN-OFFTGT cross-family off-target NOMINATION (NOT a clearance). Ranks candidate sites with a
+    real-data mismatch-calibrated risk band + the real CRISOT learned score (nuclease), a pseudo-attB scan
+    (integrase), or the Perry-DMS pseudosite engine (bridge); ships the validation assay that would confirm
+    each candidate. Abstains without inputs; never fabricates sites."""
+    from pen_stack.wgenome.offtarget_predict import nominate_offtargets
+    return nominate_offtargets(writer_family, guide=guide, candidate_sites=candidate_sites,
+                               sequence=sequence, assay=assay)
+
+
 @mcp.tool()
 def generate_designs(goal: dict | None = None, candidates: list | None = None, keep: int = 25) -> dict:
     """v5.8 generative designer (verifier-as-discriminator): hazardous/illegal candidates are DISCARDED;

{pen_stack-6.9.2 → pen_stack-6.10.1}/pen_stack/api/manifest.py

@@ -46,6 +46,12 @@ _TOOLS = [
      "efficiency w/ conformal interval + auto-designed guide/att (v6.8 PEN-WRITER)",
      "input": "write request (write-type, cargo, cell type, optional target/donor seq)", "output": "WriterRanking",
      "entrypoint": "pen_stack.atlas.writer_recommend.recommend_writers", "fabricates": False},
+    {"name": "nominate_offtargets", "summary": "cross-writer-family off-target NOMINATION: rank candidate sites "
+     "with a real-data mismatch-calibrated risk band + the real CRISOT learned score (nuclease), pseudo-attB "
+     "scan (integrase), Perry-DMS pseudosite engine (bridge); nomination is NOT a clearance (v6.10 PEN-OFFTGT)",
+     "input": "writer family + guide/candidate sites (nuclease) or locus sequence (integrase)",
+     "output": "ranked off-target candidates + calibrated risk + recommended validation assay",
+     "entrypoint": "pen_stack.wgenome.offtarget_predict.nominate_offtargets", "fabricates": False},
 ]
 
 _POLICY = ("outputs outside scope are returned as `out_of_scope` (known-unknown) or `extrapolating` (OOD) and are "

{pen_stack-6.9.2 → pen_stack-6.10.1}/pen_stack/planner/immune_mhc2.py

@@ -6,13 +6,16 @@ protein itself is immunogenic** (Cas9 elicits MHC-II-presented CD4 responses, Si
 10.1038/s41467-021-25414-9; bridge recombinases / serine integrases are bacterial/phage) — yet Stage G scored
 only the capsid. v6.9 adds an MHC-II epitope-load axis over the **writer** as a distinct antigen.
 
-Method (grounded, dependency-free, allele-agnostic PROMISCUOUS-binder density): MHC-II presents a 9-mer core in an
-open groove; the **P1 pocket is deep and hydrophobic**, the single dominant anchor (M/F/Y/W/L/I/V) (Stern & Wiley,
-Nature 1994; Jardetzky 1996), with secondary pockets at P4/P6/P9. A *promiscuous* epitope (binds many HLA-DR) is
-exactly one with a strong P1 anchor + favorable secondaries (Southwood et al., J Immunol 1998). We count
-promiscuous-binder cores -> an epitope-density proxy. This is a **population-level, sequence-intrinsic PROXY (🟡)**,
-NOT a trained allele-specific predictor and NOT a patient-HLA-specific magnitude (a known-unknown). Whether those
-epitopes drive ADA depends on self-tolerance (foreign vs self) — handled in `ada_risk`.
+PRODUCTION method (v6.9.1/6.9.2): the **real, licensed NetMHCIIpan-4.0** eluted-ligand %Rank over a frequent HLA-II
+panel (residue-coverage metric), run on the VM with only the derived fractions cached (`configs/mhc_epitope_oracle.yaml`);
+`mhc2_epitope_load(seq, name)` returns the real value for a cached antigen and otherwise **abstains** (no production
+proxy). A population-level proxy (🟡), never a patient-HLA-specific magnitude (a known-unknown). Whether the epitopes
+drive ADA depends on self-tolerance (foreign vs self) — handled in `ada_risk`.
+
+OFFLINE-ONLY fallback (`mhc2_proxy_estimate`, NOT the production axis): a documented, dependency-free PROMISCUOUS-
+binder density — MHC-II presents a 9-mer core in an open groove whose **P1 pocket** is the dominant hydrophobic
+anchor (M/F/Y/W/L/I/V; Stern & Wiley, Nature 1994) with secondary pockets at P4/P6/P9 (Southwood 1998). Provided for
+offline triage where NetMHCIIpan cannot be run; the production axis uses the real tool and abstains otherwise.
 """
 from __future__ import annotations
 
