DOI: 10.5555/peerrxiv.2026.00431.v1 · STATUS: Preprint Archive (No Open Review) · ROUND: 1 · BAYESIAN QUALITY SCORE: 0/5.00 (Wilson 95% LB: 0)

Structure-Guided Engineering of Compact Cas13d Ribonucleoproteins for High-Fidelity Transcriptome Editing

Public Co-Authors & Pooled Credit Contributors:
Dr. Chidi Okafor (University of Cambridge, ORCID: 0000-0002-6610-4982, +0 Cr)

1. Abstract

Collateral trans-cleavage of bystander host transcripts limits the clinical translation of RNA-targeting CRISPR-Cas13 effectors. Using cryo-EM structure-guided mutagenesis of the HEPN1–HEPN2 catalytic cleft, we engineer a high-fidelity variant (hfCas13d-Max) that eliminates detectable transcriptome-wide collateral cleavage while preserving >88% on-target knockdown in human iPSC-derived cardiomyocytes.

2. Side-by-Side Manuscript Evolution: Before (v1) vs. After (Pending)

BEFORE MANUSCRIPT (v1 · 10.5555/peerrxiv.2026.00431.v1)

1. Cryo-EM Architecture of the HEPN Catalytic Pocket

We resolved the ternary complex at 2.6 Å resolution, identifying three electropositive residues (R295, K641, H712) that stabilize non-specific bystander RNA binding.

AFTER MANUSCRIPT (Awaiting Author Revision)

Pending author upload of revised After Manuscript.

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No peer reviews published yet (Route 1 Normal Preprint Archive).

5. Cryptographic SHA-256 Quorum Decision Certificate (qr-v2.1)

Status: NORMAL_PREPRINT · Gate A (Zero Fatal Flaws): PASSED · Round 1 Endorsements: 0/0

SHA-256 Decision Digest:

442b648e4a6177ca4536f6aa72e105c0f5843cc414b3db3d7be8d730ce0e18ac