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HDR Template Design - Usage Guide

Overview

Designs donor/repair templates for precise CRISPR knock-ins -- ssODN, long-ssDNA (Easi-CRISPR), dsDNA/plasmid, and AAV6 donors. The skill picks the format by edit size and whether the target cell cycles, sizes the homology arms, requires a guide that cuts within ~10 bp of the edit, and adds a mandatory codon-checked blocking (PAM/seed) mutation so the corrected allele is not re-cut. It frames the HDR-vs-NHEJ-vs-MMEJ pathway competition and the HITI/HMEJ/PITCh alternatives for cells where HDR is the wrong tool. Its central discipline: HDR is the minority pathway, and a donor without a blocking mutation is self-destructing.

Prerequisites

pip install biopython primer3-py
# primer3-py designs primers (primer3.bindings.design_primers); arm extraction and codon-aware
#   blocking are the skill's own BioPython code. Design arms/guide against the ACTUAL cell-line
#   sequence, not GRCh38. lssDNA production (Easi-CRISPR) and AAV donors are wet-lab methods.

Quick Start

Tell your AI agent what you want to do: - "Design an ssODN to correct this point mutation, with a blocking mutation" - "Add a GFP tag to MYC -- which donor format should I use?" - "My HDR gives mostly indels and almost no edit -- what's wrong?" - "I'm knocking in to neurons -- how should the donor differ?" - "The nearest guide cuts 25 bp from my edit -- is HDR still the right tool?"

Example Prompts

Point mutations and tags

"Design an ssODN to install the HBB sickle-cell correction. Confirm a guide cuts within 10 bp, add a synonymous PAM-blocking mutation so the allele isn't re-cut, give me both strands to test, and add phosphorothioate caps."

"Add a C-terminal FLAG tag to my gene. Pick the donor format and explain why, and design junction-genotyping primers."

Format and pathway

"I'm knocking a 1.5 kb cassette into primary T cells -- ssODN, plasmid, lssDNA, or AAV?"

"Why does dsDNA donor kill my iPSCs, and what should I use instead?"

"I need a knock-in in post-mitotic neurons -- HDR isn't working. What now?"

Troubleshooting

"Low HDR, lots of indels -- walk me through the likely cause."

"Only the silent mutation got incorporated, not my edit -- why?"

What the Agent Will Do

  1. Establish the cell type (cycling vs post-mitotic) and edit size -- this picks HDR vs HITI/HMEJ and the donor format.
  2. Confirm a guide cuts within ~10 bp of the edit; if not, recommend a closer guide or base/prime editing.
  3. Choose the format (ssODN / lssDNA / dsDNA / plasmid / AAV6) and size the arms.
  4. Add a mandatory codon-checked blocking mutation (synonymous PAM disruption preferred; silent seed mutations otherwise).
  5. For ssODN, emit both strands and symmetric/asymmetric variants to test, and recommend phosphorothioate end-protection.
  6. Suggest HDR enhancers as ranked experiments (cell-cycle timing / cold shock / M3814 / 53BP1 first).
  7. Design arm-amplification and junction-genotyping primers, and recommend screening enough clones.

Tips