@@ -75,8 +78,9 @@ def real_mhc2_load(name: str) -> dict | None:
             "n_covered": rec.get("n_covered"), "length": rec.get("length"),
             "method": f"NetMHCIIpan-4.0 EL %Rank<=2, {rec.get('metric', 'residue coverage')}, frequent HLA-II "
                       f"panel ({len(panel)} alleles): {', '.join(panel)}",
-            "status": "population-level (frequent-HLA panel; NetMHCIIpan-4.0 eluted-ligand), OOD-gated; NOT a "
-                      "patient-HLA-specific magnitude (known-unknown)", "backend": "netmhciipan_cache"}
+            "status": "population-level (frequent-HLA panel; NetMHCIIpan-4.0 eluted-ligand), coverage-gated "
+                      "(abstains for uncached antigens — NOT a distributional OOD gate); NOT a patient-HLA-specific "
+                      "magnitude (known-unknown)", "backend": "netmhciipan_cache"}
 
 
 def mhc2_epitope_load(seq: str, name: str | None = None) -> dict:

{pen_stack-6.9.2 → pen_stack-6.10.1}/pen_stack/server/api.py

@@ -208,6 +208,26 @@ def immune_endpoint(design: dict):
     return immune_profile(design)
 
 
+@app.post("/offtarget", tags=["v6.10 off-target"])
+def offtarget_endpoint(req: dict):
+    """v6.10 PEN-OFFTGT cross-family off-target NOMINATION (NOT a clearance). Body:
+    {writer_family, guide?, candidate_sites?, sequence?, accessibility?, assay?}. Returns ranked candidate
+    off-targets with a real-data mismatch-calibrated risk band + the recommended validation assay; abstains
+    without inputs and never fabricates sites."""
+    from pen_stack.wgenome.offtarget_predict import nominate_offtargets
+    return nominate_offtargets(
+        req.get("writer_family", ""), guide=req.get("guide"), candidate_sites=req.get("candidate_sites"),
+        sequence=req.get("sequence"), accessibility=req.get("accessibility"),
+        target_core=req.get("target_core"), assay=req.get("assay", "guideseq"))
+
+
+@app.get("/offtarget/assay", tags=["v6.10 off-target"])
+def offtarget_assay_endpoint(writer_family: str):
+    """v6.10 validation-assay recommendation for a writer family (the assay that would confirm a nomination)."""
+    from pen_stack.wgenome.offtarget_assay import recommend_assay
+    return recommend_assay(writer_family)
+
+
 @app.post("/generate", tags=["v6.1 AI surface"])
 def generate_endpoint(req: dict):
     """v5.8 generative designer: verifier-as-discriminator. Body: {goal?, candidates?, keep?}. Hazardous/illegal

{pen_stack-6.9.2 → pen_stack-6.10.1}/pen_stack/twin/position_effect.py

@@ -150,7 +150,8 @@ class PositionEffectModel:
         lo, hi = self.conformal.interval(yhat, sigma=widen)
         return {"yhat": yhat, "lo": lo, "hi": hi, "ood_widen": widen,
                 "nominal_coverage": 1 - self.conformal.alpha,
-                "interval_kind": "trained split-conformal (chromosome-Mondrian), OOD-widened"}
+                "interval_kind": "trained split-conformal (chromosome-blocked OOF; per-chromosome Mondrian qhats "
+                                 "computed, GLOBAL qhat served — a query has no chromosome at serve time), OOD-widened"}
 
     # ---- persistence ----------------------------------------------------------------------------
     def save(self, path: str | Path) -> str:

{pen_stack-6.9.2 → pen_stack-6.10.1}/pen_stack/validate/immune_calibration.py

@@ -96,9 +96,10 @@ AXIS_STATUS: dict[str, dict] = {
                   "rates (and induced post-dose-1 anti-PEG is a separate dynamic)."},
     "mhc2_writer": {        # v6.9 G-WS1 — CD4/MHC-II epitope load over the writer enzyme
         "status": "mechanistic_proxy",
-        "label": "mhc2_writer: mechanistic/population proxy — NOT outcome-validated",
-        "reason": "sequence-intrinsic promiscuous MHC-II binder density (P1-anchor; Stern & Wiley 1994) is a "
-                  "presentation potential, NOT a trained allele-specific predictor or a patient-HLA magnitude."},
+        "label": "mhc2_writer: population proxy — NOT outcome-validated",
+        "reason": "real NetMHCIIpan-4.0 eluted-ligand MHC-II epitope load over a frequent-HLA panel (v6.9.2; the "
+                  "v6.9.0 P1-anchor proxy was replaced) — a population-level presentation potential, NOT a "
+                  "patient-HLA-specific magnitude or an observed-ADA-validated number."},
     "ada_writer": {        # v6.9 G-WS2 — ADA-risk (MHC-II x foreignness, self-tolerance filtered)
         "status": "mechanistic_proxy",
         "label": "ada_writer: mechanistic/population proxy — NOT outcome-validated",

