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Prime Editing Design - Usage Guide

Overview

Designs pegRNAs and nicking guides for prime editing (PE) -- scarless point mutations, small insertions/deletions, and any of the 12 base conversions without a double-strand break or donor. The skill chooses the nick/strand, designs the PBS and RTT as a per-locus panel (there is no universal optimum), selects the PE system (PE2/PE3/PE3b/PE4/PE5/ PEmax/PE7), adds the free wins (PAM-disrupting and MMR-evading silent edits, a tevopreQ1 3' motif), ranks with PRIDICT/DeepPrime, and routes large insertions to twinPE/PASTE. Its discipline is that PE efficiency is a cellular-genetics problem (flap resolution + mismatch repair), so the system choice and a tested panel matter more than any single oligo.

Prerequisites

pip install biopython
# PrimeDesign: Docker-only (no pip) -- docker run ... pinellolab/primedesign primedesign_cli ...
#   Edit is encoded inline: (ref/edit) substitution, (+ins) insertion, (-del) deletion.
# PRIDICT2.0 / DeepPrime: web servers / authors' code for ranking pegRNA panels.

Quick Start

Tell your AI agent what you want to do: - "Design a pegRNA to correct the G551D mutation in CFTR" - "Give me a PBS x RTT panel to test for this edit, not just one pegRNA" - "My pegRNA edits at 2% -- what system or design changes would help?" - "Should I use PE3 or PE3b here, and how do I avoid indels?" - "Insert a 2 kb reporter -- is prime editing the right tool?"

Example Prompts

Point mutations and small edits

"Design pegRNAs to install the protective APOE Christchurch variant. Give me a PBS x RTT panel, add a PAM-disrupting silent edit if possible, and tell me which PE system to use in MMR-proficient iPSCs."

"Correct the sickle-cell mutation (HBB E6V) with prime editing -- and explain whether a base editor would be a better choice for this conversion."

System and efficiency

"My PE3 design gives good editing but lots of indels. How do I fix that?"

"This locus is GC-rich -- how should that change my PBS length?"

"We're in HCT116 -- do I still benefit from MLH1dn (PE4/PE5)?"

Large edits

"I need to knock in a 1.5 kb cassette -- route me to the right method."

What the Agent Will Do

  1. Establish the edit, cell type, MMR status, and delivery (expressed vs synthetic pegRNA).
  2. Decide whether a base editor (transition) or integrase method (large insert) is the better tool before committing to PE.
  3. Choose the nick/strand and design a PBS x RTT panel, enforcing the 5'-G-prepend and don't-start-on-C rules.
  4. Add the free wins: a PAM-disrupting silent edit and 1-2 MMR-evading silent edits.
  5. Select the PE system by the axis the problem needs (MMR -> PE4/PE5; indels -> PE3b; stability -> epegRNA/PE7).
  6. Rank the panel with PRIDICT2.0/DeepPrime (a prior, not a verdict) and recommend testing.
  7. For PE3/PE3b, design the second nicking guide at ~40-100 bp on the appropriate strand.

Tips