STRC Research

STRC PE Phase 3_5 STRCP1-Aware Redesign

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STRC PE Phase 3.5 STRCP1-Aware Redesign

Phase 3.5 was queued after STRC PE Phase4 STRCP1 Paralog Off-Target showed 0/61 Phase-3 pegRNAs discriminate STRC from STRCP1 paralog. The Phase 3.5 task: retry candidate selection with ≥2 seed-region mismatches vs STRCP1 required. Result: STRCP1 paralog 18 bp core at the variant locus is 100% identical to STRC. Geometric impossibility, not a filter deficit.

Input

  • STRC c.4976A>C (E1659A) on-target window: chr15:43,600,548-43,600,567 (+/-40 bp flanks)
  • STRCP1 paralog off-target window: chr15:43,700,346-43,700,363 (+/-40 bp flanks)
  • Phase-4 guide set: 61 pegRNA candidates across 6 Cas PAM variants, all failed Phase 4 on STRCP1 cross-hybridization

Method

Pure-numpy sequence alignment + seed mismatch count per Cas variant:

  1. Pull both genomic windows from hg38_chr15.fa.
  2. For each of the 61 Phase-4 protospacers: slide across STRCP1 window (+ and - strand), find best-aligning position.
  3. Count mismatches restricted to the PAM-proximal seed region (positions 9-20 for SpCas9/SpG/enCas9/SpRY/SpCas9NG, positions 10-21 for SaCas9).
  4. Gate: ≥ 2 seed-region mismatches required to discriminate.

Result

STRCP1 vs STRC core sequence identity

STRCP1 core (chr15:43,700,346-43,700,363): CCCCACCTGCTATGGTGC
STRC best-aligning slice:                    CCCCACCTGCTATGGTGC
Mismatches: 0 / 18 — 100% identity in the 18 bp variant window

Zero divergence between paralog and gene at the PE target locus.

Per-guide seed-mismatch distribution vs STRCP1

Seed mm countn guides
061
≥10

Every single Phase-3 discriminating protospacer has 0 seed mismatches against STRCP1. Not a “filter would have saved some” situation — the design space is empty at this locus.

Why this is the clean kill

The STRC STRCP1 GTEx Expression Check (2026-04-22) already showed STRCP1 is transcribed in all 54 GTEx tissues — so “STRCP1 silent in OHC” escape path is false. Now Phase 3.5 shows the sequence-level escape path is also closed: at the PE target locus, STRCP1 is not merely paralogous to STRC, it is identical in the 18 bp window + flanking seed region.

This means:

  • No pegRNA targeting c.4976 in STRC can avoid simultaneously editing STRCP1.
  • PE3b strategy (which requires mismatch-based strand discrimination) relies on seed-region discrimination — impossible here.
  • The only remaining PE escape paths are (a) accept dual-edit + characterise STRCP1 consequences, or (b) move to an entirely different STRC target locus outside the identity window, which loses the E1659A variant specificity.

Escape paths (all require abandoning c.4976 or accepting off-target)

Path A — Accept dual-edit, prove STRCP1 is functionally silent

  • PE will edit both STRC c.4976 and STRCP1’s equivalent position simultaneously.
  • If STRCP1 produces no protein (Ensembl biotype: transcribed_unprocessed_pseudogene), dual-edit is harmless.
  • Verification required: functional assay showing STRCP1 does not translate. GTEx transcription ≠ translation. Needs ribosome-profiling or mass-spec of STRCP1 peptides.
  • Risk: if STRCP1 produces low-abundance truncated protein that assembles into cochlear ECM, dual-edit creates a new haploinsufficiency + chimeric transcript.

Path B — Target a different STRC window

  • Find a locus in STRC CDS that IS divergent from STRCP1 ≥ 20 bp, and which still provides pegRNA access to c.4976. Likely impossible — the whole exon 19 region (where c.4976 sits) is in the high-identity zone.
  • Alternative: edit regulatory / splicing regions of STRC upstream of the variant. But this doesn’t fix E1659A — would only modulate expression, which is the wrong mechanism.

Path C — Abandon PE for Misha, work on Mini-STRC AAV

  • This is the sensible call given STRC Mini-STRC Single-Vector Hypothesis at S-tier with Pasteur/Holt/Regeneron validation, and the existing Shanghai Shu Yilai lab collaboration building a c.4976 humanized knock-in mouse.
  • PE stays academic interest only — study for other STRC loci in different patients, not Misha’s variant.

Ranking delta

Prime Editing for STRC (#7): Tier B → C.

Scoring:

  • Mechanism 4 → 3: PE chemistry sound but Misha’s c.4976 locus is not tractable
  • Delivery 2 → 2: unchanged (still dual-AAV + pegRNA challenge if ever pursued)
  • Misha-fit 2 → 1: STRCP1 identity disqualifies his specific variant
  • min(3, 2, 1) = 1 → C-tier (or D — borderline with the kill list)

Status: paused. Candidate pool = 0 at the c.4976 window. Not killed outright because Path A (characterize STRCP1 translation) is a clean unblocker if a collaborator wants to pursue it — could resurrect PE for all DFNB16 patients, not just Misha. Requires ribosome-profiling or mass-spec evidence. Not computational — wet-lab or collaborator-dependent.

STRC ASO Exon Skipping (#8) stays C — same STRCP1 identity blocker applies for splice-switching ASOs, per STRC ASO Phase2 STRCP1 Paralog Cross-Hybridization. The morpholino chemistry escape path from that note is the only ASO direction left (RNase-H-free steric block).

No other hypothesis affected.

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