pen_stack-6.10.1/pen_stack/wgenome/offtarget_assay.py

@@ -0,0 +1,55 @@
+"""Validation-assay recommender for off-target nomination (v6.10 PEN-OFFTGT, E-WS3).
+
+Nomination is NOT a clearance — it ships with the empirical assay that would CONFIRM the candidates. This maps a
+writer family to the appropriate unbiased genome-wide assay(s) + their documented applicability, grounded in the
+validated assay literature. For families with no published genome-wide off-target assay (bridge recombinases) it
+says so honestly and recommends targeted confirmation, never implying a clearance exists.
+"""
+from __future__ import annotations
+
+# assay applicability by writer family (validated citations in offtarget_data.ASSAY_PROVENANCE)
+_NUCLEASE_ASSAYS = [
+    {"assay": "GUIDE-seq", "setting": "cell-based (in cellulo)", "doi": "10.1038/nbt.3117",
+     "use": "captures off-targets in the actual chromatin/cell context (dsODN tag at DSBs)"},
+    {"assay": "CHANGE-seq", "setting": "in vitro, high-throughput", "doi": "10.1038/s41587-020-0555-7",
+     "use": "most sensitive genome-wide nomination; pair with a cell-based assay to filter chromatin-masked sites"},
+    {"assay": "CIRCLE-seq", "setting": "in vitro (cell-free)", "doi": "10.1038/nmeth.4278",
+     "use": "highly sensitive in vitro confirmation; over-nominates vs cell context"},
+]
+
+
+def recommend_assay(writer_family: str) -> dict:
+    """Recommend the empirical validation assay(s) for a writer family + the documented expected sensitivity, or
+    an honest 'no genome-wide assay exists' for data-thin families. Always frames nomination as not a clearance."""
+    fam = (writer_family or "").lower()
+    if "cas9" in fam or "nuclease" in fam or fam in {"spcas9", "sacas9", "cas12a", "ascas12a", "nickase"}:
+        return {"family": writer_family, "writer_class": "RNA-guided nuclease (DSB)",
+                "recommended": _NUCLEASE_ASSAYS,
+                "strategy": "nominate in vitro (CHANGE-/CIRCLE-seq, high sensitivity) THEN confirm the survivors "
+                            "in the target cell type (GUIDE-seq) — chromatin masks a fraction of in vitro sites",
+                "available": True,
+                "note": "nomination ranks CANDIDATES; an empirical assay is required for clearance."}
+    if "integrase" in fam or "paste" in fam or "passige" in fam or "bxb1" in fam or "phic31" in fam:
+        return {"family": writer_family, "writer_class": "large serine integrase",
+                "recommended": [{"assay": "Cryptic-seq / HIDE-seq", "setting": "unbiased LSI off-target discovery",
+                                 "doi": "10.1101/2024.08.23.609471",
+                                 "use": "the genome-wide unbiased assay for serine-integrase cryptic attB sites "
+                                        "(Tome Biosciences, 2024 preprint)"}],
+                "strategy": "scan cryptic pseudo-attB (this engine) THEN confirm by Cryptic-seq/HIDE-seq; "
+                            "quantitative prediction (IntQuery) is paper-only (no public weights)",
+                "available": True,
+                "note": "LSI off-target assays are recent preprints; coverage is single-company / largely Bxb1."}
+    if "bridge" in fam or "is110" in fam or "is621" in fam or "seek" in fam or "iscro4" in fam:
+        return {"family": writer_family, "writer_class": "bridge recombinase (IS110/IS621 RNA-guided)",
+                "recommended": [{"assay": "targeted amplicon / capture sequencing at nominated pseudosites",
+                                 "setting": "targeted", "doi": None,
+                                 "use": "confirm individual nominated pseudosites (no genome-wide unbiased "
+                                        "off-target assay exists for bridge recombinases yet)"}],
+                "strategy": "nominate pseudosites with the Perry-DMS engine THEN confirm by targeted sequencing; "
+                            "an unbiased genome-wide bridge off-target assay is an open need",
+                "available": True,
+                "note": "HONEST GAP: bridge-recombinase off-target is essentially unmodeled — there is NO "
+                        "published genome-wide unbiased assay or predictor (verified). Treat nominations as "
+                        "high-uncertainty / extrapolative; do not read as clearance."}
+    return {"family": writer_family, "available": False,
+            "note": f"no off-target assay applicability rule for family {writer_family!r}"}